a yay ss odes bo pa a pias Sri CORNELL UNIVERSITY. THE Roswell MP. Flomer Library THE GIFT OF ROSWELL P. FLOWER FOR THE USE OF THE N. Y. STATE VETERINARY COLLEGE. 1897 Cornell University Library QH 581.H46 germ-cell cycle in animals. ! THE GERM-CELL CYCLE IN ANIMALS EN ee THE MACMILLAN COMPANY NEW YORK - BOSTON + CHICAGO - DALLAS ATLANTA + SAN FRANCISCO MACMILLAN & CO., Limirep LONDON + BOMBAY + CALCUTTA MELBOURNE THE MACMILLAN CO. OF CANADA, Lr. TORONTO THE GERM-CELL CYCLE IN ANIMALS BY ROBERT W. HEGNER, Pu.D. ASSISTANT PROFESSOR OF ZOOLOGY IN THE UNIVERSITY OF MICHIGAN AUTHOR OF “AN INTRODUCTION TO ZOOLOGY” AND “COLLEGE ZOOLOGY” New Bork THE MACMILLAN COMPANY 1914 All rights reserved Yip, 4 14 f- Zz CoprnigHt, 1914, . By THE MACMILLAN COMPANY. & x Ope zk Set up and electrotyped. Published September, rgrq. cm + Nortosobd Press J. 8. Cushing Co. — Berwick & Smith Co. Norwood, Mass., U.S.A. PREFACE Tuts book is the result of a course of lectures delivered during the past school year before a class in Cellular Biology at the University of Michigan. Many of the most important recent additions to our knowledge of heredity have resulted from the study of the germ cells, especially those of animals. This study is now recognized as one of the chief methods of attacking certain problems in genetics and must be employed in correlation with animal breeding before we can hope to obtain an adequate explanation of the results of hybridization. For- tunately the cytological studies of the germ cells, both observational and experimental, have kept pace with the rapid advances in our knowledge of plant and animal breeding which have been made since the rediscovery of Mendel’s investigations in 1900. The term ‘‘Germ-Cell Cycle”? is meant to include all those phenomena concerned with the ori- gin and history of the germ cells from one genera- tion to the next generation. The writer has, with few exceptions, limited himself to a consideration of the germ cells in animals because the cycle is here more definite and better known than in plants. It is obvious to any one familiar with this subject that only a few of the many interesting phases of v vi PREFACE the problems involved can be considered in a work of this size, and those for which space can be found must be limited in their treatment. For this reason some periods in the germ-cell cycle are only briefly mentioned, whereas others are more fully discussed. The latter are naturally those in which the writer is most interested and with which he is best ac- quainted. Furthermore, the attempt is made to present the data available in such a way as to make it intelligible to those who have not been able to follow in detail the progress of cytology during the past few years. This can only be accomplished by introducing many facts that are well known to cytologists and zodlogists in general, but are neces- sary for the presentation of a complete account of the subject. Much of the recent cytological work done on germ cells has emphasized the events which take place during the maturation of the eggs and spermatozoa, that is, the periods of odgenesis and spermatogenesis. These are, of course, very important phases of the germ-cell cycle, but they should not be allowed to overshadow the rest of the history of the germ cells. Contrary to the usual custom, the period that is emphasized in this book is not the maturation of the germ cells, but the segregation of the germ cells in the developing egg and the visible substances (keimbahn-determinants) concerned in this process. It has been impossible to include in this book as much illustrative material as desirable, but the bib- liography appended indicates what data exist and PREFACE vii where they may be obtained. This list of publica- tions has been arranged according to the method now in general use among zodlogists; the author’s name and the date of the appearance of the contri- bution in question are bracketed in the text wher- ever it has been considered necessary, and reference to the list at the end of the book will reveal the full title and place of publication of the work, thus avoiding cumbersome footnotes. The figures that have been copied or redrawn are likewise referred in every case to the original source. Many of them have been taken from the writer’s previous publica- tions and a few have been made especially for this work. The writer has likewise drawn freely upon the text of his original investigations already published. Ann Arzor, MICHIGAN, April 16, 1914. TABLE OF CONTENTS CHAPTER I PAGE INTRODUCTION . ; é ; 1 The Cell, 2; Cell Division, 13; Methods of Repro- duction, 17; The Germ Cells, 19; The Life Cycles of Animals, 22. CHAPTER II GENERAL ACCOUNT OF THE GERM-CELL CYCLE IN ANI- Protozoa, 25; Metazoa, 28. CHAPTER III Tue GerM-ceLL CyYcLE IN THE Pa#DOGENETIC FLy, Miastor . 2 , ‘ : : : » 6b CHAPTER IV Tue SEGREGATION OF THE GERM CELLS IN SPONGES, Caz- LENTERATES, AND VERTEBRATES. . 69 1. Porifera, 69. 2. Coelenterata, 80. 3. Verte- brata, 98. CHAPTER V THE SEGREGATION OF THE GERM CELLS IN THE ARTHRO- PODA ‘i : 3 : : - 3 - 106 1. The Keimbahn in the Insects, 106; Diptera, 107; Coleoptera, 109 (In Chrysomelid Beetles, 109; Origin of Nurse Cells, 119; Cyst Formation in Testis, 125; Amitosis, 133; Differentiation of Nuclei in Egg, 141) ; Hymenoptera, 143. 2. The Keimbahn in the Crusta- cea, 163. ix x TABLE OF CONTENTS CHAPTER VI Tue SEGREGATION OF THE GERM CELLS IN NEMATODES, Sacirra, AND OTHER MeEtTAzoa 1. The Keimbahn in the Nematodes, 174. 2. The Keimbahn in Sagitta, 179. 3. The Keimbahn in Other Animals, 183. CHAPTER VII Tue Germ CELLS oF HERMAPHRODITIC ANIMALS CHAPTER VIII KeEIMBAHN-DETERMINANTS AND THEIR SIGNIFICANCE A. The Genesis of the Keimbahn-Determinants, 211 (a, Nuclear, 213; b, Cytoplasmic, etc., 224; c, Dis- cussion, 228). B. The Localization of the Keimbahn- Determinants, 235. C. The Fate of the Keimbahn- Determinants, 240. CHAPTER IX Tur CHromosomMEs AND MitocuHonpria oF GERM CELLS The Chromosome Cycle in Animals, 245. The Mito- chondria of Germ Cells, 275. CHAPTER X Tue Germ-pLasm THEORY REFERENCES TO LITERATURE . InpEx oF AUTHORS . INDEX OF SUBJECTS . 189 211 290 311 337 341 THE GERM-CELL CYCLE IN ANIMALS GERM-CELL CYCLE IN ANIMALS CHAPTER I INTRODUCTION Since the enunciation by Harvey of the aphorism Omne vivum ex ovo in the seventeenth century, the statement has frequently been made that every animal begins its individual existence as an egg. While this is not strictly true, since no eggs occur in the life history of many one-celled animals (PRo- Tozoa), and a large number of multicellular animals (Merazoa) are known to develop from buds or by fission, still the majority of animals arise from a single cell — the egg (Fig. 4, A). In most cases this egg, or female sex-cell, is unable to develop in nature unless it is penetrated by a spermatozoén or male sex-cell (Fig. 4, B). The single cell resulting from the fusion of an egg and a spermatozoién is known as a zygote. One of the most remarkable of all phenom- ena is the development of a large, complex organism from a minute, and apparently simple, zygote. According to the older scientists, a miniature of the adult individual was present in the egg, and devel- opment consisted in the growth and expansion of B 1 2 GERM-CELL CYCLE IN ANIMALS rudiments already preformed. This belief could not continue to exist after Caspar Wolff’s brilliant researches proved that adult structures arise grad- ually from apparently undifferentiated material ; that is, development is epigenetic. Epigenesis, however, does not explain development; it simply maintains that it occurs. During the years since the theory of epigenesis was proposed a new theory of preformation has entered into our conception of development, a theory which we may designate as predetermination. We know from our microscopical studies that the germ cells possess a certain amount of organization, and that the zygote contains certain structures con- tributed by the egg and other structures brought into the egg by the spermatozoén. Hence, to a certain extent, development is predetermined, since the initial structure of the zygote determines the characteristics of the individual that arises from it. On the other hand, development is also epigenetic, and our modern conception includes certain features of each theory. Tue Ceiu. A brief account of the structure, physics, and chemistry of the cell will serve to give us some idea of the condition of the zygote from which the individual arises, and will help us to understand certain events in the germ-cell cycle to be discussed later. The cell is the simplest particle of matter that is able to maintain itself and reproduce others of its kind. The term ‘cell’ was applied by Hooke in 1665 to the cell-like compartments in cork. Cells filled INTRODUCTION 3 with fluid were slightly later described by Malpighi. In 1833 Robert Brown discovered nuclei in certain plant cells. What is known now as the Ceti THEORY is usually dated back to the time of the botanist Schleiden (1838) and the zodlogist Schwann (1839), whose investigations of the cellular phenomena in animals and plants added greatly to the knowledge of these units of structure. At this time the cell- wall was considered the important part of the cell, but continued research proved this idea to be erro- neous. Schleiden called the substance within the cells plant slime. Later (1846) von Mohl gave the term protoplasm to the same substance. The substance within the animal cell was named sarcode by Du- jardin. The similarities between the protoplasm of plants and the sarcode of animals were noted by Cohn, and animal cells without cell-walls were observed by Kolliker (1845). It was not, however, until 1861 that Max Schultze finally established the fact that plant protoplasm and animal sarcode are essentially alike, and defined the cell as a mass of protoplasm containing a nucleus. Schultze’s re- searches serve as the starting point for modern studies of cellular phenomena, but the definition furnished by him must be modified slightly, since we now know that many cells exist without definite nuclei. These cells, however, are provided with nuclear material scattered throughout the cell body (the so-called distributed nucleus). Our definition must be changed to read, a cell is a mass of proto- plasm containing nuclear material. Changes like- A GERM-CELL CYCLE IN ANIMALS wise have taken place in the Cell Theory; we no longer consider cells as isolated units and the multi- cellular animal as equivalent to the sum of its con- stituent cells, but recognize the influence of the cells upon one another, thus reaching the conclusion that the metazoén represents the sum of the individual cells plus the results of cellular interaction. Cells vary considerably in size, ranging from those we call Bacteria, which may be no more than 33359 of an inch in length, to certain egg cells which are several inches long; the latter, however, owe their enormous size to the accumulation of nutritive sub- stances within them. An average cell measures about gz55 of an inch in diameter. Cells vary in shape as well as in size; egg cells are frequently spherical, but most cells are not, since they are sur- rounded by other cells which press against them. A diagram of a typical cell is shown in Fig. 1. Authorities are not agreed as to the structure of protoplasm; to some it appears, as shown in Fig. 1, to consist of a network of denser fibers called spon- gioplasm (s) traversing a more liquid ground substance, the hyaloplasm. Others consider proto- plasm to be alveolar in structure, thus resembling an emulsion, whereas another group of zodlogists maintain that while protoplasm may appear to be fibrillar or alveolar, its essential basis consists of multitudes of minute granules. Wilson’s view is the one usually adopted at the present time; that is, the protoplasm of the same cell may pass suc- cessively ‘“‘through homogeneous, alveolar, and INTRODUCTION 5 fibrillar phases, at different periods of growth and in different conditions of physiological activity,” and that “apparently homogeneous protoplasm is a complex mixture of substances which may assume Fic. 1.— Diagram of o cell. as =attraction-sphere; c = centrosome; ch =chromatin reticulum; cr = chromidia; cc = ectoplasm; cn = en- doplasm; / = karyosome; J = linin; m = mitochondria; me = mcta- plasm; nm=nuclear membrane; p= plastid; p/ = plasmosome or nucleolus; s = spongioplasm; » = vacuole. various forms of visible structure according to its modes of activity.” The physical properties of protoplasm are not well known, since most of our studies have been made with fixed material. We know that protoplasm may exist as a gel or a sol, and that it is intermediate between true solids and true liquids, with many of 6 GERM-CELL CYCLE IN ANIMALS the properties of each and a number of properties peculiar to itself. No doubt the protoplasm differs in its physical nature in different cells. In the egg of the starfish, Asterias, Kite (1913) has shown that the cytoplasm is a translucent gel of comparatively high viscosity and is only slightly elastic; pieces become spherical when separated from the rest of the egg. Scattered throughout this gel are minute granules (microsomes) about 7/55 mm. in diameter which cannot be entirely freed from the matrix. What appear to be alveoli contain globules which possess many of the optical properties of oil drops; these are suspended in the living gel. The cyto- plasm of the starfish egg is not therefore alveolar in structure as usually stated, but is rather of the nature of a suspension of microsomes and globules in a very viscous gel. The nuclear membrane is a highly translucent, very tough, viscous solid, and not a delicate structure as ordinarily conceived. The nucleolus is a quite rigid, cohesive, granular gel suspended in the sol which makes up the rest of the nuclear material. Dividing male germ cells of cer- tain insects (squash bugs, grasshoppers, and crickets) revealed the fact that the chromosomes are the most highly concentrated and rigid part of the nuclear gel; that the spindle fibers are elastic, concentrated threads of nuclear gel; and that the metaphase spindle fibers seem to be continuous with the ends of the chromosomes. The ground substance of the nucleus is a sol termed nuclear sap or karyolymph. In the so-called ‘rest- INTRODUCTION 7 ing’ nucleus a network of fibers may be observed similar to the spongioplasm in the cytoplasm; these consist of a substance named linin because it usually occurs in threads (Fig. 1, 1). Distributed along the linin fibers are granules of a substance which stains deeply with certain dyes, and for this reason is known as chromatin (ch). These chromatin gran- ules may unite to form larger spherical masses, the karyosomes or chromatin-nucleoli (/), and during mitotic nuclear division constitute the chromosomes (Fig. 3, C). In many cells one or more bodies resembling the karyosomes somewhat, but differing from them chemically and physiologically, are pres- ent; these are the true nucleoli or plasmosomes (Fig. 1, pl). Embedded in the cytoplasm near the nucleus may often be seen a granular body, the centrosome (c), which is thought to be of great importance during mitotic cell division. The pro- toplasm surrounding the centrosome is usually a differentiated zone, the attraction-sphere (as), con- sisting of archoplasm. The chromatin which may be seen in the cytoplasm of certain cells is as a rule in the form of granules called chromidia (er). Cer- tain other cytoplasmic inclusions that have attracted considerable attention within the past fifteen years exist as granules, chains, or threads, and are known as mitochondria, chondriosomes, plastosomes, etc. (m). Various sorts of plastids (p), such as chloroplastids and amyloplastids, may be present, besides a varying number of solid or liquid substances, collectively designated as metaplasm (me) or paraplasm, which 8 GERM-CELL CYCLE IN ANIMALS are not supposed to form part of the living sub- stance; these are pigment granules, fat globules, excretory products, vacuoles (v), ete. It has been found possible to explain many cellular activities and even the results obtained by experi- mental animal breeding by studies of the physics and chemistry of protoplasm. An exhaustive ac- count of the subject is impossible and even unneces- sary here, but the importance assigned to the physico- chemical explanation of life phenomena requires a brief statement. Kossel has separated the cellular constituents into two main groups. (1) Primary constituents are those necessary for life; these are water, certain minerals, proteins, nucleoproteins, phosphatides (lecithin), cholesterin, and perhaps others. (2) Secondary constituents are not essen- tially necessary and do not occur in every cell; they are usually stored up reserve material or meta- bolic products representing principally what we have termed metaplasm. Water which constitutes about two-thirds of the animal is necessary for the solution of various bodies, the dissociation of chemical compounds, the exchange of materials, the removal of metabolic products, etc. Mineral substances are present in all animal tissues, and different tissues are characterized by the presence of different minerals. The principal ones are potassium, sodium, calcium, magnesium, iron, phosphoric acid, sulphuric acid, and chlorine. The other constituents are of a colloidal nature, and its richness in colloids is one of the chief charac- INTRODUCTION 9 teristics of protoplasm. To understand the activi- ties of protoplasm we must therefore know something of the physics and chemistry of colloids. Colloids (from colla = glue) do not diffuse, or diffuse very slowly, through animal membranes; in this respect they differ from crystalloids, which diffuse comparatively rapidly through animal mem- branes. Wolfgang Ostwald recognized two sorts of colloids: (1) suspension colloids, which are mix- tures of solid and liquid phases, are non-viscous, and easily coagulated by salts, e.g. a mixture of finely divided metal and water; and (2) emulsion colloids, which are composed of two liquid phases, are viscous, and coagulated by salts with difficulty. Protoplasm is rich in emulsion colloids; these may exist as liquid sols, or more solid gels. In either case they consist of fine colloidal particles. Accord- ing to another classification colloids may be separated into reversible and irreversible; the former may change from the sol to the gel state and back again, but the latter are unable to do this. Protoplasm is a reversible colloid, and many cellular structures appear to originate through the gelation of liquid colloids. Since protoplasm is a sol or gel due to water, it is a hydrosol or hydrogel, and because of its water content is said to be hydrophylic. It contains crystalloids and its chemical reactions take place in a dilute solution of electrolytes; these are substances which dissociate, at least in part, into their constituent ions when in solution, and the ions are electrically charged. For example, NaCl disso- 10 GERM-CELL CYCLE IN ANIMALS ciates into electro-positive Na ions (cations) and electro-negative Cl ions (anions). Colloidal par- ticles are likewise electrically charged, those of acid colloids usually negatively and those of alkaline colloids positively. The union and separation of particles and their consequent rearrangement cause gelation, liquefaction, etc.; it is thus evident that many physiological activities may be due to the electrical charges of ions instead of the chemical nature of the particles themselves. Cellular struc- tures therefore depend upon the tendency of col- loidal particles to form aggregates (gelation, coagula- tion), and more or less upon the electrically charged nature of the particles. The most characteristic chemical constituents of protoplasm are the proteins. The most common proteins in the body show on the average the follow- ing percentage of elements : — Carbon mts +s oo . 50 -55 % Hydrogen . . . . . - 6.5- 7.3% Nitrogen. . . . . hte - 15 -17.6% Oxygen io ee - 19 -24 % Sulphur... . . 2. ‘ 3- 24% Proteins may be separated into three groups: (1) simple proteins, such as protamines, albumins, and globulins; (2) conjugated proteins, the glucopro- teins, nucleoproteins, and chromoproteins; and (3) the products of protein hydrolysis, infraproteins, proteoses, peptones, and polypeptides. These have been studied both by microchemical and macro- chemical methods. In the former method reagents are applied to the microscopic objects and the INTRODUCTION 11 changes in color, etc., indicate its constitution ; e.g., iron and phosphorus may be detected in this way. Parts showing affinity for acid stains like eosin are said to be acidophile or oxyphile; those showing affinity for basic dyes, like methylene blue, are called basophile. The chromatin is _ basophile, whereas the linin and cytoplasm are oxyphile. In macrochemistry large quantities of the substances are collected and examined by ordinary laboratory methods. Because of the importance that has been assigned to the chromatin, this substance is particularly interesting. Chromatin consists of nuclein, which is a conjugated protein containing nucleic acid, the latter being an organic acid, rich in phosphorus; it is hence called nucleoprotein. Nucleoproteins are found chiefly in the nucleus but also occur in the cytoplasm. They may differ from one another in their protein content as well as in the character of their nucleic acid constituent. When treated with dilute acids nuclein is obtained, and when this is further subjugated to caustic alkali it decomposes into protein and nucleic acid. The nucleic acids which have been principally studied are those de- rived from the thymus gland, and from the sperma- tozoa of salmon, herring, and other fish; they are probably all the same. Levene (1910) recognizes three sorts of nucleic acid, of which the most complex is termed thymonucleic acid. This consists of two purine bases, guanine and adenine; two pyrimidine bases, thymine and cytosine; 12 GERM-CELL CYCLE IN ANIMALS a hextose (carbohydrate) ; and phosphoric acid. Its formula, according to Schmiedeberg, is CoHs6 NyO;,.2 P:O;, and according to Steudel, CusHs7 N,;0...2 P:O;. Considerable progress has been made, especially by Emil Fischer and his students, in the synthesis of protein-like bodies. Many com- plex polypeptides have been built up which resemble peptones in many of their reactions and when in- jected into living organisms appear to be utilized in metabolism in much the same way as are native proteins. We are still, however, very far from an adequate understanding of the nature of chromatin. Della Valle (1912), for example, after an exhaustive study of the physico-chemical properties of chromatin both in the resting nucleus and in the dividing cell, has concluded that this substance resembles that of fluid crystals. ‘‘Consequently all of the pheno- mena presented by the chromosomes; their mode of origin, differences in size, state of aggregation, form, structure, colorability, optical characteristics, varia- tions in form, longitudinal division and the phenom- ena which follow this mode of scattering, demon- strate that the chromosomes are crystalloids.” Two other primary constituents of protoplasm may be mentioned briefly. The phosphatide, lecithin, belongs with cholesterin to a group of compounds called lipoids. It consists of glycerophosphoric acid plus certain fatty acid radicles, such as stearic acid, oleic acid, etc., and a nitrogenous base (cholin). It INTRODUCTION 13 probably plays some part in cell metabolism, may furnish material for building up nucleins, and ac- cording to Fauré-Frémiet is concerned in the forma- tion of mitochondria. Cholesterin is considered a waste product of cell life, although it is known to in- hibit hemolysis produced by certain bodies and is thus a protective against toxins, and may have other functions. We should look forward with great interest to the results of investigations that are now being carried on by biochemists, since we depend upon them for an explanation of many of the phe- nomena of life, cellular differentiation, and heredity. We even hope that they may be able to create compounds in the laboratory that we may consider living organisms. However, the task does not seem to be so simple to the biochemist, who should know, as it does to the biologist. Nevertheless, as Jacques Loeb has said, we should “‘either succeed in producing living matter artificially, or find the reasons why this should be impossible.” Creuu Diviston. Cells may increase in number by direct (amitotic) or indirect (mitotic or karyokinetic) division. There is no doubt that mitosis occurs, but not all investigators are convinced that cells ever divide amitotically. Direct division was once considered the only method of cell multiplication. It was described as a simple division of the nucleus into two parts (Fig. 2), preceded by a division of the nucleolus into two, and succeeded by a constriction of the entire cell; the result was two daughter cells each with one nucleus containing one-half of the 14 GERM-CELL CYCLE IN ANIMALS nucleolus. As we shall see later (Chapter V), amitosis has been described in cells of the germ-cell cycle, and must therefore be reckoned with in any discus- sion of the phys- ical basis of heredity. Mitosis or ka- ryokinesis in- volves a rather complicated series of pro- cesses which cannot be fully discussed here but will be out- lined very briefly with the aid of Fig. 3. Fie. 2.— Amitosis. A. Division of blood-cells (@) Durin 8 in the embryo chick, illustrating Remak's the pro phase quote, ot mente toee ¢ Ariiee ihe chromatin division in the follicle cells of a cricket’s egg. granules which (From Dahlgren and Kepner, 1908.) are scattered through the nucleus in the resting cell (4) become arranged in the form of a long thread or spireme (B). At the same time the centrosomes move apart (4, ¢; B, a), and a spindle arises between them (C). While this is going on, the nuclear membrane generally disintegrates and the spireme segments into a num- ber of bodies called chromosomes (C); these take a position at the equator of the spindle, halfway be- INTRODUCTION 15 tween the centrosomes (D, ep). The stage shown in Fig. 3, D, is known as the amphiaster; at this time Fic. 3.— Mitosis. Diagrams illustrating mitotic cell division. (From Wilson.) A, resting cell; B, prophase showing spireme and nucle- olus within the nucleus and the formation of spindle and asters (a); C, later prophase showing disintegration of nuclear membrane, and breaking up of spireme into chromosomes; D, end of prophases, showing complete spindle and asters with chromosomes in equatorial plate (ep); E, metaphase — each chromosome splits in two; F, ana- phase — the chromosomes are drawn toward the asters, if = inter- zonal fibers; G, telophase, showing reconstruction of nuclei; H, later telophase, showing division of the cell into two. all of the mechanism concerned in mitosis is present. There are two asters, each consisting of a centrosome 16 GERM-CELL CYCLE IN ANIMALS surrounded by a number of radiating astral rays, and a spindle which lies between them. The chromo- somes lie in the equatorial plate (ep). (b) During the second stage, the metaphase, the chromosomes split in such a way that each of their parts contains an equal amount of chromatin (F, ep). As we shall see later, this is one of the most significant events that takes place during mitosis. (ec) During the anaphase (F) the chromosomes formed by splitting move along the spindle fibers to the centrosomes. As a result every chromosome present at the end of the prophase (D) sends half of its chromatin to either end of the spindle. The mechan- ism that brings about this migration is as yet some- what in question. Fibers are usually left between the separating chromosomes; these are known as interzonal fibers (F, if). (d) The telophase (G, H) is a stage of reconstruction from which the nuclei emerge in a resting condition ; the chromatin becomes scattered through the nucleus, which is again enveloped by a definite membrane (H); the centrosome divides and, with the centro- sphere, takes a position near the nucleus. Finally the cycle is completed by the constriction of the cell into two daughter cells. There are a number of differences between the sort of mitosis just described and that which occurs during the maturation of the egg and spermatozoun ; these and certain other phases of cell division will be considered in their appropriate places in succeed- ing chapters. INTRODUCTION 17 Metuops oF Repropuction. In the beginning paragraph of this chapter it was stated, with reserva- tions, that every individual develops from an egg. Before we can discuss the germ-cell cycle intelli- gently, however, we must consider the exceptions to this rule, and outline as briefly as possible the various methods of reproduction which are known to occur among animals. Reproduction is the forma- tion of new individuals by division ; this is frequently preceded by conjugation (in the Protozoa) or fertil- ization (in both the Protozoa and the MeErtazoa). Three principal methods of reproduction occur in the Protozoa. (1) Binary fission appears to be the most primitive. The individual divides into two parts which are similar in size and structure; these grow into cells like the original parent. Many Cruiata, FuaGeLuata, and Raurizopopa normally reproduce in this way. (2) Budding occurs when a small outgrowth or bud separates from the parent cell. This method occurs among the Suctorta, Rapiouaria, Heniozoa, Cmiata, and Myxosro- RIpIA. (3) Sporulation results from the division of the nucleus of the parent into many daughter nuclei and a subsequent division of the cell into as many “spores” as there are nuclei. This process is characteristic of the Sporozoa and also is found among the Ruizopopa. Conjugation is of frequent occurrence in the Protozoa. Two or more indi- viduals may become connected without fusion of nuclei or cytoplasm, thus forming colonies; a pair of individuals may unite either temporarily or per- Cc 18 GERM-CELL CYCLE IN ANIMALS manently with fusion of the cytoplasm only; or both cytoplasm and nuclei of such a pair may fuse or be interchanged. Merazoa reproduce either sexually or asexually. Asexual reproduction is reproduction without the aid of sex cells. It takes place as a rule by means of buds or by fission as in many polyps, sponges, flat-worms, segmented round-worms, and bryozoans. Even the tunicates, which occupy an advanced posi- tion in the animal series, form buds. Some of the sponges produce internal buds called gemmules, and certain bryozoans form similar bodies known as statoblasts. Sexual reproduction requires that the individual develop from a mature egg. Asarule the egg must be fertilized by the union with it of a spermatozooén, thus forming a zygote; but the eggs of many animals develop without being fertilized; that is, they are parthenogenetic. In rare cases such parthenogenetic eggs may be produced, as in the fly Miastor (see Chapter III), by immature individ- uals. When this occurs, reproduction is said to be pedogenetic. The sex of an animal is judged by the kind of sex cells it produces, — eggs by the female and sperma- tozoa by the male, — and when the individuals of a single species are differentiated as either males or females, the species is said to be diecious and the individuals gonochoristic. In many species there is but a single sort of individual which produces both eggs and spermatozoa; such species are monecious, and the individuals are hermaphroditic. INTRODUCTION 19 Tue Germ Criis. Eggs and spermatozoa differ from each other both morphologically and physiolog- ically. Eggs are usually spherical or oval in shape (Fig. 4), although they may vary greatly from the typical form and may even be ameboid as in certain coelenterates. In size they range from that of the mouse, which is only about 0.065 mm. in diameter, to that of birds, which are several inches long. The large volume of the latter is due to the presence of an enormous amount of nutritive material, and the general statement may be made that the size of an egg does not depend so much upon the size of the animal as upon the amount of yolk stored within it. The egg nucleus, which is frequently very large and clear, is known as the germinal vesicle; and its nucleolus has often been referred to as the germinal spot. Embedded within the cytoplasm of the ovum are several bodies besides the yolk globules. A “volk nucleus” may be present; mitochondrial granules or rods may occur; and special inclusions, which become associated with the primordial germ cells and have been named keimbahn-determinants, have been recorded in many cases. Considerable evidence has accumulated that the egg substance is not a homogeneous, isotropic mixture, but is def- initely organized, and that this organization is related to the morphology of the embryo which is to develop from it; hence we speak of the promor- phology of the egg. Eggs are said to possess polarity, and even the odgonium as it lies in the ovary is definitely oriented with respect to its chief axes. 20 GERM-CELL CYCLE IN ANIMALS The principal poles are dissimilar; the end of the egg containing most of the cytoplasm and nearer which lie the nucleus and centrosome is known as the animal pole; the other end, which is often crowded Fie. 4.—Germ cells. Ovarian ovum of a cat just before maturity. c.m, =cell membrane; mics. = microsomes; ncl = nucleolus; n. m= nuclear membrane; yk. al.=yolk alveoli. (From Dahlgren and Kepner.) with the yolk globules, is called the vegetative pole. The subject of the organization of the egg will be referred to more in detail later (Chapter VIII). The male sex cells or spermatozoa differ very strikingly from the eggs. They are usually of the INTRODUCTION 21 flagellate type (Fig. 4a), consisting of a head, largely made up of chromatin, a middle piece, and a vibratile tail. Spermatozoa are comparatively minute, rang- ing in size from those of Amphioxus, which are less than 0.02 mm. long, to those of the amphibian, Dvscoglossus, which reach a length of 2.0 mm. According to Wilson it would take from 400,000 to 500,000 sea urchin spermatozoa to equal in volume the egg of the same species. It is not surprising, therefore, to find that the num- ber of spermatozoa produced by a single male may be hundreds of thousands times as great as the number of eggs developed | inafemale. Eggs are, as a rule, incapable of locomotion, but spermatozoa are active, swim- ming about by means of their tails until they reach the passive eggs which they are to fertilize. i ; Fic. 4a.— Diagram of a Since generally only one sperm- flagellate spermatozodn. atozodn fuses with an egg, it is "72" Wilson, 1900.) obvious that most of them never perform the function for which they are specialized; but apparently an enormous number are formed to make the fertiliza- tion of the eggs more certain. The experiments of Loeb and Bancroft (1912) on spermatozoa have shown that when the living Apical body or acrosome, Nucleus. End-knob. Middle-piece. Envelope of the tail. Axial filament, End-piece, 22 GERM-CELL CYCLE IN ANIMALS spermatozoa of the fowl are placed in a hanging drop of white of egg or in yolk they undergo a transfor- mation into nuclei. The possibility that a sperma- tozoén may give rise to an embryo without the help of an egg is recognized, but this has not yet been accomplished. Ture Lire Cycues or ANIMALS. The life cycle of an animal has considerable influence upon the course of the germ-cell cycle. In all animals that are produced by the sexual method the beginning stage in the life cycle is a mature egg, either fertilized or unfertilized according to the species. Animals which develop asexually, on the other hand, begin their cycle with the first recognizable evidence of budding or fission. As a rule budding or fission are sooner or later interrupted by the formation of sex cells, hence the life cycle of such animals may be considered to extend from the mature egg to that stage in the life history of the species when mature eggs are again produced. Such a life cycle consists really of two or more simple life cycles represented by individuals differing from one another in both structure and method of reproduction. As examples of some of the principal types of life cycles we may select certain insects and ccelenterates. A very simple life cycle is that of the wingless insects of the order ApTERA. The young, when they hatch from the egg, are similar in form, structure, and habits to the fully grown individual and undergo no perceptible changes, except increase in size, until they become sexually mature adults. In INTRODUCTION 23 certain other groups of insects, such as the grass- hoppers, the newly hatched young resemble the adult in many ways, differing principally in the absence of wings. The young Rocky Mountain locust (Melanoplus spretus), for example, changes its exoskeleton (molts) five times before the adult condition is attained. After each molt there are slight changes in color, structure, and size, the most notable difference being the gradual acquirement of wings. In still other orders of insects a larva hatches from the egg; this larva, on reaching its full growth, changes in shape and structure, becoming a quiescent pupa, from which after a rather definite interval an adult emerges. A combination of two simple life cycles to form one complex cycle occurs in certain hydroids. The eggs of these species produce free-swimming em- bryos which become fixed to some object and de- velop into polyps. These polyps form other polyps like themselves by budding, but finally give rise to buds which become jelly-fishes or medusee. In- stead of remaining attached to the parent colony the medusz, as a rule, separate from it and swim about in the water; they later give rise to eggs which, after being fertilized, develop as before into polyps. There are thus in this species two life cycles com- bined, that extending from the egg to the time when the colony forms medusa-buds, and that beginning with the medusa-bud and ending with the mature egg. Such an alternation of an asexual and a sexual generation is known as metagenesis. 24 GERM-CELL CYCLE IN ANIMALS There is another sort of alternation which nor- mally occurs in many species, and that is the alterna- tion of individuals developing from parthenogenetic eggs with those from fertilized eggs. In the aphids, or plant lice, for example, the race in the northern part of the United States passes the winter in the shape of fertilized eggs. All of the individuals which hatch from these eggs in the spring are females called stem-mothers. The stem-mothers produce broods of females from parthenogenetic eggs, and these in turn give rise to other broods of females in the same manner. Thus throughout the summer, generation after generation of parthenogenetic females appear; but as autumn approaches females develop whose eggs must be fertilized, and males are also pro- duced. The eggs of these females are fertilized by spermatozoa from the males, and the zygotes thus formed survive the winter, producing stem-mothers the following spring. CHAPTER II GENERAL ACCOUNT OF THE GERM-CELL CYCLE IN ANIMALS It will be impossible to present in this chapter even a general account of all the variations in the germ- cell cycle that are known to occur in animals. It will be necessary, therefore, to restrict ourselves to the series of events that occurs in the majority of animals, mentioning as many of the more notable variations and exceptions as possible without causing confusion. It also seems advisable to consider the germ-cell cycles in the Protozoa and the Mrta- ZOA separately. Protozoa. Weismann, in his classical essays on the germ-plasm, argues in favor of the view that the Protozoa are potential germ cells, and, since new individuals arise by division of the parent cell into two or more parts, that natural death does not occur. The Protozoa are consequently also potentially immortal. The Merazoa, on the other hand, possess a large amount of somatic substance which always dies a natural death. It has often been pointed out that a Prorozoon, although consisting of but a single cell, performs most of the physiological activities characteristic of the larger, complex Metazoa, and that certain parts of the Protozo6n 25 26 GERM-CELL CYCLE IN ANIMALS are recognizably concerned with the performance of certain definite functions. The fundamental difference, then, between the one-celled and the many-celled animals is that the differentiated struc- tures in the former are not separated from one another by cell walls as in multicellular organisms. Whether all Protozoa possess a body which can be considered as specialized and set aside for reproduc- tion purposes, as the germ- plasm theory requires, is a salts \ question upon a ' which author- a Se ities differ. In certain cases it seems pos- Fic. 5.—Reproduction in Arcella vulgaris. A. For- sible to distin- mation of secondary nuclei. Ch = chromidia; nm =secondary nuclei; N=primary nucleus. guish between (From Hertwig, 1899.) B.Twogametes. (From ‘ Elpatiewsky, 1907.) germinal and somatic proto- plasm without any difficulty. The life history of the fresh water rhizopod, Arcella vulgaris (Fig. 5), will serve to illustrate this (Hertwig, 1899; Elpatiewsky, 1907; Swarczewsky, 1908; Calkins, 1911). The single nucleus of the young Arcella divides to form two primary nuclei (NV); chromatin from these mi- grates out and forms a layer near the periphery (Ch) —the “ chromidial net”’ of Hertwig. This chromatin substance in the mature individual produces hundreds of secondary nuclei (7), each of which is cut off, with ACCOUNT OF THE GERM-CELL CYCLE 27 a small amount of the surrounding cytoplasm, from the others, thus becoming a swarm spore. The swarm spores escape from the mouth of the parent cell; whereas the two primary nuclei and a portion of the cytoplasm not used up in the forma- tion of the swarmers die. The swarmers are not all alike, being of two sizes; the larger, which may be called macrogametes, and which correspond to the eggs of the Mrrazoa, fuse with the smaller micro- gametes. The zygotes which result develop into normal Arcelle. The swarmers may be supposed to represent the germinal protoplasm, of which, as in metazoan germ cells, the chromatin content may be considered the essential portion. The conditions during reproduction in other Protozoa may also be explained in this way, so that germinal and somatic protoplasm can be distinguished as in the Mrtazoa. The discovery of the chromidia in Protozoa led to the formulation of the hypothesis of binu- clearity. Believers in this hypothesis maintain that each cell contains both a somatic and propaga- tory nuclear material which, as a rule, are united into one amphinucleus. The somatic nuclear ma- terial controls vegetative functions; the propaga- tive portion serves only for the propagation of new individuals. Separation occurs rarely except in certain Protozoa, where, as in Paramecium, the propagative substance is represented by the micronu- cleus, the somatic by the macronucleus. Since the chromatin is the essential substance concerned in the binuclearity hypothesis, the term dichroma- 28 GERM-CELL CYCLE IN ANIMALS ticity has been suggested as more appropriate, and the two kinds of chromatin involved have been called idiochromatin, which is reproductive in function, and trophochromatin, which is vegetative in function. The hypothesis has not gained many adherents and is considered of doubtful value by eminent proto- zodlogists (Dobell, 1908). Merazoa. If we consider the mature egg, either fertilized or parthenogenetic, as the starting point of the germ-cell cycle in the MrtTazoa, we may recognize seven or eight distinct periods as follows : 1. The segregation of the primordial germ cells; z.e., the formation of one or more primordial germ cells during the segmentation of the egg; 2. Early multiplication of the primordial germ cells; 3. A long period of “rest”’ characterized by cessa- tion of cell division, either active or passive change of position, separation of the germ cells into two groups which become the definitive germ glands, accompanied by the general growth of the embryo until the larval stage is almost attained ; 4. Multiplication by mitosis of the primitive odgonia or spermatogonia to form a definite number (Miastor and perhaps others) or indefinite number (so far as we know) of odgonia or spermatogonia ; 5. In some cases the differentiation of odgonia into nurse cells and ultimate odgonia, and the spermatogonia into Sertoli cells and ultimate sper- matogonia ; 6. The growth of the ultimate odgonia and sper- ACCOUNT OF THE GERM-CELL CYCLE 29 matogonia to form primary odcytes and primary spermatocytes ; 7. Maturation ; 8. Fertilization (if not parthenogenetic). 1. THE SEGREGATION OF THE PRIMORDIAL GERM Cretts. This phase of the germ-cell cycle is espe- cially emphasized in this book (see Chapters III to VI) and need be referred to only casually here. The mature eggs of animals are organized both mor- phologically and physiologically ; that is, differenti- ations have already taken place in their protoplasmic contents before they are ready to begin develop- ment. This organization determines what sort of divisions the egg will undergo during the cleavage stages. During cleavage certain parts of the cell contents become separated from other parts and thus the differentiated substances of the egg are localized in definite parts of the embryo. The contents of the cleavage cells likewise become differentiated as development proceeds, until finally the cells produced form two or three more or less definite germ layers. In some cases the egg always divides in the same way, and the history or “cell lineage”’ of the cells can be followed accurately, and the parts of the larva to which they give rise can be established. This is known as determinate cleavage in contrast to the indeterminate type in which there appears to be no relation between the cleavage cells and the structure of the egg or larva. The degree of organization of the egg no doubt ac- counts for the differences in cleavage; those of the 30 GERM-CELL CYCLE IN ANIMALS determinate type being more fully organized than those of the indeterminate type. The period when the primordial germ cells are es- tablished is probably due in part to the state of organization of the egg when development begins, and it is not strange, therefore, that the primordial germ cell may be completely segregated in certain eggs as early as the four-cell stage; whereas in others germ cells have not been discovered until a late larval condition has been reached. An ever increasing number of species of animals is being added to those in which an early segregation of the germ cells has already been recorded. Neverthe- less, there are certain zodlogists who still question the general occurrence of an early segregation of the germ cells, but more careful investigations will probably establish the fact of early segregation in species in which this has not yet been demonstrated. 2. Earty MULTIPLICATION OF THE PRIMORDIAL Germ Cris. The number of germ cells present at the time of their first appearance in the embryo varies in different species. There may be one, as in the majority of cases, for example the fly, Muastor (Fig. 17), the nematode, Ascaris (Fig. 51), the crustacean, Cyclops (Fig. 48), and the arrow worm, Sagitta (Fig. 54) ; or a number, as in chrysome- lid beetles (Fig. 36), certain parasitic HYMENOPTERA (Fig. 44), and vertebrates (Fig. 6). As a rule the primordial germ cell or cells increase in number by mitosis soon after they are segregated, and then cease to divide for a considerable interval. For ACCOUNT OF THE GERM-CELL CYCLE 31 example, in Miastor the single primordial germ cell produces eight; in the beetle Calligrapha multi- punctata the original sixteen undergo two divisions resulting in sixty-four; and in the chick Swift (1914) has counted as many as eighty-two at this stage. We shall see later that the primordial germ cells are often characterized by the presence of certain cytoplasmic inclusions (the keimbahn-determinants) which are absent from the other cells of the embryo. These inclusions appear to be equally divided be- tween the daughter cells so that each of the eight or sixty-four, as the case may be, is provided with an equal amount of the keimbahn-determinants. 3. Pertop oF “Rest” anp Micration. By rest here is really meant cessation of division. During this period the germ cells either actively migrate or are passively carried by surrounding tissues to the position the germ glands occupy in the larva. In species possessing two germ glands the germ cells separate to form two groups, with, at least in some cases, an equal number in each group. Thus in Miastor the number in each group is four (Fig. 22) and in Calligrapha, thirty-two (Fig. 37). There is evidence that an active migration of germ cells occurs both in vertebrates and invertebrates. Figure 6 shows the positions of the germ cells in four species of vertebrates during their change of position. That the germ cells at this time are actively migrat- ing by ameboid movements is the general opinion of investigators, since frequently these cells are ameboid in shape and the distance between the place 32 GERM-CELL CYCLE IN ANIMALS of origin and the germinal ridge is too great to be traversed in any other way. ; Professor B. M. Allen, who has made extensive studies of the germ cells of many species of verte- Lepidosteus I SITY 6 ThA [Tf ety wit. End, Fic. 6.— Diagrams showing the paths of migration in 4A, a turtle, Chrysemys marginata; B, a frog, Rana pipiens; C, a fish, Lepidos- teus osseus, and D, the dog-fish, Amia calva. (From Allen, 1911.) Arch = archenteron; Int. =intestine; Lat. Mes =lateral plate of mesoderm; Mes = mesentery; Meson = mesonephros; Myo = myo- tome; Noto = notochord; P. card = post cardinal vein; S.C = sex- cells; S.Gl=sex gland; Vit. End =vitelline endoderm; W.D= Wolffian duct. brates, makes the following statement regarding this phase of the germ-cell cycle: “The sex-cells are migratory to a high degree. The path and time of their migration may vary greatly within a given group of animals, as illus- ACCOUNT OF THE GERM-CELL CYCLE 33 trated by the case of Amia and Lepidosteus. While in the forms that I have studied they are first to be observed in the entoderm, I am quite open to convic- tion that in other forms they may migrate from this layer into the potential mesoderm before the two layers are separated, as shown by Wheeler in Petro- myzon.” Swift (1914) has recently obtained evidence which seems to prove that not only do the germ cells of the chick migrate by ameboid movements but they enter the blood vessels and are distributed by the blood stream to all parts of the embryo and vascular area. The migration of the germ cells has been noted in many invertebrates and has been fully described in chrysomelid beetles (Hegner, 1909a). In these insects the primordial germ cells are segregated at the posterior end of the egg at the time the blasto- derm is formed (Fig. 36, C). The blastoderm is never completed just beneath them, but a canal, called the pole-cell canal, remains. Through this at a later embryonic stage the germ cells migrate by means of ameboid movements. *“As soon as the germ cells of Calligrapha have passed through the pole-cell canal, they lose their pronounced pseudopodia-like processes and become nearly spherical (Fig. 37, E); nevertheless, they undergo a decided change in position. They move away from the inner end of the pole-cell canal, and creep along between the yolk and the germ-band. Thus two groups are formed near the developing D 34 GERM-CELL CYCLE IN ANIMALS coelomic sacs; each group probably contains an equal number of cells. The smallest number I have counted in one group at this time is thirty; the largest number, thirty-four. As there is some difficulty in obtaining an accurate count, it seems probable that the sixty-four germ cells are equally divided and that each germ gland receives thirty-two. Some of the germ cells migrate not only laterally along the germ gland but also back toward the posterior end of the egg, where we find them forming narrow strands in the last abdominal segments. From this stage on, the germ cells are not very active ; they move closer to one another to form the compact germ glands. I was unable to determine whether the later movements of the germ cells are due to an active migration or to the tensions created by the growth of the surrounding tissues; the latter seems the more probable” (Hegner, 1909a, p. 280). It is thus evident that during the blastoderm stage the germ cells of this beetle are actually outside of the egg. How well this illustrates the theory of primary cellular differentiation, that is, the differentiation of germ cells from somatic cells, since the two sorts are here completely separated, the former constitut- ing a group in contact with but not connected with the somatic cells. Later, as the germinal con- tinuity hypothesis demands, the germ cells migrate into the embryo, there to be nourished, transported, and protected by the body until they are ready to separate from the somatic cells, and thus to give rise to a new generation. ACCOUNT OF THE GERM-CELL CYCLE 35 4. PeRiop or Mu.tiIpLicaTion. Soon after the germ cells aggregate to form more or less rounded groups lying in the position of the definitive germ glands mitotic division is resumed. At about this time also, the sex of the individual can often be determined by the shape of the germ-gland. Then both the testes and the ovaries acquire envelopes of the follicular cells, and frequently testicular cysts and ovarian tubes or chambers develop. The ques- tion of the origin of the follicular cells is still un- settled, but the evidence in most cases seems to favor the view that they are mesodermal. The multiplication of the germ cells by mitosis continues rapidly from this time on. In only one case, so far as I am aware, do we know the actual number of germ cells produced by the primordial germ cell; this is in Miastor, where typically sixty- four odgonia are formed (Fig. 26). As the germ cells multiply they become smaller in size and the substances present in the primordial germ cell become divided among a large number of progeny. Thus at the beginning of the growth period each germ gland contains many odgonia or spermatogonia, and each of these contains a small fraction of the material present in the primordial germ cell, plus whatever substances may have been assimilated during the period of multiplication. 5. THe Oricin or Nurse CELLS AND SERTOLI Crtts. Germ cells receive nourishment during the growth period in many ways, e.g., from nurse cells, follicle cells, or directly from the blood. The origin 36 GERM-CELL CYCLE IN ANIMALS of the nurse cells and follicle cells is important since in a few cases the germ cells themselves are known to give rise to them. There is thus a second differ- entiation whereby somatic cells (follicle cells or nurse cells) become differentiated from germ cells (odgonia or spermatogonia). In some species, such as Miastor, we can prove without question that both the nurse cells and follicle cells are of mesodermal origin, and that the germ cells give rise only to germ cells. On the other hand, there are instances in both vertebrates and invertebrates of a common origin of germ cells and somatic cells from odgonia and spermatogonia. Perhaps the most striking examples are the differentiation of the nurse cells and ultimate odgonia in the water beetle, Dytiscus, and the differentiation of the Sertoli cells and ulti- mate spermatogonia in man. (See Chapter V.) Haecker (1912) distinguishes’ between a somato-ger- minative period and a true germinative period; the former is that during which the primordial germ cells are established and the latter that of the differentia- tion of nurse cells and ova. 6. Tut GrowtH Prriop. The last divisions of the odgonia and spermatogonia are followed by the growth of these cells. The extent of this growth depends, in the case of the female, upon whether or not the mature egg is to be supplied with an abundance of nutritive material. Nurse cells, fol- licle cells, and circulating fluids may all assist in the enlargement of the odgonia. If the eggs are small, sufficient nutriment is supplied by surrounding ACCOUNT OF THE GERM-CELL CYCLE 37 liquids and no special nurse cells are required; but larger eggs either become surrounded by follicle cells which nourish them and with which they are often intimately connected by protoplasmic bridges, or special nurse cells are provided. In the primitive type of ovary, such as exists in most ccelenterates, any of the cells surrounding the odgonium may function as nurse cells and even neighboring odgonia are engulfed by the odgonium that is successful in the struggle for development. A more definite mechan- ism exists in higher organisms, where one or more cells become differentiated for the special purpose of supplying nutriment consisting of either their own substance or of material elaborated by them and then transferred to the egg. The egg of the annelid, Ophryotrocha, for example, is accompanied by a single nurse cell; that of Myzostoma is provided with two, one at either end; and the eggs of certain insects are more or less intimately connected with groups of cells in definite nurse chambers (Fig. 46). The growth of an odgonium may be well illus- trated by that of the potato beetle. The general arrangement of the cells in the ovary of an adult beetle is shown in Fig. 7. The terminal chamber of the ovarian tubule contains three kinds of cells: (1) nurse cells (n.c), (2) young odcytes (y.o) and growing odcytes, and (3) epithelial cells. The nurse cells and odcytes are both derived from the odgonia; the epithelial cells are of mesodermal origin. The positions of the stages to be described are indicated in the diagram (Fig. 7) and the nuclear 38 GERM-CELL CYCLE IN ANIMALS Fic. 7.— Leptinotarsa de- cemlineata. Diagram of an ovarian tubule showing various stages in the de- velopment of the odcyte. The capital letters refer to the positions f cellsshown in Fig. 8. cy = cytoplasm; es = egg string; n.c = nurse chamber; doc = odcyte; y.o = young oocyte. and cytoplasmic structures are shown in Fig. 8. Two odcytes and a neighboring epithelial cell from position A in Fig. 7 are shown in Fig. 8, A. The nuclei of the odcytes are large and contain a dis- tinct spireme; the cytoplasm is small in amount and ap- parently homogeneous. After a short period of growth the odcytes form a linear series in the ovarian tubule and become connected with the spaces between the nurse cells by means of egg strings (Fig. 7, e.8) through which the nu- tritive streams flow into the odcytes. One of the young- est of these odcytes is repre- sented in Fig. 8, B (position B in Fig. 7). The nucleus is no larger than in those of the earlier stage; its chromatin forms a reticulum, and a dis- tinct nucleolus is present. The cytoplasm, on the other hand, has trebled in amount and within it are embedded a number of spherical bodies ACCOUNT OF THE GERM-CELL CYCLE 39 H Fic. 8.— Leptinotarsa decemlineata. A-H, Stages in the growth of the odcyte from positions indicated in Fig. 7. a—c = amitotic nuclear division of nurse cells. ch =chorion; f.ep = follicular epithelium. 40 GERM-CELL CYCLE IN ANIMALS which stain with crystal violet after Benda’s method, and appear to be mitochondrial in nature. At a slightly later stage (Fig. 8, C; position C in Fig. 7) the nucleus is larger and contains several small spherical chromatic bodies besides the nucleolus. The cytoplasm has increased more rapidly in volume and a corresponding increase in the number of mito- chondrial granules has also taken place. Further growth results in an increase in the volume of both nucleus and cytoplasm (Fig. 8, D; position D in Fig. 7), and a slight increase in the number of mito- chondria. Whether these bodies developed de novo or by division of the preéxisting granules could not be determined. In succeeding stages growth is very rapid. The cytoplasm (Fig. 8, E; position E in Fig. 7) still remains homogeneous except for the mitochondria, which increase slightly in size and become situated as a rule near the periphery. The nucleus at this time contains a large number of chromatin granules and a diffuse reticulum. Part of an older odcyte is shown in Fig. 8, F (position F in Fig. 7); the cyto- plasm has assumed a reticular appearance; the mitochondrial granules are present in greater num- bers, and the nucleus is larger, oval in shape, and contains a distinct reticulum with many chromatin bodies of various sizes. A still older odcyte (Fig. 8, G; position G in Fig. 7) is interesting particularly because of the rapid increase in the mitochondria and the localization of these near the periphery. From this stage on the character of the contents changes ACCOUNT OF THE GERM-CELL CYCLE 41 until, as shown in Fig. 7, the central part of the odcyte consists of homogeneous cytoplasm (cy), and the outer region of the cytoplasm is crowded with granules and spherical bodies of various sizes. Apparently the mitochondria lying near the periphery (Fig. 8, H) increase in size, gradually losing their affinity for the crystal violet stain and swelling up until they constitute the large yolk globules so numerous in the mature egg. All stages in the evolution of these bodies are illustrated at this time as represented in Fig. 8, H. In the meantime material is brought into the egg through the egg string from the nurse cells, thus probably adding several sorts of granules to the contents of the odcyte. The growth period in the male germ-cell cycle is not so striking as in the female, since many sperma- tozoa of small size are produced, whereas only comparatively few large eggs develop. An increase in the size of the ultimate spermatogonia may occur, however, but the multiplication and growth periods are not nearly so distinct as in the case of the odgonia. In testes which are composed of cysts of spermato- gonia there is evidence in some cases that all of the germ cells in a single cyst are descendants of a single spermatogonium. The proof for this seems certain in the potato beetle, where I have been able to follow the formation of the cysts by means of an uninterrupted series of stages (Hegner, 1914a). 7. Maturation. Maturation or the ripening of the eggs and spermatozoa comprises a series of events which results in a reduction in the number 42 of GERM-CELL CYCLE IN ANIMALS chromosomes and the amount of chromatin in the germ cells. Typically, both male and female germ cells divide twice during the process of matura- PRIMORDIAL GERM-CELL MULTIPLICATION PERIOD SPERMATOGONIA_.--—~ X GROWTH PERIOD PRIMARY SPERMATOCYTE OTT SECONDARY SPERMATOCYTES MATURATION PERIOD SPERMATIDS SPERMATOZOA Fic. MULTIPLICATION PRIMORDIAL GERM=CELL, OUT PERIOD OOGONIA ger ‘~\ GROWTH PRIMARY = PERIOD oocyTe SECONDARY oocyTEs (OVARIAN EGG ~~ MATURATION ‘AND POLAR. BODY) PERIOD MATURE EGG AND 0 -=--~———=---- POLAR BODIES 9.— Diagrams illustrating (above) the stages of spermatogenesis and (below) of oédgenesis. The primordial germ cell is represented as possessing four chromosomes. ACCOUNT OF THE GERM-CELL CYCLE 43 tion, and as shown in Fig. 9 these divisions result in the production of four functional spermatozoa in the male, and one functional egg and three polar bodies (abortive eggs) in the female. This increase in the number of cells is not, however, the most im- portant phase of the maturation process, since a large part of our knowledge of the physical basis of heredity has been derived from studies of the be- havior of the chromatin at this time. This subject will be dealt with more fully in Chapter IX, and for the present only a brief account of events need be given. The first thing to be noted is that the mitoses leading to the division of the germ cells during mat- uration differ from those of ordinary cell multiplica- tion. The germ cells, when they are ready for the maturation divisions, are known as primary odcytes and primary spermatocytes. The nuclei of these cells possess the complete or diploid number of chromosomes, characteristic of somatic cells; but after maturation the eggs and spermatozoa con- tain only one-half of the original diploid number, or the haploid number. These mitoses are conse- quently called reducing or meiotic. The details of these mitoses differ in male and female germ cells and in different species of animals. During and at the close of the growth period in the male the chromatin granules form a spireme which condenses at one side of the nucleus, a condition known as synizesis. After a time the spireme again spreads throughout the nucleus, but is now 44 GERM-CELL CYCLE IN ANIMALS divided into segments, the chromosomes, which are only haploid in number. The reduction from the diploid to the haploid number is brought about by the union of the chromosomes in pairs, a condition called synapsis. Each of the haploid chromosomes thus consists of two of the diploid chromosomes and is said to be bivalent. That one of the chromo- somes of each pair is of maternal origin, 2.¢., is a descendant of a chromosome present in the egg at the time of fertilization, and the other of pater- nal origin, 7.e., a descendant of one brought into the egg by the spermatozoén, seems to be well established. The final act of fertilization, therefore, occurs at this point in the germ-cell cycle — an act of much greater significance than that of the union of the egg and spermatozoén. Furthermore, there is considerable evidence that the chromo- somes differ one from another and that in synapsis corresponding (homologous) chromosomes unite. The importance of such a union from a theoretical standpoint will be discussed later. The nuclei now prepare for the two maturation mitoses. In many nematodes, annelids, and arthro- pods these are characterized by the formation of tetrads. Divisions of this sort may be illustrated as in Fig. 10. The diploid number of chromosomes is for convenience supposed to be four, as in the sper- matogonium A. During the spermatogonial divi- sions these divide as in B, so that each daughter cell receives the diploid number, four. After synapsis, however, each of the haploid chromosomes of the ACCOUNT OF THE GERM-CELL CYCLE 45 <=> al) F ff Fic. 10.— Diagrams showing the essential facts of reduction in the male. The somatic number of chromosomes is supposed to be four. A, B, division of the spermatogonia, showing the full number (four) of chromosomes. C, primary spermatocyte preparing for division; the chromatin forms two tetrads. D, E, F, first division to form two secondary spermatocytes, each of which receives two dyads. G, H, division of the two secondary spermatocytes to form four spermatids. Each of the latter receives two single chromosomes and a centrosome which passes into the middle piece of the spermatozo6n. (After Wilson.) 46 GERM-CELL CYCLE IN ANIMALS primary spermatocyte is seen to be divided into four parts, thus forming in this case two tetrads (C). During the division of the primary spermatocyte, as shown in D, E, and F, half of each tetrad, or two dyads, passes to each daughter cell. The division of the daughter cells, which are known as secondary spermatocytes (G H), results in the separation of the two parts of each dyad so that each of the four spermatids (H): receives one member of each original tetrad or two monads. Thus the chromosomes (monads) of the spermatids (H) are already formed in the primary spermatocytes (C) by two divisions ; whereas the nuclear and cell divisions do not occur until later. The spermatids (H), which proceed to metamorphose into spermatozoa, possess, there- fore, only two chromosomes, 7.e., one-half of the number present in the spermatogonia (A) and so- matic cells. Tetrad formation does not occur in most animals; but usually the members of the bivalent chromosomes become separated on the first maturation spindle, the pairs appearing U-, V-, or ring-shaped, as in Fig. 62. Each secondary spermatocyte receives one-half of each haploid, bivalent chromosome. The second maturation mitosis then ensues, during which each daughter cell is provided with one-half of each chromosome as in ordinary mitotic division. Be- cause of the peculiar behavior of the chromosomes the first division is often called the heterotype, whereas the second is known as the homotype divi- sion, The final results are the same whether tetrads ACCOUNT OF THE GERM-CELL CYCLE 47 are formed or not, each spermatid containing the haploid number of chromosomes. The maturation of the egg differs in no very im- portant respects from the process as it has been described in the male cells. Tetrads may or may not be formed according to the species, and the mature egg and polar bodies each contain the haploid number of chromosomes. Two phases of the matura- tion of the egg may be referred to here: (1) when the nucleus of the primary odcyte prepares for divi- sion a considerable amount of chromatin separates from the chromosomes and is lost in the cytoplasm. The size of the chromosomes is thus diminished, but no entire chromosomes are lost. (2) The cellular divisions are very unequal, the polar bodies being very small as compared with the rest of the egg. The chromatin content of the polar bodies, however, is equal to that of the much larger egg. In the male all of the four spermatids are functional, but in the female only the egg survives, the polar bodies de- generating. Asarule two polar bodies are produced, but in certain cases of parthenogenesis (rotifers, Cuapocrera, Ostracopa, and aphids) only one is formed. Rarely the first polar body divides into two. 8. Frertinization. Eggs that develop partheno- genetically are ready to begin a new germ-cell cycle as soon as they become mature; but the eggs of the majority of species must be fertilized before they are able to develop. Fertilization may be de- fined as the fusion of an egg with a spermatozoén and the resulting processes of rearrangement of the egg 48 GERM-CELL CYCLE IN ANIMALS contents which result in the formation of a uninuclear cell, the zygote. As a rule one spermatozoén only enters the egg (monospermy); but in a few species (certain insects, selachians, tailed amphibians, reptiles, and birds) many spermatozoa may normally fuse with the egg (physiological polyspermy). The sper- matozoon, which consists usually of three rather dis- tinct parts, the head, the middle piece, and tail, may become entirely embedded within the egg sub- stance, or the tail may be left outside, or, in excep- tional cases, only the head succeeds in entering. The union of the egg and spermatozodn may occur before, during, or after the polar body formation (Fig. 11). If the spermatozoén enters before the maturation of the egg is completed (A), its head transforms into a nucleus equal in size to that of the egg (C); the middle piece dissolves, giving rise to a centrosome which inaugurates the formation of a spindle with asters (B); and the tailpiece ap- parently takes no active part in the fertilization processes. The middle piece also does not seem to be necessary for the formation of the centrosomes and asters. The nucleus of the spermatozoén and that of the mature egg approach each other and ~ecome into contact between the asters (C). Then the nuclear walls dissolve; a spireme which segments into the haploid number of chromosomes is produced by each nucleus, and the first cleavage spindle of the developing egg results. This spindle bears the haploid number of chromosomes from the spermato- zoon and a like number from the egg nucleus ACCOUNT OF THE GERM-CELL CYCLE 49 and thus the diploid or somatic number of chromo- somes is regained. When the spermatozoén enters an egg which has completed polar-body formation, the head does not Fic. 11.— Diagrams of two principal types of fertilization. I. Polar bodies formed after the entrance of the spermatozoa (annelids, mollusks, flat-worms). II. Polar bodies formed before entrance (echinoderms). A, sperm-nucleus and centrosome at ¢; first polar body forming at 2. B, polar bodies formed; approach of the nuclei. C, union of the nuclei. D, approach of the nuclei. £, union of the nuclei. F, cleavage-nucleus. (After Wilson.) have time to transform into a nucleus as large as the egg nucleus, but nevertheless fuses with the latter (Fig. 11, D, E, F). Although the two nuclei are very unequal in size, they possess an equal amount of chromatin and furnish an equal number of chromo- somes to the first cleavage spindle. E 50 GERM-CELL CYCLE IN ANIMALS As already indicated, perhaps the most essential phase in the fertilization process does not occur until the homologous maternal and paternal chromosomes unite during synapsis, when the germ cells of the new individual become mature. The immediate results of fertilization are: (1) the inauguration of the development of the egg, (2) the increase of the chromosomes from the haploid to the diploid (so- matic) number, and (3) the union of hereditary substances from, as a rule, two individuals. This completes the last stage in the germ-cell cycle of animals. Many extremely important and interesting phases of the subject have had to be omitted from the account. Certain of these will be more fully discussed in succeeding chapters, es- pecially those concerned with the early history of the germ cells during embryological development, but for the details of the nutrition, growth, matura- tion, and fertilization of the germ cells, the reader must be referred to other sources (Wilson, 1900; Jenkinson, 1913; Kellicott, 1913). CHAPTER III THE GERM-CELL CYCLE IN THE PADOGENETIC FLY, MIASTOR Tuus far in only one genus of animals has the history of the germ cells from one generation to the next been followed in detail through the entire cycle. This is a genus of flies, Miastor, of the family Cecidomyide. One species, Miastor metraloas, oc- curs in Europe and has there been studied especially by Leuckart (1865), Metschnikoff (1865, 1866), and Kahle (1908), and the only other species that has been investigated is M. americana (Hegner, 1912, 1914). Peedogenesis in Miastor was discovered by Wagner in 1862, and was confirmed by Meinert in 1864. In 1865 the first investigations of its embryological development were published by Leuckart and Metsch- nikoff. These were the earliest accounts of the keimbahn in any animals. Only a glance at Metsch- nikoff’s report is necessary to convince one of the favorableness of Miastor as material for germ-cell studies. The primordial germ cell is shown to be established at a very early period in the cleavage of the egg, and the descendants of the primordial germ cell are quite easily distinguishable from other cells in the body even in in toto preparations. In spite of 51 52 GERM-CELL CYCLE IN ANIMALS the work of the above named investigators there were many who were not convinced that peedogenesis occurs in the genus, and the larvae which were known to develop within the bodies of other larve were considered by these skeptics as parasites. How- ever, the results of Kahle’s (1908) studies, which have been decisively confirmed (Hegner, 1912, 1914a), have finally settled the question in favor of peedogen- esis. Previous to 1910 no specimens of the genus Miastor had been recognized in this country, but on Oct. 5 of that year, Dr. E. P. Felt found them in great abundance, living in the partially decayed inner bark and in the sapwood of a chestnut rail. With material supplied by Dr. Felt, the writer has been able to follow the entire keimbahn in these insects. Predogenetic reproduction normally oc- curs during the spring, summer, and autumn, multi- plication being arrested during the cold winter months. This method of reproduction is interrupted in midsummer by the appearance of male and female adults. The larva of Miastor possesses two ovaries, one on either side of the body in the tenth or eleventh segment. Each ovary (Fig. 12) consists of typically thirty-two odcytes (odc.n); these are inclosed in a cellular envelope (en). Associated with each odcyte is a group of mesoderm cells which function as nurse cells (n.c.) and together with the odcyte are sur- rounded by a follicular epithelium (f.ep). The nurse cells furnish nutrition to the growing odcytes, THE PADOGENETIC FLY, MIASTOR — 53 gradually becoming reduced as the odcytes increase in size. Finally the odcyte (and accompanying nurse cells), still surrounded by the follicular epithelium, Fic. 12.— Miastor americana. Longitudinal section through an ovary. en=envelop; fep = follicular epithelium; n.c = nurse chamber; n.e.n = nurse-cell nucleus; 0.7 = mesoderm; odc.n = odcyte nucleus. Fie. 13.— Miastor americana. Longitudinal section through a nearly full-grown odcyte. g.v = germinal vesicle; n.c= nurse chamber; pFPl = pole-plasm. 54 GERM-CELL CYCLE IN ANIMALS becomes separated from the rest of the ovary and is forced by the movements of the larva into some other part of its body. Here it continues its growth and development at the expense of the tissues of the mother-larva. Not all of the odcytes (thirty-two in each ovary) complete their development, since usually only from five to seventeen young are produced by a single mother-larva. Those odcytes that do not perish pass through the stages described in the following paragraphs. Figure 13 represents the condition of an odcyte just before the initiation of the maturation processes. The nucleus, or germinal vesicle (g.v.), is eccentrically placed and nearer the anterior than the posterior end of the cell. The nurse chamber has greatly decreased in volume. The contents of the odcyte are not homogeneous, but several distinct regions can be distinguished. Near the nurse chamber is a body of cytoplasm evidently elaborated by the nurse cells, and at the posterior end is an accumulation which we may call the pole-plasm (pPl) and which is of particular interest since it is intimately associated with the formation of the primordial germ cell. The maturation division occurs soon after the stage just described has been attained. The ger- minal vesicle, which lies near the periphery of the odcyte, breaks down, and the chromatin contained within it becomes aggregated into about twenty chromosomes. Asa result of the maturation division (Fig. 14) a polar body (p.b) and the female pronucleus THE PHDOGENETIC FLY, MIASTOR — 55 (f.n) are produced. The nucleus of the polar body divides by mitosis and the two nuclei thus formed Fig. 14.— Miastor americana. Longitudinal section through mature egg. c=cytoplasm; f.n =female nucleus; n.c = nurse chamber; p.b = polar bodies; pPl = pole-plasm. remain within the egg substance near the periphery for a considerable period (Fig. 14), but finally 56 GERM-CELL CYCLE IN ANIMALS disintegrate and disappear, apparently without performing any function. As in most other animals, these polar bodies may be considered abortive eggs. The female pronucleus moves into the central an- terior part of the egg where it becomes em- bedded in the cytoplasmic mass near the nurse chamber. It may now be designated as the cleavage nucleus, since the eggs of Miastor develop without — ferti- lization and hence no male pronucleus is present tounite with it. The Fic. 15.— Miastor metraloas. Three of the four cleavage di- division figures (I, III, IV) of the four- to eight- _+ + tak cell stage represented. cMp = chromosome visions ake middle plate; n.c = nurse chamber; p.b= polar place b mi- body; pPl=pole-plasm. (From Kahle, 1908.) Pp 7 y tosis, and, as in most of the ARTHROPODA, the early cleavage nuclei are not separated by cell walls, but simply move apart after each successive division. The egg during this period is thus a syncytium within which the limits of the cells are difficult to define. THE PAHDOGENETIC FLY, MIASTOR 57 The nuclei present at the four-cell stage occupy rather definite positions and may be numbered for convenience by the Roman numerals I, II, III, and IV, as indicated in Fig. 15. The division from the four- to the eight-cell stage is a very important one, since it is at this time that the primordial Fic. 16.— Miastor metraloas. Stages in the chromatin-diminution process. (From Kahle, 1908.) germ cell is established. Each of the four nuclei divides by mitosis, but nuclei I, I, and III undergo a chromatin-diminution process during which a large part of their chromatin remains in the cyto- plasm when the daughter nuclei reform. The details of such a process are indicated in Fig. 16. Nucleus IV, on the other hand, divides as usual (Fig. 15) and each daughter nucleus receives one-half of its chroma- tin. One of these daughter nuclei becomes embedded in that peculiar mass of cytoplasm at the posterior 58 GERM-CELL CYCLE IN ANIMALS end which we have called the pole-plasm, and ap- parently all of the pole-plasm, together with this Fig. 17.— Miastor americana. Longitudinal section of egg with one germ cell (p.g.c.) and nuclei undergoing chromatin-diminution pro- cess. c=cytoplasm; cMp=chromosome middle plate; cR= chromatin remains. nucleus, is then cut off from the egg (Fig. 17). This cell, as has been conclusively proven by studies of THE PADOGENETIC FLY, MIASTOR 59 later stages, is the primordial germ cell. At this time, then, the egg consists of one primordial germ cell provided with a nucleus with an undiminished amount of chromatin, and a syncytium containing seven nuclei of which the sister nucleus of the primor- dial germ cell contains a complete supply of chroma- tin, whereas the other six nuclei have lost part of this chromatin material. Reference to the diagram on page 65 will assist in making more clear this stage and the stages yet to be described. The next developmental process is the mitotic division of the seven nuclei in the syncytium thus producing a fifteen-cell stage (Fig. 17). The sister nucleus of that of the primordial germ cell now under- goes a chromatin-diminution process and the other six nuclei in the syncytium pass through a second chromatin-diminution process. As a result every nucleus in the egg has lost a part of its chromatin except that of the primordial germ cell which still contains a complete amount. The further history of the somatic nuclei does not differ essentially from that of the somatic nuclei in other insects. They increase in number by mitosis, migrate to the periphery, and there are cut off by cell walls forming a single layer of cells over the entire surface except where interrupted at the posterior end by the primordial germ cells. Next, a thickening of the cells occurs on the ventral surface, thus forming the ventral plate. From this plate most of the embryo arises; it lengthens until the anterior or cephalic end almost reaches the anterior end of the 60 GERM-CELL CYCLE IN ANIMALS egg, and until the posterior or tail end has been pushed around for a considerable distance on the dorsal surface. A broadening and a_ shortening of this germ-band then takes place so that the pos- terior end of the embryo coincides with the posterior end of the egg and the edges of the embryo grow laterally around the egg until they meet in the median dorsal line. Meanwhile various changes have taken place within the embryo, among which is the formation of the germ glands or ovaries. Returning now to a consideration of the germ cells, we shall see that it is possible to trace the descendants of the primordial germ cell with comparative ease. This cell divides by mitosis, forming two odgonia approximately equal in size (Fig. 18). These two then produce four odgonia of the second order (Fig. 19), and these in turn increase by mitosis, forming eight odgonia of the third order (Fig. 20). When this stage is reached a period sets in during which the odgonia do not divide, but are apparently passively carried about by the somatic tissues as shown in Fig. 21, where they occupy a position near the end of the tail fold. One of the most satisfactory conditions in the keimbahn of Miastor is the comparatively large size and peculiar structure of the primordial germ cells leaving in the mind of the observer no doubt as to the identity of the cells concerned. Through- out the entire embryonic development of this insect the germ cells are considerably larger than any of the somatic cells. The nuclei are correspondingly THE PHDOGENETIC FLY, MIASTOR 61 large and are characterized by the possession of a number of spherical chromatin granules which are evenly scattered about in the nuclear sap. Fia. 18.— Miastor americana. Longitudinal section through an egg with two odgonia (0691). bc=blastoderm nucleus; ¢R = chro- matin remains. Fie. 19.— Miastor americana. Longitudinal section through an egg with four odgonia (092). GERM-CELL CYCLE IN ANIMALS 62 Even under the lower powers of the compound micro- scope the germ cells stand out with great distinct- could not possibly be confused with any ness and other cells the embryo. in Bedoarobplyal hee Doel CI He Longitudinal section through an egg Sagittal section through embryo show- cR = chromatin remains. ing odgonia (odg3) near end of tail fold. with eight odgonia (0093). Fie. 20.— Miastor americana. Fig. 21.— Miastor americana. THE PAIDOGENETIC FLY, MIASTOR — 63 During the shortening and broadening of the germ band the group of eight odjgonia of the third order becomes separated into two rows of four each — one row on either side of the body in the region of the eleventh segment (Fig. 22). Each group of four odgonia then becomes surrounded by a layer of mesoderm cells and forms a more or less spherical body which may now be called an ovary (Fig. 23). Soon after this occurs, the odgonia begin to divide again (Fig. 23, a) and by successive mitoses there are formed oégonia of the fourth, fifth (Fig. 24), and sixth orders. This completes the number of odgonia, which is typically thirty-two in each ovary, and provides us with the only case thus far on record where the number of odgonial divisions during the multiplication period in the history of the germ cells is known (Fig. 26). There are then six of these odgonial divisions between the formation of the single primordial germ cell and the production of the complete number of odgonia in the two ovaries. Some of the odgonia of the fifth order may be prevented from dividing, in which case of course there are less than thirty-two germ cells in each ovary. And not all of the odgonia in the ovary succeed in developing into odcytes and larvee, since a struggle for supremacy takes place among the germ cells resulting in the survival of only a few offspring, as may be determined by the fact, already referred to, that one larva gives rise as a rule to only from five to seventeen daughter larve. Each odgonium that succeeds in developing becomes 64 GERM-CELL CYCLE IN ANIMALS Fic. 22.— Miastor americana. Frontal section through posterior end of embryo showing oégonia (0693) forming two rows of four each. Fig. 23.— Miastor americana. Ovary containing sixteen odgonia (0694), one dividing by mitosis (a). m = mesoderm. Fic. 24.— Miastor americana. Ovary containing thirty-two odgonia (069s). m = mesoderm. Fic. 25.— Miastor americana. Young odcyte (odc) with nurse cells (n.c). Fic. 26.— Miastor americana. Diagram illustrating origin and history of germ cells from one generation to the next. cl.n = cleavage nucleus; ez.chr = extruded chromatin; oég = oégonia; p.b= polar body; p.g.c = primordial germ cell; p.o= primary odcyte; p.pl = polar plasm. st.c = stem cell. (65) F 66 GERM-CELL CYCLE IN ANIMALS provided with a group of about twenty-four meso- derm cells which form a syncytium at the anterior end and may be called nurse cells (Fig. 25, n.c), since they furnish food material to the odcyte. Another group of mesoderm cells forms a cellular layer about the odcyte and nurse cells, and thus constitutes a follicular epithelium. At this stage the odcytes break away from the ovary and become distributed in various parts of the body of the mother-larva. Several facts regarding the germ-cell cycle of Miastor deserve special emphasis: (1) There is no stage in the entire keimbahn when the germ cells cannot be distinguished without the least difficulty ; (2) the number of odgonial divisions has been defi- nitely established, and so it is no longer necessary to make the general statement that the germ cells pass through n divisions during the period of multi- plication, since here n is undoubtedly six; (3) the descendants of the primordial germ cell are only germ cells, z.e., the primordial germ cell does not give rise to both odgonia and nurse cells as seems to be the case in most other insects; (4) chromatin- diminution processes take place during the mitotic divisions of the nuclei from the four- to the eight- cell stage and form the eight- to the fifteen-cell stage of such a nature that all of the cells in the embryo finally are deprived of part of their chromatin with the exception of the primordial germ cell which retains the complete amount of this substance; (5) the primordial germ cell is established at the eight-cell stage and is the first complete cell formed in THE PHDOGENETIC FLY, MIASTOR 67 embryonic development; and (6) the contents of the primordial germ cell consist of the nucleus with undiminished chromatin and of all of the pole- plasm and apparently no other part of the egg sub- stance. The fact that only the primordial germ cell re- ceives a complete amount of chromatin is of particu- lar interest, since a similar condition has long been known in the case of Ascaris as we shall see later. It may also be noted in this place that the cyto- plasmic substance in the primordial germ cell may be recognized as the pole-plasm in the growing odcyte. Attempts have been made to determine the origin of this pole-plasm, but so far without success. It may be distinguished from the rest of the egg con- tents by its position at the posterior end and because of its affinity for certain dyes. It appears shortly before the maturation division is initiated, but no transition stages have been discovered — it has been either present or entirely absent in the preparations thus far studied. If we consider the history of this substance from the formation of the primordial germ cell to the growth period of the odcytes pro- duced by this primordial germ cell, we may conclude that at the time the multiplication period ends the pole-plasm has become equally distributed among the sixty-four odgonia. Then ensues the growth period during which the pole-plasm cannot be distinguished. Later, however, just before maturation, pole-plasm substance reappears which is equal in amount to that contained in the primordial germ cell of the 68 GERM-CELL CYCLE IN ANIMALS preceding generation or to that contained in all of the sixty-four odgonia which descended from that primordial germ cell. That is, the pole-plasm of the odcyte under discussion has in some way increased until its mass is sixty-four times as great as that of the odgonium before the growth period began. How this increase has taken place can only be conjectured. The pole-plasm in the o6gonium may have produced new material of its own kind either by the division of its constituent particles or by the influence of its presence. In any case a localization of this substance occurs at the posterior end of the egg just before maturation. Therefore, although we can follow the germ cells in Miastor throughout their entire cycle without difficulty, there are certain problems, such as the history of the pole-plasm during the growth period of the odcytes, which still remain unsolved. CHAPTER IV THE SEGREGATION OF THE GERM CELLS IN PORIFERA, CCQELENTERATA, AND VERTEBRATA Tue history of the germ cells has not been seriously investigated in a number of groups of animals, but, as will be demonstrated in Chapters V and VI, there are many species belonging to widely separated groups in the animal series in which the germ-cell cycle is almost as well known as in Miastor. On the other hand, the three phyla to be discussed in this chapter have been carefully studied for many years, but an early segregation of germ cells has not yet been established in them to the satisfaction of a majority of investigators. It seems strange because of the uncertainty of the morphological continuity of the germ cells in these animals that one of these groups, the C@LENTERATA, should have furnished the material upon which Weismann based his elabo- ration of the germ-plasm theory. 1. PoriIrERA Sponges reproduce asexually by budding and by the formation of gemmules, and sexually by means of ova and spermatozoa. Budding occurs in almost all sponges. In most cases the buds remain attached to the parent (continuous budding); but in some 69 70 GERM-CELL CYCLE IN ANIMALS species the buds become free (discontinuous bud- ding). Gemmules are groups of cells (statocytes) which occur at certain times of the year in the bodies of fresh-water sponges and in many marine species. These gemmules acquire a resistant covering and serve to preserve the race during the winter in the north or the dry season in the south. The peculiar “ budding” observed in Tethya by Désé (1879, 1880) may be a sort of gemmule formation (see p. 76). The eggs and spermatozoa are situated in the middle layer (so-called mesoderm) and in most cases seem to become ripe at different times in the same sponge. Fertilization is apparently similar to this process in other Mrtazoa. The fertilized ovum is holoblastic; the free-swimming ciliated larva becomes fixed, and then metamorphoses into a young sponge. The body wall of the sponge consists of two distinct layers, an outer dermal layer and an inner gastral layer, and an intermediate jelly-like stratum con- taining ameboid wandering cells. The various sorts of cells in these layers are indicated in the table on page 71 (from Minchin, 1900, p. 62). The reproductive cells lie in the jelly-like middle layer, but all of the cells in this layer are not repro- ductive. The origin of the archeocytes from which the re- productive cells arise can easily be pointed out in the comparatively simple development of Clathrina blanca (Minchin, 1900). In this species a ciliated PORIFERA, COELENTERATA, VERTEBRATA 71 TaBLE OF THE Various C1LassEs OF CELLS IN SPONGES 1. Pinacocytes (epithelial cells) 2. Myocytes I. Epithelial stratum (contractile cells) . Gland cells . Spongoblasts . Pore cells . Scleroblasts . Collencytes TII. Skeletogenous (stellate cells) stratum . Desmacytes (fiber cells) 9. Cystencytes (bladder cells) Gastral Layer IV. Gastral epithelium ; 10. Choanocytes (collar cells) 11. Phagocytes (ingestive cells) 12. Trophocytes (nutritive cells) Dermal Layer; II. Porocytes f “EOD Or He 09 oo V. Amebocytes (wan- Archzeocytes dering cells) 13. Thesocytes (primordial (st lI. on storage cells) 14. Statocytes le cell VI. Tokocytes (repro- ; 15 ee en ductive cells) (sexual cells) blastula-like larva is formed (Fig. 27, A). At the posterior pole two blastomeres (posterior granu- lar cells, p.g.c.) remain undifferentiated; they are much larger than the other cells, are granular, and possess vesicular nuclei. The larva becomes fixed by the anterior pole, and during the metamorphosis that then takes place, the two posterior granular cells, the archeocytes, multiply rapidly, forming a large number of minute cells which resemble certain leucocytes. These are known as amebocytes. By 2 GERM-CELL CYCLE IN ANIMALS the fourth day the amebocytes become separated into wandering cells or their derivatives and repro- ductive cells or tokocytes as indicated in the table. The primordial archeocytes do not always occur in the Clathrinide as in Clathrina blanca. In some a PEC Fic. 27.— A. Clathrina blanca. Blastula stage showing posterior gran- ular cells (p.g.c.). (From Minchin, 1900.) B. Odgonium of a sponge containing inclusions in the cytoplasm. (From Jérgensen, 1909.) C. Two oégonia in the ectoderm of Hydra fusca, each with a cytoplasmic inclusion. (From Downing, 1909.) species there is only one; in others four or more appear; and sometimes they are entirely absent. This last condition results from the formation of amebocytes before the fixation of the larva. In many other sponges the archeocytes migrate in at the posterior pole and partially or entirely fill up the segmentation cavity. Comparatively little is known about the embryology of Hexactinellida and PORIFERA, CHELENTERATA, VERTEBRATA 73 Demospongize, and few observations have been made upon their archeocytes. These archeocytes are of the greatest importance since they give rise to the amebocytes and tokocytes (reproductive cells). According to Weltner (1907) both amebocytes and tokocytes are only physiological states of one and the same kind of cell. Many authors have em- phasized the importance of the amebocytes, such as Gorich (1904), who maintains that this class of cells gives rise not only to the gonocytes, statocytes, and trophocytes, but also to certain pinacocytes. Weltner (1907) goes further than this when he states from studies upon the fresh-water sponge that the sponge could not exist without amebocytes. The earlier investigators almost invariably con- sidered the germ cells as mesodermal in origin. Lieberkiihn (1856) discovered the eggs in Spongilla and later (1859) in Sycandra raphanus. Sponge eggs were also observed by Kolliker (1864). Haeckel (1872) thought that the eggs were derived from the flagellated cells of the gastral epithelium. Schulze (1875), on the contrary, maintains that they lie deep in the so-called mesoderm; and Fiedler (1888) concludes that in Spongilla only certain cells of the middle layer may become germ cells. Maas (1893) distinguished two sorts of cells in the middle layer; one characterized by uniform, fine- granuled cytoplasm and an oval nucleus containing a very fine net-work of chromatin; the other filled with coarse-granuled cytoplasm and a spherical nu- cleus containing a deeply staining nucleolus and 74 GERM-CELL CYCLE IN ANIMALS chromatin aggregated into large masses. Only from the latter do the sex cells arise. These two kinds of cells could be distinguished in larval stages and the early separation of germ cells from somatic cells was pointed out. Maas, however, does not insist that there is here a demonstrated continuity of germ cells, since the cells which become sex-cells are separated from the egg by a long series of genera- tions. The recent investigations of Jérgensen (1910) on Sycon raphanus and S. setosa have added consider- ably to our knowledge of the origin, structure, and early history of the germ cells of sponges. Jérgensen does not agree with Maas (1893) regarding the early segregation of the germ cells from somatic cells, but finds no particular difference between so-called mesoderm cells and wandering or egg cells. It is worthy of note, however, that the youngest recog- nizable odgonia were found to contain several distinct bodies in their cytoplasm (Fig. 27, B). The method of formation of the gemmules has engaged the attention of many investigators, but several important points concerning it are still in doubt. Gemmule formation is of particular interest since the cells (amebocytes), which by most authori- ties are said to give rise to the germ cells, are also considered the cells which form the reproductive portion of the gemmules. At least four views have been held concerning the origin of the gemmule cells: (1) Carter (1849) believed that the gemmule is derived from a single cell, the “ovi-bearing cell”; PORIFERA, COKLENTERATA, VERTEBRATA 5 (2) Goette (1886) maintains that the gemmule con- sists of cells from several germ layers; (3) Carter believed at one time that the gemmule was made up of only one kind of cell; and (4) several authors (Marshall, 1884; Wierzejski, 1886; Zykoff, 1892; Weltner, 1892) believe that a number of cells belonging to several classes are concerned in the origin of the gemmule. Evans (1900) has described in detail the formation and structure of the gemmules of Ephydatia blembin- gia. In this species the first sign of the formation of a gemmule is the presence of “single cells or groups of cells scattered about chiefly in the dermal mem- brane; the strands of tissue which support the dermal membrane; and in the tissues situated immediately below the subdermal cavity” (p. 89). No mitotic figures were discovered in these cells and conse- quently the reproductive part of the gemmule is probably not derived from one mother-cell. These cells wander “through the dermal membrane, and strands of tissue which support the membrane, and become aggregated in groups situated either deep in the tissues of the sponge or even in the strands of tissue above mentioned.” Whether the reproductive cells of the gemmule arise from a single cell by proliferation or represent an aggregation without a common origin is still unsettled, but the latter view is held by most in- vestigators. If they do arise from a single cell, as H. V. Wilson (1902) admits is a possibility, the gemmule formation may be considered a kind of 76 GERM-CELL CYCLE IN ANIMALS parthenogenesis. If, on the other hand, the re- productive cells of the gemmule are of multiple origin, they may either be looked upon as true germ cells which form a group physiologically equivalent to the morula stage in the development of an egg, or as a collection of regenerative cells capable of producing a new individual. In this connection should be mentioned the bud- ding of Tethya (Désé, 1879-1880) which develops from a group of amebocytes (Maas, 1910) and the gemmules of Tedania and Esperella (Wilson, 1902) and of hexactinellids (Ijima) which become ciliated larvee. Wilson has shown “that silicious sponges, when kept in confinement under proper conditions degenerate in such a manner that while the bulk of the sponge dies, the cells in certain regions become aggregated to form lumps of undifferentiated tissue. Such lumps or plasmodial masses, which may be exceedingly abundant, are often of a rounded shape resembling gemmules, more especially the simpler gemmules of marine sponges (Chalina, e.g.), and were shown to possess in at least one form (Stylo- tella) full regenerative power. When isolated they grow and differentiate, producing perfect sponges ” (1907, p. 295). These “lumps of undifferentiated tissue’” have also been noted by F. E. Schulze (1904) and recognized as probably reproductive; they have been named by this author, “ sorites,”’ and have been called by several authors “artificial gemmules.”” The process involved in their forma- tion is termed ‘regressive differentiation.”” The PORIFERA, CGELENTERATA, VERTEBRATA 77 undifferentiated tissue of which they are composed, undoubtedly consists largely, if not entirely, of amebocytes (Weltner, 1907). These amebocytes are, however, of heterogeneous origin (Maas, 1910), since some of them represent transformed pore cells, whereas the rest are wandering cells. Even more interesting than these reproductive bodies are the artificial plasmodia produced by Wil- son (1907, 1911) in Microciona, Lissodendoryx, and Stylotella and by Miiller (1911) in the Spongillide. The method and results from a study of Microciona as stated by Wilson (1911) are briefly as follows. Branched specimens are cut up and strained into a dish of water through fine bolting cloth. The cells, which are dissociated in this way, “settle down on the bottom of the dish like a fine sediment.” Three classes of cells are present: (1) ‘‘the most con- spicuous and abundant” are unspecialized granular “ameboid cells of the sponge parenchyma (amcebo- cytes)”; (2) ‘“‘a great abundance of partially transformed collar cells”; and (3) ‘‘more or less spheroidal cells ranging from the size of the granular cells down to much smaller ones.” “Fusion of the granular cells begins imme- diately and in a few minutes’ time most of these have united to form conglomerate masses which at the surface display both blunt and elongated pseu- dopodia. These masses (plasmodia) soon begin to incorporate the neighboring collar and hyaline cells.” “The small conglomerate masses . . . early begin to fuse with one another,” and if the tissue is strewn 78 GERM-CELL CYCLE IN ANIMALS sparsely over a slide, in the course of a week it will be found that the slide is covered with a thin in- crusting sponge provided with pores, oscula, canals, and flagellated chambers.” Many, at the end of two months, had ‘developed reproductive bodies (eggs or asexual embryos?) ...”? Whether these reproductive bodies arose from eggs or masses of cells was not determined. ‘“‘ When the plasmodia have metamorphosed and the canals and chambers have developed, the skeleton makes its appearance.” Experiments with Lissodendoryx and _ Stylotella were not quite so successful, but plasmodial masses were formed in every case. Further experiments proved that “when the dissociated cells of these two species [Microciona and Lissodendoryx] are intermingled, they do not fuse with one another, but fusion goes on between the cells and cell masses of one and the same species.” A similar result was obtained by intermingling dissociated cells of Micro- ctona and Stylotella. Discussion AND SuMMARY. The foregoing ac- count of the origin of the germ cells in sponges shows conclusively that these cells arise in the so- called mesoderm from wandering cells (amebocytes) and that amebocytes are descended from archzo- cytes which may be distinguished in certain cases very early in embryological development (Fig. 27, 4, p.g.c). Odgonia and spermatogonia have not been recognized by most investigators except in the adult, but Maas (1893) has observed them in the planula. Jorgensen (1910), who has made the most careful PORIFERA, CQELENTERATA, VERTEBRATA 79 study of the development of the odgonia, states that the youngest recognizable odgonia lie in the mesoderm, and his figure (Fig. 27, B) shows that they may be distinguished from neighboring cells by certain characteristics, among which is the presence of a darkly staining inclusion. In the adult sponge the amebocytes from which the oégonia and sperma- togonia arise occur in the middle layer of all regions of the body, but, as pointed out by Korschelt and Heider (1902), the odgonia and spermatogonia may develop in only certain definite regions (Plakina monolopha), or in groups (Aphysilla violacea) which contain a more or less definite number of cells and occupy a similar position in each individual (Eu- spongia). Such an aggregation is the most primitive form of ovary. Some of the amebocytes of the sponge are un- doubtedly germ cells (tokocytes) and are able to develop into oégonia or spermatogonia, or to form aggregations (gemmules, “ artificial gemmules,”’ “ so- rites,” etc.) which can “regenerate” an entire sponge, but whether the amebocytes that produce odgonia and spermatogonia are the same as the reproductive cells of the gemmules, the regenerative cells of the “artificial gemmules,”’ and amebocytes which form the buds in Tethya is still uncertain. It seems probable that they are all alike potentially but develop differently because of the effects of different environmental factors. The distribution of ame- bocytes with reproductive powers throughout the entire sponge-body accounts for the great regenera- 80 GERM-CELL CYCLE IN ANIMALS tive ability of these animals and must also account for the development of plasmodia formed by dis- sociated cells (Wilson, 1911; Miiller, 1911) into adult sponges with all specific characteristics in- cluding reproductive bodies. It therefore seems possible that there may exist in the sponges a continuity of the germ-plasm and that the germ-cell material is distributed among thousands of cells (tokocytes, see Table, p. 71) which are derived from archeocytes, and that under proper conditions these tokocytes may produce odgonia or spermatogonia, or may aggregate to form gemmules or regenerative bodies. This wide distribution of the germ cells is what might be expected in such lowly organized animals. Figure 28 shows the probable history of the germ cells in the Portrera from one generation to the next. 2, CG@LENTERATA The origin of the germ cells in the C@LENTERATA has been a much debated subject among zodélogists for three-quarters of acentury. As early as 1843 van Beneden undertook to determine the germ layer from which the germ cells arise and concluded that the ova originate in the entoderm and that the spermatozoa come from the ectoderm. F. E. Schulze (1871) claims that in Cordylophora both the ova and spermatozoa are of ectodermal origin. Kleinenberg (1872), working on Hydra, announced that the germ cells are interstitial in origin and, since the interstitial cells arise from the ectoderm, PORIFERA, CQHLENTERATA, VERTEBRATA 81 Zygote Archeocytes Cells of dermal and gastral layers Tokocytes (Reproductive cells) Amebocytes Gonocytes (Sexual cells) Statocytes (Gemmule cells) Spermatozoon Zygote Fic. 28.— Diagram illustrating the probable history of the germ cells in sponges from one generation to the next. G 82 GERM-CELL CYCLE IN ANIMALS are therefore also ectodermal. Van Beneden (1874), from investigations on Hydractinia, Clava, and Cam- PANULARID#, confirms his earlier results and again maintains that the ova arise in the entoderm. The brothers Hertwig (1878) decided that the germ cells of HypromEpvus2 arise from the ectoderm and those of the ScypHomEDUs® and ANnTHOzoA from the entoderm. In a second paper, Kleinenberg (1881) reports the ova of Eudendrium as of ectodermal origin. Varenne (1882) maintains that both the ova and the spermatozoa of half a dozen species examined arise from entoderm cells of the young blastostyle before the appearance of medusa buds. The results of Weismann’s extended studies were published in a monograph (1883), and later (1884) a brief general account appeared. From this time until the present day almost every year has witnessed one or more contributions to the subject of the origin of the germ cells in ccelenterates, and a perusal of this mass of literature shows that the problem is not yet solved. Hypra. The fresh-water polyp, Hydra, has been employed for germ-cell investigations more often than any other ccelenterate, and a number of de- tailed papers have appeared within the past ten years upon this genus. Among the earlier workers who actually saw the egg should be mentioned Trembley (1744), Résel V. Rosenhoff (1755), Ehren- berg (1836) and Leydig (1848). The processes involved in odgeneses were not clearly determined, however, until Kleinenberg’s classic investigations PORIFERA, CQELENTERATA, VERTEBRATA 83 in 1872, upon which most of the accounts in our zoological textbooks are still based. Kleinenberg’s researches were followed by those of Korotneff (1883), Nussbaum (1887), Schneider (1890), and Brauer (1891). Investigations of the germ cells of Hydra then almost ceased until 1904, when another period of activity in this field began and papers quickly followed one another (Guenther, 1904; Downing, 1905; Hadzi, 1906; Hertwig, R., 1906; Tannreuther, 1908, 1909; Downing, 1909; and Wager, 1909). The following account is based chiefly upon the researches of Downing (1905, 1908, 1909), Tannreuther (1908, 1909), and Wager (1909). The origin of the male germ cells has been carefully investigated by Downing (1905) and Tannreuther (1909). Previous to Downing’s researches all in- vestigators, beginning with Kleinenberg (1872), considered the sex cells as interstitial in origin. Downing, however, believes that germ cells and in- terstitial cells may be distinct. The sex cells, according to this investigator, are distinguished “by their very large nuclei, extremely granular, and often by the presence of a Nebenkern”’ (Fig. 27, C). “The characters of the sex cells .. seem constant, and my conclusion would be that at some stage of the embryonic development certain cells are stamped with these characters and that they and their progeny form the sex cells distinct through- out the life of the individual . . . the germ-plasm is then continuous in Hydra” (p. 413). This tentative 84 GERM-CELL CYCLE IN ANIMALS opinion is expressed with more certainty in a later paper (Downing, 1909), since the “distinctive charac- ter of the germ cell is more marked in theovary than in the spermary” (p. 311). Tannreuther (1909), on the other hand, claims that the male germ cells are interstitial in origin, and “the progenitors of the spermatozoa have no special characters by which they can be recognized as germ cells.” The origin of the eggs of Hydra is better known than that of the male germ cells. The ova have by most investigators been considered modified intersti- tial cells. Downing (1908, 1909) disagrees in several respects with the results of Tannreuther and Wager. His most important difference is regarding the ques- tion of the origin of the ova directly from interstitial cells or from definite propagative cells that are set aside for reproductive purposes at some stage in the animal’s embryonic development. He believes “that in the adult Hydra the odgonia (and spermatogonia) are distinctly differentiated as a self-propagating tissue” (p. 310). Wager (1909), on the contrary, claims that it is impossible to prove that eggs do not arise from ordinary interstitial cells; whereas Tannreuther (1909) finds that the primitive ova can be distinguished from interstitial cells “by their large nucleus, nucleolus, and abundance of chromatin, even before the growth of the ovary begins”’ (p. 205), especially during the breeding season, and admits that “If these sex cells could be distinguished during the budding season as well, it would at least suggest specificity of the germ cells’ (p. 205). PORIFERA, CQELENTERATA, VERTEBRATA 85 By far the most important question arising from a study of the origin of the germ cells of Hydra is whether these cells arise from ordinary interstitial cells, as is clatmed by most investigators, or whether they originate from cells that are set aside for re- productive purposes at some stage of development, as Downing maintains. If the latter be true, “the germ-plasm is then continuous in Hydra’ (Downing, 1905, p. 413). Wager (1909) thinks the presence of special prop- agation cells to be “extremely improbable”’ and Tannreuther (1909) does not believe the known facts warrant the view that there is continuity of the germ-plasm in Hydra. This is, of course, a matter that may never be decided definitely, and at least not until some method of distinguishing the primordial germ cells, if these be present, from ordinary interstitial and other cells, has been found. Furthermore, if the germ-plasm is continuous, primor- dial germ cells must be present in buds, in adults at all times of the year, and in pieces of tissue that are capable of regenerating sexually reproductive adults. That such primordial germ cells exist seems to me to be quite possible. Hyprozoa. Many Hyprozoa besides Hydra have furnished material for germ-cell studies. Thus Weismann (1883) reported upon about forty species belonging to a number of different families. The results of the researches of the various investigators do not agree in many instances. In order to indicate the variety of the opinions expressed, the data re- 86 GERM-CELL CYCLE IN ANIMALS garding the germ cells in the following genera is considered below: (1) Eudendrium, (2) Hydractinia, (3) Pennaria, and (4) Clava. EvupENpRium. Five species of this genus have been investigated. In E. racemosum, according to Weismann (1883), the ova arise in the ectoderm and the male germ cells originate either from entoderm cells or from ectoderm cells that later migrate into the entoderm. Ischikawa (1887) asserts that the germ cells arise in the ectoderm and migrate into the en- toderm, and Hargitt (1904a) found ova in both the ectoderm and entoderm, but, since those in the entoderm were always the smaller, he concludes that they may have wandered into that layer from the ectoderm, though such a migration was not ob- served. In E. capillare Hargitt found ova in the entoderm except in one case where they occurred in the ecto- derm. This author also reports the female germ cells of E. tenue and E. racemosum from the entoderm only. The ova of the EuDENDRID2 when first dis- tinguishable “are slightly larger than the ordinary cells of the surrounding tissue, and differ also in shape, being generally ovoid or spherical and with comparatively conspicuous nuclei. .. . Growth at this period would seem to take place in situ, through the direct nutritive activity of the surrounding tissue cells... . As growth continues, the ova become more or less amceboid, migrating toward the gono- phore region, where they seem to aggregate in con- siderable numbers, the presence of which may act as a PORIFERA, CQELENTERATA, VERTEBRATA 87 stimulus from which results the formation of the gonophore” (Hargitt, 1904 a, pp. 261-262). Hypractinia has been investigated by van Beneden (1874), Weismann (1883), Bunting (1894), and Smallwood (1909). Weismann considered the ectoderm of the blastostyle to be the probable place of origin of the germ cells in this genus. Bunting (1894) was unable to trace the ova to this layer, although she found them to be quite abundant in the entoderm of the blastostyle, even before the gono- phore appeared. According to this author the ova apparently arise in the entoderm of the blasto- style, and ‘“‘reach maturity on the outside wall of the spadix, lying between the endoderm and the inner layer of the bell nucleus. The spermatozoa arise from the inner layer of the bell nucleus; we see that they are, therefore, ectodermal in origin” (p. 228). These results are not confirmed by the researches of Smallwood (1909), who finds that the eggs arise in the entoderm in any region of the polyp, at the base, the side of the polyp, or in the gonophore. They may be distinguished from other entoderm cells by the larger size of the nucleus. In Pennaria cavolini the germ cells arise in the ectoderm, according to Weismann (1883), and this conclusion is confirmed for the ova by Hargitt (1904b). In P. tiarella the germ cells are likewise of ectodermal origin (Smallwood, 1899, Hargitt, for the ova, 1904b). The eggs of this species arise in the ectoderm of the manubrium and grow by 88 GERM-CELL CYCLE IN ANIMALS engulfing other primitive ova. Only six or eight, rarely more, of the eggs survive. In Clava, according to van Beneden (1874), the ova arise in the entoderm. Weismann (1883) was not able to determine whether they originated in the entoderm or migrated into that layer from the ectoderm, but he was certain that the male germ cells were ectodermal. This conclusion regarding the male germ cells was confirmed by Thallowitz (1885). Harm (1902) was able to trace the primitive germ cells back to a very early stage, and could distinguish them in even young hydranths. The odcytes dif- fered from the remaining ectoderm cells in the pos- session of a larger amount of cytoplasm, a larger nucleus with a big nucleolus, and an ameboid shape. Hargitt (1906), working on Clava leptostyla, comes to conclusions different from those of Harm on C. squamata. He says “that eggs probably never arise in the ectoderm but always in the entoderm of the peduncle of the gonophore, or in that of the polyp very near the base of the gonophore. ... Clava, like other Hydroids, has its breeding season, during which the germ cells are extremely abundant, and at other times these cells are either entirely absent or very scarce” (p. 208). Concerning the early origin of germ cells Hargitt says, “it may not be im- possible that ‘Urkeimzellen’ should perhaps exist in undifferentiated stages, still the probability is so extremely remote as to render doubtful to a degree any but the most thoroughly substantial claims ” (p. 209). PORIFERA, CQKELENTERATA, VERTEBRATA 89 One more Hyprozoon may be mentioned — Gonothyrea lovent— since Wulfert (1902) traced the germ cells of this species back to the planula stage where they arise from the interstitial cells of the ectoderm and later undergo characteristic migrations. Our knowledge of the origin of the germ cells in other ccelenterates is very fragmentary and even less decisive than that of the Hyprozoa. For this reason a consideration of the subject is omitted here. Discussion. As in the Porirera we are here confronted with the question whether or not there is continuity of the germ-plasm in the C@LENTERATA. There is sufficient evidence for the belief that the cells which develop into germ cells are not derived from the ectoderm or the entoderm but belong to a special sort of propagative cells which are scattered about among the other cells throughout the body and which give rise to ova or spermatozoa under certain environmental conditions differing in the different species. This conclusion is based partly upon the results of Downing (1905, 1908, 1909), who still holds, as stated in his published papers, that there is continuity of the germ-plasm in Hydra; and upon the fact that germ cells have been recog- nized in the young hydranths of Clava (Harm, 1902) and in the planula of Gonothyrea (Wulfert, 1902). It seems certain that more careful studies of the early stages of ccelenterates with special regard to the origin of the germ cells and with the use of many and varied stains would result in the discovery 90 re Fig. 29. — Diagram to illustrate the phylogenetic shifting back of the origins of the germ cells in medusoids and hydroids. A composite picture. A, branch of a polyp-colony; P, polyp-head with mouth (m) and tentacles; St, stalk of the polyp; M, medusoid-bud with the bell (Gi); T, marginal tentacle; m, mouth; Mst, ma- nubrium; GphK, a gonophore-bud; GH, gas- tric cavity; ekt, ectoderm; ent, endoderm; st, supporting lamella. The germ cells (kz) arise in the medusoid in the ectoderm of the manubrium — first phyletic stage— where they algo attain maturity. In the gonophore-bud (GphK) they arise in the ectoderm (kz’), or further down in the stalk of the polyp at kz” — third phyletic stage — or in the ectoderm of the branch from which the polyp has arisen, at kz’’’ — fourth phyletic stage of the shunting of the originative area of the germ cells. In the last two cases the germ cells migrate until they reach their primitive place of origination in the medusoid, or in the corresponding layer of the medussid gonophore, as may be more clearly seen in Fig. 30. (After Weismann, 1904.) GERM-CELL CYCLE IN ANIMALS of these cells in younger em- bryos than yet recorded, and might even dis- close charac- teristics which would enable us to trace the keimbahn in some species back into the early cleavage stages. In discussing the germ cells of ccelenterates, it is necessary to refer to the work of Weis- mann who has added so much to our knowl- edge of this subject. Weis- mann’s position may best be presented in his own words (The Evolution The- ory, Vol. I, pp. 413-415, 1904). PORIFERA, CCHELENTERATA, VERTEBRATA 91 “In the hydroid polyps and their medusoids the germ-cells always arise in the ectoderm; in species which produce sexual medusoids by budding, the germ cells arise in the ectoderm of the manubrium of these medusoids (Fig. 29, M, kz). But in many species these sexual stages have degenerated in the course of phylogeny into so-called gonophores, that is, to medusoids which still exhibit more or less complete bells, but neither mouth (m) nor marginal tentacles (7), and which no longer break away from the colony to swim freely about, to feed in- dependently, and to produce and ripen germ-cells. The degeneration of the ‘gonophores’ often goes even farther; in many the medusoid bell is repre- sented only by a thin layer of cells, and in some even this token of descent from medusoid ancestry is absent, and they are mere single-layered closed brood-sacs (Fig. 30, Gph). “The adherence of the sexual animal to the hydroid colony has, however, made a more rapid ripening of the germ-cells possible, and nature has taken advan- tage of this possibility m all cases known to me, for the germ-cells no longer arise in the manubrium of the mature degenerate medusoid, that is, of the gonophore, but earlier, before the bud which becomes a gonophore possesses a manubrium. The birth- place of the germ-cells is thus shifted back from the manubrium of the medusoid to the young gonophore- bud (Fig. 29, M, kz). The same thing occurs in species in which the medusoids are liberated, but live only for a short time, for instance, in the genus 92 Fic. 30.— Diagram to illustrate the migra- tion of the germ cells in hydromeduse from their remotely shunted place of origin to their primitive place of origin in the gonophore, in which they attain to ma- turity. The state of affairs in Eudendrium is taken as the basis of the diagram. mu, mouth; ma, gut-cavity; ¢t, tentacle; Sta, stem; A, a branch of the polyp-colony; SP, lateral polyp; Gph, a medusoid-bud completely degenerated into a mere gono- phore; Hi, ovum; GH, gastric cavity; st, supporting lamella. The originative area of the germ cells lies in the stem of the principal polyp at kz’’”’, whence the germ cells first migrate into the endo- derm of the branch (A) at kz’’’, creeping within which they reach kz” in the lat- eral polyp (blastostyle), finally reaching the gonophore (kz) and passing again into the ectoderm. (After Weismann, 1904.) GERM-CELL CYCLE IN ANIMALS Podocoryne. Al- though perfect medusoids are formed, these have their germ- cells fully devel- oped at the time of their liberation from the hydroid colony. But in species in which the medusoid- buds have really degenerated and are no longer lib- erated, the birth- place of the germ- cells is shifted even farther back, and in the first place into the stalk (St, kz’’) of the polyp from the gonophore- buds. This is the case in the genus Hydractinia. In the further course of the process the birthplace of the germ-cells has PORIFERA, CQQALENTERATA, VERTEBRATA 93 shifted as far back as to the branch from which the polyp has grown out (Fig. 29, A, kz’’’); and finally, in the cases in which the medusoid has degenerated to a mere brood-sac (Fig. 30, ph), even to the generation of polyps immediately before, that is, into the polyp-stem from which the branch arises that bears the polyps producing the gonophore-bud (Fig. 30, kz’’’). Then we find the birthplace of the germ-cells still further back (Fig. 30, kz’’’’), for the egg and sperm cells arise in the stem of the principal polyps (the main stem of the colony). The advantage of this arrangement is easily seen, for the principal polyp is present earlier than those of the secondary branches, and these again earlier than the polyp which bears the sexual buds, and this, finally, earlier than the sexual bud which it bears. Thus this shunting backwards of the birthplace of the germ-cells means an earlier origin of the primordium (Anlage) of the germ-cells, and consequently an earlier maturing of these. ‘But none of these germ-cells come to maturity in the birthplace to which they have been shifted, for they migrate independently from it to the place at which they primitively arose, namely, into the manubrium of the medusoid, which is still present even when great degeneration has occurred, or even — in the most extreme cases of degeneration — into the ectoderm of the brood-sac. This is the case in the genus Eudendrium, of which Fig. 30 gives a diagram- matic representation. “The most interesting feature of this migration of 94 GERM-CELL CYCLE IN ANIMALS the germ-cells is that the cells invariably arise in the ectoderm (kz’’’’), then pierce through the sup- porting lamella (sf) into the endoderm (kz’’’), and then creep along it to their maturing-place. Once there, they break through again to the outer layer of cells, the ectoderm (kz), and come to maturity (£7). That they make their way through the endoderm is probably to be explained by the fact that they are there in direct proximity to the food-stream which flows through the colony (GH = gastric cavity), and they are thus more richly nourished there than in the ectoderm. But, although this is the case, they never arise in the endoderm; in no single case is the birthplace of the germ-cells to be found in the endoderm, but always in the ectoderm, no matter how far back it may have been shunted. Even when the germ-cells migrate through the en- doderm, their first recognizable appearance is in- variably in the ectoderm, as, for instance, in Podo- coryne and Hydractinia. The course of affairs is thus exactly what it would necessarily be if our supposition were correct, that only definite cell- generations —in this case the ectoderm-cells — contain the complete germ-plasm. If the endoderm- cells also contained germ-plasm it would be hard to understand why the germ-cells never arise from them, since their situation offers much better con- ditions for their further development than that of the ectoderm-cells. It would also be hard to under- stand why such a circuitous route was chosen as that exhibited by the migration of the young germ-cells PORIFERA, CCHELENTERATA, VERTEBRATA 95 into the endoderm. Something must be lacking in the endoderm that is necessary to make a cell into a germ-cell: that something is the germ-plasm.” Several important contributions have appeared within recent years which seem to deprive Weis- mann’s contentions of much of their importance. For example, Goette (1907) has found that the germ cells of many Hypromepts.£ may arise in the en- toderm or in the ectoderm, and that in Clara multt- cornis the germ cells are transformed half-entoderm cells. After a long series of studies on ccelenterate development C. W. Hargitt (1911) has attacked Weismann’s position in the following words: ‘‘ That there is any such region as may be designated a “Keimzone’ or ‘Keimstiatte’ may be at once dis- missed as absolutely without warrant as a general proposition. Furthermore, that the germ cells have their origin in the ectoderm alone in hydromeduse may be similarly denied and dismissed as unworthy of further inquiry or doubt. And still further, I am thoroughly convinced that the still more recent controversy as to the hypothesis of the ‘germ-plasm,’ if not as clearly a delusion as the preceding, is yet without the slightest support from the ontogeny of the group under review. “Tt is a matter of easy demonstration that in many species of hydroids the egg may be followed in every detail from its origin as an ectoderm or an entoderm or interstitial cell through its gradual differentiation and growth to maturation, as a distinct individual cell, without the slightest tendency to multiplication.” 96 GERM-CELL CYCLE IN ANIMALS “Tt is passing strange that he should ignore the body of facts concerned in regeneration, and among them the reproductive organs. And it is still more strange that in support of this he should cite in detail the Hyprozoa as illustrating and supporting the hypothesis, ignoring the well-known facts that among these are abounding evidences which afford insuperable objections to just these assumptions. The present author has, in many cases, shown that gonads may be as readily regenerated by hydroids and meduse as any other organs; and that not for once or twice, but repeatedly in the same specimen, and that de novo and in situ; not the slightest evi- dence being distinguishable that any migration through preéxisting ‘germ-tracks’ occurred. The assumption that in these animals the gonads have “been shifted backwards in the course of phylogenetic evolution, that is, have been moved nearer to the starting point of development’ seems so at variance with known facts as to be difficult to appreciate or respect.” Professor Hargitt finally concludes with the fol- lowing sentence: “I believe the foregoing facts must suffice to show that, both as to origin, differen- tiation, and growth, the germ-cells of the HypRozoa, so far from sustaining the doctrine of the germ- plasm, afford the strongest and most direct evidence to the contrary.” G. T. Hargitt (1913) has also discovered facts regarding the history of the germ cells in ccelenter- ates which are decidedly opposed to Weismann’s PORIFERA, CQALENTERATA, VERTEBRATA 97 views. He finds that “The egg cells of Campanularia flexuosa arise in the entoderm of the pedicel of the gonophore, by the transformation of a single epithelial cell, or from the basal half of a divided cell, the distal half of which remains an epithelial cell and retains its epithelial functions. Therefore the egg cells have come from differentiated body- cells (so-called) and there is no differentiation of the germ-plasm in the sense that germ-cells are early differentiated and set aside and do not partici- pate in the body functions. Any cell of the ento- derm of Campanularia fleruosa may become an egg cell if it is in the position of the developing gono- phore”’ (p. 411). In spite of these attacks upon the germ-plasm theory as applied to ccelenterates, the possibility and even probability of such a condition seems to the writer to exist, and he is inclined to accept Downing’s position in the matter. Weismann’s views must, however, be modified, since the germ cells are not ectoderm cells, as he claims, nor do they belong to any germ layer. They are, according to the view adopted here, set aside as a separate class of cells at some stage during early development, are scattered about among the cells of the ectoderm or entoderm, depending upon the species, or lie in the mesoglea. We know that external conditions may stimulate reproductive activity in certain coelenterates (Frischholz, 1909) and consequently the development of germ cells, and we must conclude that these germ cells are present at all times in a H 98 GERM-CELL CYCLE IN ANIMALS more or less dormant condition, just as they are in more complex animals. Furthermore, the germ cells must be widely scattered, as has been shown by Harm (1902) in the young hydranths of Clava, by Wulfert (1902) in the planula of Gonothyrea, and by Small- wood (1909) in the polyp of Hydractinia. This wide distribution of primitive germ cells accounts for the reproductive powers of regenerated pieces of hy- droids. 8. VERTEBRATA Efforts have been made by many investigators to trace the keimbahn in vertebrates, but thus far no method has yet been devised which will enable us to distinguish germ cells from other cells in the early embryonic stages. That we shall be able to recognize germ cells in still earlier stages of development than has yet been accomplished seems certain, and the recent contributions of Rubaschkin (1910), Tschasch- kin (1910), von Berenberg-Gossler (1912a) and Swift (1914) have already made considerable ad- vances by the use of some of the more modern cyto- logical methods. Three principal theories have been advanced regarding the origin of the germ cells in vertebrates, and these will be briefly stated before the histories of the germ cells in special cases are discussed. The germinal epithelium theory was advanced by Waldeyer in 1870. At that time nothing was known regarding the migration of germ cells during the embryonic development of vertebrates, and it is PORIFERA, COELENTERATA, VERTEBRATA 99 not strange that he should have come to the con- clusion that the primordial ova arise from the epithelial cells of the genital ridge among which they were observed. Although this theory was accepted - by most embryologists, it has gradually been aban- doned until now it has very few supporters. The gonotome theory resulted from the studies of Riickert (1888) and Van Wijhe (1889). The germ cells appeared to these investigators to arise in a part of the segmental mesoblast of the embryo to which the latter applied the term ‘gonotome.’ From the gonotome they become embedded in the peritoneum. Thus the same cells are recognized as germ cells by the adherents of both theories, but a difference exists regarding their origin. The theory of early segregation has become the most prevalent view.of the origin of the germ cells of vertebrates, although there are many who still hold one of the other hypotheses. According to this theory the germ cells are set aside during the early embryonic stages before definite germ layers are formed, and they later arrive at the germinal ridge either by their own migration or by changes in the position of the tissues during development. The germinal epithelium theories have little if any evidence in their favor, since no one has actually ob- served a transformation of peritoneal or mesoblast cells into germ cells. On the other hand, there is an abundance of proof that these cells migrate from some distance into the position of the sex glands. According to Dustin (1907), Firket (1914) and 100 GERM-CELL CYCLE IN ANIMALS several others there are two methods of origin, and primary and secondary sex cells are produced. The former are probably derived from the blastomeres ; whereas the secondary sex cells are entirely inde- pendent and arise from the ccelomic epithelium. The first statement of the theory of early segre- gation was made by Nussbaum (1880), who studied the history of the germ cells in the trout. Following Nussbaum, Eigenmann (1892, 1896) contributed to the support of the theory by his investigations on the viviparous teleost, Cymatogaster. This proved to be excellent material for such studies and led Eigenmann to the conclusion that the germ cells are set aside in this fish during the early cleavage stages of the egg, probably at the thirty-two cell stage. In other cases it has been impossible to trace the germ cells back to such an early embryonic condition, but nevertheless the evidence has been almost uniformly in favor of early segregation. Some of those who have advocated such an early origin of germ cells are Wheeler (1900) in the lamprey, Beard (1900, 1902) in Raja and Pristiurus, Nussbaum (1901) in the chick, Woods (1902) in Acanthats, Allen (1906, 1907, 1909, 1911) in Chrysemys, Rana, Amia, and Lepidosteus, Rubaschkin (1907, 1909, 1910, 1912) in the chick, cat, rabbit, and guinea-pig, Kuschakewitsch (1908) in Rana, Jarvis (1908) in Phrynosoma, Tschaschkin (1910) in the chick, von Berenberg-Gossler (1912) in the chick, Schapitz (1912) in Amblystoma, Fuss (1912) in the pig and man, and Swift (1914) in the chick. This is by no PORIFERA, CQALENTERATA, VERTEBRATA 101 means a complete list but indicates the range of forms studied and the current interest in this subject. Some of the characteristics by means of which germ cells can be distinguished in vertebrate embryos are as follows: (1) the presence of yolk, (2) an ameboid shape, (3) large size, and (4) slight staining capacity. By sectioning embryos of various ages the changes in position of the germ cells can be fol- lowed with considerable accuracy. Most investi- gators agree that the movement of the germ cells from the tissues where first observed to the genital ridge is caused by ameboid activities of the cells themselves and by changes in the position of the organs of the embryo. The paths of migration of four verte- brates, a turtle, Chrysemys, a frog, Rana, the gar pike, Lepidosteus, and the fresh-water dogfish, Amia, are shown in Fig. 6. For example: ‘In Lepidosteus the sex-cells [Fig. 6, 3, SJ] first seen in the ventral and lateral portions of the gut- entoderm [Int] migrate to occupy a position in the dorsal portion of it, from which they pass dorsally into the loose mesenchyme that forms the substance of the developing mesentery [Mes]. As the mesen- tery becomes more narrow and compact, owing to the increase in size of the body cavity, the sex cells migrate to its dorsal portion and laterally to the sex-gland anlagen (Fig. 6, 4, Sc). Roughly speaking, one-half of the total number of sex-cells reach the sex-gland anlagen, the remainder being distributed between the intestinal entoderm, the mesodermal layers of the intestine, the mesentery, 102 GERM-CELL CYCLE IN ANIMALS and the tissues at and dorsal to the root of the intestine” (Allen, 1911, p. 32). Of the more recent investigations, facts discov- ered by Dodds (1910), Rubaschkin (1910, 1912), Tschaschkin (1910), von Berenberg-Gossler (1912), and Swift (1914) are especially worthy of mention. Dodds (1910) found that in the teleost, Lophius, the germ cells in the embryos cannot be definitely distinguished previous to the appearance in their cytoplasm of a body which stains like a plasmosome (Fig. 31, A). Germ cells are undoubtedly segregated before this period, but they exhibited no characteris- tics with the methods employed which rendered them distinguishable. Dodds believes that this cyto- plasmic body is extruded plasmosome material, probably part of one of the two plasmosomes pos- sessed by many of the cells at this period. Rubaschkin, in 1910, announced the results ob- tained with the eggs of the guinea-pig by certain methods designed to bring into view the chondrio- somes. He shows that the chondriosomes of the undifferentiated cells are granular, and that as differentiation proceeds, these granules unite to form chains and threads (Fig. 31, B). The sex cells, however, retain the chondriosomes in their primitive granular form, and remain in an undiffer- entiated condition situated in the posterior part of the embryo among the entoderm cells. Tschaschkin (1910), in the same year, came to a similar conclusion from studies made with chick embryos. Rubaschkin (1912) has also extended his investigations on guinea- PORIFERA, CHLENTERATA, VERTEBRATA 103 pig embryos. The accompanying diagram (Fig. 32) shows the fertilized egg and the early cleavage cells all alike (in black) ; some of their descendants become differentiated into the somatic cells of the germ Fic. 31.— Germ cells of vertebrates. A. From embryo of the teleost, Lophius, with plasmosome (?) extruded into cytoplasm. (From Dodds, 1910.) B. One germ cell and four somatic cells from a guinea-pig embryo. (From Rubaschkin, 1912.) C. Germ cell of chick showing ‘‘ Netzapparat.’’ (From von Berenberg-Gossler, 1912.) D. Primordial germ cell (g) and blood cell (6) in lumen of blood vessel (1) of a nineteen somite chick embryo. a = attraction-sphere. (From Swift, 1914.) layers (circles), but others (in black) remain in a primitive condition and are recognizable as the primordial germ cells ; these remain at rest for a considerable period, but finally multiply and become part of the germinal epithelium (g.ep). 104 GERM-CELL CYCLE IN ANIMALS Von Berenberg-Gossler (1912) considers the ‘‘ Netz- apparat” in the primitive germ cells of the chick of particular importance (Fig. 31, C), comparing it with the “ wurstformige Kérper” described by Hasper gep—O 0000 O00 €6@00C@066 Wd } G Jd Fic. 32.— Diagram to show the history of the germ cells in the embryo of the guinea-pig. g.ep = germinal epithelium. (From Rubasch- kin, 1912.) (1911) in Chironomus (p. 108, Fig. 33). The ap- pearance of this structure in ‘‘Keimbahnzellen”’ is thought to be due to the long period during which these cells do not divide. Duesberg (1912), however, after an exhaustive review of the literature on this PORIFERA, CQELENTERATA, VERTEBRATA 105 structure concludes that it is not a special cell organ but an artifact. Kulesch (1914), on the contrary, finds it to be a constant organ in the eggs of the cat, dog, and guinea-pig. The evidence of a continuous germ-cell cycle in the vertebrates is more convincing than in the sponges and ccelenterates, and leads us to predict that it will not be long before the gap still existing during which germ cells cannot be recognized will be filled in to the satisfaction of the majority of investigators. CHAPTER V THE SEGREGATION OF THE GERM CELLS IN THE ARTHROPODA 1. Tae KEIMBAHN IN THE INSECTS THE insects have furnished a very large proportion of the data upon which many of our biological conceptions are now based, and they are becoming more and more popular for studies of the physical basis of heredity, and for purposes of animal breeding. It was in insects (Miastor) that the early segrega- tion of the germ cells in animals was first definitely established. The accessory chromosome was dis- covered in insects by Henking in 1891, and our knowledge of the chromosomes, which has increased so remarkably within the past fifteen years, is due principally to the study of odgenesis and spermato- genesis in insects. In this chapter the chromosomes will only be considered incidentally, a more detailed account being deferred until later (Chapter IX). The early history of the germ cells in insect develop- ment has not been slighted, for there are many reports based on this subject alone and still more data hidden away in contributions on general em- bryology. It will be necessary here to select from this abundance of material those reports that give us the clearest pictures of the keimbahnen. As 106 GERM CELLS IN THE ARTHROPODA 107 usual, certain species or groups of species have proven more favorable than others for germ-cell studies, especially those belonging to the orders DipTERA, COLEOPTERA, and HyMENOPTERA. Diptera. Robin, in 1862, described what he called “globules polaries”’ at one end of the nearly transparent eggs of the crane fly, Tipulides culici- formes, and the following year Weismann (1863) re- ported the formation of similar cells, the ‘“Pol- zellen”’ at the posterior end of the eggs of the midge, Chironomus nigroviridis, and the blow fly, Calliphora (Musca) vomitoria. It remained for Leuckart (1865) and Metchnikoff (1865, 1866), however, to identify the pole cells (in Miastor) as primordial germ cells; their results were confirmed for Chironomus by Grimm (1870) and Balbiani (1882, 1885). Pole cells have also been described among the Diptera, in Musca by Kowalevsky (1886), Voeltz- kow (1889), and Escherich (1900); in Calliphora by Graber (1889) and Noack (1901); in Chironomus by Ritter (1890) and Hasper (1911); in Lucilia by Escherich (1900); in Mvastor by Kahle (1908) and Hegner (1912, 1914a), and in Compsilura by Hegner (1914). Four genera of flies will serve to illustrate the methods of germ-cell segregation in this order: (1) Chironomus (Ritter, 1890; Hasper, 1911), (2) Cal- liphora (Noack, 1901), (3) Muastor (Kahle, 1908; Hegner, 1912, 1914a), and (4) Compsilura (Hegner, 1914a). Since Miastor has been discussed in detail in Chapter III it will be only briefly referred to here. 108 GERM-CELL CYCLE IN ANIMALS We owe the first accurate account of the germ cells in Chironomus to Ritter (1890), who, by means of the section method, showed that the “yolk granules” described by Weismann (1863) in the pole cells are derived from a disc-shaped mass of substance situated near the posterior end of the egg and termed by him the ‘“Keimwulst.” Hasper (1911) was able to confirm this discovery, to add other interesting facts, and to correct several of Ritter’s errors. The “Keimwulst” of Ritter is called by Hasper the “ Keimbahnplasma.”’ Ritter advanced the idea that the cleavage nucleus of Chironomus divides within the “Keim- wulst”’ and that here the first cleavage division occurs, one daughter nucleus remaining in the “‘ Keim- wulst”? and becoming the center of the primordial germ cell, the other giving rise to somatic nuclei. This is probably the basis for Weismann’s (1904) statement regarding his conception of the germ- plasm that, “If we could assume that the ovum, just beginning to develop, divides at its first cleavage into two cells, one of which gives rise to the whole body (soma) and the other only to the germ-cells lying in this body, the matter would be theoretically simple. ... As yet, however, only one group of animals is known to behave demonstrably in this manner, the Diptera among insects... .” There is, however, nothing in the literature to warrant the above statement, since Ritter’s hypothesis has been disproved by Hasper. According to Hasper one of the cleavage nuclei GERM CELLS IN THE ARTHROPODA 109 at the four cell stage becomes separated from the rest of the egg, together with all of the Keimbahn- plasma as the primordial germ cell (Fig. 33 B, p.g.c.). The Keimbahnplasma is apparently equally divided between the daughter cells when the primordial germ cell divides. Later the nuclei of the germ cells increase in number without an accompanying division of the cell, thus producing binucleated cells (Fig. 33, C). The history of the pole cells during embryonic development will be more fully described in the CoLEOPTERA, since in the beetles the Keimbahn is much more distinct. The origin and nature of the Keimbahnplasma was not determined by Hasper, but it was found to persist in certain cases even until the larval stage was reached (Fig. 33, D). In Calliphora Noack (1901) described a dark granular disc at the posterior end of the egg (Fig. 34) which he termed the ‘‘Dotterplatte’ and which, like the pole-plasm of \Wiastor and the Keimbahn- plasma of Chironomus takes part in the formation of the primordial germ cells. The eggs of the parasitic fly, Compstlura concinnata, were also found by the writer (Hegner, 1914a) to possess a granular pole- disc, thus adding one more species to the list of Diptera in which such a structure exists. CoteoprersA. The origin of the germ cells in beetles and their subsequent history are well known only in certain species of the family CHRYSOMELID of the genera Calligrapha and Leptinotarsa. The contributions of Wheeler (1889), Lecaillon (1898), PEs, D Fig. 33.— Chironomus. A. Longitudinal section through the posterior end of a freshly laid egg. B. Longitudinal section through egg during division of first four cleavage nuclei; at posterior end the primordial germ cell is just being formed. C. One of primordial germ cells containing two nuclei and remains of ‘‘ Keimbahnplasma.” D. Germ gland of the larva in which remains of ‘‘ Keimbahnplasma”’ stillappear. Abpl = ‘‘ Keimkahnplasma”’; p.g.c. = primordial germ cell. (From Hasper, 1911.) (110) GERM CELLS IN THE ARTHROPODA 111 Hegner (1908, 1909a, 19096, 191la, 1911b, 1914a), and Wieman (1910a, 1910b) will be referred to in the following paragraphs. Wheeler (1889) figured several primordial germ cells in an egg of Leptinotarsa with a segmented germ band and _ suspected their true nature, but did not discover them in earlier stages. Le- caillon (1898) de- scribed the pole-cells in several chrysomelid beetles, but did not make out any of the details concerning their origin, structure, and migrations. Within the last seven years the writer has devoted a consid- Fra. 34. — Calliphora. A. Longitudi- $ ‘ nal section through posterior end of erable portion of his freshly laid egg, showing ‘‘ Dotter- time to morphological platte (Dpl). B. Longitudinal sec- d . tion through posterior end of egg at an experimen tal time of blastoderm formation, showing studies of the eggs of peat Pe ay ets beetles, particularly Calligrapha bigsbyana, C. multipunctata, C. lunata, and Leptinotarsa decemlineata. The eggs of these species are peculiarly favorable for study, since they are definitely oriented in the body of the mother and various surfaces can be recognized in the newly laid egg: they can be placed under the most severe 112 GERM-CELL CYCLE IN ANIMALS experimental conditions without killing them or stopping their progressive development; and they can be killed, fixed, sectioned, and stained with comparative ease. Furthermore, the eggs of these beetles possess a well-defined pole-disc, and the primordial germ cells which arise even before the blastoderm is formed are easily distinguishable from the somatic cells and thus can be traced from the time of their appearance until they become ma- ture eggs and spermatozoa. The ova of insects have long been considered among the most highly organized of all animal eggs. That they are definitely oriented while still within the ovary was expressed by Hallez (1886) in his ‘‘Law of the Orientation of Insect Embryos” as follows: “‘The cell possesses the same orientation as the maternal organism that produces it; it has a cephalic pole and a caudal pole, a right side and a left side, a dorsal surface and a ventral surface; and these different surfaces of the egg-cell coincide to the corresponding surfaces of the embryo.”’ The orientation of an ovarian egg is indicated in Fig. 35, and here also is shown the position and surfaces of the egg at the time of deposition. When the egg is laid the beetle clings to the under surface of a leaf, and with a drop of viscid substance from the acces- sory glands of the reproductive organs, fastens the egg by its posterior end (p) tothe leaf; then with the tip of the abdomen the egg is pushed back through the are indicated by the dotted line. It is a simple matter to determine the various surfaces of eggs GERM CELLS IN THE ARTHROPODA 113 laid in this manner. Gravity apparently has no influence upon the development, since eggs in a state of nature occupy all positions with respect to this factor without becoming altered in any way. Only one case has come to the writer’s attention of an influence of gravity in insect development — the eggs of the water beetle, Hydrophilus atterimus, Fic. 35.— A diagramatic drawing of Calligrapha bigsbyana clinging to the under side of 1 willow leaf and showing the orientation of the egg in the ovarian tubule and after deposition. a@= anterior; d= dorsal; p=posterior; r=right side; 2=place where egg was marked with India ink as means of orientation after removal from leaf. according to Megusar (1906), develop abnormally if the cocoon in which they are laid is inverted. The events that precede the establishment of the primordial germ cells in chrysomelid beetles may be described briefly as follows: The egg, when laid (Fig. 36, 1), consists of a large central mass of yolk globules (y), among which are very fine strands of cytoplasm; a thin peripheral layer of cytoplasm, the ‘““keimhautblastem” of Weismann (khb/), a delicate vitelline membrane (v.m.), a chitinous shell, the chorion, and a nucleus consisting of the egg nucleus I D Fic. 36.— Calligrapha. A. Longitudinal section through an egg of C. bigsbyana four hours after deposition. B. Longitudinal section through an egg of C. bigsbyana 14 hours after deposition. C. Two germ cells just protruding from posterior end of egg of C. multi- punctata. D. The pole-disc in an egg of C. multipunctata. g.c.d. = pole-disc ; g.n. = germ nuclei fusing; kAbl = keimhautblastem ; p. = posterior end of egg; p.bl.n. = preblastodermic nuclei; v.m. = vitel- line membrane; vt. = vitcllophags;: y. = yolk. (114) GERM CELLS IN THE ARTHROPODA 115 and a sperm nucleus combined (g.n). Frequently the two polar bodies have not yet been produced when the egg is laid and thus many stages may be encountered in the newly laid eggs. Polyspermy is a normal condition in insects and several sperma- tozoa are often observed among the yolk globules. The keimhautblastem is not homogeneous through- out, for at the posterior end there is embedded in it a disc-shaped mass of darkly staining granules which I have called the pole-dise (g.c.d.) and which resembles the pole-plasm of Jiastor, the “ Keimwulst”’ or “ Keimbahnplasma” of Chironomus and the “ Dotter- platte” of Callcphora. The cleavage nucleus divides by mitosis; the daughter nuclei separate slightly, and divide; and this process is continued until nuclei, each surrounded by a small mass of cytoplasm, are scattered more or less regularly throughout the egg. Then a division of the nuclei into two groups occurs; those of one group migrate to the periphery, fuse with the periph- eral layer of cytoplasm, and are cut off by cell walls, thus forming the blastoderm; whereas the other nuclei, the vitellophags, remain behind among the yolk globules which it is their function to dissolve. The blastoderm consists of a single layer of cells, except at the posterior end where its formation has been interrupted by the process resulting in the establishment of the primordial germ cells. The primordial germ cells are formed in the fol- lowing manner. The cleavage nuclei at the posterior end of the egg that encounter the pole-disc granules 116 GERM-CELL CYCLE IN ANIMALS behave differently from those at other points, since they do not remain to form part of the blastoderm but continue to migrate until they have become entirely separated from the rest of the egg. During this process each of the sixteen nuclei that act in this way becomes surrounded by a halo of granules — part of the pole-disc. Then cell walls appear and sixteen primordial germ cells result. These form a group at the posterior end, each member of which divides twice, thus producing sixty-four germ cells in all. During these divisions, which are mitotic, the pole-disec granules appear to be equally distrib- uted between the daughter cells (Fig. 37, B). A rest period then occurs, as far as cellular multipli- cation is concerned, during which a ventral plate, which later grows into the germ band, develops on the ventral surface of the egg. Asin Miastor the germ-band pushes around on the dorsal surface and the group of sixty-four germ cells is carried along with it. In the meantime the germ cells begin to migrate from the amniotic cavity in which they lie through a sort of canal at the bottom of a groove in the germ-band and thus make their way inside of the embryo (Fig. 37, F). That the germ cells actually migrate and are not simply forced about by the surrounding tissues seems certain since they are ameboid in shape and pseudopodia extend out in the direction of their movement (Fig. 37, F). After penetrating into the embryo the germ cells become separated into two groups. It was difficult to count the number in each group, but many GERM CELLS IN THE ARTHROPODA 117 4---khbl. y. god pee Fia. 37.—Calligrapha. A. A germ cell of C. multipunctata shortly after being cut off from the egg. 8B. Division of a primordial germ cell. C. Longitudinal section through egg of C. bigsbyana at blastoderm stage; the posterior end was killed with a hot needle just after deposition. D. Longitudinal section through uninjured egg at same stage. E. Two ectoderm cells (e), two mesoderm cells (m), and two germ cells (g.c.) from an egg three days old. F. Germ cell during migration into the embryo (three days old). G.H.I. Longi- tudinal sections through eggs centrifuged for one hour, two hours, and four hours respectively. bl = blastoderm; g.c.d. = granules of pole-dise ; k = killed portion of egg; Ahbl. = keimhautblastem; p. posterior; pgc=primordial germ cells; » = vitellophags; 2.2. vesicular zone; y. = yolk. Wil 118 GERM-CELL CYCLE IN ANIMALS attempts seem to justify the conclusion that the division is equal or approximately equal, that is, each group contains about thirty-two germ cells. These groups acquire a covering of mesoderm cells, are carried by the somatic tissues to a position near the dorsal surface on either side of the body in the last two abdominal segments, and thus become germ glands situated in their definite positions. Some time before the larval stage is reached, the sex of the embryo can be determined by the shape of the germ glands; those of the male become dumb- bell shape, whereas the female organs retain the earlier pear shape and begin to acquire terminal filaments. It is interesting to note that much time and effort have been wasted by those who have attempted to influence the sex of caterpillars by over-feeding or starving. Kellogg (1907), for example, ‘“‘ dis- covered,” after an unsuccessful attempt to change the sex of silk worms by this means, that these cater- pillars already possess germ glands which are dif- ferentiated as male or female. If he, and others who have undertaken similar experiments, had examined the literature on the origin of the germ cells in insects, they would have found that as long ago as 1815, Herold published results of investiga- tions on Papilio brassica and other species of Lrpt- DOPTERA which proved that the sex of the larva is already determined before it hatches from the egg. A similar condition was reported in Bombyx pini by Suckow (1828), in Zeuzera esculi by Bessels (1867), and in Pieris brasstca by Brandt (1878). GERM CELLS IN THE ARTHROPODA 119 There now ensues a period of activity during which a large number of ovarian tubules develop in the female and testicular follicles appear in the male. A number of much debated problems exist regarding the cellular elements within the ovaries and testes of insects — problems which are of con- siderable importance in any discussion of the germ- cell cycle. Put in the form of questions, two of these are with respect to the ovary: (1) Do the nurse cells originate from the odgonia, thus becoming abortive eggs, or are they of mesodermal parentage ? (2) Does amitotic nuclear division occur in nurse cells and odgonia ? The answers to these questions differ according to the species of insects studied, and, as usual, the ob- servations and interpretations of different investi- gators do not always agree. They can be answered with certainty in the case of JMuvastor. All of the oégonia in this form are direct descendants of the primordial germ cell; the nurse cells are of meso- dermal origin; and amitotic division occurs neither in the nurse cells nor in the ojgonia. The situation is quite different in chrysomelid beetles. The nurse cells in the ovaries of the potato beetle all seem to be of germ-cell origin. That the nurse cells which are derived from odgonia are abortive eggs is the general opinion of zodlogists. Convincing evidence for this view has recently been provided by De Winter (1913) from studies of the apterous insect, Podura aquatica. In this species the proportion of eggs and nurse cells which develop from the odcytes is about 120 GERM-CELL CYCLE IN ANIMALS one toten. The odcytes that become eggs are those that chance to lie at the periphery of the ovary and hence are in a position to derive abundant nutrition from the blood. The odcytes that fail to become eggs are not, according to De Winter, “ vitello- génes”’ but true abortive eggs, representing a more primitive stage than the nurse cells of other insects which have acquired, secondarily, a nutritive func- tion. On the other hand, Govaerts (1913) argues strongly in favor of the view that the odgonia divide differen- tially, the daughter cells becoming true germ cells (the ultimate odgonia) and true somatic cells (the nurse cells). He bases his position upon the condi- tions existing in the ovaries of certain beetles of the genera Carabus and Cicindela, and upon the dis- coveries of Giardina (1901), Debaisieux (1909), and Giinthert (1910) in Dytiscus marginalis. Giar- dina established for Dytiscus the fact that the mito- ses which result in the formation of nurse cells are differential, as theoretically postulated by Paulcke (1900). During the four divisions preceding the formation of the odcyte a single obgonium gives rise to one odcyte and fifteen nurse cells (Fig. 38). A differentiation takes place in the chromatin of the odgonial nucleus, one half consisting of a condensed mass, the other half of large granules which corre- spond to the forty chromosomes of the odgonium (Fig. 38, A). During mitosis the chromosomes become arranged as an equatorial plate, and the chromatic mass forms a ring about it — the “anello GERM CELLS IN THE ARTHROPODA 121 cromatico” (B). This ring passes intact to one of the daughter cells (C), whereas the chromosomes are Fic. 38.— Differentiation of nurse cells and odcytes in Dytiscus mar- ginalis. A. Odgonium with chromatin of nucleus separating into two parts. B. Metaphase of odgonial mitosis; the ‘‘ anello croma- ” tico’’ is situated at the lower end of spindle. C. Two-cell stage; the lower cell with nucleus containing two sorts of chromatin. D. Four-cell stage; ‘‘ anello cromatico”’ in one cell. E. Eight-cell stage; cells ready to divide. F. Sixteen-cell stage; one large cell (odcyte) with chromatin from the ‘‘anello cromatico,’’ and fifteen nurse cells. (A-D, F, from Giardina, 1901; E, from Debaiseaux, 1909.) equally divided. During the succeeding mitoses similar differential divisions occur resulting in one odcyte containing the chromatic ring (Fig. 38, F) and 122 GERM-CELL CYCLE IN ANIMALS fifteen nurse cells lacking this nuclear substance. Thus as Paulcke’s theory demands, the difference between the nurse cells and the odcytes is the result of internal and not external causes. Giardina considered the formation of the chromatic ring as a sort of synapsis, and later (1902) distin- guished between a complete synapsis, such as ordinarily occurs in the germ-cell cycle, and a partial synapsis as exhibited by Dytiscus. Regarding the significance of this differential mitosis, he maintains that this phenomenon is the cause of the differen- tiation into nurse cells and odcytes, resulting in a complete amount of chromatin in the keimbahn cells and perhaps also an unequal distribution of cyto- plasmic substances. As in the case of Ascaris and Miastor, it might better be regarded as a means of depriving the nurse cells of part of their chromatin. Moreover, Boveri (1904) has compared the chroma- tin-diminution in Ascaris with Giardina’s differ- ential mitoses. Debaisieux (1909) and Giinthert (1910) have confirmed Giardina’s results, and the latter studied two other Dytiscipm, Actlius and Colymbetes, which also exhibit differential mitoses similar except in certain details. Giinthert found that the chromatic ring is composed of fine granules which may split off from the surface of the chromo- somes (compare with Ascaris and Miastor) and stain like cytoplasm. He interprets this as ‘‘ Zerfallspro- dukte” of the chromosomes. Debaisieux, on the other hand, claims that this cast-out nuclear material is nucleolar rather than chromatic in nature. GERM CELLS IN THE ARTHROPODA 123 It seems highly probable that the ‘‘anello croma- tico”’ of Giardina consists of chromatin, and Gold- schmidt (1904) and others do not hesitate to class it as an example of a “‘ Chromidialapparat.”’ Further- more it is apparently the result of a chromatin- diminution, as Boveri (1904) maintains, differing from the similar process in Ascaris and Muastor in details but not in the ultimate result. Finally, the discovery of this peculiar body in Dytiscus adds one more argument to the hypothesis that the chromatin content of the germ cells differs from that of the somatic cells quantitatively, at least in some cases, and perhaps also qualitatively. Many are the bodies that have been homologized with the “anello cromatico” of Dytiscus. Buchner (1909) claims that the nucleolar-like structure in the odgonia and young odcytes of Gryllus is homol- ogous to both accessory chromosomes of the sper- matogenesis and to this chromatin ring in Dytiscus. This “ accessorische Kérper”’ passes intact into one half of the odcytes where it disintegrates into granules of a “tropische Natur.” Foot and Strobell (1911) have also compared it with the chromatin nucleolus in the odgonia of Protenor with which it has certain characteristics in common, but no such differential divisions occur as in Dyftiscus. Govaerts (1913) was unable to find anything resembling the chromatic ring of Giardina, and con- cludes that the formation of a chromatic mass dif- ferentiating the odcytes and the nurse cells is unique in the Dytiscip™. His investigations demonstrate 124 GERM-CELL CYCLE IN ANIMALS that this phenomenon does not occur in all insects and that we must seek some larger cause than the un- equal distribution of chromatic elements. If no differential divisions are present, as in Dytiscus, what is the cause of the formation of odcytes and nurse cells? Govaerts decides that since the ultimate odgonium possesses a definite polarity marked by the localization of the “‘residu fusorial,”’ and the two kinds of daughter cells arise from op- posite ends of the mother cell, the cause of the differ- entiation resides in the polarization of the odgonium. He does not, however, account for this ‘‘ polarité pre- differentielle.”’ Haecker (1912) has described in Cyclops and Diaptomus a three-cell stage in the development of the gonad which is brought about by the delayed division of one of the germ cells of the two-cell stage, and concludes that as in Dytiscus there must be an internal difference in the cells to account for this condition. Wieman (19105) has followed the history of the odgonia in Leptinotarsa signaticollis through the larval and adult stages, but was unable to find any evidence that the nuclei inaugurate differentiation as in Dytiscus. He concludes that “the process seems to be the result of several distinct cell elements which operate together as a whole” (p. 148) and that the semi-fluid matrix which results from the lique- faction of cells at the base of the terminal chamber may exert a “specific effect on those germ cells coming under its influence, enabling them to develop GERM CELLS IN THE ARTHROPODA 125 into ova, while the more distant germ cells become nurse cells” (p. 147). My observations agree with those of Wieman; no definite relations nor nuclear evidence were discovered during the differ- entiation of the odgonia into oécytes and nurse cells. The data available do not suggest any method of differentiation not already proposed, and still leave the question whether the nurse cells should be regarded as abortive germ cells or true somatic cells one of personal opinion. A study of cyst formation in the testis of the potato beetle has revealed what seems to be a series of events in the male germ-cell cycle parallel to that in the females of Dytiscus, Carabus, and Cicindela, during which the nurse cells are produced. There are in Leptinotarsa two pairs of testes, one on either side of the body. Each testis consists of a large number of follicles radiating out from near the center. Figure 39 is a diagram of a longitudinal section made from the testis of an old larva. At the lower end is attached the sperm duct (s.d) which is con- nected with a cavity (c) within the testis. Just above this cavity is a region containing degenerating cells; above this region is a mass of spermatogonia (sg) not yet within cysts; and this mass is capped by a small group of epithelial cells (¢.c). The major part of the testis is composed of radiating follicles containing cysts of spermatogonia, spermatocytes, or spermatozoa (cy). Tn that region of the testis surrounding and under- 126 GERM-CELL CYCLE IN ANIMALS lying the terminal cap (Fig. 39, ¢. c) there are a large number of spermatogonia not yet contained in cysts. All stages in cyst formation may be observed here not only in larval testes but also in those of pup and adults. The youngest spermatogonia are those lying near the terminal cap. Figure 40, A shows a 7 few cells of the Oe terminal cap (t.c), : se some of the neigh- boring spermato- gonia (spg), and cy several of the epi- thelial cells (ep) ie that are scattered about among the sd spermatogonia. Fic. 39.— Leptinotarsa decemlineata. Longi- Cysts are formed tudinal section through testis of full-grown toward the edge larva. c=cavity; cy=region of cysts; s.d = sperm duct; sg =region of spermato- of the spermato- gonia; sp=region of spermatozoa; t.c = gonial mass away terminal cap. i from the terminal cap, and Fig. 40, A to G represent certain of the stages observed. The spermatogonia divide ap- parently exclusively by mitosis. A well-developed spindle is formed and this persists after the cell wall has separated the two daughter cells. The spindle fibers which are at first perfectly distinct (Fig. 40, B) unite into a compact strand (Fig. 40, C) which stains dense black in iron hematoxylin after fixa- tion in Carnoy’s fluid. In many cases it was im- possible to distinguish an intervening cell wall Fic. 40.— Leptinotarsa decemlineata. Stages in cyst formation in testis. A. Spermatogonia (spg), cells of terminal cap (¢.c), and epithelial cells (ep). B. Mitotic division of spermatogonium. C. Later stage in same process. D. Binucleate spermatogonial cell within epithe- lial envelope. #. Four spermatogonia connected by spindle re- mains. F. Spermatogonia from cyst containing eight cells. G. Section through cyst containing thirty-two spermatogonia. (127) 128 GERM-CELL CYCLE IN ANIMALS between the daughter nuclei (Fig. 40, D). In either case, however, the spindle remains persist, forming a basic staining strand with enlarged ends connecting the two nuclei. Since at this time and in all later stages the two or more spermatogonia may be found surrounded by an envelope of epithelial cells, it seems certain that, as Wieman (19106) maintains, the spermatozoa in a single cyst are derived from a single spermatogonium. A cyst containing four spermatogonia is repre- sented in Fig. 40, H. Here again appear the strongly basic staining spindle remains connecting the nuclei. These black strands persist until the succeeding mitotic division occurs as Fig. 40, F, which was drawn from a section of a cyst containing eight spermatogonia, shows. Spindle remains are still evident in later stages, as in Fig. 40, G, which repre- sents a cyst containing thirty-two spermatogonia, but were not observed in cysts containing more than sixty-four cells. Many investigators have figured spermatogonial divisions which result in rosette-like groups of cells similar to that represented in Fig. 40, F. Ap- parently, however, the spindle remains, if present, did not possess such a strong affinity for basic stains. Furthermore, only those of my preparations that were fixed in Carnoy’s fluid and stained in iron heemotoxylin exhibited these black strands. Similar spindle remains have been observed in Dytiscus, especially by Giinthert (1910), and Carabus (Go- vaerts, 1913), during the differentiation of nurse GERM CELLS IN THE ARTHROPODA 129 cells and odcytes from odgonia, and there can be little doubt but that the process of cyst formation in the male as described above is similar to the differ- ential divisions in the female. Thus the discovery of these distinct spindle re- mains in the spermatogonial divisions enables us to homologize one more period in the cycle of the male germ cells with a corresponding period in the cycle of the female germ cells. According to this view the ultimate spermato- gonium passes through a certain number of divisions — probably five or six—which correspond to the differential divisions so clearly exhibited by the ultimate odgonia of Dytiscus. Just as in the matura- tion processes, however, where only one female cell but all of the male cells are functional, so these earlier divisions result in the female in the pro- duction of a single odcyte and a number of nurse cells which may be considered abortive eggs, whereas in the male every daughter cell is functional. The limited period of division in the cycle of the male germ cells in man (Montgomery, 1911; von Wini- warter, 1912) is also similar to those in Dytiscus and Leptinotarsa. The Sertoli cells are intimately con- nected with the germ cells in the mammalian testis and probably perform three functions: (1) they nourish the spermatocytes; (2) they provide the spermatic fluid; and (3) they exert some chemico- tactic stimulus which serves to orient the spermato- zoa into bundles. The origin of the Sertoli cells has been for many years in doubt. Many investigators kh 130 GERM-CELL CYCLE IN ANIMALS claim that they arise from cells other than germ cells ; these writers have been called by Waldeyer (1906) “dualists.” An equal number of authorities be- lieve that both Sertoli cells and spermatogonia Fie. 41.— Stages in the formation of the Sertoli cell in man. Fic. 55.— A-D. Stages in formation of acopulationszelle blastula of A’quorea forskalea showing seg- described by Weis- regation of metanucleolus. (From Haecker, I hi 1892.) E. Oécyte of the cat containing mann and Ischi- Faas CO Com Kawain the winter eggs of certain Dapunip&, and in both cases it is considered prob- able that these peculiar bodies are restricted to the *“Keimbahnzellen” of the embryo. GERM CELLS IN NEMATODES, SAGITTA 185 In the eggs of Myzostoma, Wheeler (1897) found that the nucleolus of the germinal vesicle does not dissolve soon after it is cast out into the cytoplasm during the formation of the first maturation spindle, but remains visible at least until the eight-cell stage, at which time it lies in the large posterior macromere, a cell which “‘ very probably gives rise to the entoderm of the embryo.” Later embryonic stages were not studied. According to Wheeler ‘“‘the nucleoli are relegated to the entoderm cells as the place where they would be least liable to interfere in the further course of development and where they may perhaps be utilized as food material after their disintegra- tion” (p. 49). McClendon (19066) has likewise described a body embedded in the cytoplasm of the egg of Myzostoma clarki which he derives from the “‘accessory cells” which, as Wheeler (1896) has shown, attach them- selves to either pole of the odcytes. These ‘“acces- sory” cells are really the ‘‘Néhrzellen” of other authors. The cleavage of the egg was not studied. Buchner (19100) suggests that this body described by McClendon and the “nucleolus” of Wheeler are identical and that through them the keimbahn may be determined. Granules of various sorts have been noted in the eggs of various animals which are segregated in par- ticular blastomeres and may have some relation to the keimbahn. For example, among the mollusks, Blockmann (1881) has described the appearance of a group of granules in the early cleavage cells of 186 GERM-CELL CYCLE IN ANIMALS Neritina which finally reach the velar cells. It is also probable that Fol (1880) observed similar gran- ules in the 16-cell stage of Planorbis. In the same category, no doubt, belong the bodies figured by Fujita (1904) in the 4-cell to the 16-cell stages of Siphonaria lying at the vegetative pole, and the *“Ectosomen” described and figured by Wierzejski (1906) in Physa. These granules appear at the vege- tal pole in the blastomeres of Physa during the second cleavage; are at first embedded in the ento- derm mother cells, but finally become localized in the ectoderm cells. They periodically appear and disappear, and may, as suggested by Wierzejski, represent only ‘‘eine besondere Erscheinung des Stoffwechsels” (p. 536). Similarly in the rotifer, Asplanchna, Jennings (1896) has traced a “cloud of granules” from the eight-cell stage until the seventh cleavage, when this mass forms part of the smaller entodermal cell. In Lepas there has also been recorded (Bigelow, 1902) a segre- gation of granules in one blastomere. Many other substances granular in form have been described in the eggs of animals, some of them at least having migrated there from the somatic tissue. Blockmann (1887) discovered a number of bacteria-like rods in the undeveloped eggs of Blatta germanica; these rods multiplied by division and were considered sym- biotic bacteria. ‘“‘Bacterienartige St&bchen” were also noted by Heymons (1895) in the eggs of Pert- planata orientalis and Ectobia livida; these sink into the yolk and disappear. More recently a report of GERM CELLS IN NEMATODES, SAGITTA 187 Buchner (1912) indicates that these bodies are really organisms which seem to be symbiotic and not para- sitic, although it remains to be proved what advan- tage the host receives from their presence. Of a similar sort are the Zoéxanthelle which Mangan (1909) has shown enter the developing ovum from the parental tissues. All of these organisms become in some way embedded in the germ cells, but so far as we know never serve to distinguish the keimbahn, although a more selective distribution within the developing animal would obviously be greatly to their advantage. Vander Stricht (1911) has compared the ‘‘beson- dere Koérper” found by Elpatiewsky (1909, 1910) in the egg of Sagitta with several bodies, the ‘‘corps enigmatique,” which he discovered in the odcyte of the cat (Fig. 55, E). One or two of these ‘‘corps enigmatique” are present in the young odcyte originating from a few (one to five) cytoplasmic safranophile granules which are visible at the begin- ning of the growth period. They at first lie near the nucleus, but as the size of the odcyte increases they become situated near the periphery. Usually three parts can be recognized in the “‘corps enigmatique”’ : “granulation centrole, couche intermediaire et couche corticale foncée.”” As the term applied to them indi- cates, the functions of these bodies were not deter- mined. The following suggestion is, however, made: “il est possible que cet élément nous montre, des Vorigine, la ‘Keimbahn’ ainsi que les premieres cellules génitales constituées.”” A body stained 188 GERM-CELL CYCLE IN ANIMALS deeply by nuclear dyes which was found by O. Van der Stricht (1909) in the bat at the time of the first cleavage mitosis may be similar to the “corps enig- matique”’ of the cat. In many animals no keimbahn-determinants nor similar bodies have as yet been discovered. The best we can do in cases of this sort is to determine from what cleavage cell or cells the germinal epithelium probably originates. For example, in Arenicola, Lillie (1905) has shown that the part of the perito- neum from which the germ cells arise develops from teloblast cells which are probably derived (Child, 1900) from cell 4d. At present, however, no charac- teristics have been discovered which enable us to distinguish between the germ cells and the somatic cells in the early embryonic stages of such animals (Downing, 1911). CHAPTER VII THE GERM CELLS OF HERMAPHRODITIC ANIMALS Many of the most interesting biological problems are those connected with the phenomenon of sex. The term “‘sex”’ is applied to the soma or body of an organism; it indicates the presence of certain mor- phological and physiological characteristics, which may be separated into primary and secondary sexual characters. The primary sexual characters are those immediately connected with the reproductive organs ; the secondary sexual characters, such as the beard of man, the brilliant feathers and beautiful songs of many male birds, and the antlers of the moose, repre- sent differences between male and female individuals not directly concerned with the production of germ cells. It is customary to speak of male germ cells and female germ cells; this is not strictly proper, since in only a few special cases can we predict the sex of the individual which will develop from an egg. Moreover, every germ cell must contain the poten- tiality of both sexes since sooner or later its descend- ants will give rise, some to male, some to female or perhaps to hermaphroditic offspring. Thus the egg is an initial hermaphrodite; it may or may not be- come an eventual hermaphrodite according to the sex- ual condition of the individual to which it gives rise. 189 190 GERM-CELL CYCLE IN ANIMALS All the species of Mrrazoa may be separated into two groups. The individuals in one group of species Fic. 56.— Diagram of the reproductive organs of the earthworm, dorsal view. A, B, C, seminal vesicles; N, nerve-cord; O, ovary; OD, ovi- duct; R, egg sac; S, spermatheca; SF, seminal funnel; 7’, testes; VD, vas deferens. (From Marshall and Hurst.) possess only one sort of reproductive organs (male or female) and produce only one sort of germ cells (eggs or spermatozoa); these species are said to be dioe- GERM CELLS OF HERMAPHRODITES 191 cious or gonochoristic. In the other group both male and female reproductive organs occur in each individual; and such species are called moncecious or hermaphroditic. The majority of animals are gonochoristic, but a number of classes and orders consist almost entirely of hermaphroditic species, and probably no large group of animals is free from species which are moneecious. A study of hermaph- roditism is necessary for the elucidation of many biological problems; and some of those dealing more directly with the germ-cell cycle will be con- sidered in this chapter. There are many variations in the morphology of the reproductive organs in hermaphrodites. In some, such as the earthworm (Fig. 56), the male and female organs, consisting of all the parts typically present in gonochoristic animals, are present and entirely separate from each other. All gradations between such a state and an intimate association of male and female germ cells are known. Perhaps the most interesting series occurs among the mollusks. Here the germ gland may consist of two regions, as in Pecten maximus, one of which gives rise to ova, the other to spermatozoa; or certain cysts may contain only female germ cells and other cysts only male germ cells, or both sorts of germ cells may occur in a single cyst. Hermaphroditism has been shown to be prevalent among animals that are parasitic or sedentary, or for some other reason may become isolated from their fellows. Thus, it is of advantage for a parasite, such 192 GERM-CELL CYCLE IN ANIMALS as the tapeworm, to be able to form both male and female germ cells, since it may at any time become the only one of its species to occupy the alimentary canal of ahost. Hermaphroditism in such a case, however, is of no benefit if self-fertilization is not possible. Although there are thousands of hermaphroditic species of animals there are comparatively few whose eggs are known to be fertilized by spermatozoa from the same individual. We must therefore distinguish between morphological and physiological hermaphro- ditism and recognize the fact that the former condi- tion is much more prevalent than the latter. Among the species in which self-fertilization normally occurs are certain rhabdoccels, digenetic trematodes, ces- todes, ascidians, and mollusks. Van Baer, in 1835, claims to have observed self-copulation in the snail, Lymnea auricularia; that is, an individual with its penis inserted in its own female opening. That species of this genus fertilize their own eggs has frequently been stated by investigators. Frequently the spermatozoa of an hermaphrodite are capable of fertilizing the eggs of the same individual, but penetrate more readily the eggs of other individ- uals. Such is the case in the ascidian, Ctona in- testinalis (Castle, 1896; Morgan, 1905). Both sorts of germ cells are seldom produced at the same time by hermaphrodites. Those species in which spermatozoa mature first are called protan- dric; this is the usual condition. In a few cases eggs are formed first and later spermatozoa; in- dividuals in which this occurs are called protogynic. GERM CELLS OF HERMAPHRODITES 193 Proterogyny has been described in certain ascidians (Salpa), pulmonate gasteropods, and corals. That hermaphrodites are not sexless but really animals with double sex is well shown by the life history of a worm, Myzostoma pulminar, which passes through a short male stage during which spermatozoa are produced, then a stage when no functional germ cells are formed, and finally a female stage, characterized by the development of eggs (Wheeler, 1896). Thus, in this species, although hermaphroditic, there is no functional hermaphroditic stage. All variations be- tween this entire separation of the periods of germ- cell development and the simultaneous production of male and female germ cells have been recorded. Some degree of protandry has been observed among the sponges, ccelenterates, flatworms, segmented round-worms, mollusks, echinoderms, crustacea, and chordates. Hermaphroditism may occur in only a few families, genera, or species inaclass. This is true, for example, among the anthropods and vertebrates. Normally the insects are called dicecious, but among bees, ants, and butterflies, and more rarely other groups, individ- uals appear which exhibit male characters on one side of the body and female characters on the other, or the anterior part may be male, the posterior female, ete. (von Siebolt, 1864; Schultze, 1903; Morgan, 1907, 1913). Such a phenomenon is known as gynan- dromorphism. Several hypotheses have been pro- posed to account for this condition. Boveri has suggested that if the egg nucleus should chance to oO 194 GERM-CELL CYCLE IN ANIMALS divide before the sperm nucleus fuses with it, the latter may unite with one of the daughter nuclei of the egg nucleus; this cell with this double nucleus might then produce female structures, whereas the other cell with only a single nucleus representing one- half of the egg nucleus might give rise to male char- acters. Morgan has proposed another theory which is based on the fact that more than one spermatozoén is known to penetrate the eggs of insects. If one of these supernumerary spermatozoa should chance to divide, it might result in the formation of male structures, whereas the cells containing descendants of the egg nucleus fused with another sperm nucleus would exhibit female characteristics. There is some evidence that true hermaphroditism may exist among insects, at least during their embry- onic and larval stages. Thus Heymons (1890) has described in a young larva of the cockroach, Phyllo- dromia germanica, what appear to be rudimentary egg-tubes, and in another larva eggs were found in the testes which resembled those present in the egg- tubes of female larve of the same size (1 mm. in length). More recently, Schénemund (1912) has reported the presence of egg-tubes attached to the anterior end of the testes of stone-fly nymphs (Perla marginata). True hermaphroditism is rare in man and other mammals, but several cases have been described in the pig by Sauerbeck (1909) and Pick (1914), and in man by Simon (1903), Uffreduzzi (1910), Gudernatsch (1911), and Pick (1914). GERM CELLS OF HERMAPHRODITES 195 One of the problems connected with hermaphrodit- ism that has caused a great amount of discussion is whether the dicecious or the moneecious condition is the more primitive. The majority of zodlogists are inclined to consider the hermaphroditic condition more primitive, but a number of careful investigators have decided in favor of gonochorism. Among these are Delage (1884), F. Miiller (1885), Pelseener (1894), Montgomery (1895, 1906), and Caullery (1913). Very little is known regarding the segregation and early history of the germ cells of hermaphrodites. The principal results have been obtained from studies on Sagitta by Elpatiewsky (1909), Stevens (19100), and Buchner (1910a, 19106), and on Helix by Ancel (1903), Buresch (1911), and Demoll (1912). Boveri (1911), Schleip (1911), and Kruger (1912) have made some interesting discoveries on the chromosome cycle in nematodes, and likewise Zarnik (1911) on pteropod mollusks. To this list we may add such investigations as those of King (1910), Kuschake- witsch (1910), and Champy (1913), on amphibians. The segregation of the germ cells in Sagitta was described and figured in Chapter VI (Fig. 54). Here the first division of the primordial germ cell is probably differential; one daughter cell becomes the ancestor of all the ova, the other of all the spermatozoa in the hermaphroditic adult. None of the three investi- gators who have studied this subject in Sagitta have been able to discover with certainty any visible differ- ences between the first two germ cells, but Elpatiew- sky thinks the peculiar cytoplasmic inclusion, called 196 GERM-CELL CYCLE IN ANIMALS by him the “‘besondere Korper,” may be unequally distributed between these cells, and that the one which procures the larger portion is the progenitor of the spermatozoa, the other of the ova. The evi- dence for this view is, however, insufficient. In Helix both eggs and spermatozoa originate in every acinus of the ovo-testis; it is therefore an ex- cellent species for the study of the differentiation of the sex cells. According to Ancel (1903) the anlage of the hermaphroditic gland of Helix pomatia appears a few hours before the larva hatches; it consists of a group of cells situated in the midst of the mesoderm, from which germ layer it seems to originate. It soon loses its rounded form and becomes elongate; then a lumen appears within it, thus changing it into a vesicle whose wall consists of a single layer of cells —a true germinal epithelium. Secondary, tertiary, etc., vesicles bud off from the single original vesicle, forming the acini of the fully developed gland. Cel- lular differentiation takes place by the transformation of the germinal epithelial cells into male, nurse, and female elements. An indifferent epithelial cell is shown in Fig. 57, 4; the chromatin granules are con- densed to form irregular clumps. Some of these indifferent epithelial cells increase in size and give rise to indifferent progerminative cells; the chroma- tin clumps fuse, forming more or less spherical masses (Fig. 57, E). From cells of this sort originate both the odgonia and spermatogonia. The progermina- tive male cell passes through the stages shown in Fig. 57, B—D; part of the chromatin of the progermi- GERM CELLS OF HERMAPHRODITES 197 native cell loses its affinity for nuclear dyes; the chromatin masses become less numerous and more nearly spherical; and the entire cell increases in size, the nucleus growing much more than the cytoplasm. These progerminative male cells divide mitotically E F Fic. 57.— Helix pomatia. Stages in differentiation of male and female sex cells from indifferent cells. A. Epithelial indifferent cell. E. Progerminative indifferent cell. B-D. Stages in transformation of progerminative cell into a spermatogonium. F-G. Stages in transformation of progerminative cell into an odcyte. (From Ancel, 1903.) and then pass into the lumen of the acinus, where they may be recognized as spermatogonia of the first order. After the spermatogonia have passed into the lumen of the acinus the wall is seen to consist of two groups of cells; those of one group are central and in contact with the spermatogonia, the others are periph- 198 GERM-CELL CYCLE IN ANIMALS eral. The centrally situated cells now increase in size; but their nuclei retain the original condition ; that is, the chromatin is present in irregular clumps. These are nurse cells. After the nurse cells have formed, certain of the peripheral cells increase in volume and pass through an indifferent progermina- tive stage (Fig. 57, E). Then they transform into female progerminative cells, as shown in Fig. 57, F, G. The chromatin clumps break up and become oriented near the nuclear membrane, where they form a layer of more or less rounded bodies bearing chromatic filaments. In the meantime, both nucleus and cyto- plasm increase in amount, especially the cytoplasm. This (Fig. 57, G) represents an odcyte, which does not divide before maturation. Ancel concludes from these observations that there are three successive periods of cellular differentiation in the hermaphroditic gland of Heliz: (1) the ap- pearance of spermatogonia, (2) nurse cells, and (3) obcytes. Both spermatogonia and odcytes pass through the indifferent progerminative-cell stage, but the nurse cells do not; there are therefore two sorts of differentiation of the indifferent epithelial cells. Regarding the cyto-sexual determination, the follow- ing hypothesis is advanced: A progerminative in- different cell becomes a male or female element according to its environment at the time of its trans- formation; if it appears before the nurse cells are formed it becomes a spermatogonium; if nurse cells are already present it grows into an odcyte. The discovery of certain individuals containing only male GERM CELLS OF HERMAPHRODITES 199 elements is explained by Ancel by supposing the transformation of the cells into sex cells to cease Fig. 58.— Helix arbustorum. Stages in the differentiation of male and female sex cells. A. Nucleus of germinal epithelium. B. Nucleus of nurse cell. C. Nucleus of indifferent sex cell. D. Spermatogo- nium of first order. EH. Spermatogonium of second order. F. Grow- ing odcyte. (From Buresch, 1911.) before nurse cells are formed; thus all the sex cells would become spermatogonia. More recently Buresch (1911) has repeated the 200 GERM-CELL CYCLE IN ANIMALS work of Ancel, using Helix arbustorum for his material. He confirms many of Ancel’s results, objects to others, and adds certain new observations. The germinal epithelium is considered by Buresch to be a syncy- tium containing both in young and old specimens three sorts of cells, indifferent cells, egg cells, and nurse cells. Likewise spermatogonia are present not only in young but also in fully developed her- maphroditic glands. This is contrary to Ancel’s idea of successive transformation. Buresch’s view is indicated in Fig. 59. Here the vertical row of circles represents the nuclei of the syncytial germinal epithe- lium, some of which, as at m, change to indifferent germ cells. These may pass into the lumen of the acinus as spermatogonia of the first order (Sg. D) and divide to form spermatogonia of the second order (Sg. IT) which grow into spermatocytes (Sc); sper- matozoa are derived from these in the usual manner. Other indifferent germ cells remain in the wall, as at w, and grow into odcytes, and a third class of cells become nurse cells (n). In Fig. 58, A is shown a nucleus of the germinal epithelium about 4 microns by 6 microns in size. During differentiation into an indifferent germ cell (Fig. 58, C) the chromatin forms a nucleolus, and both nucleus and nucleolus increase in size until the former reaches a diameter of about 7 microns. Those indifferent germ cells that are to produce spermatozoa separate from the epithelium with a small amount of cytoplasm and fall into the lumen of the acinus as spermatogonia of the first order (Fig. 58, D). These divide to form spermato- GERM CELLS OF HERMAPHRODITES 201 SgE Sof a 00 0O0W@O Oc Oo Sp OO 00 Fic. 59.— Helix arbustorum. Diagram showing row of germinal epithe- lial cells some of which, as at m, become spermatogonia and drop into lumen of germ gland; others become nurse cells (mn); and still others odcytes (w). SgI = spermatogonium of first order; SgII = spermatogonium of second order; Sc = spermatocyte; St = sperma- tid; Sp =spermatozoa. (From Buresch, 1911.) 202 GERM-CELL CYCLE IN ANIMALS gonia of the second order (Fig. 58, #). Those in- different germ cells that are to form odcytes grow large, remain in the germinal epithelium, and do not divide. They possess a double nucleolus (Fig. 58, F). When a diameter of 36 microns is attained, the odcyte passes out of the hermaphroditic gland into the uterus. The nurse cells, like the o6cytes, remain in the wall and do not divide; their nuclei grow to be about 15 microns in diameter and the chromatin forms irregu- lar clumps more or less evenly distributed (Fig. 48, B). No differences could be discovered in the indifferent germ cells by means of which the future history of these cells could be determined. It was noted, how- ever, that egg cells were never present without a neighboring nurse cell, and the conclusion was reached that a favorable position with regard to a nurse cell determines whether an indifferent germ cell shall develop into a spermatogonium or an egg. If Buresch’s observations are correct, Helix is not protandric, but both sorts of germ cells mature at the same time, and the fate of an indifferent germ cell depends upon nutrition, that is, its proximity to a nurse cell. Demoll (19126) has proposed a new hypothesis regarding sex determination and has selected certain events in the odgenesis and spermatogenesis of Helix pomatia as arguments in its favor. The hypothesis is that the accessory chromosome (see Chapter IX) contains the anlagen of the male sexual characters, whereas the female sexual characters are localized GERM CELLS OF HERMAPHRODITES 203 in the autosomes. In Helix the odgonia and sperma- togonia arise from cells that are similar in size and constitution (Fig. 60, A). When the germ-cell nuclei reach the bouquet stage, a Nebenkern appears near the side against which the chromatin threads Fic. 60.— Helix pomatia. Stages in the differentiation of male and female sex cells. A. Young odcyte. B. Later stage of odcyte showing faint Nebenkern. C. Young spermatocyte. D. Later stage of spermatocyte showing well-marked Nebenkern. JZ. Still later stage of spermatocyte containing Nebenkern consisting of banana-shaped rods. (From Demoll, 1912.) become packed. This Nebenkern is probably a product of the nucleus; it appears in the female cell only as a slightly darker area of cytoplasm (Fig. 60, B) but in the male cell is more dense (D), later consisting of a number of darkly staining banana- shaped pieces (/). With the appearance of the Nebenkern the specific growth of the female cells 204 GERM-CELL CYCLE IN ANIMALS is initiated. The Nebenkern disappears in the odcyte soon after the yolk begins to form. The chromatin threads in the spermatocytes break down and lose their affinity for dyes, but later reappear. In the odcyte, on the contrary, the chromatin threads persist. Demoll concludes from these observations that the Nebenkern always determines the character of the germ cells, which, up to its formation, may be called indifferent germ cells. He further concludes, that, since in dicecious animals sex is determined by the accessory chromosomes, in Helix the sexual specificity of the Nebenkern must be determined by the accessory chromosomes. Such chromosomes were described by Demoll (1912a) in a previous contribution. A similar idea has been expressed by von Voss (1914) regarding the differentiation of indifferent germ cells in a flat-worm, Mesostoma ehrenbergt. In the embryo of this hermaphrodite the germ gland is a syncytium containing both the nuclei of future odgonia and future spermatogonia. The cytoplasm is apparently homogeneous throughout. The forma- tion of the odgonia from indifferent germ cells begins with the appearance of a “germinal-vesicle stage” ; this is followed by an increase in the amount of cytoplasm surrounding them. Since the cytoplasm appears to be similar in all parts of the syncytium, differentiation must be initiated by the nucleus, and the suggestion is made that perhaps the accessory chromosome may be responsible for the separation of the germ cells into odgonia and spermatogonia. GERM CELLS OF HERMAPHRODITES 205 The investigators whose results have been de- scribed above have thus furnished three theories re- garding the differentiation of male and female germ cells in hermaphrodites: (1) In Sagitia, according to Elpatiewsky, it is an unequal distribution of the ‘“besondere Kérper,”’ (2) in Helix, according to Ancel and Buresch, it is due to the presence or absence of a nurse cell in the immediate neighborhood, and (3) in Helix, Demoll considers it a result of the influence of the accessory chromosome. It is perfectly obvious that hermaphrodites offer exceptionally fine material for the study of the differentiation of germ cells, but that thus far the results have not furnished an ade- quate explanation of the phenomenon. The investi- gations of Boveri (1911), Schleip (1911), and Krueger (1912) on the chromosomes in hermaphroditic nema- todes may be discussed more profitably during the consideration of the chromosome cycle in the next chapter. Certain morphological and experimental studies on the germ glands of amphibians are of interest be- cause both odgonia and spermatogonia are sometimes more or less closely associated in a single individual during the developmental stages, and may persist even in the adult germ glands of a number of species which are commonly considered dicecious. Pfliiger, for example, was able to separate the young of the frog, Rana temporaria, into three groups, males, fe- males, and hermaphrodites; the hermaphrodites developed into either males or females. Similar results were obtained by Schmidt-Marcel (1908) 206 GERM-CELL CYCLE IN ANIMALS and Kuschakewitsch (1910), who refer to the her- maphroditic individuals as intermediates. There is no consensus of opinion regarding the origin of the germ cells in amphibians; one group of investigators, including Allen (1907) and King (1908), recognize a definite keimbahn, whereas many others (Semon, 1891; Bouin, 1900; Dustin, 1907; Kuschakewitsch, 1910; Champy, 1913) believe they arise from the germinal epithelium or near-by cells. Very few students have attempted to determine the stages in or causes of the differentiation of male and female cells from the primordial germ cells. Kuscha- kewitsch (1910) concludes from his extensive studies on the history of the germ cells in frogs that at first all of the germ cells are indifferent but subsequently become differentiated in two directions. Champy (1913) has studied this differentiation in a number of amphibians and has concluded that if the charac- teristically lobed or polymorphic nuclei of the pri- mordial germ cells in Bufo, Hyla, and Rana temporaria lose their original shape and become spherical and clear, the germ gland will form an ovary; but if the nuclei retain their primitive condition, a testis will result. Champy believes with Kuschakewitsch that both sorts of germ cells arise from sexually indifferent cells, that is, sex is not irrevocably fixed in the fer- tilized egg. Furthermore Champy’s observations have led to the conclusion that the germ cells in the sexually indifferent germ glands are morphologically identical with primitive spermatogonia. These in- different germ cells become differentiated into ova GERM CELLS OF HERMAPHRODITES 207 or spermatozoa as a result of various causes, some general and others local in nature, which probably are most influential at certain definite stages in the cellular activity. A new equilibrium is thereby es- tablished between the different cell organs which initiates new processes resulting in differentiation. The undifferentiated cells in the testis of the adult appear also to be identical with the primitive sper- matogonia, and have still the power of producing either ova or spermatozoa. Thus the male amphib- ians are also females “en puissance,” but the re- verse is not true. This accounts for the numerous discoveries of ova in the testes of these animals. Reports of so-called hermaphroditism in amphib- ians are abundant in the literature. Cases have been reported in frogs by Cole (1895), Friedmann (1898), Gerhartz (1905), Ognew (1906), Yung (1907), Schmidt-Marcel (1908), Youngman (1910), Hooker (1912), and many others. Hooker has re- viewed the literature of the subject. Hermaphrodit- ism in other amphibians is more rare, but it has been noted in salamanders by La Vallett St. George (1895) and Feistmantel (1902). Usually the condi- tion spoken of as hermaphroditism consists in the presence of ova in the testis, and it is probable that true hermaphroditism is rare in these animals as it is in other vertebrates. In the toad, however, a condition exists which is of particular interest. The genital ridge of every toad tadpole 15 to 18 days old becomes visibly differentiated into two regions, an an- terior portion which develops into Bidder’s Organ, and 208 GERM-CELL CYCLE IN ANIMALS a posterior region which becomes an ovary or testis. Bidder’s Organ persists in the adult of males, where it lies just anterior to the testis, but in the females of Bufo variabilis, B. cinereus, B. clamita, and B. lentiginosus it disappears at the end of the second year. Bufo vulgaris seems to differ from the other species since here Bidder’s Organ persists, becom- ing small and shrunken during the winter (Ognew, 1906) and regenerating during the summer months (Knappe, 1886). At first the cells in both the anterior and posterior portions of the genital ridge are similar, all possessing a polymorphic nucleus, and dividing mitotically, but later those of Bidder’s Organ begin to divide amitotically and assume the characteristics of young odcytes with rounded nuclei. Knappe (1886) claims that these cells never become functional ova because they are unable to form yolk. King (1908), however, does not consider this prob- able, but traces their differentiation to irregularities in the synizesis stage. By most investigators Bidder’s Organ is regarded as a rudimentary ovary. Others believe that the AMPHIBIA were derived from hermaphroditic ances- tors and that in the male it is a rudimentary ovary and in the female a rudimentary testis. This seems more probable than Marshall’s suggestion that this organ is the result of degenerative processes proceed- ing backward from the anterior end of the genital ridge, or than that it represents the remains of a sex gland possessed by the larve of ancestral toads when they were pedogenetic, as Axolotl is at the GERM CELLS OF HERMAPHRODITES 209 present time. Champy (1913) has found that the cells of Bidder’s Organ in Bufo pantherina pass through stages in their transformation similar to those of the primitive germ cells of Rana esculenta which become ova, and is inclined to the view that the principal difference between the toad and the intermediate type of young frogs lies in the fact that in the former the oviform cells are localized in Bidder’s Organ, whereas in the frog they are scattered throughout the germ gland. The development of the germ glands in the hag- fish, Myxine glutinosa, resembles that in the toad in many respects. Cunningham (1886) and Nansen (1886) considered Myxine to be a protandric her- maphrodite. Schreiner (1904), however, was able to show that every adult is functionally male or female with a rudimentary ovary anteriorly situated and a posterior, mature testis, or a functional ovary ante- rior to arudimentary testis. These results were con- firmed by Cole (1905). Similar conditions have been found by Okkelberg (1914) in the young of the brook lamprey, Ento- sphenus wilderi. Of fifty larve ranging from 7} cm. to 20 cm. in length, 46 per cent were true females, 10 per cent were true males, and 44 per cent were hermaphrodites. Since male and female adults are approximately equal in numbers, it was concluded that the juvenile hermaphrodites become adult males. In favor of this conclusion is also the fact that the adult males frequently possess ova in their gonads which resemble those present in the her- maphroditic larve. P 210 GERM-CELL CYCLE IN ANIMALS Regarding the differentiation of the germ cells in hermaphrodites then we may recognize two principal views: (1) that there is some material within the cell which initiates specialization, or (2) that differ- entiation is due to general or local causes outside of the germ cells. The former is favored by Elpatiew- sky (1909, 1910) from studies on Sagitta and by Demoll (1912) from studies on Helix. The second view is more widely advocated. The conclusions derived by Kuschakewitsch (1910) and Champy (1913) on amphibians, and of Ancel (1903) and Buresch (1911) on Helix agree in their essential fea- tures. All of these investigators maintain that the sex cells pass through an indifferent stage and are differentiated into odcytes or spermatocytes because of influences external to themselves. Buresch and Champy also believe that even in the fully developed germ glands of the adult these primitive cells are present. The causes of their differentiation, how- ever, have not been definitely determined. CHAPTER VIII KEIMBAHN-DETERMINANTS AND THEIR SIG- NIFICANCE Iv is customary to be suspicious of any peculiar bodies revealed to us in fixed and stained material under high magnification. There can be no doubt, however, that most, if not all, of the cytoplasmic inclusions mentioned in the preceding chapters are realities and not artifacts. Some of them have been seen in the living eggs; most of them have been de- scribed by several investigators; they occur after being fixed and stained in many different solutions ; and their presence is perfectly constant. The genesis, localization, and fate of these bodies are difficult to determine, and their significance is prob- lematical ; but the writer has attempted in the follow- ing pages to draw at least tentative conclusions from the evidence available and to indicate what still needs to be done. A. Tue GENESIS OF THE KEIMBAHN-DETERMINANTS The writers who have discussed the origin of the keimbahn-determinants have derived them from many different sources. In afew cases they are known to be nuclear in origin, consisting of nucleolar or chro- matic materials; they are considered differentiated 211 212 GERM-CELL CYCLE IN ANIMALS parts of the cytoplasm by some investigators; in some species they are extra-cellular bodies, such as nurse cells. The accompanying table indicates the number and diversity of the animals in which keimbahn-determi- nants have been described, and shows the increasing interest that has been given to this subject within re- cent years, over half of the papers listed having been published since 1908. Several cases have been re- ferred to in the text, but omitted from the table be- cause of insufficient evidence regarding their connec- tion with the primordial germ cells. The list as given includes representatives of the C@LENTERATA, CuzatocnatHa, Nematopa, ARTHROPODA, and VER- TEBRATA. The terms applied to the various sub- stances have been chosen evidently because of their genesis, position in the egg, or supposed function. TaBLeE oF PrincipaL Cases OF VISIBLE SUBSTANCES CON- CERNED IN DIFFERENTIATION OF GERM CELLS (IN CHRON- OLOGICAL ORDER) NAME oF SPECIEs, Name APPLIED TO GEnvs, or Group SUBSTANCE AUTHORITY Date Chironomus nigro- | Dotterkérnchen | Weismann 1863 viridis Miastor Dottermasse Metchnikoff 1866 Moina rectirostris | Richtungskérper | Grobben 1879 Chironomus Keimwulst Ritter 1890 Daphnidee Paracopulations- | Weismann and | 1889 zelle Ischikawa AE quorea Metanucleolus Haecker 1892 Ascaris megaloce- | Chromatin Boveri 1892 phala KEIMBAHN-DETERMINANTS 213 A. lumbricoides A. rubicunda Chromatin O. Meyer 1895 A. labiata Cyclops Aussenkérnchen | Haecker 1897 Ektosomen Haecker 1903 Calliphora Dotterplatte Noack 1901 Dytiscus Anello cromatico | Giardina 1901 Apis mellifica Richtungskorper | Petrunkewitsch | 1902 Parasitic Nucleol Silvestri 1906 Hymenoptera eee ac 1908 Chrysomelidee Pole-disc Hegner 1908 Miastor metraloas | polares Plasma | Kahle 1908 Sagitta besondere Elpatiewsky 1909 Korper Guinea-pig Chondriosomes_ | Rubaschkin 1910 Chick Chondriosomes | Tschaschkin 1910 Lophius extruded Dodds 1910 plasmosome : Ascaris Plasmadifferen- | Boveri 1910 zen Chironomus Keimbahn- Hasper 1911 plasma Copepoda Ectosomen Amma 1911 Polyphemus Nahrzellenkern | Kiihn Re Sagitta Keimbahn- Buchner 1910 chromidien Man Sertoli cell Montgomery 1911 determinant Chick Attraction- Swift 1914 sphere Parasitic Keimbahn- Hegner 1914 Hymenoptera chromatin a. Nucuear. Nucueour. It seems certain that bodies of a nucleolar nature behave as keimbahn- determinants. There are three or more kinds of bodies that are spoken of as nucleoli. Of these may be mentioned (1) the true nucleoli or plasmosomes, (2) karyosomes or chromatin-nucleoli, and (3) double-nu- 214 GERM-CELL CYCLE IN ANIMALS cleolt, consisting of usually a single principal nucleolus (Hauptnucleolus of Flemming), and one or ‘more accessory nucleoli (Nebennucleoli of Flemming). Many nucleoli have been described that may perhaps represent intermediate stages in the evolution of one of the types mentioned above into another. The young ovarian egg of most animals contains a single spherical nucleolus (‘‘Keimfleck,”’ or “germi- nal spot”’), but the number may increase greatly dur- ing the growth period. Usually during the formation of the first maturation spindle the nucleolus escapes from the nucleus into the cytoplasm, where it dis- appears, often after breaking up into fragments. Many theories have been advanced regarding the origin, function, and fate of the nucleoli of the germi- nal vesicle. They are considered by some of chro- matic origin, arising as an accumulation of the chro- matin, or from the chromatin by chemical trans- formation. Others consider them extra-nuclear in origin (Montgomery, 1899). Many functions have been attributed to the nu- cleoli; of these the following may be mentioned: (1) They function as excretory organs (Balbiani, 1864; Hodge, 1894); (2) nucleoli play an active role in the cell, since they serve as storehouses of material which is contributed to the formation of the chromosomes (Flemming, 1882; lLubosch, 1902; Jordan, 1910; Foot and Strobell, 1912) and may give rise to kinoplasm (Strasburger, 1895) or “‘Kine- tochromidien” (Schaxel, 1910); (3) nucleoli are passive by-products of chromatic activity; they KEIMBAHN-DETERMINANTS 215 become absorbed by active substances (Haecker, 1895, 1899) ; (4) nucleoli represent nutritive material used by the nucleus into which it is taken from the cytoplasm (Montgomery, 1899). Undoubtedly the various bodies known as nucleoli originate in different ways, have different histories, and perform different functions. In the particular cases to be discussed here the nucleoli are not temporary structures, as is usually true, but persist fora comparatively long interval after the germinal vesicle breaks down. What seemed to be the most important and convincing evidence of the functioning of a nucleolus as a keimbahn-determi- nant is that furnished by Silvestri (1906, 1908) in parasitic Hymenoptera. As shown in Chapter V, however, the ‘“‘nucleolo”’ of Silvestri is really not a nucleolus but consists of chromatin. As we have already noted, in a few instances the nucleolus does not disappear during the maturation divisions but persists for a time as a ““metanucleolus”’ (see p. 183). These metanucleoli are evidently of a different nature from the usual type and are hence saved from immediate disintegration in the cyto- plasm. The localization of the metanucleolus in the egg is the result of either its own activity, or that of the surrounding cytoplasm, or a combination of these. Gravity can have no decided effect upon it (Herrick, 1895), since its position is constant, whereas the posi- tion of the egg with respect to gravity is not. It also seems hardly possible that oxygenotactic stimuli are the cause of its localization, as has been suggested 216 GERM-CELL CYCLE IN ANIMALS by Herbst (1894, 1895) for the migration of the blastoderm-forming cells from the center to the sur- face of the eggs of certain arthropods. Haecker (1897) has suggested that the “‘Aussen- kérnchen” which appear in the egg of Cyclops during the formation of the first cleavage spindle may be nucleolar in nature. Later (1903) this idea was withdrawn, and more recently Amma (1911) has likewise been unable to sustain this hypothesis. The most convincing data furnished by Amma are that in an allied form, Diaptomus ceruleus (Fig. 49, H), these granules appear before the cleavage spindle is formed and before the nucleoli of the pronuclei have disap- peared. The remaining forms in which nucleoli have been considered as keimbahn-determinants are merely suggestive. In A/quorea, Haecker (1892) traced the metanucleolus, which arises from the germinal vesicle, into certain cells of the blastula. Similar bodies appear in Mitrocoma (Metchnikoff, 1886), Tiara (Boveri, 1890), Stephanophyes (Chun, 1891), Myzo- stoma (Wheeler, 1897), and Asterias (Hartmann, 1902), but their ultimate fate has not been determined. Meves (1914), however, has traced the middle piece of the sperm of the sea urchin, Parechinus miliaris, into one of the cells of the animal half of the egg at the thirty-two-cell stage. This middle piece is of a plastochondrial nature. It seems probable that in all these cases the same influences may be at work regulating the time, the place, and the method of localization of the nucleoli. KEIMBAHN-DETERMINANTS Q17 The writer can only conclude (1) that the metanu- cleoli differ in nature from ordinary plasmosomes, chromatin-nucleoli, and double-nucleoli; (2) that these bodies are definitely segregated in a certain part of the egg or in a certain blastomere, probably by protoplasmic movements; (3) and that their disin- tegration and the distribution of the resulting frag- ments or granules are controlled by reactions between them and the substances in which they are embedded. Curomatin. In two genera of animals the differ- entiation of the primordial germ cells is accompanied by a diminution of the chromatin in the nuclei of the somatic cells, so that eventually the nucleus of every germ cell is provided with the full complement of chromatin, whereas the nucleus of every somatic cell lacks a considerable portion of this substance, which remains behind in the cytoplasm when the daughter nuclei are reconstituted. These two genera are Ascaris and Miastor. This diminution process was described by Boveri (1892) in the former and confirmed by O. Meyer (1895) and Bonnevie (1902), and by Kahle (1908) in Miastor and confirmed by Hegner (1912, 1914a). For details of these processes reference should be made to Figs. 15-16, 51-52, and pp. 57 and 174. It may be pointed out here that although the final results are similar the process differs in the two genera. In Ascaris both ends of each chromosome are split off, whereas in Miastor approxi- mately one-half of each daughter chromosome is left behind to form the ‘‘Chromosomenmittelplatte”’ (Fig. 16) and later the “Chromatinreste” (Fig. 18). 218 GERM-CELL CYCLE IN ANIMALS The elimination of chromatin during the matura- tion and early cleavage divisions of the egg, as well as during the mitotic divisions of other kinds of cells, has often been recorded. For example, Wilson (1895, p. 458) estimates that only about one-tenth of the chromatin in the germinal vesicle of the star- fish is retained to form the chromosomes during the first maturation division, and Conklin (1902) finds that ‘“‘in Crepidula the outflow of nuclear material occurs at each and every mitosis” (p. 51). Further- more, Rhode (1911) argues that chromatin-diminu- tion is a normal histological process, and describes such phenomena in blood cells, nerve cells, and cleavage cells of several AMPHIBIA, comparing con- ditions with the chromatin-diminution in Ascaris and Dysticus.+ Diminution processes similar to those in Ascaris and Miastor have not been discovered in other ani- mals, although investigators have been on the watch for such phenomena and have studied allied species, e.g., the work of Hasper (1911) on Chironomus and my own work on the chrysomelid beetles (see pp. 108 1His conclusion is as follows: ‘In der Histogenese der allerver- schiedensten Gewebe tritt uns also die Erscheinung entgegen, dass die sich entwickelnden Zellen, bzw. Kerne einen Teil ihres Chromatins abstossen, d. h. also eine Chromatindiminution erfolgt, wenn auch die Befunde selbst im speziellen von den bisher beobachteten in der Einleitung beschriebenen Fallen der Chromatindiminution etwas ab- weichen. “Eine Chromatindiminution tritt also nicht nur am Anfang und Ende der Keimbahn, wie es bisher angegeben worden ist, sondern in den ver- schiedensten Entwicklungsstadien und bei den verschiedensten Geweben und Tieren ein, sie hat also offenbar eine allgemeine Bedeutung.” (p. 25.) KEIMBAHN-DETERMINANTS 219 to 118). If, therefore, there is a similar difference in all animals in chromatin content between the germ cells and somatic cells, the elimination of chromatin from the latter must take place by the transformation of the basichromatin of the chromosomes into oxy- chromatin which passes into the cytoplasm during mitosis, or else by the more direct method advocated by the believers in the chromidia hypothesis. The causes of the diminution of chromatin in As- caris and Miastor are unknown. Recently Boveri (1910) has concluded from certain experiments on the eggs of Ascaris (see p. 177) that in this form it is the cytoplasm in which the nuclei are embedded that determines whether or not the latter shall undergo this process. Kahle (1908) does not explain the cause of the diminution in Miastor. To the writer it seems more important to discover why the nuclei of the keimbahn cells do not lose part of their chro- matin, since the elimination of chromatin during mitosis is apparently such a universal phenomenon. I would attribute this failure of certain cells to under- go the diminution process not to the contents of the nucleus alone but to the reaction between the nucleus and the surrounding cytoplasm. As stated in a former paper (Hegner, 1909a), “In Calligrapha all the nuclei of the egg are apparently alike, potentially, until in their migration toward the surface they reach the ‘Keimhautblastem’; then those which chance to encounter the granules of the pole-disc are differentiated by their environment, 2.e., the granules, into germ cells. In other words, whether or 220 GERM-CELL CYCLE IN ANIMALS not a cell will become a germ cell depends on its posi- tion in the egg just previous to the formation of the blastoderm.” Similarly in Ascaris the cleavage nuclei are con- ceived as similar so far as their “ prospective potency” is concerned, their future depending upon the char- acter of their environment, 7.e., the cytoplasm. In the egg of Miastor cleavage nucleus IV (Fig. 15) does not lose part of its chromatin because of the character of the reaction between it and the substance of the “‘polares Plasma.’’ In chrysomelid beetles (Hegner, 1908, 1909, 1914a) and Chironomus (Hasper, 1911), however, although no diminution process has been discovered in the nuclei that encounter the pole-disc or ““Keimbahnplasma,” the other nuclei in the egg, so far as known, are similar in this respect. The nuclei of the primordial germ cells, however, may be distinguished easily from those of the blastoderm cells in chrysomelid beetles, proving conclusively that a differentiation has taken place either in one or the other. This differentiation probably occurs in the nuclei that take part in the formation of the blastoderm, since the nuclei of the germ cells retain more nearly the characteristic features of the pre- blastodermic nuclei, whereas those of the blastoderm cells change considerably. In some cases the eliminated chromatin may have some influence upon the histological differentia- tion of the cell, since it is differentially distributed to the daughter cells, but in Ascaris and Miastor no mechanism exists for regulating the distribution KEIMBAHN-DETERMINANTS 221 of the cast-out chromatin, and there is consequently no grounds for the hypothesis that “‘in Ascaris those cells which become body cells are the ones that in- clude the cast-off chromosome ends in their cyto- plasm, and it will probably be found that these ejected chromosome parts engender such cytoplasmic differentiations as characterize the body cells” (Montgomery, 1911, p. 192). Curomipia. To several of the bodies listed in the table on page 88 as keimbahn-determinants has been ascribed an origin from the chromatin of the germinal vesicle. Many cases of the elimination of chromatin from the nuclei of growing odcytes are to be found in the literature. Blochmann (1886) dis- covered a process of “budding” in the odcytes of Camponotus ligniperda resulting in the formation of ‘‘Nebenkerne.” These appear first as small vacuoles lying near the nucleus; later they contain small staining granules and acquire a membrane. The “‘Nebenkerne” grow in size and increase in num- ber, while the nucleus of the odcyte becomes smaller. Stuhlmann (1886) described a similar phenomenon in about a dozen different species of HYMENOPTERA. The odcyte nucleus in all species examined becomes localized near the anterior end; then the small nuclear-like bodies form around it at its expense. The time of their production varies in the different species; in some they appear in the very young eggs; in others not until a much later stage has been reached. Sometimes’ they fuse to form a large ““Dotterkern”’ lying at the posterior pole of the egg ; 222 GERM-CELL CYCLE IN ANIMALS or they may remain separate and later become scat- tered. Paulcke (1900) also noted nuclear-like bodies near the odcyte nucleus of the queen bee, and Mar- shall (1907) has likewise found them in Polistes pallipes. In this species the nuclear-like bodies form a single layer around the nucleus; later they come to lie near the periphery of the odcyte and finally disappear. Loewenthal (1888) has described what appears to be chromatin in the cytoplasm of the egg of the cat, and an elimination of chromatin was noted by van Bambeke (1893) in the ovarian egg of Scorpena scrofa. In none of these species, however, have keimbahn-determinants been dis- covered. According to Buchner (1910) the “‘besondere Korper”’ in the egg of Sagitta, and in fact keimbahn- determinants in most other animals are of a chromid- ial nature, representing the tropho-chromatin de- manded by the binuclearity hypothesis. The term chromidia was introduced by R. Hertwig in 1902 and applied to certain chromatin strands and granules of nuclear origin in the cytoplasm of Actinospherium. Goldschmidt (1904) transferred the chromidia hy- pothesis to the tissue cells of Ascaris. Since then chromidia have been described in the cells of many animals, including both somatic and germ cells. Thus far the group of zodlogists that favor the chromidia idea have not received very extensive backing, but the fact remains that chromatin particles are in some cases cast out of the nuclei in the odcytes of certain animals and continue to exist KEIMBAHN-DETERMINANTS 223 as such in the cytoplasm for a considerable period. It is also possible that, as Buchner (1910) maintains, the keimbahn-determinants may be in reality ‘‘ Keim- bahnchromidien.” This view was suggested by the writer in 1909 (p. 274) to account for the origin of the pole-disc granules in the eggs of chrysomelid beetles. It was thought that here as in the Hymenoptera (Bloch- mann, 1886; et al.) chromatin granules might be cast out of the nuclei of the odcytes, and that these granules might gather at the posterior end to form the pole-disc. It was also suggested that chromatin granules from the nurse-cell nuclei might make their way into the odcyte and later become the granules of the pole-disc. It should not be forgotten, moreover, that these granules stain like chromatin. Finally, mention should be made of the “anello cromatico” of Giardina (1901) which is associated with the differentiation of the odcytes in Dytiscus (see p. 120, Fig. 38), and the keimbahn-chromatin which I have recently described (Hegner, 19146) in the eggs of the parasitic hymenopteron, Copidosoma (p. 151, Figs. 46-47). Conciusion. Certain keimbahn-determinants may consist of nucleolar material which is derived from the germinal vesicle and persists until the primordial germ cells are established. In some cases the keimbahn cells are characterized by the posses- sion of the complete amount of chromatin in con- trast to the somatic cells which lose a part of this substance. Since, however, the chromatin-diminu- 224 GERM-CELL CYCLE IN ANIMALS tion process does not occur in many species, it is probably not a universal phenomenon, and conse- quently cannot be of fundamentalimportance. Most of the evidence, on the other hand, points toward the conclusion that all of the cleavage nuclei are qualitatively alike, and that the cytoplasm is the controlling factor. b. Cytoplasmic oR EXTRACELLULAR NUTRITIVE Supstances. It was pointed out on a preceding page (p. 101) that one of the characteristics used to distinguish primordial germ cells from other embry- onic cells is the presence within them of yolk material. In many vertebrates the yolk globules persist in the primordial germ cells until a comparatively late stage, and indeed are often so numerous as to practi- cally conceal the nuclei of these cells. A large num- ber of the keimbahn-determinants that have been described are supposed to consist of nutritive sub- stances. Some of the earliest investigators were aware of the yolk content of the primordial germ cells. For example, in Chironcmus Weismann (1863) found four oval nuclei lying in the ‘‘ Keimhautblas- tem” at the posterior end of the egg, each of which is associated with one or two yolk granules; these are the ‘“Polzellen.” In another Dipteron, Simula sp., Metchnikoff (1866) records four or five pole- cells which possess fine yolk granules in their cell substance. The same author (1866) also states that when the pseudovum in the pzedogenetic larva of Miastor contains twelve to fifteen nuclei, one of these, together with the dark yolk-mass in which it KEIMBAHN-DETERMINANTS 225 lies, is cut off as a cell which gives rise to the pole- cells. In certam Dapnnipm, Weismann and Ischikawa (1889) describe a “Paracopulationszelle’’ which is derived from the contents of the germinal vesicle (see p. 163) ; but the recent work of Kiihn (1911, 1913) renders it probable that this body is nothing but the remains of a nurse cell. The “Dotterplatte” discovered by Noack (1901) at the posterior end of the egg of Calliphora (Fig. 34) is considered by this investigator to consist of yolk elements. In previous communications (Hegner, 1908, 1909, 1911) the writer has discussed the probability that the pole-disc in chrysomelid eggs consists of nutritive material, and Weiman (1910a) also has offered arguments for this view. The granules segregated in certain cleavage cells of Nerttina (Blochmann, 1881), Asplanchna (Jennings, 1896), Lepas (Bigelow, 1902), Siphonaria (Fujita, 1904), and Physa (Wierzejski, 1906) may be of a nutritive nature, and these cells may be the stem cells from which the germ cells of these animals eventually arise. The hypothesis that the nucleoli consist of food substance also argues in favor of the idea that the keimbahn-determinants are nutritive. The importance of these nutritive substances to the primordial germ cells can be stated with some degree of certainty. According to some authorities the primordial germ cells remain in the primitive condition and do not undergo differentiation at the same time, or at least at the same rate, as do the Q 226 GERM-CELL CYCLE IN ANIMALS other embryonic cells. On this account their yolk contents are not at first utilized, since their meta- bolic activities are so slight. This is more especially true of the vertebrates in which, it has been sug- gested (Hegner, 1909a, p. 276), the yolk contents of the germ cells are transformed into the energy of motion during the characteristic migration of these cells into the germinal epithelium. Why these nutritive substances are segregated in the primordial germ cells is more difficult to answer. Finally, it is interesting to note that the differentiation of the indifferent germ cells of Helix arbustorum into sper- matogonia or odgonia has been found to depend upon nutrition (Buresch, 1911).1 Yotx Nuctgeus. There are many bodies in the cytoplasm of growing odcytes that have been called yolk nuclei and that may be responsible for the origin of the keimbahn-determinants. Some of these bodies have already been considered, but the term ‘yolk nucleus’ has been applied to so many different cytoplasmic inclusions (Munson, 1912) that no attempt will be made here to describe them nor to trace their history. Mitocuonpria. The condition of the chondrio- somes in the primordial germ cells of certain verte- brates (Rubaschkin, 1910, 1912; Tschaschkin, 1910; Swift, 1914) and the theories that have been pro- 1“Qb aber eine indifferente Geschlechtszelle sich in mannlicher oder weiblicher Richtung weiter entwickeln wird, das kénnen wir schon sehr friih sagen, nimlich nach der Lage dieser Zelle niher oder weiter von einer Nihrzelle ” (p. 327). KEIMBAHN-DETERMINANTS 227 posed regarding the réle of these bodies in heredity make it necessary to refer to them briefly here. At the present time it is difficult to make any definite statement regarding the origin, nature, and signifi- cance of the various cytoplasmic inclusions that have been grouped under the general title of mitochondria. It seems probable that we are concerned with a num- ber of different sorts of inclusions, and with various stages in their evolution. Inthe guinea pig (Ru- baschkin, 1910, 1912) and chick (Tschaschkin, 1910) the chondriosomes of the cleavage cells are spherical and all similar, but, as development proceeds, those of the cells which become differentiated to produce the germ layers unite to form chains and threads, whereas those of the primordial germ cells remain in a spherical and therefore primitive condition (Fig. 31, B). Swift (1914) has found, however, that in the chick the mitochondria in the primordial germ cells are not at all characteristic, resembling those of the somatic cells. The germ cells neverthe- less can be distinguished from the latter by the pres- ence of an especially large attraction-sphere (Fig. 31, D). This distinction between the primordial germ cells and the surrounding somatic cells may enable us to trace the keimbahn in vertebrates back into cleavage stages — something that has not been accomplished as yet. An examination of the various keimbahn-deter- minants listed in the table (p. 212) has led the writer to conclude that none of them is of a mitochondrial nature, but the results obtained by the special methods 228 GERM-CELL CYCLE IN ANIMALS employed by students who are studying mitochondria give us good reason to hope that other substances may be made visible which will help to clear up the problem of primary cellular differentiation. Merapo.ic Propucts. Among the most difficult cases to explain are those of Sagitta and certain cope- pods, since here the keimbahn-determinants ap- parently arise de novo in the cytoplasm. Buchner’s (1910) contention that the ‘“‘besondere Ké6rper”’ of Sagitta is the remains of the “‘accessory fertiliza- tion cell” of Stevens (1904) is not sustained by either Stevens (1910) or Elpatiewsky (1910). The idea of the nucleolar nature of the “‘Aussenkérnchen”’ in Cyclops has been discarded by Haecker (1903) and the conclusion reached that these granules are similar to nucleoli in one respect, namely, they are by-products of activities within the cell. Amma (1911) has considered this subject at some length, and after rejecting the possibilities of these being of (1) chromatic, (2) nucleolar, (3) chromidial, and (4) mitochondrial origin likewise concludes that they are transitory by-products. In this way the keimbahn-determinants in copepods are satis- factorily explained, and a similar explanation may be applied to Sagitta, although with less certainty. c. Discussion. A review of the literature on the keimbahn-determinants and the investigation of these substances in the eggs of insects force me to conclude that the fundamental organization of the eggis respon- sible for the segregation of the primordial germ cells, whereas the visible substances simply furnish evi- KEIMBAHN-DETERMINANTS 229 dence of this underlying organization. As I have stated elsewhere (Hegner, 1908, p. 21) regarding the keimbahn-determinants in beetles’ eggs, ‘“‘the granules of the pole-disc are therefore either the germ- cell determinants or the visible sign of the germ-cell determinants.” The writer’s experiments have thus far failed to determine the exact function of these granules. When the posterior end of a freshly laid beetle’s egg is pricked with a needle, not only the pole-dise granules flow out, but also the cytoplasm in which they are embedded (Hegner, 1908). If a small region at the posterior end is killed with a hot needle, the pole-disc is prevented from taking part in the development of the egg, but so also is the sur- rounding cytoplasm (Fig. 37, c). Eggs thus treated continue to develop and produce embryos without germ cells, but as a rule a part of the posterior end of the abdomen is also absent (Hegner, 1911a). The pole-disc granules and the cytoplasm containing them is moved by centrifugal force toward the heavy end of the egg and is proved to be quite rigid, but eggs thus treated do not develop sufficiently normally to enable one to decide whether the pole-dise pro- duces germ cells in its new environment or not. That the germ cells of Chironomus arise from a pre- localized substance was stated by Balbiani (1885) in these words, ‘‘the genital glands of the two sexes have an absolutely identical origin, arising from the same substance and at the same region of the egg.” Ritter (1890) expressed the opinion that the ‘‘Keimwulst”’ of Chironomus consists of fine 230 GERM-CELL CYCLE IN ANIMALS granulated protoplasm, an opinion concurred in by Hasper (1911), who terms it “ Keimbahnplasma.” The similar material in Miastor metraloas, the “‘polares Plasma,’’ is considered a special sort of protoplasm by Kahle (1908), and I can confirm this for Miastor americana. Further evidence of the protoplasmic nature of the substances which be- come segregated in the primordial germ cells is fur- nished by Boveri’s experiments on Ascaris. In 1904 this investigator concluded from a study of dispermic eggs that the diminution process is con- trolled by the cytoplasm and not by an intrinsic prop- erty of the chromosomes, and that the chromosomes of nuclei lying in the vegetative cytoplasm remain intact, whereas those of nuclei embedded in the animal cytoplasm undergo diminution. This con- clusion has been strengthened by more recent experi- mental evidence (Boveri, 1910) both from observa- tion on the development of dispermic eggs and from a study of centrifuged eggs (see p. 178, Fig. 53). Boveri’s results furnish a remarkable confirma- tion of the conclusions reached by the writer from a morphological study of the germ cells of chrysomelid beetles and expressed in the following words: “All the cleavage nuclei in the eggs of the above-named beetles (Calligrapha multipunctata, etc.) are poten- tially alike until in their migration toward the periph- ery they reach the ‘keimhautblastem.’ Then those which chance to encounter the granules of the pole- disc are differentiated by their environment, 7.e., the granules, into germ cells; all the other cleavage KEIMBAHN-DETERMINANTS 231 products become somatic cells.” Here, however, the pole-disc granules were considered the essential substance. The appearance of the keimbahn-determinants at a certain time and in a certain place, and their deter- minate segregation, point unmistakably to an under- lying regulating mechanism. These phenomena have some definite relation to the fundamental organiza- tion of the egg and require an investigation of our present knowledge of this subject. The isotropism of the egg as postulated by Pfliiger and the “cell interaction” idea especially developed by O. Hertwig and Driesch have given way before the beautiful researches tending to uphold the hy- pothesis of ‘‘ germinal localization” proposed by His and championed by so many investigators within the past two decades. The starting point for embry- ological studies has shifted from the germ layers to the cleavage cells and from these to the undivided egg. Organization, which Whitman (1893) main- tains precedes cell-formation and regulates it, is now traced back to very early stages in the germ-cell cycle and held responsible for the cytoplasmic lo- calization in the egg. One of the fundamental characteristics of the egg is its polarity. It has been known for about thirty years that the eggs of insects are definitely ori- ented within the ovaries of the adults. Moreover, gravity and the action of centrifugal force have no effect upon the polarity of insect eggs (Hegner, 19096). Giardina (1901) has found that during the divisions 232 GERM-CELL CYCLE IN ANIMALS of the odgonia in Dytiscus a rosette of sixteen cells is produced of which one is the ojcyte and the other fifteen nurse cells. The rosette thus formed possesses a definite polarity coincident with the axis of the odcyte which is identical with that which was present in the last generation of odgonia. Similarly in Muastor (Fig. 12) the polarity of the odcyte is recog- nizable as soon as the mesodermal cells, which serve in this species as nurse cells, become associated with it. The germ cells of other animals also possess a precocious polarity, as evidenced by their implanta- tion in the germinal epithelium (e.g., Wilson, 1903; Zeleny, 1904, in Cerebratulus), the position of the nucleus, the formation of the micropyle (Jenkinson, 1911), etc. This is true not only for the inverte- brates, but, as Bartelmez (1912) claims, ‘“‘the polar axis persists unmodified from generation to genera- tion in the vertebrates and is one of the fundamental features of the organization of the protoplasm”’ (p. 310). Furthermore, experiments with centrifugal force seem to prove that the chief axis of the egg is not altered when substances are shifted about, but is fixed at all stages (Lillie, 1909; Morgan, 1909; Conklin, 1910). Bilaterality also is demonstrable in the early stages of the germ cells of many animals, and, like polarity, seems to be a fundamental charac- teristic of the protoplasm. It is somewhat difficult to harmonize the various results that have been obtained, especially by experi- mental methods, from the study of egg organization. As the odcytes grow, the apparently homogeneous KEIMBAHN-DETERMINANTS 233 contents become visibly different in some animals, and when the mature eggs develop normally these “organ-forming substances” are segregated in def- inite cleavage cells and finally become associated with definite organs of the larva. Conklin (1905) has shown ‘‘that at least five of the substances which are present in the egg (of Cynthia) at the close of the first cleavage, viz., ectoplasm, endoplasm, myoplasm, chymoplasm, and chordaneuroplasm, are organ-forming sub- stances.” Under experimental conditions “they develop, if they develop at all, into the organs which they would normally produce; and, conversely, embryos which lack these substances, lack also the organs which would form from them.”’ ‘“‘Three of these substances are clearly distinguishable in the ovarian egg and I do not doubt that even at this stage they are differentiated for particular ends”’ (p. 220). “‘The development of ascidians is a mosaic work because there are definitely localized organ- forming substances in the egg; in fact, the mosaic is one of organ-forming substances rather than of cleavage cells. The study of ctenophores, nemer- tines, annelids, mollusks, ascidians, and amphibians (the frog) shows that the same is probably true of all these forms and it suggests that the mosaic principle may apply to all animals” (p. 221). The same writer has also proved from his study on Phallusia (1911) that these various substances exist even when they are not visible in the living egg. It is interesting also to note that Duesberg (1913) finds the ‘myo- 234 GERM-CELL CYCLE IN ANIMALS plasm” of Cynthia to be crowded with plasmosomes, differing in this respect from other egg regions. Experiments, especially those of Lillie (1906, 1909), Morgan and Spooner (1909), Morgan (1909a), and Conklin (1910), have shown that in many eggs the shifting of the supposed organ-forming substances has no influence upon development, and leads to the conclusion that these visible substances play no fundamental réle in differentiation, but that the invisible ground substance is responsible for de- terminate development. The eggs of different ani- mals, however, differ both in time and degree of organization, and the conflicting results may be accounted for by the fact that specification is more precocious in some than in others. The most plausible conclusions from a considera- tion of these observations and experiments are that every one of the eggs in which keimbahn-determi- nants have been described consists essentially of a fundamental ground substance which determines the orientation; that the time of appearance of keimbahn-determinants depends upon the _ preco- ciousness of the egg; that the keimbahn-determi- nants are the visible evidences of differentiation in the cytoplasm ; and that these differentiated portions of the cytoplasm are definitely localized by cytoplas- mic movements, especially at about the time of maturation. KEIMBAHN-DETERMINANTS 235 B. Tue Locauization oF THE K®EIMBAHN-DETER- MINANTS One of the characteristics of the keimbahn- determinants is their regular appearance at a certain stage in the germ-cell cycle according to the species in which they occur, and their constant localization in a definite part of the egg, or in one or more definite cleavage cells. Keimbahn-determinants are recog- nizable in many insects’ eggs before fertilization is accomplished, and even before the odcyte has reached its maximum size. We know that in Chironomus the ‘‘Keimwulst” (Ritter, 1890) or “‘Keimbahn- plasma’’ (Hasper, 1911) is present when the egg is laid, at which time the pronuclei as a rule have not yet fused. This is true also of the “Dotter- platte” in Calliphora (Noack, 1901). There can be little doubt, however, that these substances are present as such in the eggs before fertilization, judging from our knowledge of the history of similar materials in the eggs of other insects. The “‘pole- disc” in the eggs of chrysomelid beetles (Hegner, 1908; Wieman, 1910a) and the “polares Plasma” in Miastor (Kahle, 1908; Hegner, 1912, 1914a) are recognizable some time before fertilization and cannot therefore arise because of any influence exerted by the spermatozoén. Moreover, in Miastor the eggs thus far examined have all been parthenogenetic. In parasitic Hymenoptera the Keimbahn-chromatin appears in both fertilized and parthenogenetic eggs at an early growth period. In only one animal not 236 GERM-CELL CYCLE IN ANIMALS an insect has a similar occurrence been noted, namely, in Polyphemus, where, according to Kiihn (1911, 1913), the keimbahn-determinants consist of the remains of one or more nurse cells (Fig. 50). In the DapHnip& (Weismann and Ischikawa, 1889) the *“Paracopulationszelle”’ arises from material cast out by the germinal vesicle; in 4/quora (Haecker, 1892) the “‘Metanucleolus”’ is likewise derived from the germinal vesicle; in Ascaris (Boveri, 1892) chroma- tin-diminution occurs during the two- to four-cell stage; in Cyclops (Haecker, 1897, 1903) and other copepods (Amma, 1911) the ‘“Aussenkérnchen”’ or *“Ectosomen” become visible soon after fertiliza- tion (Diaptomus), but usually not until the pro- nuclei fuse (other species); in Sagitta the “be- sondere Kérper” (Elpatiewsky, 1909, 1910) or “Keimbahnchromidien”’ (Buchner, 1910) appear to arise de novo after fertilization, although if Buchner’s contention that they are the remains of the accessory fertilization cells is correct, they should be classed with the ‘“Nahrzellenkern” described by Kiihn (1911, 1913) in Polyphemus. It is thus evident that the keimbahn-determinants become visible, wherever they have been described, either just before or just after the eggs are fertilized, or, in parthenogenetic forms, shortly before matura- tion and cleavage are inaugurated. The localization of the keimbahn-determinants at the time of their appearance seems to be predeter- mined. In insects the posterior end of the egg is invariably the place where these bodies occur. In KEIMBAHN-DETERMINANTS 237 species whose eggs undergo total cleavage they are, under normal conditions, segregated in one definite blastomere from the two-cell stage up to the thirty- two-cell stage, as a rule, and are then distributed among the descendants of the single primordial germ cell. In Ascaris it is normally the cell at the posterior (vegetative) pole that fails to undergo the diminution process. It seems therefore that there must be some mechanism in the egg which definitely localizes the keimbahn-determinants. The segregation of these substances in one blas- tomere at the first cleavage division is a result of their previous localization, but in later cleavage stages events are more difficult to interpret. Both Haecker (1897) and Amma (1911) have attempted to explain the distribution of the ‘‘Ectosomen”’ in copepods by postulating a dissimilar influence of the centrosomes resulting in the segregation of these granules at one end of the mitotic spindle in the dividing stem cell. According to Zeigler’s hypothesis the centrosomes during unequal cell divison are heterodynamic, and Schonfeld (1901) believes that the synizesis stage is due to the attraction of the chromosomes by the centrosomes. It is well known that in many cases where unequal cell division occurs one aster is larger than the other, and this may be the true interpretation of the phenomena, but to the writer it seems more probable that the entire cell contents undergo rearrangement after each cell division, possibly under the influence of the material elab- orated within the nucleus and set free during mito- 238 GERM-CELL CYCLE IN ANIMALS sis. Elpatiewsky (1909) also believes in the unequal attractive force of the centrosomes in Sagttta.! In Ascaris, certain copepods, Sagitta, Polyphemus, and certain DapHnip the keimbahn-determinants are segregated in one cleavage cell until about the thirty-two-cell stage, but their substance is dis- tributed at the next division between the daughter cells. The insects such as Chironomus, Miastor, and chrysomelid beetles, where, on account of the super- ficial cleavage the keimbahn-determinants are not segregated in blastomeres, the primordial germ cells from the beginning consist almost entirely of the keimbahn material or this material plus the matrix in which it is embedded. Hence in these cases the keimbahn-determinants are localized at a determined point during each cleavage stage instead of being carried about by the movements of the egg contents or of the blastomeres, but, as in the eggs that undergo total cleavage, the determi- nants are distributed between the daughter cells as 1“Nach der vierten Teilung kommt der besondere Korper in den Wirkungskreis eines Zentrosomos, namlich desjenigen, welcher niher der Polarfurche liegt. Fast die ganze ‘Energie’ dieses Zentrosomas wird fiir die Ueberwindung der vis inertiae des besonderen Korpers ver- braucht; dieser wird dem Zentrosoma genihert und umschliesst es wie mit einer Kappe, so dass er im optischen Durchschnitt stets Hufeisen oder Sichelform aufweist. Infolge davon wird die wirkung dieses Zentroso- mas auf das Zellplasma nur sehr schwach, dieses Zentrosoma kann nur einen kleinen Plasmateil beherrschen, und die resultierende Zelle wird viel kleiner, als die Schwesterzelle. Diese kleine Zelle, die den beson- deren Korper bekommen hat, liegt niher zum yegetativen Poles, als die griéssere Schwesterzelle, und stellt die erste Urgeschlechtszelle G(d'"), die gréssere Schwesterzelle die erste Urentodermzelle E(d") vor"’ (p. 231). KEIMBAHN-DETERMINANTS 239 soon as the primordial germ cells are established. The reason for this appears to be that localizations occur in holoblastic eggs at each cleavage and that not until the thirty-two-cell stage or thereabouts does the keimbahn material become entirely sep- arated from other organ-forming substances and segregated in a single cell. When this point is finally reached, this keimbahn material must neces- sarily become divided between the daughter cells. In practically all known cases the daughter cells of the primordial germ cells are equal in size and each receives an equal portion of the keimbahn-de- terminants (Fig. 37, B). This is certainly to be expected from their constitution and future history. Sagitta, however, differs in this respect, for the remains of the “‘besondere Kérper” appear to be unequally distributed between the two daughter cells of the primordial germ cells (Fig. 54) and both Elpatiewsky (1909, 1910) and Stevens (1910), therefore, consider this as probably a differential division whereby in this hermaphroditic animal the substance of the male primordial germ cell is separated from the female. More work is necessary to make certain of this point. Conctuston. Keimbahn-determinants are def- initely localized in the egg and in definite cleavage cells. This localization is first observable just before or just after the eggs are fertilized, or, in parthenogenetic forms, shortly before maturation and cleavage are inaugurated. Some mechanism in the egg must be responsible for this localization. Heterodynamic centrosomes may have some influence 240 GERM-CELL CYCLE IN ANIMALS so far as the segregation of the keimbahn-determi- nants in cleavage cells is concerned, but the move- ment of the egg contents seems to be a more probable cause of localization. C. Tue Fate or THE KEIMBAHN-DETERMINANTS It is unfortunately impossible to trace the keim- bahn-determinants throughout the entire germ-cell cycle. The question of their fate, however, is an important one. As we have seen, they become vis- ibly apparent shortly before or just after the inaugu- ration of the maturation divisions, and remain intact for a brief period during the early cleavage stages. They persist in insects as definitely recognizable granules (Fig. 37, F) for some time after the primor- dial germ cells are segregated; then they gradually break up into finer particles, leaving no trace of their existence behind except in so far as they give the cytoplasm of the germ cells a greater affinity for certain dyes. In Chironomus they may still form distinct masses after the definitive germ glands have been formed (Fig. 33, D). The ectosomes in the copepods are temporary bodies which appear to rise de novo during the formation of each mitotic figure in the early cleavage stages, then break down and disappear. Practically all of the other keim- bahn-determinants persist during early cleavage and then disappear as distinct visible bodies as soon as the primordial germ cells are definitely segregated. What becomes of them during the comparatively long period between their disappearance in the primordial KEIMBAHN-DETERMINANTS 241 germ cells and their reappearance in the odcytes or mature eggs can only be conjectured. They seem to disintegrate into very fine particles which become thoroughly scattered within the cell body and mixed with the cytoplasm. It has been suggested (p. 68) that they may retain their physiological characteris- tics and become concentrated again in the growing odcytes into morphologically similar bodies, in- creasing in the meantime, by multiplication or in some other way, until they equal in mass those of the preceding generation of germ cells. On the other hand, they may all, like the ectosomes of copepods, be temporary structures produced at a certain time and place under similar metabolic conditions, and, becoming associated with particular parts of the cell contents, thus be constant in their distribution. Several ideas have been advanced regarding the fate of the eliminated chromatin in Ascaris. The ends of the chromosomes which are cast out into the cytoplasm are not equally distributed among the daughter cells nor does there appear to be any mech- anism for their definite unequal division. These facts argue against the theory that these cast-out chromatin bodies serve as determinants and also make improbable the hypothesis that they enable the somatic cells to differentiate, whereas the germ cells which do not undergo the diminution process remain in an indifferent condition, since their cyto- plasm lacks this material (Montgomery, 1911, p. 792). However, the fact that during the early cleavage divisions in some animals (see p. 218) large amounts R 242 GERM-CELL CYCLE IN ANIMALS of chromatin escape from the nucleus and are dif- ferentially distributed to the daughter cells is evidence that nuclear material may play some important réle in the progressive changes of cleavage cells. It has been shown that in many animals the germ cells do not multiply for a considerable period during the early developmental stages. This period coincides also with that during which the keimbahn- determinants, as a rule, disappear. For example, the germ cells of chrysomelid beetles multiply until there are about sixty-four present, at which time they constitute a group at the posterior end of the egg and the embryo has just started to form; no further increase in number occurs until the larval stage is reached and the definitive germ glands are established. As soon, however, as the embryo has reached a certain developmental stage, the germ cells migrate into it, and it looks very much as though they remain quiescent until the somatic cells are “‘able to protect, nourish, and transport”’ them. The number of primordial germ cells during the ‘period of rest”’ is perhaps most definitely known in Miuastor, where, as one group of eight and later as two groups of four each, they are present throughout a large part of embryonic development. In vertebrates also a long period exists during which division of the primordial germ cells does not take place (Fig. 6) and at least in several species certain cell contents (the mitochondria) remain in an indifferent condition (Rubaschkin, 1910; Tschasch- kin, 1910; Fig. 31, B). These facts all indicate that KEIMBAHN-DETERMINANTS 243 these cells remain in a primitive condition and do not undergo the histological differentiations charac- teristic of somatic cells, a view which, however, has ~ been objected to (Eigenmann, 1896). The disap- pearance of the keimbahn-determinants and the yolk globules of vertebrates during this period have suggested that these substances are nutritive in function, furnishing energy to the migrating germ cells. The fact of this long rest period, followed by rapid multiplication of the odgonia and spermatogonia during which no important specializations occur, and later succeeded by the remarkable changes that occur in both the odcytes and spermatocytes, has led to the suggestion (Montgomery, 1911, pp. 790-792) that in the germ-cell cycle there is a series of changes parallel with that of the somatic cycle. In the development of both cycles preformation and epi- genesis proceed at the same time. The chromosomes seem to be the preformed elements of the germ cells, since they are apparently the most stable constitu- ents. The cytoplasm, on the other hand, undergoes a series of epigenetic changes such as the formation of an idiozome, the development of mitochondria, the appearance of a sphere, and the metamorphosis of the spermatozoén. Finally we must inquire into the fate of the keim- bahn-determinants in the male germ cells. Does the keimbahn material in these cells increase in amount as has been suggested for the odcytes and is it localized in the spermatogonia, spermatocytes, or spermatozoa 244 GERM-CELL CYCLE IN ANIMALS as a definite, visible substance ? We know from the investigations of Meves (1911) that the plastosomes in the spermatozoén are carried into the egg, in the case of Ascaris, and there fuse with the plastosomes of the ovum. Whether keimbahn-determinants act in a similar manner is unknown. There are, how- ever, certain cytoplasmic inclusions in the male germ cells that have been compared with similar structures in the odcytes, for example, the chromatic body described by Buchner (1909) in the spermato- genesis of Gryllus (see p. 88), and the plasmosome which is cast out of the nucleus of the second sperma- togonia in Periplaneta and disintegrates in the cy- toplasm (Morse, 1909). That keimbahn-determi- nants from the spermatozo6n are not necessary for the normal production of germ cells is of course evi- dent, since some of the species with which we are best acquainted, for example, Miastor, are partheno- genetic. CHAPTER IX THE CHROMOSOMES AND MITOCHONDRIA OF GERM CELLS No account of the germ-cell cycle in animals can be considered complete without at least a brief reference to the history of the chromosomes and mitochondria of germ cells. The chromosomes have for many years been recognized as the most important visible bodies in the cell, and their behavior during the germ- cell cycle has convinced most zodlogists that they may also be regarded as the bearers of hereditary factors. The mitochondria, on the other hand, are cellular constituents which have only compara- tively recently come into prominence in cytological literature, and ideas concerning their nature and functions are still in a very chaotic condition. Tue CHROMOSOME CYCLE IN ANIMALS A few general statements regarding the behavior of the chromosomes during cell division, maturation, and fertilization are contained in Chapters I and II. We may recognize a rather definite chromosome cycle as a part of the germ-cell cycle, and it is to certain events in this chromosome cycle that our attention will be directed in the following paragraphs. It is best to begin our discussion, as in the general review 245 246 GERM-CELL CYCLE IN ANIMALS of the germ-cell cycle (Chapter II), with the par- thenogenetic or fertilized egg after the maturation processes have been completed, and to exclude all references to the accessory chromosome until later. It may be pointed out first that the number of chromosomes in the cells of any individual of a species is, with few exceptions, constant. Thus the thread worm of the ‘horse, Ascaris megalocephala var. univalens, has two; eececece Fact TR@ A Cee fil 8g — Seesese8 8 B Won dh oer WY 3 Oncopeltus ¢ ofe gees sage Mi Fig. 67.— Maturation in Oncopeltus. Male above. Female below. Lettering as in Fig. 65. (From Morgan’s Heredity and Sex, pub- lished by the Columbia University Press.) CHROMOSOMES AND MITOCHONDRIA 263 Ascaris 3 Aye Ay © of = ff We bu A © = 2s WN Ny WN m 2 A ¢ au Fic. 68.— Maturation in Ascaris. Male above. Female below. Let- tering as in Fig. 65. (From Morgan's Heredity and Sez, published by the Columbia University Press.) 264 GERM-CELL CYCLE IN ANIMALS Reduction Ripe Eggs Division Polar Body oO 6 2 Fertilized oy) NO) Fertilized Egg Oégonium Spermat- ogonium Sperm Fic. 69.— Diagrams showing the behavior of the chromosomes during maturation and fertilization in the starfish, Echinus. One kind of spermatozo6n is formed, but the ripe eggs differ, one containing a large X-element, the other asmall Y-element. (From Schleip, 1913.) x © jG — o Protenor, Anasa Syromastes, Homo Ascaris lumbricoides X 00 00 & 09 e ve Nazara Euschistus Nazara Thyanta viridula coenus hilaris calceata ee ey Rocconota, Prionides, Gelastocoris Acholla Fitchia Sinea multispinosa Fig. 70.— Diagram showing the number and size relations of the X- and Y-chromosomes in a number of animals. (From Wilson, 1911.) CHROMOSOMES AND MITOCHONDRIA 265 and the drones parthenogenetically. The history of the chromosomes has here been worked out by Nachtsheim (1913). The primary odcyte contains sixteen chromosomes in the form of eight tetrads; the mature egg and polar bodies are each provided with eight chromosomes (Fig. 71, E); the inner half of the divided first polar body fuses with the second polar body, forming a “‘Richtungskopulationskern” (Fig. 71, F) which does not give rise to the male germ cells as Petrunkewitsch (1901) claimed, but degenerates. The cleavage nucleus in the parthenogenetic egg which produces the male shows sixteen chromosomes which divide to form thirty-two or sixty-four in the somatic cells, but do not increase in number in the spermatogonia. The first maturation division is unequal, and a “‘polar body”’ without any chroma- tin is pinched off (Fig. 71, A-C, Rk,). The sperma- tids are likewise of two sorts; the smaller (Fig. 71, C, Rk.) contain as many chromosomes as the larger (16), but degenerate, while the larger transform into spermatozoa. The fertilized (female) eggs possess the same number of chromosomes as the partheno- genetic eggs, plus an equal number which is brought in by the spermatozodn. The cleavage nucleus exhibits thirty-two chromosomes which may become sixty-four in the somatic cells, but unite two by two to form sixteen in the odgonia. Phylloxera caryecaulis will serve to illustrate the chromosome cycle in a species with a life cycle composed of parthenogenetic females which alter- nate with sexual males and females (Morgan, 1909, 266 GERM-CELL CYCLE IN ANIMALS 1910). The eggs laid by the stem-mother (see Chap- ter Fia. I, p. 24) in the spring possess four ordinary and A B F 71.— Stages in the spermatogenesis and odgenesis of the honeybee. A, B. First maturation division in the male. C. Second matura- tion division in the male. Three cells are produced: the first (RKi) without chromatin; the second (RK:2) with chromatin, but small and functionless; and the third a functional spermatid. (After Meves, 1907.) D. First maturation division in the female showing polar body with eight dyads, and secondary odcyte with eight dyads. EF. Sec- ond maturation division in the female showing the divided first polar body, the second polar body, and female pronucleus each with eight monads. F. Outer end of first polar body disintegrating ; inner half of first polar body uniting with second polar body, and female pronucleus. (After Nachsheim, 1913.) two sex chromosomes. These eggs give rise to parthenogenetic females with the same number of CHROMOSOMES AND MITOCHONDRIA 267 chromosomes, and generation after generation of such females appear during the summer; but in the autumn, females, whose eggs must be fertilized before they will develop, and males are produced. The chromosomes of these eggs are distributed during maturation as shown in the diagram (Fig. 72). The eggs that develop into the females possess the usual number of chromosomes, but those that give rise to males cast out in the polar body one chromo- some that fails to divide, and hence are provided with one chromosome less than the others. During the maturation of the germ cells of these males two sorts of spermatozoa are formed, one with three chromosomes, the other with only two; the latter degenerate. Therefore, since only one sort of spermatozoa is functional, the fertilized winter eggs are all alike and all give rise to females (stem- mothers) the following spring. The chromosome distribution in certain nema- todes resembles somewhat that of the phylloxerans. Here, however, we have to deal with organisms that are peculiar in several respects. Maupas (1900) has shown that in the genus Rhabditis the number of males per 1000 females ranges from 45.0 to 0.15 according to the species; and that these few males do not copulate with the females and hence are func- tionless. Furthermore, the females are not true females, but hermaphrodites. Kruger (1912) dis- covered that in Rhabditis aberrans the nuclei of the spermatozoa did not fuse with that of the egg, except in one instance, but disappeared in the cytoplasm ; 268 GERM-CELL CYCLE IN ANIMALS hence the spermatozoa simply initiate development. The chromosome cycle of Rhabditis nigrovenosa has been studied by Boveri (1911) and Schleip (1911). PHYLLOXERA CARYECAULIS Fotar Plate @@eo ion X Jie) O|}@@! ree soe ||| Il LT 10 sae a — lg Wi Staual tale Oo CPw aur = we 68) ee Fela oe ~ Be &3 6 ee oe Fic. 72.— Chromosome cycle in Phylloxrera caryeca © os Mor- gan’s Heredity and Sex, published by the Columbia University Press.) CHROMOSOMES AND MITOCHONDRIA 269 This nematode is a parasite in the lung of the frog for part of its life cycle; during this period it re- sembles the female, but is really hermaphroditic. These hermaphrodites give rise to free-living indi- viduals which are true males and females; the eggs of the latter when fertilized develop into para- sitic hermaphrodites. The odgonia and sperma- togonia of the hermaphroditic parasites possess twelve chromosomes (Fig. 73, A). The nucleus of the mature egg is provided with six (B). Two sorts of spermatozoa are formed, one-half with six chromo- somes, the other half with five; the latter result from the casting out of one chromosome (F) in a manner similar to that described above in Phylloxera. The eggs fertilized with the spermatozoa containing six chromosomes (F) produce free-living, true fe- males, whereas those fertilized by the spermatozoa with five (G) develop into free-living, true males. The hermaphroditic condition is regained as follows: The free-living females give rise to eggs all with six chromosomes; the males, whose spermatogonia contain eleven chromosomes, produce spermatozoa with six or five chromosomes; those with the latter number, however, are not functional, hence all fertilized eggs must be provided with twelve chromo- somes and develop into the hermaphroditic parasites. The chromosome cycle in pteropod mollusks as worked out by Zarnik (1911) seems even more re- markable than that described for nematodes. The hermaphroditic species, Creseis acicula, possesses twenty chromosomes, sixteen large ordinary chromo- 270 GERM-CELL CYCLE IN ANIMALS E Fic. 73.— Rhabditis nigrovenosa. Stages in maturation, fertilization, and cleavage. A. Odgonium with twelve chromosomes. B. Sec- ond maturation division. Pronucleus and second polar body each with six chromosomes. C. Primary spermatocyte. D. Division of primary spermatocyte. FE. Second spermatocyte division; one chromosome delayed. F. Two spermatozoa each with six chromo- somes. G. Cleavage spindle of egg showing two groups of chromo- somes; one with six contributed by the egg, the other with five contributed by the sperm. (After Schleip, 1911.) CHROMOSOMES AND MITOCHONDRIA 271. somes (shown in black in Fig. 74), two large sex- chromosomes (dotted), and two small sex-chromo- Spermato- gonium. Spermato- cyte 1. Ordn. Spermato- cyte 2. Ordn. Reifes Hi. Fie. 74.— Diagrams showing the chromosome cycle in the pteropod mollusk, Creseis acicula. In order to simplify the diagrams each black chromosome is made to represent eight ordinary chromosomes. (After Zarnik, 1911.) somes (dotted). The spermatogonia enter the mat- uration period in this condition. The number of 272 GERM-CELL CYCLE IN ANIMALS chromosomes is reduced in the first division, resulting in two secondary spermatocytes each with eight large ordinary chromosomes, and one large and one small sex-chromosome. During the second division the small sex-chromosome does not divide, but passes intact into one spermatid; thus two sorts of sperma- tozoa are formed, one with eight large ordinary and one sex chromosome and the others with eight large ordinary chromosomes and two large sex- chromosomes. The spermatozoa with only one sex chromosome is not functional. The odgonia differ from the spermatogonia and somatic cells in the possession of sixteen large ordinary chromosomes and four small sex-chromosomes; two of the latter arise by the diminution of the chromatin in two of the large sex-chromosomes. The maturation divi- sions are of the usual sort, and all of the eggs are alike, containing eight large ordinary chromosomes and two small sex-chromosomes. Fertilization, as indicated in Fig. 74, always results in a zygote with sixteen large ordinary chromosomes, two large sex- chromosomes, and two small sex-chromosomes, which develop into a hermaphroditic individual. Although we know very little about the chromo- somes of man, the data available seem to indicate that here also there are chromatin bodies concerned with sex-determination. The following table indi- cates the state of our knowledge at the present time. Guyer (1910) was the first to announce the dis- covery of accessory chromosomes in man. He found twenty-two chromosomes in the spermatogonia, CHROMOSOMES AND MITOCHONDRIA 273 TaBLE SHOWING THE NUMBER OF CHROMOSOMES IN MAN ACCORDING To Various INVESTIGATORS ee Haptoiw NuMBER INVESTIGATOR DatE Bardeleben 1892 Q4 Flemming 1897 18 (15 or 19)! | Wilcox 1900 12 Duesberg 1906 32 Farmer, Moore, and Walker | 1906 16 Moore and Arnold 1906 12 or 10 Guyer 1910 12 or 10 Montgomery 1912 24( ?) Gutherz 1912 47 23 or 24 Winiwarter 1912 34 (33, 38) Wieman 1913 which became ten bivalent and two accessories in the primary spermatocytes. The latter pass un- divided to one pole (Fig. 75, 4), and hence two classes of spermatozoa result, one with ten ordinary chromo- somes, and the other with ten ordinary and two accessory chromosomes. Winiwarter (1912), on the other hand (Fig. 75, D-E), reports forty-seven chromosomes in the spermatogonia and two classes of spermatozoa, one with twenty-three and the other with twenty-four. The number in the female, according to Winiwarter, is probably forty-eight, and hence all mature eggs are alike so far as chromo- some number is concerned, each being provided with twenty-four. If these data are confirmed, it is evident that sex in man is determined at the time of fertilization and cannot be influenced by changing the environment. 1 Wilcox doesn’t state whether this is the reduced or diploid number. T Q74 GERM-CELL CYCLE IN ANIMALS The above illustrations indicate that there is some internal mechanism which controls sex, and that certain chromosomes are, in at least many cases, E Fic. 75.— Chromosomesinman. A. First spermatocyte division show- ing two accessories passing early to one pole. B. Two contiguous spermatids, one without and the other with two accessories. C. Two secondary spermatocytes; the one above with an accessory. D. Sec- ond spermatocyte with twenty-four dyads. £. Second spermatocyte with twenty-three dyads. (A-B, from Guyer, 1910; C-E, from Wintwarter, 1912.) factors in sex-determination. Several hypotheses have been suggested as to the relation of these chromosomes to sex, such as that sex is determined by the quantity of chromatin present in the zygote. CHROMOSOMES AND MITOCHONDRIA 275 No view, however, has won general acceptance, but it seems probable that there are fundamental inter- relations between the different parts of the cell which regulate the behavior of the chromosomes. We must, therefore, look further for an explanation of sex-determination. It has been suggested that differences in metabolism may be responsible for the fundamental differences between the sexes. Ac- cording to this view changes in metabolism may control the behavior of the sex-chromosomes, or the presence of the sex-chromosomes in every cell in the body may influence the metabolism “in such a way that the organism is caused to become of one sex rather than of the other, in consequence of its type of metabolism ” (Doncaster, 1914, p. 515). Tur MirocHonpria oF GERM CELLS The study of the relative importance of the nucleus and the cytoplasm in heredity has been given a new impetus within recent years by the more accurate examination and description of certain cytoplasmic inclusions of both germ cells and somatic cells known as mitochondria, chondriosomes, plastosomes, chro- midia, etc. Some of the best recent evidence that part of the germ-plasm may be located in the cyto- plasm is afforded by the work of Benda, Meves, Regaud, Duesberg, and others on the history of these mitochondrial bodies during maturation, fer- tilization, early cleavage, and cellular differentiation. As long as forty years ago the cytoplasm of the germ cells was known to contain bodies other than 276 GERM-CELL CYCLE IN ANIMALS the nucleus; these bodies have been given various names such as sphérules (Kunstler, 1882), cytomi- crosomes (La Valette St. George, 1886), bioblasts (Altmann, 1890), and ergastoplasm (Bouin, 1898). In 1897 and 1898 Benda noticed the constant pres- ence of certain granules in the male germ cells of a number of vertebrates and was able to trace their history from the spermatogonia until they formed the spiral filament in the tail of the spermatozoa. These observations were extended the following year (1899) so as to include all stages in the development of the eggs and spermatozoa of many vertebrates and invertebrates and also various tissue cells such as striated muscle-fibers, leucocytes, marrow-cells, etc. This work attracted wide attention chiefly for two reasons: (1) the history of the granules was carefully worked out and the various stages accurately described, and (2) special, rather com- plicated, staining methods were devised which were supposed to color the mitochondria so that they could be distinguished from all other cell inclusions. From 1899 until the present time an ever increasing number of investigators have attacked the problems presented by the mitochondria, or referred to these structures incidentally when working upon other his- tological or cytological problems. The study of mito- chondria received its greatest impetus, however, in 1908, when Meves published a paper on these structures in the chick embryo entitled ‘‘Die Chon- driosomen als Traiger erblicher Anlagen.” In this paper the chick embryo is described from the fifteen- CHROMOSOMES AND MITOCHONDRIA 277 hour stage up to the three-days-nine-hour stage. The cells of the earliest stage studied contained mito- chondria (Fig. 76) which were differently arranged in the germinal layers: the ectoderm and entoderm cells contained, for the most part, rods and threads, the granules being scarce, and the mesoderm cells were characterized by numerous granules and few rods and threads. At the three-day stage the mito- chondria of the neuroblasts became difficult to stain by the usual method, but did stain like neuro- fibrils. These and other observations led Meves to the conclusion that the mitochondria are of con- siderable importance in cellular differentiation and are in fact the bearers of hereditary Anlagen. Since this paper of Meves appeared, the zodlogical periodicals have been flooded with the results of in- vestigations of the mitochondria in almost every sort of germ and somatic cell, both normal and abnormal, and in Protozoa and Mertazoa, In- VERTEBRATES and VERTEBRATES. No report on spermatogenesis, odgenesis, or early embryonic de- velopment is complete without reference to the mito- chondria. In plants, also, cellular bodies have been described of a mitochondrial nature (Meves, 1904; Duesberg and Hoven, 1910; Guilliermond, 1911). A large number of new terms have been coined for the purpose of describing these cytoplasmic in- clusions. Some of them are as follows: (1) mito- chondria, applied by Benda (1897, 1898) to certain granules with definite staining reactions; (2) chon- driosomes, proposed by Meves (1908) for both single 278 GERM-CELL CYCLE IN ANIMALS Fie. 76.— Mitochondria in the embryonic cells of the chick. A. In cells of the primitive streak. B. In dividing connective tissue cells. C. In connective tissue cells. D. In a cartilage cell. 2. In osteo- blasts and bone cells. F. In cells of Wolffian body. (From Dues- berg, 1913; A, B, C, E, after Meves; D, F, after Duesberg.) CHROMOSOMES AND MITOCHONDRIA 279 granules and chains of granules; the latter were also called chondriokonts; (3) plastosomes (plastochon- dria, plastokonta), employed by Meves (1910) be- cause of their supposed réle in histogenesis; (4) éclectosomes, selected by Regaud (1909) as a general physiological expression for chondriosomes ; (5) chon- driotaxis, used by Giglios-Tos and Granata (1908) to describe the parallel arrangement of chondrio- konts; (6) chondriodiérése, proposed by the same authors for the division of the chondriokonts during cell division; (7) karyochondria, coined by Wildman (1913) for cytoplasmic inclusions derived from the basichromatin of the nucleus; (8) chromidia, a term considered by Goldschmidt (1904) and others to in- clude the mitochondria. We are here especially interested in the mitochon- dria of the germ cells, their origin, fate, and signif- icance, but our ideas regarding the importance of these bodies in heredity depend somewhat upon their behavior in somatic cells. As already stated, Benda (1903) observed mitochondria in both germ cells and somatic cells. Since then they have been recorded in Protozoa, in almost every sort of somatic cell in Mrazoa, and in many plant cells (Fig. 77). Excellent reviews have been published by Benda (1903), Fauré-Frémiet (1910), Prenant (1910), and Duesberg (1912). These reviews have led to the conclusion already expressed by Regaud (1909, p. 920) that ‘it is probable that they (mitochondria) exist in all cells, at least at certain stages in their activities.”” Among the somatic differentiations to 280 GERM-CELL CYCLE IN ANIMALS which mitochondria are supposed to give rise are neurofibrils and myofibrils. Meves (1907, 1908) considered it probable that neurofibrids were trans- formed chondriosomes, and Hoven (1910) seemed to have proved it, but Marcora (1911) and Cowdry (1914) find that the neurofibrils arise independently, Fic. 77.— Mitochondria in the cells of a plant, Pisum sativum. A. Young germ cell. B. Young germ cell dividing. C. Old cell containing vacuoles. (From Duesberg and Hoven, 1910.) although mitochondria are present in the nerve cells. Duesberg (1910) is quite positive that the myofibrils of striated muscle fibers are produced by the metamorphosis of chondriosomes from em- bryonic muscle cells, and has recently (Duesberg, 1913) strengthened his position by the discovery that the myoplasm described by Conklin (1905) in the egg of the Ascidian, Cynthia, is well supplied with chondriosomes. Mitochondrial structures have been studied in both living and preserved cells. Fauré-Frémiet (1910) describes them in living cells (Fig. 78, D) as CHROMOSOMES AND MITOCHONDRIA 281 small, transparent, slightly refringent granules of a pale gray tint, either homogeneous or else vesicular with fluid contents and a thin, denser, refringent periphery. Rod-like mitochondria were likewise observed by Montgomery (1911) in the living male germ cells of Huschistus (Fig. 78, A—B) which had been teased out in Ringer’s solution; and this in- Fig. 78.— Division of mitochondria. A-B. Mitochondrial rods divid- ing during first maturation division in Euschistus. C. Stages in division of mitochondrial body in Hydrometra. D. Simultaneous division of micronucleus and mitochondria in Carchesium (in vivo). (A-B, from Montgomery, 1911; C, from Wilke, 1918; D, from Fauré-Frémict, 1910.) vestigator concluded that in preserved material ‘‘we have been working with images that are very close to the living... .”’ More recently Lewis and Lewis (1914) have made careful studies of mitochon- dria in living cells from chick embryos. Granules were here seen ‘‘to fuse together into rods or chains, and these to elongate into threads, which in turn anastomose with each other and may unite into a complicated network, which in turn may again break down into threads, rods, loops, and rings.” Even more remarkable are the movements within the ? &® 9 o Solio e MMs ON o ? e % D 282 GERM-CELL CYCLE IN ANIMALS cell described by the same investigators. “The mitochondria are almost never at rest, but are con- tinually changing their position and also their shape. The changes in shape are truly remarkable, not only in the great variety of forms, but also in the rapidity with which they change from one form to another. A single mitochondrium may bend back and forth with a somewhat undulatory movement, or thicken at one end and thin out at the other with an appear- ance almost like that of pulsation, repeating this process many times. Again, a single mitochondrium sometimes twists and turns rapidly as though attached at one end, like the lashing of a flagellum, then suddenly moves off to another position in the cytoplasm as though some tension had been re- leased.”” Mitochondria may also be stained intra vitam, especially with dahlia violet and Janus green. Most of the fixing solutions ordinarily used for cyto- logical purposes destroy the mitochondria. The methods which seem to give the best results have osmic acid or formalin as a basis, such as those de- vised by Altmann (see Lee, 1905, p. 43), Benda (Lee, 1905, p. 223), Meves (1908), and Regaud (1908, p. 661). Benda (1903) claimed that all cellular structures which stained violet by his method were of a mitochondrial nature ; but this has not been found to hold true. Undoubtedly the many bodies which have been discovered in cells are of several sorts, and only by a thorough study of their staining qualities, morphological aspects, and biological réles can they be identified. Benda’s crystal violet CHROMOSOMES AND MITOCHONDRIA | 283 stain seems to be more selective than any other for mitochondria and is of great value for this reason. Mitochondria most often appear as_ spherical or elongated granules about 0.001 mm. diameter. These granules may become arranged in a series, thus forming a chain, and the granules in a chain may fuse into a homogeneous rod. Different forms are present in different kinds of cells or even in the same cell at various stages in its evolution or func- tional activity. Some investigators (Prenant, 1910) maintain that the homogeneous rod is the primitive condition and that the granules are formed by the disintegration of such rods; to others just the reverse seems to be true (Rubaschkin, 1910; Dues- berg, 1912). The chemical: constitution of the mitochondria has been studied by a number of investigators. Regaud (1908) has shown that the mitochondria of the ~seminal epithelium are not histochemically identical. He distinguishes three sorts of granules: (1) those which resist the action of acetic acid and are stainable without being previously immersed in a solution of potassium bichromate, (2) granules which resist acetic acid but require intense chromisa- tion, and (3) granules which do not resist acetic acid and demand chromisation. Fauré-Frémiet, Mayer, and Schiffer (1909) have studied the mito- chondria by microchemical and comparative methods and reached the conclusion that they are lecithal- bumins. Mitochondria have been noted in all stages of 284 GERM-CELL CYCLE IN ANIMALS the male germ-cell cycle, especially in mammals, mollusks, and insects, and appear to be continuous from one generation of cells to the next. During Fic. 79.— Behavior of the mitochondria during the fertilization and early cleavage of the egg of Ascaris. A. Egg into which a sperma- tozoén has penetrated. B, C. The mixing of the mitochondria of the egg and spermatozoén. D. Division stage of the first two blas- tomeres. (After Meves, 1911 and 1914.) mitosis the plastosomes lie outside of the spindle (Fig. 79, D); they may divide autonomously as claimed by Fauré-Frémiet (1910) in Protozoa (Fig. CHROMOSOMES AND MITOCHONDRIA 285 78, D) and Wilke (1912) in the spermatocytes of Hydrometra or en masse, as in the spermatogenesis of Euschistus (Fig. 78, A-B), thus undergoing a sort of paramitosis (Montgomery, 1911) and Notonecta (Browne, 1913). In the former cases each daughter cell is supposed to receive one-half of each granule; in the latter the distribution is largely by chance, but apparently equal (Cowdry, 1914). According to certain observers the centrosomes exert an in- fluence upon the mitochondria as indicated by the aggregation of these bodies around the asters (Fauré- Frémiet, 1910; Meves, 1914); but others have been unable to find any confirmatory evidence in their material (Montgomery, 1911). Duesberg (1908) has pointed out that since there is no rest period between the two maturation divisions there must be a quantitative reduction of plastosomes in the sper- matids; a quartering of the mitochondria could not, however, be observed by Montgomery (1912) in Pertpatus. Montgomery (1911) has suggested that the relative amount of the mitochondrial sub- stance received “‘might determine the sex-prepon- derance character of the sperm, a matter unfor- tunately very difficult to test.” Fauré-Frémiet recognizes four types of mitochon- drial distribution in the germ cells: (1) filaments or masses that do not undergo profound morphological changes (Fig. 80); (2) one or more masses which transform into a definite morphological element, the Nebenkern; (3) masses which only partially change into a Nebenkern or yolk nucleus; (4) bodies 286 GERM-CELL CYCLE IN ANIMALS which transform entirely or in part into deuto- plasmic granules of a fatty nature. The origin of the mitochondria in male cells can- not be stated definitely, since certain investigators (Goldschmidt, Buchner, Wassilieff, etc.) claim that they arise from thenucleus; others (e.g., Meves, Wilke, Duesberg) consider them to be integral parts of the cytoplasm ; and athird group (Montgomery, Browne, Wildman) looks upon some of them as the results of chemical interaction between the nucleus and the cytoplasm. Less is known con- Fic. 80.— Four stages in the formation ‘ * of the spermatozoén of Enterorenos cee the mitochon- sponrng th sition of he miter dria. during ob genesis than during sperma- togenesis, but certain bodies have been described in the ova of a number of animals which exhibit all of the characteristics of the mitochondria of male cells. As in the latter, they have been considered chromidial by some and of cytoplasmic origin by others. The importance of the mitochondria depends largely upon their functions. Those of the egg have been observed by Russo (1907), Loyez: (1909), Fauré-Frémiet (1910), Van Durme (1914), Hegner (1914a), and others to transform directly into yolk globules. According to Van der Stricht (1904), Lams (1907), etc., they produce yolk elements in- CHROMOSOMES AND MITOCHONDRIA 287 directly; and it is the opinion of Meves, Duesberg, and their followers that they play an important réle in fertilization. Likewise in the spermatozoa ideas differ regarding their functions. Benda (1899) believed them to be motor organs; Koltzoff (1906), from a study of the spermatozoa of Decapods, maintains that they represent elements which form a sort of cellular skeleton; Regaud (1909) claims that they are the particular cellular organs which exercise a “fonction éclectique,”’ extracting and fixing substances in the cell, and should therefore be called “‘éclectosomes”; and Meves (1907, 1908) holds that they are cytoplasmic constituents cor- responding to the chromosomes of the nucleus. Meves (1907, 1908) came to the conclusion that there must be hereditary substances in the cytoplasm, and by the method of elimination decided in favor of the mitochondria. In his studies on fertilization and cleavage in Ascaris (Meves, 1911, 1914) he has shown that granules from the spermatozoén (Fig. 79) fuse with similar granules in the egg, as described previously by L. and R. Zoja (1891), and that these granules are plastosomes. The distribution of the fused granules is followed until the amphiaster is formed in the two-cell stage; here the plastosomes are mainly grouped about the centrosomes, although a few are scattered about in the cytoplasm (Fig. 79, D). Although there are many who believe Meves and his followers to be correct in their contention that the plastosomes are the bearers of hereditary charac- 288 GERM-CELL CYCLE IN ANIMALS teristics in the cytoplasm, just as the chromosomes are the bearers of hereditary characteristics in the nucleus, still there are many objections to this view, such as the fact that part or all of the plastosomes may be cast out of the spermatid (e.g., in the opos- sum, Jordan, 1911; and in Pertpatus, Montgomery, 1912). It is obvious from the foregoing account that there are a number of opposing views regarding the origin, nature, and réle of the various cytoplasmic inclusions which have been considered mitochondria. Are they constant, necessary constituents of the living protoplasm, or are they inactive lifeless bodies which may be included under the term metaplasm ? Tf they constitute a part of the living protoplasm, do they form the skeleton of the cell, do they take part in the metabolic activities of the cytoplasm or nucleus, or do they play a réle in the process of differentiation, and should they be considered as the hereditary substance of the cytoplasm? If they are simply metabolic products, are they excretory in nature, or reserve materials set aside for the later use of the cell? And finally, do they arise from the nucleus, are they strictly cytoplasmic, or do they originate through the interaction of nucleus and cytoplasm? It is impossible in a short space to give an adequate account of the arguments pro and con, and so we must refer the reader to the compre- hensive reviews mentioned above. The conclusion, however, is perfectly safe that we shall have to await the results of further investigations before we can come to a definite decision. In the meantime we CHROMOSOMES AND MITOCHONDRIA 289 should thank the mitochondria for focusing the attention of cytologists upon the cytoplasmic ele- ments, since the belief is becoming more and more general that hereditary phenomena are the result of interactions between nucleus and cytoplasm and that the latter may play a more important rdéle than is usually supposed. CHAPTER X THE GERM-PLASM THEORY In discussing the germ-plasm theory it is necessary to distinguish between this hypothesis and that of the morphological continuity of the germ cells. The facts and theories involved have grown up to- gether. Owen (1849) was perhaps the first to point out the differences between germ cells and body cells. “‘Not all of the progeny of the primary impreg- nated germ cell,’’ he writes, ‘‘are required for the for- mation of the body in all animals; certain of the de- rivative germ cells may remain unchanged and become included in that body which has been composed of their metamorphosed and diversely combined and confluent brethren; so included, any derivative germ cell or the nucleus of such may commence and repeat the same processes of growth by imbibition, and of propagation by spontaneous fission, as those to which itself owed its origin. . . .”” Galton (1872) was among the earliest to recognize the necessity for two sorts of materials in the individual metazoén, ‘one of which is latent and only known to us by its effects on his posterity, while the other is potent, and constitutes the person manifest to our senses.” He at that time believed in the inheritance of ac- quired characters and conceived the egg as a struc- 290 THE GERM-PLASM THEORY 291 tureless body from which both the body and the ova of the individual evolve; and considered these ova to consist of contributions partly from the egg and partly from the body which developed from the egg. Later Jager (1877) stated the idea of germinal con- tinuity more definitely. He maintained that part of the germ-plasm (Keim Protoplasma) of the animal forms the individual, and the rest is re- served until sexual maturity, when it forms the repro- ductive material. The reservation of this phylo- genetic substance he termed the “continuity of the germ-plasm” (“‘Continuitit des Keimproto- plasmas’). To Weismann (1885) is usually given the credit for originating the germ-plasm theory, but while we are undoubtedly indebted to him for the great influence the hypothesis of germinal con- tinuity has had upon the trend of biological investi- gations within the past thirty years, we must con- sider Jager as the first to clearly enunciate the idea. Jager (1878) also expressed a belief in the mor- phological continuity of the germ cells of succeed- ing generations, but this idea was first definitely stated by Nussbaum (1880), whose investigations of the germ cells in the trout and frog led him to conclude that the cleavage cells form two groups independent of each other. One group contains the cells which multiply and differentiate and thus build up the body of the individual, but do not pro- duce germ cells; the other group takes no part in the formation of the body and undergoes no differen- tiations, but multiplies by simple division. The germ 292 GERM-CELL CYCLE IN ANIMALS cells are thus not derived from the individual in which they lie, but have a common origin with it. The segregated germ cells or species substance is therefore distinct and independent of the individual ; this accounts for the constancy of the species. We may distinguish between the two ideas by defining them as follows: (1) Germinal continuity, or the germ-plasm theory. “In each ontogeny a part of the specific germ-plasm contained in the parent egg-cell is not used up in the construction of the body of the off- spring, but is reserved unchanged for the formation of the germ cells of the following generation” (Weismann, 1891, p. 170). (2) Morphological continuity of the germ cells. The developing egg produces by division two sorts of cells, germ cells which contain the germ-plasm and somatic cells which protect, nourish, and transport the germ cells until they leave the body to give rise to the succeeding generation. No case of a complete morphological continuity of germ cells has ever been described. Such an occurrence would necessitate the division of the egg into two cells, one of which would give rise to all of the body cells and nothing else, the other only to germ cells. The behavior of the germ-plasm in such a case would be as follows (Weismann, 1904, p. 410) : “The germ-plasm of the ovum first doubles itself by growth, as the nuclear substance does at every nuclear division, and then divides into two similar halves, one of which, lying in the primordial somatic THE GERM-PLASM THEORY 293 cell, becomes at once active and breaks up into smaller and smaller groups ‘of determinants corre- sponding to the building up of the body, while the germ-plasm in the other remains in a more or less ‘bound’ or ‘set’ condition, and is only active to the extent of gradually stamping as germ cells the cells which arise from the primordial germ cell.” According to Weismann this actually occurs in Dipterous insects, but there is no evidence in the literature to warrant this statement. It is conse- quently necessary to imagine the germ-plasm as present but not definitely localized in a germ cell until some time after the two-cell stage has been reached. Thus in hydroids Weismann explains the situation as follows: “‘Here the primordial germ cell is separated from the ovum by a long series of cell-generations, and the sole possibility of explaining the presence of germ-plasm in this primordial germ cell is to be found in the assumption that in the divisions of the ovum the whole of the germ-plasm originally contained in it was not broken up into determinant groups, but that a part, perhaps the greater part, was handed on in a latent state from cell to cell, till sooner or later it reached a cell which it stamped as the primordial germ cell.” Evidence that the germ-plasm does become sooner or later localized in the primordial germ cell has accu- mulated rapidly within recent years. In the pzedo- genetic fly, Miastor (see Chapter III), the first cell to be cut off from the egg is the primordial germ cell (Fig. 17, p.g.c.), although at this time there are 294 GERM-CELL CYCLE IN ANIMALS eight nuclei. in the egg. As determined by Kahle (1908) and confirmed by the writer (Hegner, 1912, 1914a), this primordial germ cell gives rise to sixty- four odgonia and to no other cells. This is the nearest approach to a complete morphological continuity of the germ cells that has yet been described, and since this primordial germ cell must contain the germ- plasm of the succeeding generation, the condition in this fly is really comparable to that of the hypo- thetical case cited above, only in Miastor the cell set aside for reproductive purposes is much less than one-half of the egg, the somatic part of the egg being not a single cell, but a syncytium containing seven nuclei. We may therefore look for the germ-plasm of Muastor in the primordial germ cell. So far as we know there are only two sorts of materials in this cell, that contained in the nucleus, and the darkly staining part of the egg which becomes recognizable just before maturation occurs, is situated at the pos- terior pole, and has been termed the pole-plasm (Fig. 13). If the primordial germ-cell multiplies by simple division and if there is an equal distribution of the contents at every mitosis, then the sixty- four odgonia must each possess one sixty-fourth of both the nucleus and the pole-plasm of the primordial cserm cell plus any materials that have been added during the period of multiplication. An enormous enlargement occurs during the growth period both of the nucleus and of the cell. The pole-plasm cannot be recognized at this time, but again becomes THE GERM-PLASM THEORY 295 evident just before maturation; it has increased in amount to approximately sixty-four times its former mass. How this increase has been brought about is not known, but it has been suggested (p. 68) that preéxisting particles of pole-plasm may grow and divide, or the dilution of the pole-plasm caused by the growth of the egg might start into action some catalyst which would cause the production of more substance like the pole-plasm and cease its activity when the amount of pole-plasm characteristic of the mature egg had accumulated and brought it to a state of equilibrium. In the midge, Chironomus, the primordial germ cell is segregated even earlier than in Mvastor, namely, at the four-cell stage. The later history of the germ cells is not so well known in this species, however, as in Miastor. The data presented in Chapters V and VI prove that a definite and early segregation of germ cells is known in a sufficient number of groups to indicate that the process is quite general among animals. The morphological continuity of the germ cells, however, cannot be established with such a degree of certainty in the vertebrates, and although most investigators believe that the germ cells are con- tinuous, still the entire keimbahn has never been traced as accurately as it has in many invertebrates. Fortunately almost every new investigation contains additional data and more refined methods which lead us to hope that some time in the near future the primordial germ cells even here may be traced back to early cleavage stages. 296 GERM-CELL CYCLE IN ANIMALS One of the distinguishing features of many primor- dial germ cells is the presence within their cytoplasm of certain stainable bodies to which I have applied the term “keimbahn-determinants.”’ Although, as pointed out in Chapter VIII, these inclusions do not appear to consist of the same sort of material in the eggs of different species and hence their signif- icance is problematical, still they seem to be asso- ciated with that particular part of the egg sub- stance which becomes the cytoplasm of the primor- dial germ cells. For this reason, if for no other, the keimbahn-determinants are of the greatest value, since they enable us to determine the position of this germ-cell substance during the stages before the primordial germ cells are established. It is therefore possible to trace the germ-cell substance in such eases as Sagitia (Fig. 54), where there is no morphological continuity of the germ cells. What relation the keimbahn-determinants have to the germ- plasm is not yet definitely known. There have, of course, been many objections to the germ-plasm theory. The history of the germ cells in the Ccelenterata, upon which Weismann (1882) based a large part of his argument, is consid- ered by Hargitt (see p. 95) to be directly opposed to the hypothesis. According to some zodlogists there is no essential difference between the repro- ductive cells and the various sorts of somatic cells; they have all arisen as the result of division of labor, and the germ cells have been differentiated for pur- poses of heredity just as the muscle cells have been THE GERM-PLASM THEORY 297 differentiated for causing motion and the nerve cells for receiving and conducting stimuli. That the germ cells remain in a primitive condition during a large part of the embryonic period is accounted for by the fact that they become functional at a compara- tively late stage in ontogeny (Eigenmann, 1896). Asexual reproduction by means of fission or budding has seemed to some to invalidate the theory of ger- minal continuity, but as Montgomery (1906, p. 82) has pointed out, “Perhaps in all cases products of asexual generation contain germ cells. If this were so, it might then be the case that the incapacity of any part of the body of an animal to reproduce asexually, or even to regenerate, would be due to the absence of germ cells in it — but this is merely a suggestion.” The probability that the regenerat- ing pieces of ccelenterates and the artificial plas- modia formed by dissociated sponge cells contain germ cells has already been noted (p. 79), but there are cases of the regeneration of sex organs that are not so easily explained. For example, Janda (1912) has found that if the anterior part of the hermaph- roditic annelid, Criodrilus lacuum, is removed, a new anterior end will regenerate containing both ovaries and testes, although not always in their normal positions. The study of the germ cells in the cestode Moniezia expansa convinced Child (1906) that germ cells may develop from tissue cells. In this species the germ cells are derived from the parenchymal syncytium, which has undergone a considerable degree of cytoplasmic 298 GERM-CELL CYCLE IN ANIMALS differentiation and therefore consists of real tissue cells. Those parenchymal cells that encounter certain conditions become germ cells. Later (1906) the same author gave an account of the development of spermatogonia in the same animal from the dif- ferentiated musclecells. These studies, together with the results from experiments on regeneration, have led Child (1912) to the belief “that this germ-plasm hypothesis and the subsidiary hypotheses which have grown up about it are not only unnecessary and constitute an impediment to biological thought, which has retarded its progress in recent years to a very appreciable extent, but furthermore, that they are not in full accord with observed facts and can be maintained only so long as we ignore the facts.” He further maintains that if protoplasm is a physico- chemical substance it is capable of changing its con- stitution in any direction according to the conditions imposed upon it, and that therefore the continuous existence of a germ-plasm with a given specific constitution is unnecessary. The evidence in favor of the germ-plasm theory is so strong that the arguments thus far advanced against it have had but little influence. If, then, we accept germinal continuity as a fact and consider the germ-plasm to be a substance that is not con- taminated by the body in which it lies, but remains inviolate generation after generation, we should next inquire as to the nature of this substance. The generally accepted idea is that the chromatin of the nucleus represents the physical basis of heredity. In THE GERM-PLASM THEORY 299 favor of this view are the facts that during mitosis the number and shape of the chromosomes are con- stant in every species (variations sometimes occur) and the complex series of processes in indirect nuclear division seems to be for the sole purpose of dividing the chromosomes equally between the daughter cells; even during the intervals (interkinesis) be- tween successive mitoses the chromosomes may be recognized in certain species as prochromosomes (see Digby, 1914, for review of literature). During the maturation of the germ cells chromosomes seem to play the most important réle, uniting in synapsis, and separating in the reducing division. The chromosomes of the minute, motile sperma- tozoa equal in number those of the comparatively enormous, passive egg; the spermatozodn consists almost entirely of chromatin, and this is the only substance present in the zygote that is equally contributed by both egg and spermatozodn. The processes following the penetration of the spermato- zoon into the egg bring about a combination of the chromosomes of the two gametes into a_ single nucleus; in certain animals at least some characters depend upon the presence of a certain chromosome, the X-chromosome; in certain cases of polyspermy the addition of extra male chromosomes seems to be the cause of the abnormal development of the egg. These and many other facts of chromosome be- havior that have been discovered by observations and experiments have convinced most biologists that the chromatin is the germ-plasm. 300 GERM-CELL CYCLE IN ANIMALS It is becoming more and more evident, however, that the cytoplasm cannot be entirely excluded. As noted in Chapter IX, the mitochondria appear to be constant cell elements and may actually constitute a part of the essential hereditary substance. Even if these particular cytoplasmic bodies do not repre- sent germ-plasm, still, as pointed out by Guyer (1911) and others, cytoplasm as well as nuclear material is necessary to explain the phenomena which we call heredity. It was shown in Chapter I that the most important primary constituents of protoplasm are the proteins, and the idea is rapidly becoming general that the mechanism of heredity consists of (1) fun- damental species substances, probably mainly pro- tein in nature, together with (2) equally specific enzymic substances which regulate the sequences of the various chemical and physical processes incident to development (Guyer, 1911, p. 299). The chro- mosomes have been suggested as enzymatic in nature (Montgomery, 1910), but enzymes are sup- posed merely to accelerate reaction already initiated, and hence the substrate must beof as great importance as the enzymes which work upon it. But the sub- strates must be extremely numerous to supply each species with its specific proteins. That there are enough configurational differences in corresponding protein molecules to supply the number for the thousands of animal species is certain, since some comparatively simple proteins may possess thousands of millions of stereoisomers. Thus the study of heredity substance involves primarily a knowledge THE GERM-PLASM THEORY 301 of the nature and reactions of the chemical constitu- ents of protoplasm, for, as Wilson (1912, p. 66) says, “The essential conclusion that is indicated by cyto- logical study of the nuclear substance is, that it is an aggregate of many different chemical components which do not constitute a mere mechanical mixture, but a complex organic system, and which undergo perfectly ordered processes of segregation and dis- tribution in the cycle of cell life.” Some of the strongest evidence that the germ- plasm must include cytoplasmic constituents is afforded by the observations and experiments dealing with the differentiation of the germ cells, especially during early embryonic development. The writer’s morphological and experimental studies of chrysom- elid beetles seem to prove that the nuclei during the cleavage stages are all potentially alike and that it is the cytoplasm which decides their fate. Boveri’s experiments on the eggs of Ascaris likewise show that the cytoplasm determines the initiation of the chromatin-diminution process and controls the differ- entiation of the germ cells. Furthermore, much of the data in the preceding chapters indicates that the non-nuclear substance which will become segregated within the primordial germ cell is present in a more or less definite region in the undivided egg, being gradually localized and separated from the other egg substances as cleavage progresses. The position of this germ-cell substance can in many cases be deter- mined because of the presence of inclusions of vari- ous sorts, but whether these keimbahn-determinants 302 GERM-CELL CYCLE IN ANIMALS constitute an important part of the germ-plasm or play a minor réle in heredity is still uncertain. Modern cytological studies and the results of ex- perimental breeding both help to solve the prob- lems of the combination and subsequent distribution of the determiners or factors within the germ-plasm. In fact, it has been maintained by certain geneticists that “The modern study of heredity has proven itself to be an instrument even more subtle in the analysis of the materials of the germ cells than actual observations on the germ cells themselves ” (Morgan, 1913, p.v). Those who do not wish to commit themselves as to the physical or chemical nature of the germ-plasm are content to speak of determiners, factors, or genes without connecting them with any particular substances. The behavior of the chro- mosomes, however, enables us to explain so many of the facts of heredity that, as stated above, these bodies are generally considered to constitute the essential hereditary substance. The study of heredity was wonderfully stimulated by the recognition in 1900 by Correns, Von Tscher- mak, and de Vries of the results of Mendel’s (1866) investigations on plants. One of the simplest of Mendel’s experiments is that which he performed with differently colored peas (Fig. 81). A pea bear- ing green seeds was crossed with a pea bearing yellow seeds. The first (F1) generation of peas resulting from this cross all bore yellow seeds. When the in- dividual plants of this generation were inbred, three- fourths of the resulting (Ff) generation were yellow THE GERM-PLASM THEORY 303 and one-fourth green. This proved that the seeds of the first generation (F;), although yellow, still possessed within them the factor for greenness in a latent condition. Green was therefore called a re- PARENTS GAMETES Eces OO © wn Fe QO QO Fs Fic. 81.— Diagram to illustrate Mendel's law of segregation. Individ- uals (zygotes) are represented by superimposed circles, whose colors stand for the factors involved. Gamctes (germ cells) are represented by single circles. (From Morgan, 1914.) cessive character and yellow a dominant character. As a result of breeding the (F:) second generation it was found that all of the green seeds produced plants which bore green seeds; that is, these plants were pure green and “homozygous” as regards color; whereas the plants which bore yellow seeds could be 304 GERM-CELL CYCLE IN ANIMALS separated into two groups; one, containing on the average one-third of these plants, was pure yellow and homozygous as regards color; the other two-thirds, although yellow, contained green in a latent condi- tion and were therefore impure yellows and “‘hetero- zygous”’ as regards color. The conclusion reached was that the eggs and spermatozoa produced by the first (F,) generation (see Fig. 81) were pure yellow or pure green and that chance combinations during fertilization resulted in the three classes of individ- uals in the second (F;) generation; that is, one-fourth pure yellow, one-fourth pure green, and one-half with dominant yellow and green recessive. Evidently the factors for yellow and green repulsed each other during the maturation so that they became localized in different germ cells. Such a characteristic as the color of the seeds of these peas is known as a unit character, and the sepa- ration of the factors of such a character during maturation is referred to as the principle of segrega- tion. Mendel further discovered that if the seeds were also wrinkled or round, such characters behaved independently of the color characters. These and other experiments described by Mendel opened the way for new lines of investigation which have yielded results of vast importance from the stand- point of heredity and evolution.! Soon after Mendel’s results were “rediscovered” 1 For more detailed accounts of experiments and theories that have been published within the past fourteen years the reader is referred to the books of Bateson (1909, 1913) and Punnet (1911). THE GERM-PLASM THEORY 305 it was pointed out by Guyer (1902), Sutton (1903), and others that the distribution of the adult char- acteristics of hybrids which were found by Mendel to reappear in the offspring in rather definite propor- { SSe77 ye De he Fic. 82.— Diagrams to show the pairs of chromosomes and their be- havior at the time of maturation of the egg. Three pairs of chromo- somes are represented ; three from one parent, three from the other. The six possible modes of separation of these three are shown in the lowest line. (From Morgan, 1914.) tions, could be explained if these characteristics are located in the chromosomes. During synapsis, as already explained (p. 44), homologous maternal] and paternal chromosomes are supposed to pair and then separate in the reduction division. It seems probable that the pairs of chromosomes do not occupy any x 306 GERM-CELL CYCLE IN ANIMALS definite position on the spindle at this time, but, as indicated in Fig. 82, the distribution of the maternal and paternal chromosomes to the daughter cells is entirely a matter of chance. If the homologous maternal and paternal chromosomes really are dis- tributed by chance to the eggs and spermatozoa following synapsis, then the number of combinations possible are as follows (Sutton, 1903) : Somatic Series | Repucep SERIES Coe ae AN HomaiNaens 1. 2 1 Q | 4 4 g 4 16 8 4 16 256 16 8 256 65536 24 12 4096 16777216 36 18 262144 68719476736 The only direct evidence that such distribution of chromosomes takes place is that furnished re- cently by Carothers (1913) from a study of the spermatogenesis of three Orthopterous insects, Brachystola magna, Arphia simplex, and Dissosteira carolina. Miss Carothers, while working in Pro- fessor McClung’s laboratory, discovered a tetrad in the first spermatocytes of these insects which consists of two unequal dyads (Fig. 83). During the two mat- uration divisions the four parts of this tetrad pass to the four spermatozoa, and consequently two sorts of spermatozoa are produced so far as this chromo- some is concerned, one-half with one of the larger elements of the tetrad and one-half with one of the THE GERM-PLASM THEORY 307 smaller elements. These differently sized dyads are considered by Carothers as “distinct physiological individuals, representing respectively the paternal and maternal contribution to the formation of some character or characters; and, as each can be iden- tified, they furnish an excellent means of tracing the process of segregation and recombination ”’ (p. 499). It was at first assumed that each of the pairs of chromosomes which unite in synapsis was responsible for a single adult character, but a— the number of qd &? CD Mendelian char- Cc hy Po OF acters is known . Fig. 83.— Arphia simplex. Chromosomes ot to be greater m first spermatocyte. a= accessory chremo- certain cases than some. 6 = unequal dyad. (From Carothers, 1913.) the number of chromosomes. Fortunately, it has been found that the characters, instead of undergoing independent as- sortment, may become linked so that certain of them almost always occur together in the offspring. The relation of these facts to the constitution of the chromosomes may best be illustrated by reference to the studies of Morgan and his students on the fruit- fly, Drosophila. Over one hundred mutants of this species have been discovered by these investigators. So far as studied, the characters of these flies seem to form three groups. ‘‘The characters in the first group show sex-linked inheritance. They follow the sex- chromosomes. The second group is less extensive. Since the characters in this group are linked to each 308 GERM-CELL CYCLE IN ANIMALS other, we say that they lie in a second chromosome. The characters of the third group have not as yet been so fully studied, except to show that they are linked. We place them in the third chromosome without any pretensions as to which of the pairs of chromosomes are numbered II and III. “The arrangement of these characters in groups is based on a general fact in regard to their behavior in heredity, viz., A member of any group shows linkage with all other members of that group, but shows inde- pendent assortment with any member of any other group.” If the factors which determine these groups of characters are situated in the chromosomes, as the hypothesis demands, we should expect each group to act as a unit in heredity. Occasionally, however, the characters of a group appear to act independently, and there must thus be an interchange of factors at the time of synapsis. As already stated (p. 254), an interchange of substances between chromosome pairs during synapsis is possible and even probable. Mor- gan explains the degree of crossing over of characters in the following way: The factors which determine the characters are arranged in the chromosomes in a linear series; those factors that are near together will have less chance of being separated than those that lie farther apart. The relative distances be- tween these factors can be judged by the frequency of interchange as determined by breeding experi- ments. It has thus been possible to locate certain factors in the chromosomes more or less accurately and to predict with some degree of certainty the re- THE GERM-PLASM THEORY 309 sults of hybridization. Thus if the position of a newly discovered factor is determined by comparison with another particular known factor, it is possible to ‘“‘calculate the results for all other known factors in the same chromosome.” Morgan’s ideas regard- ing the organization of the chromosomes coincide with those expressed by Weismann in one respect, that is, they are assumed “‘to have definite structures and not to be simply bags filled with a homogeneous fluid.” Wilson (1912, p. 63) also regards the chro- mosomes as “compound bodies, consisting of differ- ent constituents which undergo different modes of segregation in different species.” Students of genetics now consider the individual as built up of a number of unit characters represented in the germ-plasm by factors, and when two different germ-plasms unite (amphimixis) the factors do not mix, but remain uncontaminated. The germ-plasm of offspring which develop from fertilized eggs is supposed to consist of an assortment of factors brought about during synapsis and reduction as indi- cated in Fig. 84. The factors (or genes) in the germ- plasm occur in pairs called allelomorphs,' and one of the pair may be regarded as dominant, the other re- cessive, as, for example, the yellow and green colors of pea seeds. Thus the appearance of the individual depends upon the character of its dominant factors. Any attempt to account for the origin of new species 1 According to some investigators, especially in England, the presence of a factor should be considered one allelomorph and its absence as the contrasting factor. 310 GERM-CELL CYCLE IN ANIMALS ae oe ‘Bb Aa ee ba — oS oe = SS ew ce a ce Be Oo Fic. 84.— Diagrams illustrating the union © two stocks with paired factors A, B, C, D, anda, b, c, d, to form pairs Aa, Bb, Cc, Dd. Their possible recombinations are shown in the sixteen smaller circles. 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Die Entwicklung der Gemmulae der Ephydatia fluviatilis. Zool. Anz. Bd. 25. INDEX OF AUTHORS All numbers refer to pages. An asterisk (*) after a page number in- dicates that the title of a contribution by the author will be found on that page. Allen, B. M., 32, 100, 102, 206, 311.* Altmann, 276, 282, 311.* Amma, 140, 163 f., 216, 228, 311.* Ancel, 195-197, $11.* Baer, van, 192. Balbiani, 107, 214, 229, 311.* Baltzer, 261. Bambeke, van, 222, 311.* Bancroft, 21. Bardeleben, 273, 311.* Bartelmez, 232, 311.* Bateson, 312.* Beard, 100, 312.* Beckwith, 135, 312.* Benda, 40, 276, 277, 279, 282, 312.* Beneden, van, 80, 82, 87, 88. Berenberg-Gossler, 98, 100, 312.* Bessels, 118, 312.* Bigelow, 172, 186, 225, 312.* Blockmann, 185, 221, 225, 312.* Bonnevie, 177, 286, 313.* Bouin, 206, 276, 313.* Boveri, 174 ff., 184, 193, 195, 217, 230, 268, 301, 313.* Brandt, 118, 313.* Brauer, 83, 313.* Brown, 3. Browne, 285, 313.* Buchner, 123, 140, 180, 187, 195, 222, 286, 313.* Bunting, 87, 314.* Buresch, 195, 199 ff., 226, 314.* 102, Calkins, 26, 314.* Carothers, 306, 307, 314.* Carter, 74, 314.* Castle, 192, 314.* Caullery, 195, 314.* Champy, 195, 206, 209, 314.* Child, 136, 188, 297, 314.* Chun, 184, 315.* Cohn, 3. Cole, 207, 209, 315.* Conklin, 218, 232, 233, 234, 315.* Correns, 302, 315.* Cowdry, 280, 285, 315.* Cunningham, 209, 315.* Debaisieux, 120, 121, 122, 315.* Delage, 195, 315.* Della Valle, 12, 315.* Demoll, 195, 202 ff., 316. Désé, 70, 76, 316.* Dickel, 144, 316.* Digby, 299, 316.* Dobell, 28, 316.* Dodds, 102-103, 316.* Doncaster, 275, 316.* Downing, 83-85, 97, 188, 316.* Driesch, 161, 231, 316.* Duesberg, 104, 233, 273, 280, 283, 316.* Dujardin, 3. Durme, van, 286, 316.* Dustin, 99, 206, 317.* Ehrenberg, 82, 317.* Eigenmann, 100, 243, 297, 317.* Z 337 338 Elpatiewsky, 26, 140, 179 ff., 195, 228, 317.* Escherich, 107, 317.* Evans, 75, 317.* Farmer, 273. Fauré-Frémiet, 13, 279, 283, 285, 317.* Feistmantel, 207. Felt, 52, 317.* Fiedler, 73, 317.* Firket, 99, 317.* Fischer, 12. Flemming, 214, 273, 318.* Fol, 186, 318.* Foot, 123, 137, 214, 318.* Friedmann, 207, 318.* Frischholz, 97, 318.* Fuchs, 163, 169, 318.* Fujita, 186, 225, 318.* Fuss, 100, 318.* Galton, 290, 318.* Gardiner, 157, 318.* Gates, 160, 318.* Gerhartz, 207, 318.* Giardina, 120-122, 223, 231, 318.* Giglios-Tos, 279, 319.* Goette, 75, 95, 319.* Goldschmidt, 222, 279, 286, 319.* Gorich, 73, 319.* Govaerts, 120, 123, 128, 319.* Graber, 107, 319.* Granata, 279. Grimm, 107, 310.* Grobben, 163, 170, 319.* Gross, 137, 319.* Gudernatsch, 194, 319.* Guenther, 83, 319.* Giinthert, 121, 122, 128, 319.* Guilliermond, 277, 319.* Gutherz, 273, 320.* Guyer, 272, 300, 305, 320.* Hadzi, 83, 320.* Haeckel, 320.* INDEX OF AUTHORS Haecker, 36, 73, 124, 140, 163 ff, 184, 215, 320.* Hallez, 112, 320.* Hargitt, C. W., 86, 88, 95, 296, 320.* Hargitt, G. T., 96, 320.* Harm, 88, 89, 98, 320.* Harman, 136, 320.* Harmer, 161, 321.* Hartmann, 216, 321.* Harvey, 1. Hasper, 104, 107, 110, 140, 218, 230, 235, 321.* Hegner, 33, 51, 107, 140, 219, 225, 235, 286, 294, 321.* Heider, 79. Henking, 106, 256, 321.* Herbst, 216, 321.* Herold, 118, 321.* Herrick, 215, 322.* , Hertwig, O., 82, 231, 322.* Hertwig, R., 83, 222, 322.* Heymons, 186, 194, 322.* His, 231. Hadge, 214, 322.* Hogue, 179, 322.* Holmes, 137, 322.* Hooke, 2, 207, 322.* Hoven, 277, 280, 322.* Tjima, 76. Ischikawa, 86, 163, 170, 322.* Jager, 291, 322.* Janda, 297, 322.* Janssens, 254, 255, 322.* Jarvis, 100, 322.* Jenkinson, 50, 232, 323.* Jennings, 186, 225, 323.* Jordan, 214, 288, 323.* Jérgensen, 72, 74, 78, 323.* Kahle, 51, 107, 140, 230, 235, 294, 323.* Kellicott, 50, 323.* Kellogg, 118, 323.* King, 195, 206, 208, 323.* INDEX OF AUTHORS Kite, 6, 323.* Kleinenberg, 80, 82, 83, 161, 323.* Knappe, 208, 323.* Kolliker, 373. Koltzoff, 323.* Korotneff, 83, 323.* Korschelt, 79, 324.* Kossel, 8. Kowalevsky, 107, 324.* Kruger, 195, 267, 324.* Kiihn, 140, 163 ff., 225, 236, 324.* Kulesch, 105, 324.* Kiinstler, 276, 324.* Kuschakewitsch, 100, 195, 206, 324.* Lams, 286, 324.* Lang, 157, 324.* LaValette St. George, 207, 276, 324.* Lecaillon, 109, 111, 324.* Lewis, 281, 324.* Leuckart, 51, 107, 324.* Levene, 11. Leydig, 82, 325.* Lieberkiihn, 73, 325.* Lillie, 188, 232, 234, 325.* Loeb, 13, 21, 325.* Loewenthal, 222, 325.* Loyez, 286, 325.* Lubarsch, 130, 325.* Lubosch, 214, 325.* Maas, 73, 76, 78, 325.* McClendon, 172, 185, 325.* McClung, 256, 325.* McGregor, 134, 135, 326.* Malpighi, 3. Mangan, 187, 326.* Marchal, 161, 326.* Marcora, 280, 326.* Marshall, A, 326.* Marshall, W., 75, 326.* Marshall, W. M., 222, 326.* Maupas, 267, 326.* Mayer, 283. Megusar, 113, 326.* Meinert, 51, 326.* 339 Mendel, 302 f., 326.* Metschnikoff, 51, 107, 183, 224, 326.* Meves, 134, 216, 244, 266, 284, 287, 326.* Meyer, 176, 327.* Minchin, 70, 72, 327.* Mohl, von, 3. Montgomery, 129, 131, 195, 214, 241, 285, 297, 300, 327.* Moore, 273, 328.* Morgan, 192, 232, 255, 265, 302, 307, 309, 328.* Morse, 137, 244, 328.* Miiller, F., 195, 328.* Miller, K., 77, 80, 328.* Muller-Calé, 172, 328.* Mulsow, 256, 257. Munson, 226, 329.* Nachtsheim, 143, 145, 265, 266, 329.* Noack, 107, 109, 111, 225, 235, 329.* Nussbaum, 83, 100, 291, 329.* Ognew, 208, 329.* Okkeberg, 209, 329.* Ostwald, 9. Owen, 290, 329.* Patterson, 157, 161, 329.* Paulcke, 120, 122, 222, 329.* Paulmier, 256, 329.* Payne, 138, 329.* Pelseneer, 195, 329.* Petrunkewitsch, 143, 145, 265, 330.* Pfliiger, 205, 231. Pick, 194, 330.* Prenant, 279, 283, 330.* Preusse, 137,.330.* Punnet, 330.* Rath, vom, 134, 135, 330.* Regaud, 279, 282, 330.* Rhode, 218, 330.* Richards, 136, 330.* Ritter, 107, 108, 229, 235, 330.* Robertson, 161, 330.* Robin, 107, 330.* 340 Rosel V. Rosenhoff, 72, 331.* Rosner, 161, 331.* Roux, 141. Rubaschkin, 98, 100, 103, 226, 283, 331.* Riickert, 99, 331.* Russo, 286, 331.* Samassa, 170, 172, 331.* Sauerbeck, 194, 331.* Schapitz, 100, 331.* Schaxel, 214, 331.* Schiffer, 283. Schleip, 195, 268, 331.* Schleiden, 3. Schmidt-Marcel, 205, 207, 331.* Schmiedeberg, 12. Schneider, 83, 331.* Schénemund, 194, 331.* Schonfeld, 237, 331.* Schreiner, 209, 332.* Schulze, 76, 80, 193, 332.* Schwann, 3. Selenka, 157, 332.* Semon, 206, 332.* Siebolt, von, 193, 332.* Silvestri, 143, 145, 215, 332.* Simon, 194, 332.* Smallwood, 87, 98, 332.* Spooner, 234. Steudel, 12. Stevens, 140,180, 195,228, 256, 332.* Strasburger, 214, 332.* Stricht, van der, 188, 286, 333.* Strobell, 123, 137, 214. Stuhlmann, 221, 333.* Suckow, 118, 333.* Surface, 157, 333.* Sutton, 305, 306, 333.* Swarezewsky, 26, 333.* Swift, 33, 103, 226, 333.* Tannreuther, 83, 333.* Tennent, 261. Thallowitz, 88, 333.* Trembley, 82, 333.* INDEX OF AUTHORS Tschaschkin, 98, 102, 226, 302, 333.* Tschermak, 333.* Uffreduzzi, 194, 333.* Vander Stricht, 187, 333.* Varenne, 82, 333.* Vejdovsky, 334.* Voeltzkow, 107, 334.* Vollmer, 172, 334.* Voss, von, 204, 334.* Vries, de, 302, 334.* Wager, 83, 334.* Wagner, 51, 334.* Waldeyer, 98, 130, 334.* Walker, 159, 334.* Wassilieff, 286. Weismann, 25, 82, 88, 97, 107, 113, 144, 296, 309, 334.* Weltner, 73, 75, 77, 334.* Wheeler, 33, 100, 109, 144, 157, 185, 193, 335.* Whitman, 231, 335.* Wieman, 124, 138, 225, 273, 335.* Wierzejski, 75, 186, 225, 335.* Wijhe, van, 99, 335.* Wilcox, 273, 335.* Wildman, 279, 335.* Wilke, 285, 336.* Wilson, E. B., 4, 21, 133, 224, 232, 250, 301, 309, 336.* Wilson, H. V., 75, 77, 80, 336.* Winiwarter, 129, 132, 251, 273, 336.* Winter, de, 119, 120. Woods, 100, 336.* Wolff, 2. Wulfert, 89, 98, 336.* Youngman, 207. Yung, 207, 336.* Zarnik, 195, 269, 336.* Zeigler, 237. Zeleny, 232, 336.* Zoja, 287, 336.* Zykoff, 75, 336.* INDEX OF SUBJECTS All numbers refer to pages. Words in italics are names of families, genera, species, or of higher divisions. Numbers in thick type are num- bers of pages on which there are figures. Aborting spindle, 157. Accessory chromosome, 134, 202. Acidophile, 11. Actinospherium, 222. Ageniaspis, 146. Allelomorph, 309. Alternation of generations, 23. Alveolar structure of protoplasm, 4. Ameebocyte, 71, 73, 79. Amia, 32, 33. Amitosis, 13-14, 133-139, 250. Amphiaster, 15. Amphibia, amitosis, 134-135; her- maphroditism, 205 ff. Amphimixis, 309. Amphiuma, 135. Amyloplastid, 7. Anaphase, 15, 16. Anello cromatico, 121, 123, 223. Animal pole, 20. Aptera, life cycle, 22. Arcella, 26. Archeocyte, 70-73. Archoplasm, 5, 7. Arenicola, 188. Armadillo, polyembryony in, 161. Arphia, 307. Arthropoda, 212. Ascaris, 122, 174 ff., 217 ff., 230, 241, 301; maturation in, 261, 263; mitochondria in, 284. Asexual larve, 149. Asplanchna, 186, 225. Aster, 15. Asterias, 6. Attraction-sphere, 5, 7, 227. Aurelia, 183. Aussenkérnchen, 164, 213, 216, 228. Axzolotl, 159, 208. Bacteria, 4, 186-187. Basophile, 11. Bat, 188. Besondere Korper, 180, 181 f., 213, 228, 239. Bidder’s organ, 207. Binary fission, 17. Binuclearity hypothesis, 27. Bioblast, 276. Bivalent chromosomes, 44. Blastotomy, 161. Bryozoa, 161. Budding, 17, 22, 23, 69, 161, 297. Calligrapha, 109, 111, 230. Calliphora, 107, 111 f., 235. Camponotus, 221. Canthocamptus, 165. Cat, 187. Cell, 2-16; definition, 3; division, 13-16; lineage, 29; shape, 4; size, 4; theory, 3. Centrifuged eggs, 178. Centrosome, 5, 7, 14, 15, 164, 169, 237, 238. Cerebratulus, 232. Cestoda, 136-137. 341 342 Chetognatha, 212. Characters, dominant, 303; linked, 307; recessive, 303; unit 303. Chiasmatype theory, 254. Chick, 33, 100, 103, 227, 281. Chironomus, 108-109, 110, 224, 229, 235. Chloroplastid, 7. Cholesterin, 8, 12, 13. Chorion, 113. Chondriodiérése, 279. Chondriokont, 279. Chondriosome, 7, 102, 103, 168, 227 ff., 275, 277. Chondriotaxis, 279. ; Chromatin, 5, 7, 11-12; as germ- plasm, 299; as keimbahn-deter- minants, 211 ff. Chromatin-diminution, 47, 56, 57, 139-141, 174 ff., 217 ff, 249. Chromatin-nucleolus, 5, 7. Chromidia, 26, 123, 168, 221 ff., 279. Chromidial net, 26. Chromosome, 6, 7, 14, 15, 243, 299; accessory, 106; cycle, 245-275; diploid, 43; division, 248; in fertilization, 49; haploid, 43; individuality, 255; in man, 272 f.; and Mendelism, 305; number, 246; from nucleolus, 214; in parthenogenesis, 246; univalent, 249. Chrysemys, 32. Chrysomelide, 109. Ciona, 192. Cladocera, 163 ff. Clathrina, 70. Clava, 88, 135. Cleavage, 29, 115. Cockroach, 194. Celenterata, 80-98, 212. Coleoptera, 109-143. Colloid, 9. Colony, 17. INDEX OF SUBJECTS Compsilura, 107, 109. Conjugation, 17. Copepoda, 165 ff. Copidosoma, 146 ff. Copulationszelle, 163. Corps enigmatique, 187. Crepidula, 218. Crustacea, 163-173. Crystalloid, 9. Cyclops, 124, 164 ff., 228, 247. Cymatogaster, 100. Cynthia, 233, 280. Cyst formation, 125-129. Cytomicrosome, 276. Cytoplasm, 6, 143, 179, 224 ff., 300 f. Daphnide, 163. Death, natural, 25. Determination of sex, 118. Determiner, 302. Diaptomus, 165. Differentiation, 76, 141-143. Dicecious, 18, 190. Diploid, chromosomes, 248. Diplotene, 252. Diptera, 107. Dispermic, 177, 178. Dominance, 303. Dotterplatte, 109, 115, 225, 235. Drosophila, 307 ff. Dyad, 45, 46, 306, 307. Dytiscus, 120-124, 121, 223. Dzierzon theory, 143. Earthworm, 161, 190, 191. Eclectosome, 279. Ectosome, 166, 167 ff., 213, 237. Egg, 19, 20. Encyrtus, 145. Enzyme, 300. Ephydatia, 75. Epigenesis, 2, 243. Ergastoplasma, 276. Eudendrium, 86. INDEX OF SUBJECTS Euschistis, 281. Evolution, 310. Factor, 302, 309. Female sex, 18. Fertilization, 44, 47-49, 256, ff. Fission, 22. Frog, hermaphroditism in, 205 ff. Fusion, of chromosomes, 254; of odcytes, 152, 155 ff. Gel, 5, 6, 9. Gemmule, 18, 74-75, 76, 79. Genes, 302, 309, 310. Genetics, 309. Genetic-continuity of chromosomes, 255. Germ cell, 19-22, 101, v.s. somatic cell, 296-297. Germ-cell cycle, 28-49. Germinal continuity, 292. Germinal epithelium theory, 98. Germinal localization, 231. Germinal spot, 214. Germinal vesicle, 19, 20, 54. Germ-plasm, in Ascaris, 177, in Hydra, 83-85 ; in Miastor, 293; in polyembryony, 162; in sponges, 80. Germ-plasm theory, 290-310, Gonochorism, 18, 191. Gonocyte, 71, 73. Gonothyrea, 89. Gonotome theory, 97. Graffilla, 157. Gryllus, 123, 244. Guinea-pig, 102, 103, 104, 227. Gynandromorph, 193-194. Haploid, 247. Hauptnucleolus, 214. Helix, 195, 196 ff., 226. Hemiptera, amitosis in, 137. Hermaphrodite, 18, 189-210, 269. Heterocope, 165. 343 Heterotypic mitosis, 46, 252, 253. Heterozygous, 304. Homologous chromosomes, 253. Homotypic mitosis, 46. Homozygous, 303. Honey-bee, 143-144, 261 ff., 266. Hyaloplasm, 4, 5. Hydra, 82-85, 159. Hydractinia, 87. Hydroid, life cycle of, 23. Hydrophilus, 113. Hydrozoa, 85-98. Hymenoptera, 143-163, 221, 235. Idiochromatin, 28. Individuality of 255. Interkinesis, 299. Isotropism, 231. chromosomes, Jelly-fish, 23. Karyochondria, 279. Karyokinesis, 13, 14, 15. Karyolymph, 6. Karyosome, 5, 7, 213. Keimbahn, in Hquorea, 183, 184; Amphibia, 206 ff.; Cladocera, 163 ff.; Copepoda, 165 ff.; insects, 106-163; nematodes, 174-179; Sagitta, 179 ff. Keimbahn-determinants, 19, 211- 244, 296, 301; genesis, 211-234; localization, 234-240; fate, 240- 244, Keimbahnchromidien, 223. Keimbahnchromatin, 152 ff., 223. Keimbahnplasma, 108, 110, 115, 230, 235. Keimbahnzelle, 104. Keimfleck, 214. Keimhautblastem, 113, 114. Keimstitte, 95. Keimwulst, 108, 110, 115, 235. Keimzone, 95. 344 INDEX OF Kinetochromidia, 214. Kinoplasm, 214. Lamprey, 100, 209. Larva, 23. Lecithin, 8, 12. Lepas, 172, 225. Lepidoptera, 118. Lepidosteus, 32, 33, 101. Leptinotarsa, 37-41, 111, 125-129, 138-139. Leptotene, 251, 252. Life cycles, 22 ff. Linin, 5, 7. Linked characters, 307. Locust, 23. Lophius, 102. Lygeus, 259. Lymnea, 192. Macrogamete, 27. Male, 18. Man, chromosomes of, 272 f.; hermaphroditism in, 194. Maturation, 41-47, 129, 256 ff. Medusa, 23. Mesostoma, 204. Metabolism, and sex, 275; and Keimbahn-determinants, 228. Metagenesis, 23. Metanucleolus, 183, 215. Metaplasm, 5, 7, 8. Metaphase, 15, 16. Metazoa, 1, 18. Miastor, 51-68, 107, 217 ff., 235, 293-294. Microgamete, 27. Microsome, 6. Middle piece, of sperm, 21, 216. Migration, of germ cells, 31-34, 101-102, 116, 226. Mitochondria, 5, 13, 39, 40, 226 ff., 275-289; methods, 282-283; Ascaris, 284; chick, 278; divi- sion of, 281, 284; function of, SUBJECTS 286 ff.; in living cells, 280, 281; in plants, 277, 280; reduction of, 285; and sex, 285. Mitosis, 13, 14-16. Mitrocoma, 183. Mixochromosomes, 251. Moina, 163. Mollusk, 185, 191. Monad, chromosome, 45, 46. Moniezia, 136, 297. Moneecious, 18, 191. Monospermy, 48. Mosaic development, 233. Moulting, 23. Musca, 107. Myofibril, 280. Myzine, 209. Myzostoma, 37, 185, 193. Nahrzellenkern, 170. Nebenkern, 203, 221, 285. Nebennucleolus, 214. Nematodes, chromosomes of, 267 ff. Nepa, 137. Neratina, 186, 225. Netzapparat, 103, 104. Neurofibril, 280. Nuclear sap, 6. Nucleic acid, 11. Nuclein, 11. Nucleolo of Silvestri, 145 ff. Nucleolus, 5, 6, 13, 167, 213 ff. Nucleoprotein, 8, 11. Nucleus, 3, 13-16. Nurse cells, 35-36, 53, 119-121, 150, 151, 201, 202. Nutritive substances, 225 ff. Cnothera, 160. Oncopeltus, 261, 262. Odcyte, 38, 39, 40-41. Odgenesis, 42, 256 ff. Oébpthora, 145, 146. Ophryotrocha, 37. Opossum, 288. INDEX OF Organ-forming substances, 233. Organization of egg, 19, 29, 228 ff. Oxyphile, 11. Pachytene, 252. Peedogenesis, 18, 52. Paracopulationszelle, 212, 225. Paramecium, 27. Paranucleus, 163. Paraplasm, 7. Parasitism, 191-192. Parasynapsis, 254. Parthenogenesis, 18, 47, 145, 246, 265. Pea, 302, 303. Pecten, 191. Pennaria, 87. Peripatus, 285, 288. Petromyzon, 33. Phallusia, 233. Phosphatid, 8, 12. Phylloxera, 265 ff. Physa, 186, 225. Pig, 194. Planocera, 157. Planorbis, 186. Plasmodia, artificial, 77-78. Plasmosome, 5, 7, 102, 103, 213. Plastid, 5, 7. Plastochondria, 279. Plastokonta, 279. Plastosome, 7, 244, 275, 279. Polar body, 47, 143-144. Polares Plasma (sce pole-plasm). Polarity, 19, 107, 124, 179, 231 ff. Pole-cell, 110, 111, 117. Pole-disc, 109, 114, 117, 142, 219, 225, 229, 235. Pole-plasm, 53-55, 228, 230, 235, 294-295. Polistes, 222. Polycherus, 157. Polyembryony, 145 ff., 161. Polyp, 23. Polyphemus, 170 ff., 236. x SUBJECTS 345 Polyspermy, 48, 115, 299. Porifera, 69 ff. Potato beetle (see Leptinotarsa). Preblastodermic nuclei, 114. Predetermination, 2. Preformation, 2, 243. Prochromosome, 299. Progerminative cell, 196, 197. Promorphology, 19. Prophase, 14. Protandry, 193. Protein, 8, 10. Protenor, 123, 258. Protogyny, 192-193. Protoplasm, 3-13. Protozoa, 1, 17, 25. Pteropod, 269, 271. Pupa, 23. Pyrrhocoris, 256. Rana, 32. Recessive character, 304. Reduction of chromosomes, 253. Regeneration, 79-80, 297. Reproduction, 17-18. Rotifera, 186. Rhabditis, 267, 270. Richtungscopulationskern, 144. 43, Sagitta, 179 ff., 195, 228. Salamandra, 134. Sarcode, 3. Scorpena, 222. Sea urchin, 216. Secondary sex characters, 189. Segregation of germ cells, 29. Self-copulation, 192. Self-fertilization, 192. Sertoli cell, 35, 129-133. Sex, 18, 189. Sex chromosome, 255 ff. Sex determination, 274. Sol, 5, 6, 9. Sorite, 76, 79. 346 INDEX OF SUBJECTS Spermatogenesis, 42, 256 ff. Spermatogonia, 127. Spermatozoon, 19-22, 48. Sphérule, 276. Spireme, 14, 15. Spongilla, 73. Spongioplasm, 4, 5. Sporulation, 17. Squash bug, 256. Starfish, 6. Statoblast, 18. Statocyte, 70, 71, 73. Stem-cell, 175. Stone-fly, hermaphroditic, 194. Synapsis, 44, 122, 250 ff., 305. Synaptene, 251, 252. Synizesis, 43, 237, 251, 252. Tenia, 136, 137. Telophase, 15, 16. Telosynapsis, 254. Testis, 41. Tethya, 70, 76, 79. Tetrad, 44, 45. Tipulides, 107. Toad, hermaphroditic, 207-208. Tokocyte, 71, 73, 79. Trophochromatin, 28. Unit character, 304. Uterine spindle, 157. Vacuole, 5, 8. Vegetative pole, 20. Vertebrate, 32, 95-105, 212. Vitelline membrane, 113, 114. Vitellophag, 114. X-chromosome, 255 ff., 264, 299. Y-chromosome, 259 ff., 264. Yolk, in germ cells, 101, 224. Yolk nucleus, 19, 226, 285. Zygosome, 251. Zygote, 1, 48. HE following pages contain advertisements of books by the same author or on kindred subjects An Introduction to Zodlogy By ROBERT W. HEGNER, Ph.D. Assistant Professor of Zoélogy in the University of Michigan A TEXT-BOOK INTENDED FOR THE USE OF STUDENTS IN COLLEGES AND UNIVERSITIES Illustrated, 12mo, $ 1.90 net ‘‘There are some interesting distinctive features in this new introduc- tion to zodlogy. Only a few types are studied (all of them Invertebrates) ; they are discussed so as to illustrate the principles of the science; the morphological aspect is not especially emphasized, but is codrdinated with the physiological aspect (which, of course, includes the study of interrelations and behavior).” “The author shows a keen educative instinct ; there is a marked freshness and individuality of treatment, and the assistance of a number of experts, who have read particular chapters, has secured an enviable freedom from mistakes. There is a very useful bibliography, and a glos- sary.” “Tt is a work which it has been a pleasure to read, and which de- serves a career of much usefulness.” — Nature. “The book is cordially recommended as giving a thorough prepara- tion for advanced courses in the subject.” — American Journal of Science. “The attempt is made to present the newer zodlogy to the beginner. Here we find the figures of Jennings, Yerkes, Morgan — in fact, it may be called an American product from cover to cover. Consequently, the student finds himself at home at once among American forms and Ameri- can names. It is not to be understood, however, that the view is circum- scribed and that the data from foreign sources are eliminated.” “It may be said that the result is excellent in the light of the labor set before the author. The book-making is good, the illustrations are carefully selected, and there is a unity in the volume which appeals very strongly to the reviewer.” — Science. THE MACMILLAN COMPANY Publishers 64-66 Fifth Avenue New York COLLEGE ZOOLOGY By ROBERT W. HEGNER, Ph.D. Assistant Professor of Zodlogy in the University of Michigan Mlustrated, Cloth, 12mo, xxiv+733 pp., $2.60 net This book is intended to serve as a text for beginning students in uni- versities and colleges, or for students who have already taken a course in general biology and wish to gain a more comprehensive view of the animal kingdom. It differs from many of the college text-books of zodlogy now on the market in several important respects: (1) the animals and their organs are not only described, but their functions are pointed out; (2) the animals described are in most cases native species; and (3) the relations of the animals to man are emphasized. Besides serving as a text-book, it is believed that this book will be of interest to the general reader, since it gives a bird’s-eye view of the entire animal kingdom as we know it at the present time. Within the past decade there has been a tendency for teachers of zodlogy to pay less attention to morphology and more to physiology. As a promi- nent morphologist recently said, “‘ Morphology . . . is no longer in favor . .. and among a section of the zodlogical world has almost fallen into disgrace” (Bourne). The study of the form and structure of animals is, however, of fundamental importance, and is absolutely necessary before physiological processes can be fully understood; but a course which is built up on the “ old-fashioned morphological lines” is no longer adequate for the presentation of zodlogical principles. The present volume has not been made by merely adding a description of the vertebrates to the author’s “Introduction to Zodlogy” (for a brief description of which see the preceding advertisement). On the contrary, it is a new work throughout, although the same general method of treatment, which proved so successful in the earlier book, has been employed in this one. Similarly, in the preparation of this book the author has submitted the manuscript of each chapter to a scholar and teacher of unquestioned authority in the particular field. The criticisms and suggestions thus se- cured have greatly increased both the accuracy and the practicability of the text. THE MACMILLAN COMPANY Publishers 64-66 Fifth Avenue New York Genetics. An Introduction to the Study of Heredity By HERBERT EUGENE WALTER Associate Professor of Biology, Brown University Cloth, 12mo, $1.50 net In his “ Genetics ’’ Professor Walter summarizes the more re- cent phases of the study of heredity and gives to the non-technical readers a clear introduction to questions that are at present agitat- ing the biological world. Professor Walter’s conception of sexual reproduction is that it is a device for doubling the possible variations in the offspring, by the mingling of two strains of germ plasm. The weight of prob- ability, he concludes, is decidedly against the time-honored belief in the inheritance of acquired characters. Professor Walter also predicts that the key to this whole problem will be furnished by the chemist, and that the final analysis of the matter of the “heritage carriers” will be seen to be chemical rather than mor- phological in nature. In the practical application of this theory to human conservation or eugenics, it would follow that the only con- trol that a man has over the inheritance of his children is in selecting his wife. Professor Walter holds, if only modifications of the germ plasm can count in inheritance, and if these modifica- tions come wholly from the combination of two germ plasms, then the only method of hereditary influence is in this selection. “T find that it is a very useful study for an introduction to the subject. 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THE MACMILLAN COMPANY Publishers 64-66 Fifth Avenue New York An Outline of the Theory of Organic Evolution With a Description of some of the Phenomena which it explains By MAYNARD M. METCALF, Ph.D. Professor of Zoédlogy, Oberlin College, Oberlin, Ohio THIRD EDITION, FUNDAMENTALLY REVISED Cloth, 8vo, Colored Plates, $2.50 net The lectures out of which this book has grown were written for the author’s students at the Woman’s College of Baltimore, and for others in the college not familiar with biology who had expressed a desire to attend such a course of lectures. The book is, therefore, not intended for biolo- gists, but rather for those who would like a brief introductory outline of this important phase of biological theory. It has been the author’s endeavor to avoid technicality so far as possible, and present the subject in a way that will be intelligible to those unfamiliar with biological phenomena. 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(Lond. ) The First Principles of Heredity Cloth, 199 pp., Lll., 8vo, $2.00 net The purpose of this book is to supply in a simple and yet scientific man- ner all that may be desirable for the average student to know about Heredity and related questions, without at the same time assuming any previous knowledge of the subject on the reader’s part. The First Principles of Evolution By S. HERBERT Cloth, 8vo, 346 pp. containing go illustrations and tables, $1.00 net Though there are hosts of books dealing with Evolution, they are either too compendious and specialized, or, if intended for the average reader, too limited in their treatment of the subject. In a simple, yet scientific, manner, the author here presents the problem of Evolution comprehen- sively in all its aspects. CONTENTS InTRODucTION — Evolution in General. Palzontology. Section I — Inorganic Evolution. Geographlcal Distribution The Evolution of Matter. Part II — Theories of Evolution. Section II — Organic Evolution. Section III — Superorganic Evolution. Part I — The Facts of Evolution. Social Evolution. Morphology. Conciusion — The Formula of Evolution. Embryology. The Philosophy of Change. Classification. THE MACMILLAN COMPANY Publishers 64-66 Fifth Avenue New York Laie ee