jgigiiiiiii SAGE ENDOWMENT FUND THE GIFT OF HENRY W. SAGE 1891 The original of this book is in the Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://www.archive.org/details/cu31924024795027 Number ii OCTOBER, 1920 THE AMERICAN ANATOMICAL MEMOIRS Numbers i to 7 inclusive appeared as Memoirs of The Wistar Institute of Anatomy and Biology EDITED BT GEORGE S. HUNTINGTON Columbia University WITH THE collaboration OF CHARLES R. STOCKARD and HERBERT M. EVANS Cornell University Medical School University of California, Berkeley THE PIGMENTARY, GROWTH AND ENDOCRINE DISTURBANCES INDUCED IN THE ANURAN TADPOLE BY THE EARLY ABLATION OF THE PARS BUCCALIS OF THE HYPOPHYSIS p. K. SMITH ANATOMICAL LABORATOKT OF THE tTNIVEBSITT OF CALIFORNIA PUBLISHED BY THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY PHILADELPHIA, PA., U. S. A. 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Subscribers to this service receive two cards for each publication; one bearing the abstract on one side with the reference on the reverse side (main entry card to be filed alphabetically according to author), the other bearing the reference only and intended to be used as a subject index card. Extra cards may be secured. The subscription price is $5.00, for the year ending June 1, 1921. Cards already issued will be supplied. THE AMERICAN ANATOMICAL MEMOIRS THE PIGMENTARY, GROWTH AND ENDOCRINE DISTURBANCES INDUCED IN THE ANURAN TADPOLE BY THE EARLY ABLATION OF THE PARS BUCCALIS OF THE HYPOPHYSIS PHILIP E. SMITH ANATOMICAL LABOBATOBY OF THE UNIVEESITY OF CALTFOENTA 1920 PUBLISHED BY THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY PHILADELPHIA u CONTENTS 1. Introduction 5 The conditions of the experiment 7 2. Alterations in the pigmentary system 10 The chromatophores of the albinous larvae 14 The epidermal melanophores 14 The deep melanophores 18 The xantholeucophores 18 Epidermal transplants 23 The effect of various diets upon the pigment cells 27 The responses of the chromatophores to various physiological and phar- macological agents 30 3. Growth disturbances 41 The growth rate of the albino; the effect of the administration of various physiological substances upon this growth rate 44 The growth rate of thjrroidectomized larvae 67 4. Modifications in the size and structure of the endocrine organs 68 The hypophysial components 69 The thyroid 83 The adrenal cortex and medulla 90 The epithelial bodies 97 The fat-organ 99 5. Discussion 100 6. Summary 103 BibUography 109 THE PIGMENTARY, GROWTH AND ENDOCRINE DISTURBANCES INDUCED IN THE ANURAN TADPOLE BY THE EARLY ABLATION OF THE PARS BUCCALIS OF THE HYPOPHYSIS" 1. INTRODUCTION Only within the brief period of four years has the favorable character of the early amphibian tadpole for analyzing the func- tional nature and the reactions of the members of the endocrine system been recognized. This may indeed seem strange, since the value of this material, to which attention was called by Born and which has been used so advantageously by Har- rison, in the solution of those problems requiring experimental procedure upon the early embryo has long been appreciated. The early amphibian tadpole is peculiarly useful in experimental biological investigations, since in its earliest stages it is avail- able for operation and has the inherent capacity to survive the most severe mutilations. These characteristics make a special appeal in studies upon the functional interrelationships obtaining in the endocrine system, for derangements in this system can be induced by the ablation of certain of its members in their early embryonal and non-functional stages by a simple operation, in itself not harmful. The early removal of a gland will afford then knowledge not only of the essentiality of this gland per se, but also concerning the dependence of the other endocrine organs upon this gland for their full development. This interdependence may be productive of even greater struc- tural changes in the other glands than a later operation would produce, because of the lability inherent in embryonal structures. Further, the utilization of embryonal material would appear 1 Aided by a grant from the Research Board of the University of CaHfornia. 6 PIGMENTARY GROWTH AFTER ABLATION OF to be necessary in studies upon hypophysial extirpation if the prolonged survival of the animals is desired, since the complete extirpation of the epitheUal hypophysis'" in the adult invariably proves fatal within a brief period. And it would appear that only by the prolonged survival of the animal can the maximal alterations in the other members of this correlative system fully express themselves, as is shown by the structural picture pre- sented by the endocrine organs of the 'hypophysectomized' tadpole. These organs exhibit structural changes exceeding in magnitude those obtaining in the hypophysectomized mammal. Indeed, it is possible to look upon these structural alterations as the expression of a restored functional balance in the endo- crine system which has permitted the survival of the animal. One further advantage is bestowed by the use of embryonic material, for we may discover a more general influence of the endocrine organ in question on the manner or rate of develop- ment of all of the tissues and organs of the body — namely, on growth — and, as we shall see further on, growth effects are among the most prominent ones manifested by these disturb- ances. In 1912 Gudernatsch reported that frog larvae could be meta- morphosed by thyroid feeding. This remarkable result stimu- lated Adler in the same year to utilize the tadpole for the abla- tion of members of the endocrine system. By what now appears to be a crude method, Adler, in 1912, burned out the pituitary from half-grown frog larvae. Although the failures were numer- ous, the death rate high, and in the surviving successful cases the injury to the neighboring structures severe, nevertheless, Adler was able to show that the destruction of the pituitary impaired the thyroid gland and prevented metamorphosis. Subsequent and far more successful ablations of the hjrpophysis have amply confirmed Adler's findings. In 1914 the author attempted to remove the epithelial anlage of the hypophysis in the early larvae of the newt — D. torosus. As had been the experience of other investigators, this urodele material proved unfavorable for early operation. The foUow- ^ That portion of the hsrpophysis arising from the oral ectoderm. THE PARS BUCCALIS OP THE HYPOPHYSIS 7 ing season unsuccessful attempts to transport living frog eggs from the Middle West were made. Finally favorable material was seciu-ed from the rather scanty local anuran fauna by col- lecting the adult pairs of the frog, Rana boylei, at the time of laying. The larvae of this form proved ideal, wounds healed rapidly, feeding was vigorous, and growth uniform. An astonishing result of this early removal of the epithelial hypophysis was now revealed. Characteristic, silvery-colored larvae — albinos — in great contrast to their darkly pigmented normal mates, resulted. Similar results were obtained simul- taneously by B. M. Allen. In a series of papers the writer, B. M. AUen, E. R. and M. M. Hoskins, and W. C. Atwell have further confirmed and sought to analyze this discovery. Allen has subsequently studied in more detail the changes induced by an equally early ablation of the thjToid gland. No one, it would appear, has endeavored to confine his attention to the altered anatomy and physiology displayed by these pituitary- free 'albinos.' The present monograph aims to do this. While brief statements of some of its material have appeared from time to time, there has been collected here for the first time as com- plete an account as is now possible of the pigmentary upset and other correlated bodily changes, especially endocrine alter- ations called forth by this procedure. The conditions of the experiment All amphibian material is not equally favorable for work of this nature. In order to assure prompt healing, the yolk must be moderate in amount and of a cohesive nature. Thus oper- ative work upon the larvae of the common newt, D. torosus, is precluded, due to its large, abundant, and non-cohesive yolk granules, which extrude for hours through the wound, even final healing being prevented. The unfavorable character of the Ambly stoma punctatum for work of this nature has pre- viously been commented upon by Harrison. For studies on growth it is further essential that the animals be vigorous feeders and develop at a rather uniform rate. This desideratum would 8 PIGMENTARY GROWTH AFTER ABLATION OF apparently rule out one of the common California frogs, R. aurora dratonyii, as well as the newt. The frog, Rana boylei Baird, and to a limited extent the toad, Bufo boreas, were utilized in this work and have proved very satisfactory, since they re- cover quickly from the operation, are vigorous feeders, and exhibit a rather uniform rate of growth. The anlage of the epithehal hypophysis can be more easily ablated than any of the other glands of internal secretion save the thjToid. As is well known, this ectodermal invagination, lying between the brain and the pharynx, is connected with the surface epithelium in very young frog larvae,' and is conse- quently readily accessible (fiig. 12). If a transverse cut with finely ground needles be made through the surface ectoderm between the forebrain protuberance and. the pharynx* (figs. 11, 12) and these two structures gently separated, the hypophysial ingrowth is readily distinguished lying on the ventral surface of the brain, from which it can be separated without serious injury to the latter. The stage selected for the operation should neither be too young, in which case the structures are undiffer- entiated and serious injury may be done to the mouth, nor too old, in which case the hypophysis will have migrated to its deeper position, thus making its removal extremely difficult. Larvae of 33^ to 4 mm. in length, at which time the tail bud is well formed and the nasal placodes distinct, appear to be in the most favorable stage for epithelial hypophysectomy (fig. 11). Since reflex movement has not yet appeared, no anaesthetic is necessary. It is essential to furnish a continuously renewed supply of well-aerated water to the larvae. In this locality the added necessity of 'sterilizing' the water by heat at 60°C. for at least an hour, followed by cooling and reaeration, has been forced upon us (Smith, '18), because of the pathogenic organisms which ' Excellent descriptions of the development of this structure in Amphibia have been given by Orr ('89), Coming ('99), Kingsley and Thyng ('04), Atwell ('18), and others. ^ A depression at the point of invagination of the hypophysis plainly marks its position (figs. 11, 12, Hyp. p.). With later development this pit gradually dis- appears or merges into the stomodaeum. THE PARS BUCCALIS OF THE HYPOPHYSIS 9 apparently gain ingress through the city water supply. Experi- ence during the past three seasons has abundantly justified this 'sterilization,' since infection has been inversely propor- tional to the extent to which this treated water was utilized. Such treatment of the Avater appears to impair in no way the normality of the animals raised therein. With the daily feeding of special or check food substances, as has been done in this study, the necessity of removing the uneaten particles which would otherwise putrify has forced us to have special containers manufactured. Our needs were met by having vessels of two sizes cast. One size, made of a gray glazed earthenware, is 12 inches square by 6 inches deep; the other size, made of a gray porcelain, is 6 inches square by 4 inches deep. The water level in either type can be readily changed by an L-tube draining through a hole near the bottom of the container. It was found that from fifty to seventy-five speci- mens eoTild be reared in each of the larger containers without overcrowding and from ten to twenty specimens in each of the smaller ones. The daily cleaning of such containers proved not to be an onerous task when done with a sj^phon cleaning tube fitted with an appropriately shaped inlet. Various check and special food substances have been fed: liver, muscle,^ anterior lobe, posterior lobe of the hypophysis, adrenal cortex, adrenal medulla, as well as various extractives and residues of the anterior lobe of the hypophysis. As a check diet, finely ground fresh liver has proved more satisfactory than muscle, since the connective tissue of even finely ground muscle often unites fragments and thus prevents complete deglutition of partly swallowed series of food particles, resulting in the death of the animals. In all cases an abundant supply of boiled lettuce has been furnished. Although liver has proved satis- factory for the normal tadpole, yet by far the most adequate food substance for raising a vigorous, healthy albino is the fresh anterior lobe of the beef hypophysis. With this diet, which * Mendel and Osborne ('18) report that the proteins in both muscle and liver are adequate for the needs of nutrition in growth. 10_ PIGMENT AEY GROWTH AFTER ABLATION O'F as will be shown replaces the growth substance lost by hypophy- sectomy, the albinos thrived and were much more healthy, vigorous, and enjoyed a greater longevity than with any other diet. This gland proved to be a very desirable food substance for the normal tadpole as well. The alterations referable to hypophysectomy in frog or toad larvae express themselves in several ways. There early appears a pigmentary disturbance productive of a silvery tadpole^ the albino. This system then exhibits profound structural and functional modifications. An early retardation in growth is apparent which progressively becomes more marked with devel- opment. The growth curves of these animals differ not only in their magnitude, but in their character when compared to the normal. And finally, most of the other members of the internal secretory system present a structural picture differing greatly from the normal. It is thus convenient to present the matter under three head- ings: Disturbances in the pigmentary system. Alterations in the growth rate. Alterations in the other glands of internal secretion. 2. ALTERATIONS IN THE PIGMENTARY SYSTEM The striking color changes which many of the lower verte- brates exhibit under changed environmental conditions has long been known and early attracted attention to the mutual inter- play of the various components of the chromatophore groups by which this chromatic 'adaptability' was effected. In addition to these environmental color changes, it has also been shown that pronounced changes in certain of the chromatophore groups can be experimentally induced: by the injection of adrenalin into the frog, Lieben ('06) ; by the immersion of the entire animal or portions of it in certain endocrine extracts, McCord and Allen ('17), Lowe ('17), Spaeth ('13 and '18, with fish scales); by experimental operative procedures. Lister ('58), Bimmerman THE iPARS BtrCCALIS UP THE HYPOPHYSIS 11 (78), Biedermann ('92), Hooker ('12), Redfield ('16); by elec- trical stimulation, Hermann ('86), Winkler ('10), Spaeth ('16, with fish scales); by pharmacological reagents, Lowe ('17); as well as by producing numerical and physiological modifications in the pigment cells by rearing the larvae in different back- grounds, Babdk {'13) J It is not surprising that a mechanism exhibiting such a functional labihty would be modified by dis- turbances in the endocrine system; indeed, that this pigmentary mechanism is influenced by the internal secretory system has been suggested by Fuchs ('14) and Redfield ('16, '18). It was not known, however, until the reports of the writer and of B. M. Allen appeared in 1916 that the ablation of one of the endocrine glands would in itself profoundly and permanently modify the pigmentary system. It was then shown that the early ablation' of the pars buccalis of the hypophysis induced pigmentary changes leading to the formation of a 'silvery' tadpole — the albino — the most striking pigmentary alteration as yet effected in the tadpole. In this pigmentary disturbance all components of the chromatophore system have been pictured as playing a significant role by various writers. The epidermal melanophores have been shown to be dimin- ished in number (Smith, Allen), in pigment content (Smith), and to display a persistent contraction (Allen), all of which has been substantiated in the recent article by Atwell and by the author. It has further been shown that the free melanin which lies near the outer border of the peripheral layer of epi- dermal cells suffers a pronounced diminution (Smith). By this diminution in the melanin content of the epidermis, a modification which lends greater transparency to this epithelial covering, the iridescent quality of the subjacent chromatophore group — the xantholeucophores — which in the albino display a * The literature on the pigment cells is stupendous, only a glimpse of it being given here. A complete bibliography on this subject will be found in the compre- hensive article of Fuchs in Wintersteins' Handb. d. vergl. Physiol., Bd. 3, 1 Halfte, 2 Teil. ' An early operation appears to be essential for the production of a pigmentary effect, since Adler reports no striking pigmentary disturbance subsequent to hy- pophysectomy in the midlarval stages. 12 PIGMENTARY GROWTH AFTER ABLATION OP broad and persistent expansion, is permitted Ml effect. This striking and persistent expansion in the xantholeucophores or 'interference' cells appears to have heretofore escaped attention, yet in this phenomenon of albinism they play no secondary r61e, since to them is referable the silvery and iridescent quality of the albino. 8 The contraction of the deep melanophores, first described by Allen and later confirmed by Atwell, who indeed refers the pictm-e of albinism primarily to the altered physiological state of these cells, the author can corroborate for the young albinous tadpole, but repeated examinations have failed to reveal a definite contraction in the older albinous larvae of R. boylei. As will be subsequently pointed out, the physiological condition of these cells could make no significant contribution to the picture of albinism. To anticipate, then, what will be more fully shown in this section, the essential pigmentary changes" contributing to the picture of albinism in this form are three. in number: 1. A diminution in the epidermal free pigment. 2. A diminution in the number and melanin content of the epidermal melanophores (because of their paucity in number and pigment content, the contraction of these cells plays no important role in the formation of this picture). 3. A great and persistent expansion in the xantholeucophores. It is most essential in a study of this nature that the environ- mental condition be well known on account of the adaptability of the pigment system to such external factors. The standard environmental condition, which we beUeve closely approximates that obtaining in nature, was furnished by a diffuse light, a gray background and room temperature (18° to 25°C.). The ex- 8 1 have recently called attention to the significant contribution which these cells make to the picture of albinism (Proc. Soc. Exp. Biol, and Med., 43-1418, 1919). " Although the pigmentary system of a hypophysectomized tadpole 12 mm. or even less in length shows variations from the normal, nevertheless, an animal two, or better, three or more times this size has these modifications more clearly differ- entiated. The descriptions, except when otherwise stated, were made upon animals raised upon an anterior-lobe diet and which have attained a size in excess of 45 mm. and were not less than S14 months old. THE PAKS BUCCALIS OF THE HYPOPHYSIS 13 treme variations from this 'indifferent' condition have been assumed to be a) a white background with direct sunlight; b) a black background and the absence of light, temperature in all cases being noted and regulated to suit the experimental desiderata. The obser\'ations upon the pigment cells have been made both upon the well-illuminated living animal with the binocular microscope (a Zeiss instrument fitted with a water-immersion objective and no. 2 oculars; magnification, 42 diameters) and by examination of cutaneous whole mounts. By the first method the progressive changes in the pigment cells under altered environmental conditions can be noted, the observations in most cases of necessity being rapidly taken so as to exclude possible alterations resulting from the rather briUiant illumina- tion necessary for binocular observation (as, for instance, in observing a dark-adapted animal). These observations have been checked and supplemented by skin whole mounts of the fixed animal, the fixation being so rapidly effected as to preclude any physiological alterations which might take place. Observa- tions have been largely limited to the dorsal and neighboring lateral portions of the body, save in certain cases where the pigment cells lining the body cavity were noted in fixed speci- mens. Although observation of the deep melanophores of the dorsal region involves some difficulty more especially in the albino, in which they are largely masked by the expanded xan- tholeucophores, yet the observations herein reported were re- stricted to this region, since repeated observations have revealed the fact that the pigment cells exhibit regional variations both in the time and the magnitude of their response. In this section of the paper there will be presented : 1. The anatomical and physiological characteristics of the pigment cells of the albino as compared with those of the un- operated tadpole and some remarks on the development of this condition. 2. The responsibility of the endocrine system for this pig- ment fault as shown by — 14 PIGMENTARY GROWTH AFTER ABLATION OF a. Epidermal transplants. h. The effect of various pabula upon the pigment cells, c. The response of the chromatophores to various physio- logical and pharmacological agents. The chromatophores of albinous larvae As is well known, the melanophores of the tadpole are of two types. One type — the epidermal melanophore — is found in the epithelial covering of the body; the other type— rthe deep melan- ophore — lies in or around the deeper structures. These two types, then, of necessity, will be treated independently. The epidermal melanophores. The epidermal melanophore, when in an expanded condition, presents an irregularly shaped body from which branched, slender processes radiate for rela- tively long distances (fig. 13). When in a greatly contracted condition, these processes are not evident and the cell body then is of a spherical or slightly irregular shape (figs. 19 and 21). All intermediate conditions between these two extremes can be seen with proper light and temperature conditions. The melanophores of the epidermis are greatly reduced in number in the albino (figs. 13 and 14), many counts showing an average reduction of two-thirds from the normal number. Even in a greatly expanded condition these cells are usually separated by wide intervals, a condition which contrasts sharply with that obtaining in the normal animal where the expanded processes unite with, or even overlap, each other. Thirty-one counts from five specimens gave an average of 38 epidermal melanophores in a unit area of 0.36 of a sq.mm. Similar counts from normal animals reveal 119 to this unit area, thus exceeding by over three times the number present in the albino (table 1). It is emphasized, however, that the distribution of these cells in neither type of animal is uniform. Yet their number in the normal even in the areas where they are most thinly distributed exceeds very considerably their number in the albino where they are most thickly placed. Further, the actual melanin content of these cells is diminished in the albino (figs. 13 and 20). Their processes, slender and of light color, present a very differ- THE PARS BUCCALIS OP THE HYPOPHYSIS 15 ent appearance from that furnished by the course black processes of the normal melanophore.i" Not only do these superficial melanin-bearing cells exhibit differences of a quantitative nature in these two types of ani- mals, but constant physiological dissimilarity is also encountered. It may be urged that the functional states of these cells in the normal usually exhibit such wide variation not only between two or more individuals, but also in the cells of a single indi- vidual, that the determination of a normal physiological con- dition is precluded. Certain it is that not only do we find differences between the condition of the individual cells of a specimen, but under identical environmental conditions these cells in one animal may be expanded while those of its mates are partly contracted." Yet this variability is not so great as to preclude our defining the condition of these cells in a normal animal in an 'indifferent' environment as one of complete or nearly complete expansion (fig. 13). With the albino, as with the normal, but to a lesser extent, the same variability in the superficial melanophores of various individuals is exhibited. The individual cells of any one speci- men, however, usually exhibit a greater dissimilarity than in the normal animal. A very few of these cells are almost in- variably broadly expanded, a few are half expanded, while the majority are completely contracted or but slightly expanded (fig. 14). It is apparent, then, that the usual physiological '" The evidence on this point is quite clear; corroborative evidence has also re- cently been furnished by Atwell, although Allen is apparently not in agreement with these findings, for he says: "These observations and a careful study of the pigment cells convince me that there is no disappearance and bleaching of the pig- ment cells as asserted by Smith." It is worthy of mention here that the author has never mentioned a "disappearance and bleaching of the pigment cells," stating, "Counts of the epidermal melanophores — in the albinos and checks show that the number of these cells in the epidermis is reduced in the former" and — "the melano- phores of the albino specimens contain fewer pigment granules than do those of the checks and thus have a distinctly hghter appearance." The process leading to this condition was not then discussed by the author. ii This variability has led certain investigators to confine their observations in various physiological experiments to the more uniformly reacting deep melano- phores — for example, Laurens. 16 PIGMENTARY GROWTH AFTER ABLATION OF "ft. -^ Si. H S < S ^-3 Ol 1— 1 tH t II 03 C-i ■4-= § rt O tfl «•*:' i-[ IM (M ^ t- I^ CO 00 lO H T-H C^) Oi Tt< ^ ^ IC c^ -*< o OP CI rH i-H i-H i-H CM u t. o ti* S

n 01 fe; 3 " fe'S ^H Ph ^ F.4 Fh h U F-i s a a> > > > > > > > •> S • rt • ^ ■ I-( i-:i i-:i 3 1-1 3 3 3 3" ■^ (fi . T-H lO lO cb 1-- >o M ^ "^ => ^ CD CO CO J i-H (N ^ > ^> -^^^ IN T1H> O n2 -2 (a o o Q) ^_, "o — , "o _ "o — "o r-H 'O 'o o o 1 ^ 3 c8 D. 03 D, c3 O, 03 a 2 o. o a 3^ o +3 o -^ o -^ o +^ S '^ o ■« ■s-f .3 -o ^ 1 ^g ^2 o II II II 5 -2 CJ 3 o o " ^ 3 o 3 O CO " (N Oi c^ " CO CO '— 1 CO o ! >o >I5 CD tH IN "o ID Ml 1 lO ^ 4' Average of 17 l> 1> t> lO CO CO CO 00 ira ^ a) 0) > ^?- > > o o _o .2 o 3 3 ■g 'm o g s 1^ '^ ■gcij 1 -^ ■s CO o o o o O PM CM fc PL, Pi -^^ 1— t ■* lO 1— I Tf ^H T)< CO 1 ° ^ t- " CO CO (N (N ^ CO (N Si lO IM CO lO IN 52 ■* 1 ^> -^lo TH lO Tjl Th -* tH -^IJ Tjl 00 Tt< d '^ S 1—1 1—1 '"^ 1—1 T-H l-H 1—1 _2 0) > ^^ PQ o ft 13 Mi2HOm NO. 11. 18 PIGMENTAKY GKOWTH AFTER ABLATION OF condition obtaining in these cells in the albino is one of com- plete, or nearly complete, contraction. The free pigment which forms a definite, though imperfect, layer in the normal animal is much diminished in amount and is irregularly distributed in the albinous larvae (figs. 13, 14). In the skin whole mounts of the albino, it indeed forms but a scanty and irregular sheet which stands in contrast to the very distinct layer present in the normal animal. The deep melanophores. The number and melanin content of the deep melanophores apparently are not altered by the ablation of the hypophysis. This structural independence ap- pears not to lend support to those investigators who assert that both groups of melanophores, epidermal and corial, arise from a common source. ^^ That the deep melanophores exhibit a contracted condition has been noted by Allen and Atwell, the latter, indeed, referring the phenomenon of albinism primarily to their contracted con- dition. Many examinations of larvae in excess of 45 mm. in length, made both on the living animal and after rapid fixation, convince the author beyond question that in the body and head regions these cells, whether occurring in the corium, in muscle sheaths, or in the fascia of the viscera and body cavities, exhibit nearly the same physiological state in the fully developed albino of R. boylei as in the normal (figs. 13, 14) ; in the young albino, however, certain of these cells appear to be in various stages of contraction (fig. 16). The xantholeucophores. We have noted significant and con- stant changes in the superficial layer of the first group of pig- ment cells — the bearers of melanin. Changes no less significant in the phenomenon of albinism are encountered in the second group of chromatophores, the bearers of guanine and xanthine. Indeed, since the silvery and iridescent characteristics of many fishes are referable to these cells, it would seem to make them of paramount importance in the phenomenon of albinism. When viewed by transmitted light, these cells are of a grayish or brown- ie Ehrmann ('96 and earlier) refers the common origin of the melanophores to the mesoblast of the dorsal cephaUc region of the body. THE PARS BUCCALIS OF THE HYPOPHYSIS 19 ish color. It is, however, with reflected light that their great beauty and their contribution to the color of the animal becomes apparent. For one constituent, the guanine, distributed in minute crystals reflects the light and lends to it the iridescent qualities; the other constituent, the xanthine, small in amount in the tadpole, contributes to this color effect. Delicate in structure and apparently exhibiting no resistant cell membrane, these cells are prone to disintegrate, consequently appropriate and rapid fixation is essential for their preservation. This delicacy is well shown in the epidermal transplants where although but a few seconds may lapse before the graft 'takes,' yet these cells in any region where an intimate union between host and graft does not obtain, change their character and ulti- mately disintegrate. Or, indeed, even when not subject to mechanical disturbances, as following the natural death of their host, they are wont to lose quickly their cellular integrity, form- ing a diffuse layer of fine crystals, which exhibit a vigorous Brownian movement — a most striking picture with the polar- iscope. Not only is early and rapid fixation essential, but appropriate fixers must be employed. This is well shown if a specimen be dropped into formol. Such an animal after a few weeks, or even days, loses its silvery appearance and as- sumes a gray tone. Microscopic examination reveals the fact that the xantholeucophores are no longer visible. Over the dorsal body areas the xantholeucophores form two well-defined layers, each layer being composed of discrete inde- pendent cells. The peripheral layer, Ijdng just beneath the epidermal basement membrane, is usually separated by a con- siderable interval from the deeper layer which lies adjacent to but external to the corial melanophores. In those areas where the epidermis closely approximates the underlying firm body structures, these two layers of xantholeucophores, except under high magnification, may appear as a common layer. We thus see that the iridescent cells lie between the two types of melan- ophores, an arrangement the recognition of which is of no little moment in evaluating the role of the various groups of pigment cells in albinism. 20 PIGMENTARY GROWTH AFTER ABLATION OF Both layers of corial xantholeucophores are broadly expanded in the albino (figs. 14, 16, 18, 20 to 23, 55 to 58), an expansion which is singularly unamenable, more especially in the older albinos, to altered environmental conditions. To the broad expanse of the refractive cells the albino owes its metallic tone, as can be clearly shown by all procedures which contract these cells. We have thus far confined our attention to two types of tad- poles, that type in which the tadpoles have suffered the entire loss of the buccal component of the pituitary, the albino, and that type whose members have suffered no operative inter- ference, the normal. It is now desirable to consider a third type — those in which the epithelial hypophysis was incompletely ablated, the 'partial' albino (figs. 52 to 54). This type of tad- pole usually suffers a serious pigmentary disturbance, its pig- mentary system simulating that of the albino more than that of the normal. In fact, its almost typical albinism frequently makes this animal difficult and often impossible to distinguish during early development from its completely hypophysectom- ized brothers. Later in life it is readily distinguishable, since legs develop in the partially but not in the completely hypophy- sectomized tadpole. In the albinous type of partially hypo- physectomized tadpole as in the typical albino the free pigment is greatly diminished in amount and the epidermal melanophores are scanty in number. That the epidermal melanophores are slightly greater in number than in the complete albino, however, is apparent from counts. It will be recalled that thirty-one counts from five albinos gave an average of thirty-eight epi- dermal melanophores to a unit area 0.36 of a sq.mm. Seventeen similar counts from three 'partial' albinos gave an average of fifty to this unit area (table 1). The xantholeucophores appear to be no way different from those of the typical albino. They exhibit a broad and persistent expansion. Although the pig- ment system of this animal closely resembles that of the albino, yet, in contrast to the albino, it does not suffer serious structural defects in any of its endocrine organs save one, the neural com- ponent of the pituitary. Exhibiting, then, characteristics of both the albino and the normal, this 'hybrid' tadpole has been THE PARS BUCCALIS OF THE HYPOPHYSIS 21 of especial value in freeing any of the other endocrine glands which have been studied from responsibility for the pigmentary disturbance, as will be later shown. An examination of the pigmentary system of the albino has revealed that one group of chromatophores, the xantholeuco- phores, exhibits a great expansion; another group, the epidermal melanophores, is diminished both in number and pigment con- tent and displays an unusual contraction. It is necessary, then, to examine the developmental stages leading to the production of the characteristic albino if we wish to determine whether this is a progressive phenomenon; whether from the beginning the formation of the epidermal melanophores, for example, is inhibited by the endocrine fault, or whether on the other hand they form only to partially disappear later. The three groups of pigment-bearing cells make their appearance in the following sequence: deep melanophores, xantholeucophores, epidermal melanophores. A period of several days intervenes between the appearance of each of these groups, and so they may well be treated independently in a developmental study. The time of appearance of the deep melanophores apparently is identical in both the normal and hypophysectomized tadpole. In a 6 to 7 ram. stage for the normal, they have already made their appearance in the dorsal regions of the head and body. A few show the typical shape of the deep melanophore. A majority, however, have longer and more delicate processes than the typical corial melanophore. They either lie well separated from the epidermis or, especially where the epithelium closely approximates the underlying firm structures, may be in contact with the epithelial covering. From this stage they progressively increase in number and gradually become organized until they attain the typical arrangement as a corial sheet at a 10 to 12 mm. stage (fourteen to twenty days after the operative stage). The xantholeucophores make their appearance in larvae of 10 to 11 mm. (fourteen to sixteen days after 'the operative stage), appearing concomitantly both in time and number in the normal and hypophysectomized larvae. Slightly more numerous, pos- 22 PIGMENTARY GROWTH AFl'ER ABLATION OP sibly, in the hjrpophysis-free specimens, this numerical difference is so slight as to be well within the limits of variability. From their earliest appearance they are expanded in the operated tadpole (fig. 16) in contrast to their punctate character in the normal (fig. 15). The early, almost imperceptible albinous appearance which denotes a successful hypophysis extirpation is due largely to the expanded condition of these cells, since in these early stages the free epidermal pigment in both albinous and normal larvae is identical and the epidermal melanophores have not as yet formed in either type of specimen. Scarcely perceptible at first, this whiteness of the hypophysectomized tadpoles progressively increases with the increase in the number of the xantholeucophores, and is further emphasized by the reorganization and diminution in the early embryonic pigment which takes place, as will be described later. In the diminution in the epidermal melanin we find the first structural pigmentary alteration referable to hypophysectomy. Here we have to deal with the double expression of the melanin, that existing as free granules and that included in the chromato- phores. In the earliest stages free pigment is found diffusely scattered throughout the two cell layers of the epidermis, al- though occurring more plentifully in the outer of these layers and in certain cells of this outer layer. At a 10 mm. stage it has become localized in the superficial layer of the epithelium, and by the time the larvae have reached approximately a 20 to 25 mm. stage it is found only in the peripheral zone of this cell layer. '3 Identical in amount in the earlier larval stage of the operated and normal larvae (figs. 15, 16), it gradually be- comes diminished in the albino (figs. 17, 18), a diminution which aids in permitting the iridescent qualities of the xantholeuco- phores to be fully expressed and which is shown by the rapid development of the albinous picture at this time (figs. 42 to 45). The first typical epidermal melanophores make their appear- ance in about a 10 mm. normal (twenty-two days after the operative stage, fig. 15). At first scatteringly distributed and " The changes here described are nearly identical with those described by Maurer ('95). THE PARS fiUCCALIS Of THE HYPOPHYSIS 23 light in color, these cells rapidly increase in number and melanin content; by a 15 mm. stage they have numerically and struc- turally attained the mature larval condition (fig. 17, table 1). Their appearance in the albino is more tardy. They do not appear as early and are always diminished in number and melanin content as shown by figures 16 and 18, which were drawn from albinos of the same age and size as the normals from which figures 15 and 17, respectively, were drawn. A partly con- tracted condition is also evident from their earliest appearance. No evidence has accrued from these studies which suggests that the diminution in the number of epidermal melanophores suffered by the albino is affected by a migration of the epidermal melanophores into the corium as suggested by Allen. On the other hand, the evidence all points to the developmental and functional independence of the two systems of melanophores. The first melanophores to appear are those in the corium, and there is at no time any evidence that their number is augmented by a migratory process from the epidermis. The formation of the epidermal melanophores from the beginning is apparently partially inhibited in the albino, an inhibition which is expressed not only in the diminished number of cells, but in their melanin content as well. One of the remarkable features in this pigmentary disturbance is its early appearance. Whether referable to some missing hormone of the anterior lobe or to other hormonal disturbances provoked by the operation, it appears clear that hormones are already produced by the small groups of embryonic cells con- stituting the endocrine glands at a time when the latter exhibit but little if any of the structural differentiations which char- acterize the adult internal secretory organs. Epidermal transplants We have detailed at some length the pigmentary disturbances which result from the early loss of the epithelial hjrpophysis in the tadpole. It is now of interest to inquire whether this func- tional upset is due directly to an alteration in the quality of 24 PIGMENTARY GROWTH AFTER ABLATION OF the fluids bathing these cells or, on the other hand, to alterations in their nervous mechanism. Unimpeachable evidence has been secured on this point by reciprocal skin transplants. In a very considerable number of cases skin interchanges have been successfully effected between a normal tadpole and its albinous mate. Invariably, and in a period not exceeding four hours for the xantholeucophores and a somewhat longer period for the epidermal melanophores,^* these cells of the transplant assume the state characteristic for the corresponding cells of the host. The technical manipulations involved in performing these skin exchanges even in larvae exceeding 40 mm. in length are not difficult. A narcosis sufficiently deep for operative work has not readily been effected at room temperature, although serving well for ordinary examinations in which no trauma was inflicted. Of the several anaesthetics employed, ethyl eurethane (Merk) in a strength of 0.5 per cent has proved the most efficacious, especially when used at a low temperatxu-e (1° to 3°). The lighter narcosis effective at this low temperature is of no little significance, since not only is recovery more certain, but also functional changes in the pigment cells inherent in a pro- found anaesthesia are avoided. After a sufficiently deep insensibihty had been produced, the specimens were placed closely together in depressions in the wax plate lining the dish. Incisions on the dorsal epidermis mapping out an area approximately 4X6 mm. were then made on the dorsum of each specimen with the aid of the binocular. Following this the skin was rapidly freed and the interchange effected, the grafts being gently pressed down and not infre- quently tucked under the edge of the host's skin. These grafts quickly adhered, and in a short time no movement of the host could dislodge them. " This reaction of the epidermal melanophores of the albinous graft to the normal host— one of expansion— applies only to the cells originally belonging to the graft. Epidermal melanophores from the surromiding normally pigmented epithelium of the host quickly invade the albinous graft. These cells display a state of great contraction for at least a considerable interval. TfiE PARS BUCCALIS OF THE HYPOPHYSIS 25 There now was exhibited one of the most striking phenomena presented in this work. The heretofore broadly expanded xantholeucophores of the albinous graft gradually contracted under the influence of its normal host. In half an hour they were much reduced; in two hours they were punctate, even exceeding in minuteness under this new stimulus the diminutive size of the host's 'interference' cells (figs. 57 and 58). Likewise the epidermal melanophores of the albinous graft gradually expanded. This change from the altered physiological state of these cells in the albino to the state characteristic of the normal animal then persisted for the life of the host. A change as striking was exhibited by the heretofore con- tracted xantholeucophores and expanded epidermal melano- phores of the normal graft. In about the same interval the xantholeucophores of the normal graft became broadly expanded (figs. 57, 58), and after a somewhat longer interval the epidermal melanophores assumed a punctate condition. These cells, then, in this brief interval had changed from the functional state they had so long experienced to the opposite condition exhibited by them in the new host. There is, however, the possibility that this remarkable trans- formation was due to the mechanical manipulation and not to the influence of the host. This possibility can definitely be excluded by the simple test involved in an autoplastic skin graft. Identical incisions were made, the freed epidermis, how- ever, instead of being transferred to a new host, was returned to the position from which it for a brief interval had been removed. In neither the normal nor albinous larvae did these cells suffer any permanent change, but remained in the same condition as' the cells which had not been removed. Definite evidence has thus been secured which shows that the altered state of these cells in the albino has been brought about by the action of some substance present in the tissue fluids and not by alterations in the nervous mechanism, since we can conceive of no reestablishment of nervous connection in such a brief interval. Further, it would seem probable that this substance acts directly upon the pigment cell itself and not upon 26 PIGMElsrTARY GROWTH AFTER AlBLATION OF the severed nerve termination. This would appear to be the case, since if this atypical condition exhibited by the cells of the normal graft to the albinous host was due to the action of the tissue fluids upon the severed motor connections, we would expect a change in the size of these cells in that interval which must occur between the degeneration of this nerve ending and the reestablishment of a new nervous connection, a change which apparently does not occur. Because of the known potency of minute amounts of endocrine secretions, the most natural as- sumption is that this alteration has been effected through some 'hormonal' component of the plasma or lymph. Rather interesting results have been secured by submitting these operated animals to adrenalin and to 'light and heat' stimuli. These experiments, although not as complete as could be wished, have been carried out on some half-dozen albinous and normal tadpoles into which grafts from the opposite type of animal had been made. The response or failure to respond in the case of host and transplant cells has been nearly identical. The tests were carried out from twelve hours to three days subsequent to the operation and supply additional evidence that the effective force causing a response of these cells to stimuli is the direct application of the internal secretory substance to the cell bodies and is not effected through the motor sympa- thetic endings, since it is probable that any nerve ending severed as it is from the nerve fiber would have degenerated in this interval, and even after this lapse of time new connections could hardly have been established. Some rather significant evidence has been secured on the formation and migration of the epidermal melanophores in the transplants. There invariably occurred an early migration of the epidermal melanophores from the normal host into or over the albinous transplant. In a period of a few hours the sulcus intervening between the two types of skin would become filled with a dense mass of melanin-rich cells in which the pigmented epidermal cells could not readily be distinguished from the true melanophores. In twelve hours, or even less, growing points would appear from this confused scar tissue which invaded or THE PARS BUCCALIS OF THE HYPOPHYSIS 27 became incorporated gradually into the albinous graft. In this invading tissue, and indeed in those parts of the albinous graft which clearly had not suffered an invasion by the epidermis of the host, epidermal melanophores could readily be distinguished which curiously displayed, for a considerable interval, a state of great contraction. In the end the albinous transplant was as abundantly supplied with melanophores as was the normal epidermis. Although the free pigment of the albinous graft increased more slowly than did the epidermal melanophores, yet this too ultimately exhibited a normal condition. Thus, after an interval not exceeding two months, both the free pig- ment and the epidermal melanophores of the albinous trans- plant had assumed the characteristics typical of the epithelium of the normal host. The sequence of changes in a dark graft to an albinous host is of a quite different nature from the alternate just described. Although there appeared to be a slight reduction in both the amount of the free pigment and the number of the melanophores of the epidermis, yet these changes were of a minor nature. No extensive naigration of the melanophores from the normal trans- plant where they were thickly congregated into the sparsely supplied surrounding albinous epithelium occurred, nor by any other process did any great reduction in the number or pigment content of the transplanted melanophores take place during the interval that the animals were under observation (two months). These cells, once formed, appear to retain their integrity. This would appear to be the correct interpretation of the non-effect upon the pigment cells of hypophysectomy in the midlarval stages (Adler). The effect of various diets upon the 'pigment cells We have just shown by the skin exchanges that the atj^ical * physiological state of the xantholeucophores and epidermal melanophores of the albino is directly referable to an alteration in the tissue juices which in turn is probably of an 'hormonal' nature. If, then, this endocrine disturbance which is inaugu- 28 PIGMENTARY GROWTH AFTER ABLATION OF rated by an hypophysial deficiency leads to an upset in the pig- mentary system, will it not be possible with the proper endo- crine diets to abort or ameliorate this derangement? The possi- bility appears the more probable since the general growth effects inherent in the loss of the buccal hypophysis can be prevented by the administration of the beef glandular lobe, as will be shown later. Of the endocrine substances which have been fed, glandular lobe and neural lobe (with the pars intermedia) of the hypophysis, adrenal cortex, adrenal medulla, and liver, one only — the neural, or posteiior lobe — has caused a formation of epidermal melanin approaching the normal, while none have reduced the xantholeu- cophores to a normal state of contraction. It' was early seen that those specimens receiving the posterior lobe of the pituitary exhibited a darker appearance than the albinous larvae fed upon the other fresh glands (figs. 46 to 51). Although expressed feebly at first, this depth of color became more pronounced as the feeding and growth progressed. So pronounced did it become after five months of this diet that there would have been some uncertainty in identifying these as hypophysectomized specimens save for the fact that legs did not develop. An examination of the living animal with the binocular reveals that although the xantholeucophores are typical of albinos, i.e., are fully expanded, there has been a great replacement of the epidermal melanin. This replacement was readily confirmed by a study of whole mounts of the epidermis which reveal that both components of the epidermal melanin have been increased (compare figs. 21 to 23 with figs. 14, 20). The free pigment is not, as in the typical albino, distributed in diffuse aggregations, but is found in considerable amounts in all of the superficial epidermal cells. The epidermal melano- _ phores also show a depth of color never attained in the typical albino, as is most clearly shown if the processes of the broadly expanded melanophores (a condition readily obtained by the death of the animal) of the posterior-lobe-fed albino be com- pared with those of the albinos on any other diet. THE PARS BXJCCALIS OF THE HYPOPHYSIS 29 Not only are the melanophores darker in the albinos receiving posterior lobe, but the number of these cells is also increased (table 1). Instead of averaging but thirty-eight to the unit area, they average in excess of fifty to this unit area (0.36 of a sq.mm.). Thus both in number and pigment content the epi- dermal melanophores of these animals are intermediate between those of the normal specimens and the typical albinos. "'^ It is not on the structural side alone that we find changes with a posterior lobe diet. The epidermal melanophores of these posterior-lobe-fed specimens uniformly exhibit a great and persistent contraction; they are uniformly either rounded or, at most, slightly irregular in shape, appearing as intense dark dots in all areas of the dorsal epidermis (fig. 21). Moreover, this contraction is persistent. After days of the substitution of another diet, it still persists; indeed, under the most potent stimulus — light and heat — for causing an expansion of the epidermal melanophores of the albino, a considerable interval must intervene in which neural lobe is not fed in order to obtain a decisive expansion of these cells. It is cxu-ious that no increment in the epidermal melanin was produced in the unoperated tadpoles with a diet of fresh posterior lobe. Not only did no pigmentary increment result, but the contraction of the epidermal melanophores so typical of albinos on this diet did not regularly occur, or at most was but transitory. Of the other substances which have been fed, none have ap- preciably affected the epidermal melanophores. One, namely, hver, has produced unmistakable differences, however, in the appearance of the xantholeucophores as contrasted to the albinos upon other diets. This effect is not expressed in any alteration in the usual extreme expansion of these cells, but rather in their color. These specimens do not show the golden sheen of the anterior or posterior lobe or adrenal cortex fed animals, but exhibit a chalky whiteness especially noticeable in the gill regions (figs. 46, 48), an appearance probably due to a reduction in the lipochrome content of the cells. " The author has not supplied albinous larvae with posterior-lobe extracts. It is apparent from Allen's work, however, that if they are potent in causing a melanin increase, they must be fed for a considerable interval of time. 30 PIGMENTARY GROWTH AFTER ABLATION OF The responses of the chromatophores to various physiological and pharmacological agents It has been shown that the pigment cells of both the normal and the albinous tadpole are influenced by the internal secretory system. Since the endocrine system is abnormal in the albino, it is logical to inquire whether the atypical pigmentary system of these specimens will react to various stimuli as does this sys- tem in the normal animal. Although we can largely confirm Laurens' observations (made on Amblystoma) upon the irregular reactions of the epidermal melanophores, it will be recalled that in the unoperated tadpole the usual state of these cells under indifferent environmental conditions — diffuse light and a gray background — is one of two-thirds to full expansion. The xantholeucophores, on the contrary, are punctate or slightly expanded, while the deep melanophores are fully expanded. The usual condition of the epidermal melanophores of an albino in our so-called 'indifferent' environment was one of complete, or nearly complete, contraction. The xantholeuco- phores, on the contrary, are broadly expanded. If we now place a large albinous and a large normal" specimen in the dark for a period of one to two hours at room temperature, we find certain well-marked changes take place. The epidermal melanophores of the normal, if not completely expanded, become so, but those of the albino being previously greatly contracted are unaffected. The xantholeucophores of the normal become punctate; these cells in the albino remain fully expanded. The deep melanophores of both animals become greatly contracted, the animals then displaying a 'transparency' typical of the con- tracted condition of these cells. A low temperature (0° to 15°) apparently does not increase the reaction to any extent, the epidermal melanophores of the albino surely not being affected. " As complete an analysis of the reactions of the epidermal melanophores in the young normal tadpole as could be desired has not been made. The reactions here described unless otherwise stated apply only to large larvae at a time shortly pre- ceding metamorphosis (in the normal). THE PAKS BUCCALIS OF THE HYPOPHYSIS 31 If we now subject these large animals to the opposite environ- mental conditions (direct sunlight, a white background, and a high temperatiire— 33° to 35°), we see a striking change take place. The epidermal melanophores of the normal frequently gradually contract and may ultimately after an hour or even less at times assume only a slightly expanded condition (fig. 19). These cells in the albino, on the contrary, invariably expand, ultimately exhibiting a fully expanded condition (fig. 20). There is thus frequently exhibited a reversal in the reaction of these cells in the large specimens of these two types of animals, i' a reversal not evinced to its fullest extent with any stimulus tried by the author save that of heat combined with direct sunlight. Either extreme heat or light evokes this reaction to some extent, the relative importance of each factor, however, being difficult to determine. A normal specimen, if placed in the dark at 30° to 35°, may exhibit after two to three hours partly contracted superficial melanophores. If the temperature becomes normal, these cells expand; if the temperature is maintained and sun- light used, further contraction not infrequently takes place. An albino, if placed in the dark at 30° to 35°, exhibits in two 1' One of the most striking features of the experimental work upon the epidermal pigment cells of the albinous and normal larvae has been the invariable response of these cells in the former as contrasted to their uncertain response in the latter. Of the very considerable number of albinous larvae which have been subjected to the stimulus of 'light and heat' a response, one of expansion, has invariably been exhibited by the epidermal melanophores. In contrast to this stands the variable response of these cells in the normal larvae. Because of the influence which the internal secretory glands have upon these pigment cells, this invariable response in the albino would appear to be of considerable significance, since certain of the endocrine glands in this type are structurally and also presumably functionally deficients Consequently, we could expect that the response of these cells in this form would be more certain than in the normal where these cells are under the variable influence of the internal secretory products. In 1919 (Proc. Soc. Exp. Biol, and Med., 44-1419) I called attention to the op- posed reaction of the epidermal melanophores in the albinous and normal larvae. The experimental work upon which this description was based was carried out upon the older albinous and normal larvae and should have been so stated. These normal larvae very uniformly exhibited a contraction of their epidermal melano- phores when subjected to 'heat and light' as contrasted to the expansion of these cells in the albino under identical conditions. Subsequent work has revealed a more variable response of these cells in the normal, but not in the albino. 32 PIGMENTARY GROWTH AFTER ABLATION OF hours partly expanded epidermal melanophores; if the tempera- ture becomes normal, they contract to the usual condition; if the temperature is maintained and sunlight used in addition, they expand to the maximum. Thus we see that, although high temperature alone evokes the reaction, it is incomplete and slowly expressed, double the time being necessary. The same condition prevails if light alone is used. The reactions are evoked slowly and incompletely. Thus a combination of both light and heat is necessary to evoke the maximal response in the shortest time. It has been mentioned that the xantholeucophores of the normal are uniformly nearly punctate in a dark-adapted animal. If the large normal animal be subjected to high temperature and direct sunlight, these cells become broadly expanded (fig. 19), never exhibiting, however, as great an expansion as invari- ably is presented by these cells in the albino. Thus we see that in the large normal animal there is frequently a reversal in the reaction between the 'interference' cells and the epidermal melanophores — one expands and the other contracts. Such a correlated opposite reaction of the pigment-cell groups no longer holds in the case of the albino, inasmuch as the xantholeuco- phores are always maximally expanded there. However, since under the usual environment the epidermal melanophores of the albino are almost maximally contracted, these two groups of cells are consequently usually in an opposite physiological condition, as is the case in the large normal larvae. We have already given evidence for our belief that the failiu-e of albino xantholeucophores to react to stimuli is due to a tonic hormonal disturbance present in the tissue juices. The deep melanophores with a white background and light expand; in darkness they contract; thus, in both tjrpes of ani- mals their reaction is identical under all the environmental conditions employed by the author. Specimens that have been thyroidectomized at an early stage, as is well known, exhibit no pigment deficiencies. Under the conditions of hght and heat or of darkness, they react as do their unoperated brothers, with this difference: the reaction, THE PARS BUCCALIS OF THE HYPOPHYSIS 33 although of an identical nature, is more slowly evoked. In determining the possible cause for this tardy response, the work of Levy is particularly suggestive. This investigator has shown that the reponses to adrenahn are more rapid if the thyroid has been stimulated or its extract injected. Although it has not been shown to what extent these responses of the pigment cells are referable either to the adrenal or directly to the sym- pathetic nervous system, still the slower responses of thyroidless larvae may well be of considerable significance in view of Levy's work. Fm-thermore, since pigment cells are formed and react in the thjToidless specimens in an identical manner with those of the normal, it is apparent that neither their atjrpical forma- tion nor reaction in the albino can be referred to the atrophy of the thyroid effected by the hypophysectomy. It has been shown that the epidermal melanophores of the • posterior-lobe-fed hypophysectomized specimens exhibit a uni- form state of great contraction, a contraction that persists for an extended period after the substitution of some other diet. WiU these cells then respond to light and heat as do those of the typical albino? It will be recalled that in the typical albino these cells, unlike the xantholeucophores, are sensitive to brilliant illumination coincident with increased temperature, undergoing an expansion. But the epidermal melanophores of the posterior-lobe-fed albinos stubbornly maintain their contracted condition with this stimu- lus and appear incapable of responding. Lest it be supposed that the reaction of these cells has approached that frequently exhibited by normal larvae, one need only to try their response to complete darkness where normal behavior would give us full expansion. Under all circumstances, their contracted condition is maintained just as is the corresponding persistent expansion of the xantholeucophores in these hjrpophysectomized larvae. It was highly interesting to note that an abrupt change in the diet of the posterior-lobe-fed albinos, a substitution of a liver diet, led gradually to a resumption of the response char- acteristic of the typical albino. After an interval of one week in which they have received no nervous hypophysis they become MEMOIR NO. 11. 34 PIGMENTARY GROWTH AFTER ABLATION OP definitely expanded (fig. 22) when subjected to heat and direct sunlight. This response becomes more and more marked, until after five weeks we have an almost typical response (fig. 23). The xantholeucophores in these animals as in the typical albino remain broadly expanded under all environmental conditions. Posterior lobe feeding, then, which gives us an epidermal melano- phore system which approaches the normal in melanin content, does not permit these cells to exhibit normal responses to light and heat. It merely maintains them in a refractory and con- tracted state, and when the feeding is stopped they exhibit the typical physiology of the albino. The stimuli to which the two types of larvae have thus far been subjected are such as they might meet in the extremes of their normal environment. There is yet another class of stimuli which may be employed, namely, pharmacological agents, in which the larvae may be directly immersed. By this means we may be able to secure further evidence in regard to the endocrine locus responsible for the peculiar functional state of the chroma- tophore groups in the albino. Of the substances employed (pars intermedia emulsion, pituitrin, adrenalin, pineal gland emulsion), the first — pars intermedia emulsion" — from the pro- found pigmentary changes which it induces in the albino, es- pecially merits attention. Atwell has called attention to the darkening of albinous larvae when placed in emulsions or ex- tracts of this substance and has noted the response — one of expansion — of the deep melanophores to this treatment, a re- sponse which led him to refer the usual picture of albinism to a contraction of this melanophore group, and conversely the darkening of these treated albinous larvae to the expansion of these cells. We can fully confirm Atwell's observations as pertains both to the darkening of these larvae and the complete expansion of the corial melanophores when placed in the pars 1' The pars intermedia substance was secured by carefully clipping off pieces of this glandular substance from the subjacent neural lobe, as described by Herring ('14) and Atwell ('18). ^ After drying on a warm plate the powdered substance was triturated with the desired amount of tap-water. THE PARS BUCCALIS OF THE HYPOPHYSIS 35 intermedia solution.'' Such a darkening clearly occurs, but an attendant color change was unnoted by Atwell. These larvae not only become darker, but they almost completely lose their metaUic iridescent appearance so characteristic of the picture of albinism. As has been previously stated, this metallic effect is due to the broad expansion of the xantholeucophores, an effect which is displayed in its full beauty only because of the deficiency in the epidermal melanin which characterizes albinos. As might be surmised, then, these 'interference' cells have suf- fered a great contraction, causing an almost entire loss of the metalUc-like albinous picture. Still another pigment change is wrought by the pars intermedia solution. The epidermal melan- ophores undergo expansion, ultimately assuming the expanded condition typical of the normal animal under 'indifferent' en- vironmental conditions. It is thus seen that the pars intermedia emulsion can be described as bringing about a normal physio- logical state in this atypical pigment system of the albino and, as will be later pointed out, aids us in determining the endocrine locus responsible for this pigmentary disturbance.^" When large albinous or normal specimens are placed in an adrenalin^! solution, serious respiratory and cardiovascular symp- toms develop. The respiration becomes labored and slowed and the heart beat decreases in rate. Although more active for a few minutes, they soon become sluggish and rest in any posi- tion. Gradually the gills become flushed and it is noted that " Two strengths of pars intermedia solutions have been used. One made by triturating 10 mg. of the dried substance in 100 cc. of water, the other by triturating 10 mg. in 300 cc. of water. Both solutions produced identical effects, but at a different rate. The larvae would later succumb when left in the stronger solution for a period of one and a half hours or longer. ^ The immersion of larvae in Pituitrin (Parke, Davis & Co.) has produced neither constant nor pronounced changes in the pigmentary system even when used in strengths sufficient to cause the serious distress or death of the animals. An emulsion of the posterior lobe (less the pars intermedia), even when used in strengths greatly in excess of that employed with the pars intermedia, appears not to evoke this reaction. Solutions of hypophysial colloid cause no pigmentary response. ''■ Adrenahn (Parke, Davis & Co.) has been employed in strengths of 1 to 20,000 to 1 to 80,000. The stronger solutions (1 to 20,000 and 1 to 40,000 in amounts of 10 to 40 cc. per animal) have been of more value, and attention is called only to the responses evoked by tkese strengths. 36 PIGMENTARY GROWTH AFTER ABLATION OF the entire animal appears somewhat translucent. While evoked to an extent by the lower dilutions, these effects are much more clearly expressed with the stronger dosages. If the animals be examined with the binocular at five-minute intervals, the progressive alterations in the pigment cells can be followed. Especially uniform and particularly well pronounced is the con- traction of the deep chromatophores. This contraction is the most important factor in the translucent appearance which follows the administration of adrenalin. In adrenalin treatment the superficial melanophores are also affected, those of the albino more uniformly than those of the normal. In the albino these cells gradually expand and after an average period of forty to forty-five minutes a very changed state is evident. The picture presented is the reverse of that which obtains normally. Instead of these cells for the most part being in a contracted condition, they now show a state of expansion. Although only a part of this cell type is completely expanded, the percentage of completely expanded cells has definitely increased and all exhibit a more expanded state than before treatment. In the large normal animal the response of these cells when a considerable amount of fluid is used (40 cc. per specimen) is one of expansion. If only partly expanded, the processes soon present their maximum size; if already com- pletely expanded, this condition persists. With a small amount of fluid (10 cc. per animal) these effects are not so pronounced and, curiously, are not infrequently reversed. The third group of pigment cells, the xantholeucophores, also respond to adrenalin. In the albino the surface layer of these cells is the first to withdraw its processes, a response which may or may not extend to their more deeply placed brothers. Indeed, not all of these cells of the superficial stratum are af- fected, for here and there are interpolated areas of uncontracted cells, and a maximum contraction of all these cells has not been secured in the large albino, save with a lethal dose. In the normal, these cells react in a most varied manner, their reac- tion, as with the epidermal melanophores, apparently depending upon the amount of fluid used. With the larger amount there THE PARS BUCCALIS OF THE HYPOPHYSIS 37 is usually a contraction, although sometimes preceded by a preliminary expansion. With the smaller amounts these cells usually expand, there then being presented in the more favorable cases xantholeucophores of approximately the same size, both in the albino and in the normal. The deep melanophores of both albinous and normal larvae contracted by a solution of pineal glands (two beef glands in 150 cc. water), the same transparency being exhibited as after adrenalin." This contraction of the deep melanophores, which, as pointed out, can be produced by darkness as well, somewhat intensifies the albinous appearance of true albinos, but its ex- treme effect in no case renders normal larvae albinous in appear- ance. These facts are of service in the evaluation of the relative significance of the various groups of pigment cells in producing the picture of albinism. The anatomical and physiological findings which have been detailed may now be reexamined in order that we may be able to evaluate the relative importance which the various com- ponents of the pigmentary system have in creating the picture of albinism. Any explanation of structural or functional causes for the albinous appearance must keep clearly in mind the ar- rangement of these pigment cell types. It will be recalled that the deepest group of pigment cells — the corial melanophores — are overlaid by double strata (in most regions of the body) of xantholeucophores, which in turn lie beneath the epidermal pigment formed by the double component of free melanin and epidermal melanophores. With this arrangement in strata it is obvious, then, that a decrease in the color depth of an over- lying stratum will exaggerate the effect of the subjacent one, or conversely an increase in the color depth of a stratum will proportionately decrease the color effect of those lying beneath. The decrease in pigment which obtains in the superficial pig- ment layer (epidermal) of the albino then exaggerates the chro- matic effect of the abnormally expanded subjacent strata of the ^' With neither of these agents was the extreme transparency gained which Mc- Cord and Allen describe. Indeed, it is difficult to conceive how such an extreme effect could be produced with the very considerable amount of melanin which the epidermis of these specimens contains. SS PIGMENTARY GROWTH AFTER ABLATION Of xantholeucophores, whose abnormal expansion in turn masks or minimizes the effect of the underlying layer, the deep melano- phores. The mere arrangement which obtains, together with the physiological and structural modifications met in the albino, tends to exaggerate the effect of the 'interference' cell layers, and to minimize the r61e of the melanin-containing cells (the epidermal melanin being structurally deficient, the deep melano- phores being masked by the overlying, broadly expanded layers of xantholeucophores), hence the color of the albino is char- acterized by its iridescent metallic nature. This effect is different from the transparency displayed by dark adapted normal larvae or those under pineal or adrenalin treatment, procedures which contract the deep melanophores and, since the xantholeucophores are unexpanded, enable this effect to be seen in a general lighten- ing of tone (translucency) of the larvae. It is possible to still further test the validity of this reasoning by examining the results of our experiments in which larvae were submitted to various stimuli (environmental and pharma- cological) which brought about changes in the components of one or more of these pigment cell layers. A pronounced darken- ing of the albino was caused by immersion in pars intermedia solution. This change, however, does not permit us to analyze the r61e played by the various pigment strata, since all are affected (it will be recalled that the deep and superficial melano- phores expand and the xantholeucophores contract, the epi- dermal free pigment of necessity not being changed). Yet by applying a test by which the pigment cells, save for the xantho- leucophores, undergo the same change as with pars intermedia emulsion, we can determine whether the expansion of the deep and superficial melanophores has materially contributed to this darkening and loss of the metallic tone.'is Such a test is afforded by placing albinous larvae in intense light when it is seen that they do not materially darken in color, although the deep melano- phores may have been previously contracted. The translucency resulting from a maximal contraction of these cells has pre- 2= The epidermal melanophores are so scanty in number and pigment content that their expansion a priori could have little effect. THE PARS BUCCALIS OF THE HYPOPHYSIS 39 viously been noted. It thus appears evident that the role of the deep melanophores in the formation of this picture of al- binism is of no great importance. In fact, they are so completely masked in the body region in the albino by the overlying, broadly expanded xantholeucophores that from the anatomical arrange- ment alone it would be apparent that in R. boylei they could play no significant role in the formation of this picture, and most certainly not the primary role as maintained by Atwell in R. sylvatica. It is also possible to test the relative significance of the dimin- ished pigment content of the epidermis in the formation of this albinous picture. Attention has been called to the partial replacement of the pigment deficiency which takes place with a posterior lobe diet, and it was seen that these larvae became distinctly darker in color than the albinos not supplied with this gland. Or again, in the skin exchanges, the normal graft, whose xantholeucophores become broadly expanded, neverthe- less is decidedly darker than the albinous host. This color depth can be referred in both cases in part to the color directly contributed by this epidermal melanin and in part to the masking of the underljdng xantholeucophores by this pigment. It thus seems clear that the anatomical factors chiefly involved in the picture of albinism are, first, the broad expansion of the xantho- leucophores; second, the diminution in the epidermal melanin which permits full display of the refractive xantholeucophores. The greatest interest attaches to the determination of the endocrine locus responsible for the altered structural and physio- logical state of the pigment cells. Attention was called to the fact that the feeding of neither anterior lobe, adrenal cortex, adrenal medulla, nor liver deepened the color of the albino. On the other hand, a diet of posterior lobe (and associated pars intermedia) materially darkened the albino — an effect immedi- ately referable to a partial replacement of the epidermal melanin. Also the immersion of the larvae in a solution of pars intermedia alone, of all the endocrine extracts employed, produced a normal physiological condition of the chromatophore system. It would 40 PIGMENTARY GROWTH AFTER ABLATION OF thus appear from these responses that the extracts or diets of the intermediate and posterior lobes of the pituitary were inti- mately concerned in this pigmentary fault. Corroborative evi- dence is supplied by a study of the structural malformations occurring in the endocrine glands of the albinous and the par- tially hypophysectomized larvae. For, as will be detailed in a subsequent section (4), the neural hjrpophysis, thyroid, epi- thelial bodies, adrenal cortex, and adrenal medulla suffer definite and often profound structural and size alterations in the albino. In the partially hj^ophysectomized tadpole, however, whose pigment system may suffer a profound disturbance (indeed, the picture of albinism approaching that of the true albino), none of these internal secretory organs save the posterior lobe and the associated pars intermedia exhibit significant structural defects. ^'^ The evidence thus points to a deficiency in the secre- tion formed by either the posterior or intermediate lobes of the pituitary or by the 'interaction' of these two intimately asso- ciated components of the pituitary as being responsible for the pigmentary upset in the larvae." ^' Further evidence freeing the thyroid from responsibility for this pigmentary disturbance comes from the thyroideotomized specimens whose pigmentary system is not at fault. ^' Indeed, the pigmentary system in the frog, at least, appears to be very sensitive to hypophysial disturbance. This is shown in a striking way by one specimen of a considerable number (seventy) of tadpoles which suffered the extirpation of their pineal glands in an early larval stage. In this operation the aqueduct of Sylvius of one specimen was inadvertently injured, an injury leading to its complete occlu- sion. This apparently induced an internal hydrocephalus of the III and lateral ven- tricles with the consequent inevitable pressure upon the pituitary, sections revealing a flattened gland slightly reduced in size. The specimen exhibited unmistakable albinism,' a condition unquestionably referable to the compression of the pituitary gland, since it is certain that this pigmentary disturbance was not due to the loss of the pineal body, for, as has been shown by Laurens ('13), the loss of this gland produces no fault in the pigmentary system, ample confirmation of which has been made in this laboratory. I'HE PARS BtrCCALIS OP THE HYPOPHYSIS 41 3. GROWTH DISTURBANCES Little doubt can be entertained but that the pituitary gland plays a significant role in the regulation of growth. Observa- tions on clinical material which have been elucidated and ex- tended by experimental operative work, more particularly by that of Paulesco and Gushing, have shown rather clearly that a hypersecretion of this gland results in an overgrowth of the skeletal system (giantism and acromegaly), the opposite picture of a pituitary deficiency being seen in dystrophea adiposo- genitalis. Clear as such findings appear to be, it seems the more enig- matical that the administration of this gland has failed to elicit a definite growth accelerative response, indeed the response most uniformly evoked being one of retardation. The evidence so far presented is in many cases at least of such an equivocal and uncertain natm-e as to come well within the limits of experi- mental error and to even be subjected to an opposite interpre- tation. This can be said not only of the feeding experiments of different investigators, but of single investigators as well, and only to the latter do we need to address our attention for a moment. Aldrich ('12), from the administration of anterior lobe to pups, concluded that growth was not stimulated nor retarded, but later concluded from work on white rats that growth retardation occurred (Aldrich, '12). Robertson, in the most elaborate work which has yet appeared upon this subject and who has not only used large numbers of animals of the same species (white mice), but who has also applied the valuable adjuvant of refined statistical methods to his data, at first concluded (Robertson, '16), "The administration of 0.125 gms. per day per animal of fresh anterior lobe pituitary tissue to mice, beginning at 4 weeks after birth (conclusion of the second growth cycle), leads to retardation of growth during the earlier portion of the third growth cycle, between the 6 th and 20th weeks. In the latter part of the third growth cycle, how- ever, from the 20th to the 60th weeks after birth, the growth of the pituitary-fed animals is markedly accelerated, so that they 42 PIGMENTARY GROWTH AFTER ABLATION OP not only catch up to the normals, but actually, at about one year of age, come to surpass the normals in weight." Later he says (Robertson, '19): "The computation of the deviations in terms of probable error units shows that the effect of the dosage of pituitary tissue administered upon the growth of the male animals was of uncertain significance, since the observed deviations were only from one to two times the probable error. That the deviations from the normal nevertheless were real and due to the administration of the pituitary tissue is evidenced by the much greater effect of the same character upon the female, consisting of a retardation during the earlier stages of growth. In both males and females the deviations from the normal after the 30th week are of indeterminate significance; that is, the growth curves of the normal and of the pituitary-fed animals are, so far as our estimates reveal, identical after seven months of age. Hence the preliminary retardation of growth has clearly, prior to the 30th week, been succeeded by acceleration." That a transient growth retardation in the young animal results from pituitary administration seems evident not only from the work of Robertson ('16, '19), but from that of Cerletti ('07), Sandri ('09), Aldrich ('12), Wulzen ('14). But that any subsequent acceleration which may be exhibited is due to anterior lobe administration is not clear. Indeed, it seems not improbable that such an acceleration is compensatory, such as Osborne and Mendel have shown to occur with an adequate diet after an animal has been stunted by undernutrition. This seems the more probable in view of Robertson's feeding experiments with Tethelin in which he was forced to abandon the view which he had previously strongly urged — that his alcoholic anterior lobe extract (Tethelin) supplied a growth-accelerating principle. These animals, indeed, exhibited a more pronounced secondary acceleration when the Tethelin diet was withdrawn than when it was continued. The principle has been formulated by Halsted that a 'physio- logical deficit' is essential for successful organ transplantation. From analogy it seems not improbable that if a physiological deficit could be created in experimental animals, they would *HE PARS BUCCALIS OF THE HYPOPHYSIS 43 be able to 'utilize' the growth-maintaining ('accelerating') prin- ciple of the administered anterior-lobe substance as they could not do were this deficit not existent. If, then, the animals suffering from such a deficit exhibited a pronounced retardation in growth and if in other animals this retardation could be pre- vented by pituitary administration, we would obtain a com- bination of conditions giving a pronounced effect from such a dietary regime. Such indeed is the case in the hjrpophysec- tomized (albinous) frog tadpole, as has previously been pointed out by the author (Smith, '18). The growth response to an- terior lobe administration in such animals, as compared to the normal, is greatly magnified, since, 1) the hypophysectomized frog tadpole grows at a slower rate than the normal (Smith, Allen) and never attains, or attains only after an extended period, the size of the normal tadpole (Smith) and, 2) the oper- ated tadpole supplied with bovine anterior lobe not only ex- hibits a normal growth rate, but, due to the persistence of the larval condition and the consequent extension of the larval growth span, ultimately attains a size notably in excess of the normal. By this sensitiveness of the albinous frog tadpole a clear response to the growth-maintaining principle of the pituitary has been secured." The ease of securing such operated material, its prolonged sm-vival after buccal hjrpophysectomy, and the magnified re- sponse of the albinous frog tadpole to anterior-lobe adminis- trations all combine to make this material of singular value in testing various hypophysial extracts and residues for the pres- ence or absence of the growth-maintaining ('accelerating') prin- ciple. Such material alone affords an ideal way to analyze physiologically the various hypophysial substances which have been .administered in order to determine the role of this gland * It will be quite evident why the term growth- 'maintaining' substance is used in comparing the growth rate of the albinous larvae with the growth rate of the normal. The growth rate of the albino is not accelerated by the administration of anterior lobe as compared to the normal; their growth rate is maintained. Such a diet, however, clearly accelerates the growth of the albinous tadpole as compared to their albinous fellows on liver diet. 44 PIGMENTARY GROWTH AFTER ABLATION Ol' in growth." It is obvious that a determination of the presence or absence of a growth-'accelerating' principle in an extract or fraction of this gland can hardly be made with the normal animal when the administration of the fresh gland does not call forth such a response. The growth rate of the albino; the effect of the administration of various hypophysial substances upon this growth rate In order to discuss the deviations from normality of the growth rate of the hypophysectomized tadpole and the effect of the administration of fresh bovine anterior lobe and its extractives and residues upon these deviations, it will first be necessary to examine the growth rate of the normal unoperated tadpole. The growth curve of a normal tadpole with a liver-lettuce diet consists of three phases, the first two indistinctly separable, the third more pronounced. An early transient and not clearly marked period of slow growth, which perhaps should receive even less emphasis than has been accorded it (Smith, '18), shades insensibly into a protracted second phase of rapid growth during which the tadpole attains nearly its maximum size, and which is in turn terminated rather abruptly by the advent of meta- morphosis (fig. 1). The administration of bovine anterior lobe neither alters the nature of this curve nor produces quantitative results of unquestionable significance, although there appears to result in most cases from this diet a small increment in size and a somewhat earlier completion of metamorphosis. The growth curve of albinous frog larvae with a liver-lettuce diet differs in character and in magnitude from that of the nor- mals (fig. 1). I have described these changes previously (Smith, '18), and later work has amply confirmed them. A retardation in growth following the appearance of albinism is unquestioned and becomes progressively greater until approximately a 30- to 2' Caselli ('00), whole-gland glycerin extracts; Cerletti ('01), a centrifugalized aqueous-glycerin emulsion; Aldrioh ('12), fresh desiccated non-defatted anterior- lobe substance; Aldrich ('12),. fresh defatted anterior-lobe substance; Robertson ('16), fresh anterior lobe and his alcoholic extract, Tethehn, of the desiccated an- terior lobe. THE PAKS BUCCALIS OF THE HYPOPHYSIS 45 Fig. 1 Growth curves of normal frog larvae supplied with a liver diet, hypophy- sectomized frog larvae supplied with liver and with fresh anterior-lobe substance. The ordinates represent the total length of the larvae expressed in millimeters. 46 PIGMENTARY GROWTH AFTER ABLATION OF 32-mm. stage (midlarval period) is reached. At this stage the retardation becomes extremely pronounced and an abrupt change in the direction of the growth curve ensues. So definite and so characteristic is this point that it appears to indicate a sig- nificant period in the development of these albinous larvae and hereafter will be designated as the 'critical point.' Both in time and stage of development this point has been coincident in the curves plotted from data secured in 1918 and 1919. From this 'critical point' the divergence between the curves of the normal and albinous larvae increases for a time, the curves later approaching each other again due to the onset of meta- morphosis in the former and the continued growth of the latter, ^s It is now possible to inquire whether the administration of a 'substitution' or 'replacement' diet of fresh bovine anterior lobe will effect a normal rate of growth in these animals suffering from hypophysial deprivation. This can be answered for the frog tadpole in the affirmative. The albinous frog tadpoles sup- plied throughout their growing period with a continuous diet of fresh anterior-lobe substance exhibit no significant growth devia- tion from the liver-fed normals (fig. 1, table 2). Since meta- morphosis does not supervene, however, their growth extends beyond the normal larval period, and they consequently attain a size notably in excess of their unoperated normal fellows.'"' The alimentary regime of fresh bovine anterior lobe has then clearly supplied the morphogenic principle lost by buccal hypo- physectomy. It is conceivable that pituitary substance greatly in excess of the amount actually requisite to replace the growth principle 28 It will be noted from the tables that after September 7th a continuous diet of fresh bovine anterior lobe was supplied the albinos whose curve of growth is here plotted. This apparently caused an abrupt rise in their growth curve. The growth curves of a group of albinos supplied throughout their life-span with a liver diet are shown on page 18 (Smith, '18). 2' The final and rather abrupt termination in growth exhibited by the albinous specimens might conceivably be due to one of at least two factors: I) a self -limiting growth factor inherent in every organism; or, 2) a seasonal growth rhythm. That this is not attributable to the latter is shown by a group of albinous frog larvae which survived for seventeen months. No resumption of growth occurred in the second season; rather, they became progressively more lethargic, the anorexia in- creased, and death finally supervened. THE PARS BXJCCALIS OF THE HYPOPHYSIS 47 s H Si ►J gj m o o s- 7 k -^ iS <^ <^'^ ^S CH 1— I i-t "^ 1— I IN 2 2 « lO o ^"^ ^ lO "o Cl 1* ■* 7 OT 7 rH 7 CO o -^ d CO '"' ti -^ 2§ o ■ 1-1 fH 1-1 IN (N So. 2"^ 03 S ^ 1' -* s» CO 7 CO O CO 00 CO o 1 1-i I— 1 cJj -^ t^2 r^ =^ tJ. <^^ ti" 1—1 1-1 rH (N IN IN 2 -o lO 00 1-1 S^-" lO •Q o> V ^ v «- 7 o 7 CO I ^i '"' •— 1 ci-' o o ^" ^ lO S n ^1 T" '^ S^ 00 "N rH ■^ CO ^ < CD o 05 ;ij=^ CO rH 1-i rH sS (N O O I I s Fresh glandu- lar lobe once a week ^ -o J^-" in CO "^ 7 IM 00 7 m O CO «j o n o t— 1 ot) ^ i—i (N ^h%°S (N Tti 00 lO S .2 3^ V CO V CO 7 o 7 CO 7 .O i- <^ -^ 4< -^ 00 <^ i <^ 4< 1^ 1—t T— ( lO ^ 1—1 1— I , (N 1—1 / o 00 lo IN "^ IN <^ ^ 00 « 00 ci =^ o 00 f 00 "? 00 s CD t^ t^ « 00 ® o f (N CO to ? t- CO ;£, ■3? 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H. 50 PIGMENTARY GROWTH AFTER ABLATION OP lost by hypophysectomy has been supplied these albinous larvae exhibiting a normal growth rate, since it will be noted that this anterior-lobe substance was fed daily and formed their entire food supply aside from a small component of lettuce. This seems the more probable if the conception usually held in regard to 'hormonal' substances be correct, namely, that minute amounts are effective. But that this substance must be sup- plied continuously is clearly shown by three groups of albinos: 1) a group receiving anterior-lobe substance once each week, liver being fed on the other days; 2) a group receiving anterior lobe until an average length of 36 mm. (on July 18th) had been attained, a liver diet then being instituted; 3) a group (the converse of group 2) reared on a liver diet until they had attained an average length of 33 mm. (on September 7th), the liver diet then being replaced by anterior lobe. The growth curve of the first group, those receiving anterior lobe once a week, although considerably modified, resembles that of the liver- rather than that of the hypophysis-fed group (fig. 2) . Though the incidence of the early retardation is slightly deferred, nevertheless the 'critical point' appears in the usual position, though in a less pronounced manner. After a brief period of depression, the curve rises in a regular manner until the level of the normal is attained. Their curve thus resembles that of the liver-fed albinos, and the ultimate size attained by these specimens is intermediate between that of the anterior- lobe- and that of liver-fed specimens. The second group, those receiving anterior lobe until July 18th, grew at a normal rate up to and for approximately two and one-half weeks past the time when the liver regime was insti- tuted (fig. 2). A definite growth retardation then appeared, the specimens eventually attaining the size of their unoperated companions, but failing by some 10 mm. to reach the size of their operated mates supplied throughout their growing period with glandular hypophysis. The specimens of the third group, those supplied with liver until they had attained a size of 33 mm., had suffered the usual early growth retardation and the pronounced midlarval slowing THE PARS BUCCALIS OF THE HYPOPHYSIS 51 Fig. 2 Growth curves of hypophysectomized frog larvae supplied with a con- tinuous diet of fresh anterior-lobe substance, anterior lobe once a week and anterior lobe from May 6th to July 18th, then liver. The ordinates represent the total length of the larvae expressed in millimeters. 52 PIGMENTARY GROWTH AFTER ABLATION OF invariably exhibited by albinos supplied with this diet. Follow- ing the substitution of a hjrpophysial for a liver diet, a normal growth rate is exhibited for a time, the albinos, however, not attaining the large size induced by continuous anterior-lobe feeding (fig. 1). It seems clear from these three groups that neither an inter- rupted diet of anterior-lobe substance nor a full diet of this substance during merely the early or the late portions of the growing period will bring about a normal growth of the albinous larvae. It is indeed remarkable that albinous larvae, growing at a normal rate in response to anterior-lobe administration, will exhibit a growth retardation so quickly on the withdrawal of this 'substitution' diet. It may then be regarded as demonstrated that the retarda- tion in growth, resulting from the early removal of the epithelial hypophysis, can be ameliorated by a continuous diet of bovine anterior lobe, the growth curves of the albinous larvae thus treated showing no deviation from normality save by its con- tinuance beyond the normal growing period. It will likewise be granted by the reader that such material is of peculiar value for de- termining the presence of the growth-maintaining principle in any hypophysial extractive or its residue. The experimental utiliza- tion of this material should show us whether the extraction of the fresh anterior lobe with boiling alcohol or boiling water removes or destroys this growth-maintaining principle — a test which can be provided with a double check, since both the extract and its residue can be fed to similar groups of albinos. To the group of four substances (aqueous extract and its residue, alcoholic ex- tract and its residue) which were submitted to this test was added the hypophysial colloid occurring in the intraglandular (vestigial cleft) of this gland." '» In the preparation of the aqueous extract and its residue, the ground fresh anterior-lobe substance was extracted in a modified Bailey- Walker apparatus (Rob- ertson) over boiUng aq. dest. for forty-eight hours, five bovine anterior lobes being placed in each alundum thimble. After this prolonged extraction the substance was dried, powdered, and placed in vials tightly corked. Each group of specimens (20 to 24) received the dried residue from one and a half glands at a feeding. The clear, yellowish extract on evaporation yielded a dark, readily soluble amorphous THE tARS BUCCALIS OF THE HYPOPHYSIS 53 Obviously, since the aqueous and alcoholic extracts and the colloid were of slight or no nutritive value, additional food substance had to be furnished, but such food substances were only suppUed after obtaining a maximum intake of the special glandular substance After turning off the flowing water, the special substance was fed, one half-hour later the nutritional substances, liver and lettuce, being supplied. Twenty-four hours later, after removing all food particles, the process was repeated. In the accompanying table (table 3) is shown the number of frog larvae at the commencement of the special feeding and at three subsequent periods. Although the number is relatively small, yet they were average-sized, carefully selected, healthy tadpoles free from injury or malformation. Work with a larger number was precluded, due to the difficulty of securing a suf- ficient amount of the special substances, 90 to 100 bovine hypo- physes being used daily with even this number of specimens. substance giving a lipoid-like odor on burning. The extract from five glands was supplied each group of tadpoles (20 to 24) at a feeding. The feeding was done with sufficient rapidity to insure against an extensive solution of the solid substance, and from the rapidly initiated movements of the tadpoles it was certain that they were feeding directly upon this substance. The alcohohc extract (TetheUn) was suppHed by H. K. Mulford Company. In the feeding of this substance even greater care was exercised than with the aqueous extract, to guard against solution. After a period of a few seconds subsequent to the breaking of the vacuimi-sealed tube, the tadpoles would be feeding upon the substance. Fifty milligrams (the extract from five glands) was given each group at feeding. In the preparation of the alcoholic residue the method elaborated by Robertson for the extraction of Tethelin was slightly modified. Dehydration was carried out with 95 per cent alcohol instead of anhydrous sodium and calcium sulphates, since the addition of the salts would render the residue useless. Following dehydration, the tissue was thoroughly dried over an electric stove, then extracted in a Bailey- Walker apparatus with boiling absolute alcohol for forty-eight hours. The residue, freed from alcohol, and preserved in tightly corked vials, wa.s supplied in amounts equal to one and a half of the fresh glands at a feeding. The colloid was secured from the vestigial cleft, between the glandular and inter- mediate lobes. The amount obtained from the forty-five glands and administered to the twelve to fifteen tadpoles at each feeding varied considerably, but averaged 0.5 gram. Occasional very large pieces (0.675 gram) were found, many other glands reveaUng none. Usually it occurred as a firm, amorphous mass, molded by the shape of the cleft, now and then, however, gels of differing viscosity or even an aqueous-like substance was obtained. Before feeding, the amorphous masses were crushed and mixed with the more fluid portions. 54 PIGMENTARY GROWTH AFTER ABLATIOiST OP H ►J m Eh h3 1 ■^ < 3 li (N CD 10 Ttt ■» § ^is ^|2 "5 IM h; ■W S 1> t~ t^ CD O rS2 m n EH <1 li 10 m t~ CO «3 10 10 10 en C3s S •ZS •e* •Ih S» e a CO ■* IN S a. § I"' e 11 (N !>. t> CD <;a i-H 'S ^l ^ .0 m ^ g a 0] 0^ 8| N M (N T-H Oi t^ ?i B P4 pS T— 1 T— 1 3 &« e CO B CD 1 H ^ CO (M i-H ■< Q ^^•§|ss cni3 C^ , lO ■^ ^ ^ CO (N IM (N -g ,^ ^i^ 8 c; a- a- g CO I-H S 't. !^ S3S o- ty CD CO T-H r-l g 3'^ s s ^ ^ tos s ■s ;a •c^ to 1 tc CO ^ d IN ^ > ■< c THE PARS BUCCALIS OF THE HYPOPHYSIS 55 Further, it was believed that if this number of animals should show an unequivocal response to hypophysial administration, it would furnish convincing evidence of either the presence or the absence of the growth-maintaining principle in these special substances — a belief which we feel has been amply sustained. The growth curves of the albinous frog tadpoles supplied with either alcoholic or aqueous residues show the same main- tenance of growth as is exhibited by their fellows fed with the fresh anterior lobe (figs. 3, 4). No 'critical point' appears; the three curves are coincident. This is not the case with frog albinos receiving aqueous ex- tract, Tethelin, or colloid (figs. 3, 4, 5). Their curves are quite similar to that of the liver-fed albinous larvae. The same early retardation is noted, the 'critical point' is well marked, but appears at a somewhat earlier period (twelve days) and at a somewhat smaller average stage (6 mm.) than their liver-fed companions. Following the critical point, some divergence ap- pears in the three curves. The specimens supplied with aqueous extract exhibit a slight secondary acceleration in growth, but they do not attain quite the average length of their liver-fed mates. The 'Tethelin' group does not exhibit any secondary acceleration subsequent to the 'critical point,' while the colloid group exhibits an even greater retardation than its fellows sup- plied with Tethelin. In order to further test the growth effects of hypophysectomy and its response to an anterior-lobe diet, a series of experiments similar in every way to those just reported upon were carried out with toad larvae. These larvae respond in general in the same way as the similarly treated frog tadpoles. The early retardation, the usual midlarval slowing ('critical point'), fol- lowed by slow growth, appears as in the frog tadpole (fig. 6). In this, my observations are somewhat at variance with those of Allen, who reports no retardation in the velocity of growth in hypophysectomized toads. Fuller reports may explain this essential difference in our results. I wish to emphasize, how- ever, that the early retardation, the 'critical point,' and the later period of slow growth are not so distinctly shown in the 56 PIGMENTARY GROWTH AFTER ABLATION OF Fig. 3 Growth curves of hypophysectomized frog larvae supplied with fresh anterior-lobe substance, with aqueous extract, and with aqueous residue of the anterior lobe. The ordinates represent the total length of the larvae expressed in milUmeters. THE PARS BUCCALIS OF THE HYPOPHYSIS 57 Fig. 4 Growth curves of hypophysectomized frog larvae supplied with fresh anterior-lobe substance, with alcohoUc extract and with alcoholic residue of the anterior lobe. The ordinates represent the total length of the larvae expressed in millimeters. 58 PIGMENTARY GROWTH AFTER ABLATION OP Fig. 6 Growth curves of thyroidectomized frog larvae supplied with liver diet, hypophysectomized frog larvae supplied with fresh anterior-lobe substance, and with hypophysial colloid. The ordinates represent the total length of the Tarvae expressed in millimeters. The tARS BUCCALIS OF THE HYJPOPHYSIS 59 60- TOAD 53- TADPOLES. / 56* 51- /- 52- ■ 48- 46- 44- 42- 40- 1 r / ^ f 36- ^ / / 9'° 'mb'" 34- /j / ; / ^ 1 Se- ij Jj < es- 1 1 ^ 21- / A- 22- ij.iy If" 18- r 16- l/s til- Fig. 6 Growth curves of normal toad larvae supplied with fresh anterior-lobe substance, hypophysectomized toad larvae supplied with fresh anterior-lobe sub- stance and with liver. The ordinates represent the total length of the larvae ex- pressed in millimeters. 60 PIGMENTARY GROWTH AITTER ABLATION OF toad as in the albinous frog larvae. This is also true of their response to a 'substitution' diet of anterior-lobe substance. The growth rate of those toad albinos supplied with an hypo- physial diet does not quite equal that of their normal brothers.'" Neither has the administration of hypophysial extracts, residues, and colloid given as clear results as in the frog tadpole (figs. 7, 8, 9). However, an unmistakable growth retardation is shown by the albinous toad larvae supplied with the extracts arid colloid, and the administration of the residues has caused a distinct acceleration in growth rate as compared to their liver- fed albinous mates. Table 4 gives the measurements upon which the growth curves of the toad tadpole are based, and table 5, the number of specimens used in the study. From a survey of these groups receiving the various hypo- physial substances and liver it is evident that they are separable, whether frog or toad larvae be used, into two distinct cate- gories, both by the size attained and by the nature of their growth curves. On the one hand is the group formed by those larvae supplied with either the alcoholic or aqueous residues; their growth curves are identical with that of the fresh anterior- lobe-fed group; on the other hand is the group formed by the larvae receiving Tethelin, aqueous extract and colloid; these exhibit some variation in size, but their curves are similar in nature to that of the liver-fed albinos. To what factor or factors is due, 1) the normal growth rate of the albinous larvae supplied with fresh anterior lobe and the hypophysial residues, and, 2) the retardation of specimens fed with liver, hypophysial extractives, and colloid? The nor- mality of the growth rate exhibited by the specimens of the first division might conceivably be due to the superior nutritive value of the anterior lobe rather than to a morphogenic agent. If this was the responsible factor, however, we should expect as great a deviation in both the magnitude and the nature of the growth curves of normal tadpoles fed in this way. No such ^1 It is to be noted, however, that the normal specimens received a diet of fresh anterior lobe, not of liver. A slight acceleration in the normal produced by this diet may account for the divergence in the two curves. THE PARS BTJCCALIS OF THE HYPOPHYSIS 61 Fig. 7 GrowtK curves of hypophysectomized toad larvae supplied with fresh anterior-lobe substance, with alcoholic extract, and with alcoholic residue of the anterior lobe. The ordinates represent the total length of the larvae expressed in millimeters. 62 PIGMENTARY GROWTH AFTER ABLATION OF Fig. 8 Growth curves of hypophysectomized toad larvae supplied with fresh anterior-lobe substance, with aqueous extract, and aqueous residue of the anterior lobe. The ordinates represent the total length of the larvae expressed in milli- meters. THE PARS BUCCALIS OF THE HYPOPHYSIS 63 70" TOAD ^ TADPOLES. ^^^ ^ .^ M- V 1 1 w- / r 1 1 too- ' •?s- ,\* / .' / sr 0^/ /h Si- $ 1 J 1 1 /A 48- t f 1 1 1 46- 1 \< / \l> 1 / ^ c^ ' u..—^... o 1 S J f \ 44- 40- 1 r ( 4-" 1 y / / / r/ S6- 1 y .'6 . 34- 1 > 1 •1 1 / ^ 30- 1 1 ' - 1 . {' " 28 - 1 /•' r p 2b- 7 /■■ ■iA - 22- 1 -: 1 1 1 %' 18 1 */6 16 - * 1 1 1 — 1 — 1 1 3- "> 5 E> S = i t^ t Si ^ ^ a w ^ t f Fig. 9 Growth curves of thyroidectomized toad larvae supplied with liver, hypophysectomized toad larvae supplied with fresh anterior-lobe substance and with hypophysial colloid. The ordinates represent the total length of the larvae expressed in millimeters. 64 PIGMENTARY GROWTH AFTER ABLATION OF W i-l m to s s a "8 I ^ >i U , 1 < 11^ CD 7 >o 7 «= ?5 -^ s« CO « =? CO T- To o tH 1-H ^s ^s ico CO CO ft a « 5 fc;^ O CD 05 55? >» IM >o m 7 00 7 « 7 t- t CD IN "p TH "p So T— I >o 03 S "P ■* 1 T "= T ■= 7 o 7 CO 7 t> 7 o CO -■ iO -< s^ cL f^ ^ IM 4< <^ ^S 00 " o tH 1—1 l-l (N (N (N 03 "C CD t- Oi 1-i lO ai ■* 00 T* "= V =o V 00 7 o 7 CO 7 CD 7 ^ 7 '^ CO ^•1 CO '"' Jj -^ ti -- ci> '^' (:!. '^ tIh 4 1—1 tH 1-H i-H (N (M (N IN a o H h n OD U fa o go CD o 7 00 ^^'^ ^« ^'^. ^-=. It cIs-' i-H ss ?5?5 IN h n o o « u 4 Q P CO ^ w '-' 22 4. '^^ i IN i" i M o !5 3 tH 1-H (N (N IN CO z H ^ o o CD t~ 2>o g^-^ ■* 00 1— 1 ?§- S T '° 7* «= 7 (N 7 w 7 00 o o 3 ^1 CO '- ra -■ «!> ^ 2S i C^ 4. =^ li IN ?5S -J "^ i-H tH '^ IM (N (N o o n IP CD s 1-H CO 00 CO CD o 7 lO « 00 7 oi 7 IM 7 ^ CO o 7 ■* 7 CD CO '-^ Jj -I ci -> W <^ 4, =^ CO '^ tl w tH t— 1 1— ( (N IN (N CO W) H CD 7 m ^2 7- N "? 03 7 CD 7 a, CO ^ 3=9 CO -> 1—1 Ji2 i^2 2?^ 4, =^ IN J, IN (N 00 '^ ■ <0 s i5 o CO CD CO § (N CO s ^ isb 3 3 3 I-! ■< 1 THE PARS BUCCALIS OF THE HYPOPHYSIS 65 t~ OS Oi m CO t^ i T) ■* ^ 1* lO ■* >o 05 "? o g O) CO t> CO 1> t^ t~ 00 t:^ Oi 00 s Jl "= £2 40 t~ lO 2 lO a «= 1 (N CD 1 CO c!, CO 1 o I o CO 1 t^ s- Tt< ■ 05 »o o o Ira 3 CO l> 00 >o 00 00 00 00 T SI " T CD ■^ f 00 ^ o TlH l-H ? Tt< CO f lO TlH »o "T 10 o " 1— 1 CO i-H CO T-H CO 4 " 1 ^ 1 l-H ■* 1 ^?^ 1 1-H CO 1 CO CO 1 CO ci " 1 CO 1 Tt< 1 o (N i Tj( 1 Tt< =3 3 CO CO CO CO CO CO TtH ^ -* Ai <1 o o cq ^ CD CD lO on lO IN CO 10 10 ^ 00 ■* 00 ■* o t* 1—1 7 CO 'it ■* ■? o> lO o *? l-H 10 IN "? (N o" 1 00 CO 1 oc T.. 1 00 ■* i-* 1 o ^' 1 ^ tl -* 1 00 ■o 1 o >o ■i lO i'" m CO CO CO tH •* ■* ■* Th lO •a 10 s§« CD lO l> lO 00 S'^ (3- 00 c lO O! C} 10 CO CO CO ^ CO « l> ^ l> CO "? 00 5! f 1* -1 ^" 1 CO CO 1 cc CO 1 CO CO J^s u: CO 1 CO 1 00 CO 1 CD CO 1 1^ -n 1 5 cL-* CO CO CO CO CO CO CO co co CO ^■^ lO t- CI CO -* ^ cc b- o CO >o CD 10 1* o f (N •* ■* "? o ": IC 05 u: o CD l-H CD CD 00 «? 00 i^ 1 l-H Tf 4 Tt( 4 -^ ti ^ 1 ■ c 00 ot ^ c lO i lO 1 10 1 00 lO ti. "= CO CO CO CO CO CO Tj< Tt< '^ ^ '^ a> (M K> ^ lO ifl lO CO IC CO CO lO lO oc ■" 00 00 t? >o 1* ^ ^ 1" a= Th o Tfl o 1* ^ -* Ttl Ttl Tt< "1* -# i" 1 IN to CO 4 CO CO 1 CO ^co 1 oc ^ 1 -* 1 -* 1 T-H 1 oc CI 1 a- -* i ^ CO CO CO CO CO CO CO CO CO CO CO CO 1— 1 b- •^t 1-H t^ Tt l> c (N c T— t (M CO s (M 1 1 J c > c iz Ml -3 03 f^ MEMOIR NO. 1 1. 66 PIGMENTARY GROWTH AFTER ABLATION OF effect has been produced in normal larvae by such alimentary regimes. One article of diet, liver — the chief nutritive substance sup- plied — is common to the specimens of the second division. That the retardation in the growth rate of these specimens is not due to a growth-retarding substance in the liver, but to the absence of the hypophysial growth-maintaining substance, is clearly indicated by three lines of evidence: 1) we should have TABLE 5 Table giving the number of normal, thyroidectomized, and albinous toad larvae on which the accompanying growth-curves are based BUCCAL COMPONENT OP HYPOPHYSIS ABLATED THYBOID ABLATED NOEMAL Liver diet DIET OF GLANDULAR LOBE OF HYPOPHYSIS Liver diet Glandu- lar lobe diet Fresh glandu- lar lobe Alco- holic extract i Alco- .1 holic residue Aqueous extract Aqueous residue Colloid June 25 Aug. 13 Sept. 14 Oct. 20 8 7 7 6 30 23 21 20 8 7 7 4 8 8 6 6 8 6 6 8 8 8 6 8 8 7 7 14 13 13 8 18 18 ■ Killed accidentally. to likewise assume that such growth-retarding substances were in adrenal cortex, adrenal medulla, and posterior lobe, for the same growth delay exists when albinous larvae are fed with any of these substances; 2) Mendel and Osborne ('18) have shown that the proteins of liver are adequate for the needs of nutrition in mammalian growth; 3) normal frog larvae supplied with a liver diet exhibit an entirely normal rate of growth. It seems clear, then, that the normal growth rate of the al- binous specimens receiving fresh anterior lobe or its residues is due to an ahmentary replacement of the growth-maintaining THE PARS BUCCALIS OF THE HYPOPHYSIS 67 substance normally furnished by the tadpole's own gland. It ie significant of the stability of this substance that its efficacy or physiological nature has not been seriously impaired by the severe treatment accorded it in the prolonged extraction with boihng water or absolute alcohol. It would seem certain that the retardation in the growth rate of the albinos receiving liver, hypophysial extracts, and colloid is due to the absence of the morphogenic principle normally furnished by the anterior lobe of the pituitary. The growth rate of thyroidedomized larvae It has been noted by previous workers (Allen, Hoskins) that thyroidectomized larvae attained a size much in excess of the normal and that the pituitary glands of these specimens were relatively increased in size. Since the larval condition persists in the thyroidless larvae and since the hypophysis is hyper- trophied, it might be expected that these specimens would ex- hibit a more prolonged, and a more rapid rate of growth than even the anterior-lobe-fed albinous tadpole. By thyroidectomy, then, there would be obtained tadpoles which would simulate the rapid growth exhibited at times by the acromegalous human subject. Observations, however, made on two groups of thyroidec- tomized frog larvae, one group (seventy-two specimens) fed on anterior lobe, the other (fifty-nine specimens) fed on liver, show that their growth is identical in rate with that of their normal brothers or with the albinos supplied with a continuous diet of the fresh anterior lobe (fig. 5). Their large size is never attained by a more rapid growth rate, but rather, as in the anterior-lobe-fed albinos, by a prolongation of the growth period. No variation in growth rate was caused by feeding the fresh anterior lobe of the pituitary, a non-effect already reported upon by Allen ('18). The enlarged pituitary which these animals possess does not appear to have supplied any growth stimulus in excess of the normal. Nevertheless, a different condition obtains with the thyroidec- tomized toad larvae, at least when supplied with a liver diet. 68 PIGMENTARY GROWTH AFTER ABLATION OF This is well shown by the members of one group (fourteen speci- mens) whose growth rate was more rapid than that of their normal fellows supplied with anterior lobe substance (fig. 9). Moreover, they attained a size notably in excess of that reached by their hypophysis-fed albinous mates. Evidently, then, in the case of the frog larvae the enlarged hjrpophysis, which Allen and Hoskins have shown to result from thyroidectomy, has not supplied any growth stimulus in excess of the normal. To what the acceleration in the toad larvae is due has as yet been undetermined, but that an increased secre- tion from the hypertrophied pituitary may be an important factor, as suggested by Hoskins, seems not improbable. 4. MODIFICATIONS IN THE SIZE AND STRUCTURE OF THE ENDOCRINE ORGANS It seems probable that a tissue, whether its distribution be diffuse or localized, is more labile and 'adaptive' in its early formative stage than in its mature condition. An example of this is seen in the nervous system, which in its mature, highly differentiated condition loses its capacity for regeneration and 'adaptation,' a capacity which it exhibits to a surprising degree, however, in its early formative stages (Lewis), more especially when associated tissues can elicit a developmental response (Burr). It is likely, then, that if a disturbance be experienced by the endocrine system in its early and embryonic stages, greater alterations in its members will result than if this upset had been suffered later in the life of the individual. As has been pre- viously suggested, the more extensive structural changes which are manifested by the internal secretory organs of the hypophysis- free tadpole as compared to the hypophysectomized mammal are probably referable to the earher institution of this disturbance. Extensive structural modifications, as we will show, are invari- ably manifested in the thyroid, the neural portion of the hy- pophysis, and the interrenal and chromaffin components of the adrenal gland subsequent to the early loss of the epithelial hypophysis, while lesser though distinct changes are shown by the epithelial bodies. The behavior of the fat-organ in hypophysial deficiency is also described in this section. THE PARS BUCCALIS OF THE HYPOPHYSIS 69 The hypophysial components The endocrine disturbance leading to the abnormal condition of the epithelial bodies, the thyroid and adrenal glands, sub- sequently to be described, has of necessity been mediated to these distant organs by the blood stream. The neural hy- pophysis, while subjected as are these other glands to this general endocrine disturbance, has, in addition, been subjected to the loss of its intimate anatomical associate, the epithelial hypophy- sis, a loss which in itself might conceivably greatly modify the neural lobe entirely apart from the general disturbance intro- duced into this correlative system of organs by buccal hjrpo- physectomy. That the intimate anatomical association exist- ing between the neural and epithelial components, to which attention has been called by many observers, ^^ jg fundamental for the elaboration of the secretion of at least the posterior lobe, finds support not only in the anatomical evidence afforded by this relationship, but also in the recent work of Schmidt and May from the chemical side." As will be pointed out, cor- roborative evidence bearing upon the significance of this coales- cence will accrue from this study. It must be kept in mind that in this experimental work the buccal constituent of this gland has been removed prior to the union with its neural asso- ciate, which consequently remains undisturbed, and that the infundibular process at this time gives no indication of the structm-al differentiation which it is to later undergo, so that any modifications exhibited by the neural lobe (or the infundibu- lar process) are not referable to any direct operative injury inflicted upon this member. As will be subsequently pointed out more in detail, sections and models of these albinos reveal a profound reduction in size, as well as an atypical shape and '^ Herring, Trautmann, Stumpf, Kohn, Erdheim and Stumme, Vogel, Stendel, and others. '' Schmidt and May, by appropriate chemical treatment of the alcoholic hy- pophysial extract, Tethehn, have secured a substance whose action simulates the active principle of the posterior lobe, i.e., causes a contraction of the isolated uterus and produces a vasoconstriction. 70 PIGMENTARY GROWTH AFTER ABLATION OF abnormal position of the neural lobe.'^ Indeed, this malfor- mation is not limited to the neural lobe, but is participated in also by the floor of the infundibular process. This floor, nor- mally of considerable thickness, is membranous and folded in the albinous larvae. '= It is of considerable importance, in view of the conditions obtaining in the albino, to have clearly in mind the parts of the infundibular process together with their structure in the normal animal, and more especially that of the apical and ventral por- tions. The walls of this process have three divisions i''' 1) a ventromedial portion in contact with the pituitary gland, the 'pituitary' wall or floor; 2) a dorsal portion, the 'saccular' wall; and, 3) the lateral extensions of both these walls which form the shallow lateral concavities, the 'lateral processes.' The neural lobe in its early stage develops near the apex of the in- fundibular process, and in its fully formed condition is readily identified as a distinct element attached by a broad surface to the dorsal wall of the infundibular process, near its ventral border (fig. 25). An examination of the three portions of the infundibular process of the normal animal reveals the fact that these regions are of two structural types. One type, that ex- hibited by the lateral processes and saccular wall, is mem- branous and composed of a squamous ependyma with little or no ectally placed neuropilem. The other type, that forming the pituitary wall, is thickened and composed of a high columnar '* The reduction in the size of the neural lobe in the albino was reported in The Anatomical Record. In the present paper more comprehensive data have been given. ^^ The hypophysis of an anuran larva, in common with other vertebrates, con- sists of four components, three arising from oral ectoderm, one from the infundibular process. The development of those lobes derived from the buccal rudiment (the pars intermedia, the pars glandularis and its paired cephalic processes, the pars tuberahs) conforms in general to that recently described by Atwell for R. pipiens. Wax models, X133, of the pituitaries of nine normal larvae, from 17 mm. in length to the young adult stage have been made (table 6). In addition to these, the neural lobes of ten albinos, ranging in size from 16 to 51 mm., and the neural and buccal lobes of nine partial albinos were modeled. The lobes of the pituitary are readily identified and their relationship to each other is constant (fig. 25, a, b, c), compare to Atwell's figure 13, page 81, but considerable variation in their relative size is shown. '^ The terminology is essentially that of Tilney ('15). THE PARS BUCCALIS OF THE HYPOPHYSIS 71 ependyma with an ectally placed prominent stratum of neuro- pilem (fig. 28). In the albino the saccular wall and lateral processes and pituitary wall all exhibit the membranous type of structure (fig. 29). The pituitary floor, which is normally in contact with the epithelial hjrpophysis and which has been deprived of such contact by the epithelial hypophysectomy, thus exhibits the same tj^pe of structure as those portions not normally in contact with this epithelial component of the hy- pophysis. Directly attributable to the membranous character of this pituitary floor are invariably seen certain pronounced but irregular folds which appear in this structure in the albino (fig. 26), but not in the normal animal (fig. 25). Although such foldings are undoubtedly due to the technical procedure incident to the preparation of this material, yet the membranous nature of the infundibular floor of the albino is directly conducive to them, while the firmer structure of the normal infundibulum prevents them. The abnormality in the infundibular process in the albino is not limited to the pituitary floor, for it is very evident that the neural lobe in the albino as compared to the normal animal of corresponding dimensions (table 6) is also diminished in size, a diminution which varies from 40 per cent to 80 per cent and which transcends very distinctly the considerable variability which this lobe exhibits in both types of specimens.^' Asso- ciated with this diminution in size is a profound alteration in the shape and position of this lobe. Instead of the symmetrical, transversely placed, dumb-bell-shaped lobe of the normal (fig. 25), this dwarfed lobe is oval, asymmetrical in shape and posi- tion, and has no great transverse extent (fig. 26). Normally attached somewhat dorsally to the apex of the iniundibular process and thus abutting and being attached to its dorsal sur- face, in the albino it is constantly found grasping the apex of this process and extends invariably slightly onto the pituitary ^ It does not appear from figures 28 and 29 that the neural lobe of the albino is smaller than in the normal. It must be kept in mind, however, that due to the difference in the shape of this lobe in the two types that a median sagittal section passes through its smallest dimension in the normal animal, and through its max- imum sagittal diameter in the albino (figs. 25, 26). ' 72 PIGMEiSTTARY GROW^fe AFTER ABLAl'lON OS' surface (figs. 26, 29) . This lobe is thus profoundly modified by the loss of its associate. It attains normal development neither as regards size, form, nor position. 's In its response to buccal hypophysectomy the reduction of this gland thus aligns it with the thyroid and the adrenal cortex reduction. Indeed, in those animals suffering only a partial loss of the epithelial hypophysis, the thyroid and adrenal glands are usually not altered in develop- ment, but the neural hypophysis is invariably atypical. In an earlier section of the paper we have referred to the type of tadpole in which the removal of the epithelial hs^pophysis was incomplete. Since these animals usually manifest definite pigmentary alterations, we have designated them 'partial al- binos.'^' Especial interest attaches to the examination of the endocrine system, more particularly its hj^pophysial components in this type, since we have here an intermediate condition be- tween a complete deficiency of the epithelial hjrpophysis and a normal structure. A study of this form throws much addi- tional light upon the interesting relationship obtaining between the neural and epithelial components of the pituitary as well as supplying evidence bearing upon the endocrine locus responsible for the pigmentary deficiency. In the partial albinos no uniformity in shape is displayed by the vestige of the epitheUal h5rpophysis. It is usually oval in outline and of a variable thickness (figs. 27, 30). Only occa- sionally do we find it displaying the normal division into glandu- =' The findings of Allen (Abstracts, Am. Assoc. Anatomists, 1917) are distinctly at variance with those reported here. This author states, "The pars nervosa of the hypophysis forms normally in tadpoles, both Rana and Bufo, from which the anlage of the anterior lobe has been removed." As I have previously pointed out, a median sagittal section of the infundibulum usually shows a neural hjrpophysis of normal or even enlarged proportions. By modeling this component, unmis- takable evidence concerning its diminution in size and atypical form is secured, however. 2' I called attention in 1916 (Proc. Am. Assoc. Anat.) to an albinous tadpole in which a leg development took place. At that time the minute buccal fragment displayed by this animal had not been noted. Allen, in 1918, called attention to the complete metamorphosis of certain toad and frog tadpoles with an imperfect hypophysis and displaying a definite pigmentary deficiency. THE PARS BtJCCALlS OF THE HYPOPHYSIS 73 lar and intermediate components. The size"" of this vestigial gland is always considerably less than that obtaining in the normal tadpole of corresponding dimensions (table 6). As would be anticipated, this epithelial gland, subjected as it was to a varying surgical interference, leaves a vestige which shows great diversity in its position as well as in shape and size. That the position which this lobe assumes is of paramount importance in the influence it may exert upon the other glands and upon metamorphosis, is evident from a correlation of the structural changes enjoyed by the partial albino with the position which the buccal fragment has assumed. In the specimens thus far studied, three positions have been assumed by this fragment: 1) In the first case, this fragment, not separable into glandular and intermediate components, does not reach the caudal ex- tremity of the infundibular process, nor does it touch at any point the true neural lobe (figs. 27, 30). The members of this group, which includes the partial albinos (p 1, p 2, p 3, p 4, p 5), all displayed a pronounced albinism. It is to be noted that none of these animals complete metamorphosis, though, as in the other groups, their thyroid glands are not atrophic. 2) In the second case, the fragment, not separable into glandular and intermediate components, does not reach its usual caudal position, but nevertheless attains a definite though small con- tact with the true neural lobe. This class includes a smaller number of specimens (p 7, p 9, p 10), the members of which display a variable pigmentary disturbance. They complete metamorphosis, though tardily, and the thyroid glands exhibit a pronounced colloidal hjrpertrophy (colloid goiter). 3) In the third case, the epithelial fragment, which is separable into inter- mediate and glandular components, assumes the position typical of the normal animal. One specimen only fell in this class (p 8) ; it did not exhibit a pronounced pigmentary upset; it completed metamorphosis and had a normal thyroid gland. A study of a *' The metamorphic changes in all the partial albinos, except p 1, which died from infection, whose hypophyses were modeled (table 6), had ceased for some time prior to the fixation of the animal. It is certain that the metamorphosis would never have been completed nor even materially advanced had the specimens sur- vived longer. 74 PIGMENTARY GROWTH AFTER ABLATION OF 5i m X No. Of times heavier han pars aeuralis O o T-H to ■* CO 1— I t^ o> «o d T|H 1* CO -* CO +-> •» s 01 ^"..•^ 1-H to o t^ o IM en Tota eight pars itheli 00 CO IN 00 00 d d d o 1-1 T-H I— 1 CO a ^ g ^ t « hj Pa P4 G) 00 (N CO CO o 1—1 CO O 13 TH t^ CO I— ( IM o 00 1^ tH d d lO lO 00 d 6h <3 t3« a (X, ^ c3 05 O 1 Eh O Si-S m -* t^ 1—1 o i-< CD o o i6 T— t CO CD H!)H 00 Til d 1— ( (21 a 00 CO >o 1> 00 to CO t^ T— 1 "* lO Ci 00 to o o d d (N (N T— 1 IN n^ t^ tH t t^ ^ ;:h tH ^ tH o tH (U (p a; ID > > l> > > " -s > •^ -4^ O 1-:! ;3 'i2 3 3 3 ^" ;3 ■Otaaj ^ > ^ > 1-1 ^ 8= ^ g ■^ O '^ O ;z so ^ ^ ^ ^ tH ,tH ,t-l ^ tH tH tH tl =4-1 t+-( «*-! M-( t4-l d >a ■S ISs sal M to o> T-H CO lO t^ 03 1— 1 CO »—) 1—1 T-l 1—1 CO (N WW c3 It ffl o & T3 o3 O THE PARS BTJCCALIS OP THE HYPOPHYSIS 75 CD t^ Cft to '^ CO 1 1 1 1 1 1 1 1 1 CO t^ t^ Ol T— 1 00 1 1 1 1 I 1 1 1 1 T— t tH to to CO ■* lO 1 1 I 1 1 1 I I 1 (M T-^ to 1 1 1 1 1 1 1 1 1 t^ CO '^ ■* (N l> to CO (N a> I— I T-4 O: 'I' 1— 1 tH CO Oi t~ lO to to lO d d d d -< 6 d T-H CM CM i—t ^ tH t-i i~, ^ tH t4 tJ tH ^ («i fr4 ■S «, .2 „, (D > > > > > > 0) -w > s -S S -S • t-l "^ fe -fj -*^ 3 3 3 3 i-A 3 3 73 O 13 ^" —' < to Tf 05 05 ^ T— t T-H Tf i-H >H t~ lO 00 (N CO CO o o CO C35 CK m 1— I .—) I— t T-H ' ' '"^ S" S" -fj +3 bC bD C a o O O O o o t^ O ^ Fh tH tH tH Fh tH ^ ^ ti2 4^ Ch =n eS e^-H e*-t tS CO •O o O 00 CD 00 lO CO 1 ^"^ 1 T-H 1 d d (M d d o Ttl l> 00 CO o 'Jl >o t> CO O 01 (N CO SI 00 o 1 t* 00 o U3 00 OJ en CO £ CD (M CO a> CD d 6 P 1 ■a lO I-H I-H I-H CO tH T3 rt ^ T3 g tH • S c^ '53 Tl rR ^ ^ t-i u o3 X ^ ° „, fH ^ f-^ S S Q) CD o S3 -S CD HI CD Antei lobe 1 thyrc > > ;> (D -f^ > M > > !> 'h^ 3 3 1 O c:> 3 1 1^ a 3 3 IM CO 00 CO I-H 00 CO T)l Tti ira IM CO Tti Ttl IM I-H >o •* Tt< CO T— I I— I (N (M , cS >> 03 !>> 03 C^ ^ t^ lO |^^ 00 IM I-H _ to (M ._ c^ lO o --^ CO CD 05 00 CO ^ I-H . t^ -L i-i CO CO lO ^ 00 Tt< CO -* 00 CO t^ tH I-H CD ^ I-H t^ .—I ^ i-H r~i r~i I-H I-H I-H I-H CP o o O O o o O o o a a a a a d 3 3 3 o ■-H as <1 bO O "3 ^'1 be o 1 o 1 O 3 bC ^ 03 c3 c3 o3 ^-3 £ [3 '-3 *-l3 £ 1 [3 "-+3 e 3 1 ■a h| h3 H 3 o t~i H tH ^( ^ t4 ^ s s ^^ .^ a 3 -a 3 25 20 IS /O Thickness of tvsJk in fn/cn3 . . o o -# d d 1— 1 00 i-H d d .1 Fh tH h h h h a> V > > > > > (5 ■ ^ • ^ • •-* • H 3 K^ hP ij 1^ h-l a. lO (3) OS CO -* rH ■§.& S i-H o T-H U5 rH -*J -1-3 4^ •^ o CO o !> lO ^? t~J2, 055. T-H rH IN ^ ^ "= ^ '-' CD Ol _© a; V m "o ^ 'o _ "o — . "o ,-• "o r^ 'o 1 '-} a, c3 a a °' C3 Q 03 Oi OS n. a o3 E o3 Eh o -^ o -^ o -^ O *^ o -^ o ■« ^ g- ^ ^ ■^ o ^ g= ^ g= tH S~t u ^ i^ ^ «4-l t4_l U-^ ^-1 a tS 11- •-§■2 1> Oi 1—1 lO CO ,_( SoS T— I tH (N (M WbD fi5 03 o p< 'a 03 THE PARS BTJCCALIS OF THE HYPOPHYSIS 85 ?^ S to CO CO T— 1 ^ (N IN 1— I -1^ s: 00 IN o o o IN CO 3 O" g 00 IN CO CO CO ^ T-4 1— I § fe IN IN 03 T-l I— 1 o o o (N CO 1 1 o XI o 0) ■8 3 1.1 ^ -3 ,£) r/) fn -fl 73 ■ffl ?>. fl X! O -d 'S 'V ft +3 S m j:j •n &: H a n t>i -D 86 PIGMENTARY GROWTH AFTER ABLATION OiF UO tJH (N O Cq II d II § ."3 rS (^» 3 O '^ a fci^ fe 5 -a :S II J3 -+J +3 a >i >ll-l >> fi h l-l h o o ., o cc 10 CZ3 ^y^ m CO CO CO o CD CD CO CD S c:^ O tJ o O i-H o § «1 t^ 00 o (N « t^ IN (N £°'3 q3 o t^ 00 to d d IM CD 1— I d B ■ IM I— I .— I lO »o ^+i T— 1 o lO (M t- lO >o 00 oo CN 00 CO 00 CO 00 C31 00 o 1> c:3 tH oq <3> 1—1 "S* >c n CD > (> r-H tH O i> > f> Q • ^ C^ CD '-I 3 3 l-q a > s 3 3 3 •a 1^ ■*" •a 1^ c 3 e o ^ tp CO ■o^i^o r-l 1-H I— ( CM CO r^ i-H CO m §f , c3 >> o3 >% ca CO d d IN T— I 00 CO ^ 2-L '-I jT O^ CO =2, CO Si 00 51 CO J^ ■"* lO t-< ^> iO> ^ CO " lO ^ 00 '"' '^ ^ CO •i' ^ tC 1-H ^ 1— 1 1-H T-( d, GJ QJ 'cS 3 Is "3 13 "^ '~o 'o o 03 2 S. .3 "* 't. O t, o "■6 o ■■g o t o ■■g o a 03 a 03 fl c« a <:« a 03 G o3 3 £^ as as .^^ 0-3 a:s =^s fts »S o O o r ■ fin r e5 lla 00 ^ o 1— 1 ^ i> 00 c33 o T-H |8 rH I-H a I-H a" IN I-H 3. 2 Z 4 - CO « CO r« I"H ■«*< iJ 0) " CD S en t^ CO u f-H >o lO ira t^ IS o I-H o ? N o O >J > «b 1>^ IN m ^ CO CO " o o o < . ■ u s° 6 o g g o la 3 -a CD &I 3 a t; o a> o Ti< T(< o o 1-H rH K X CD O O Q C3 00 N CD 02 IM '3) ° .3 o o H H S t-H IM 1-H o -* CO d 6 a Q t4 C4-I -*j o 1 > ti C3s O ^h' s^ ! +3 1 erior 1 i to Ju en livi a 3 3 +i CD ^ 1 <1 a S is 111 t^ t^ t^ 00 00 00 T— t T-( I-H ■o ia-o © S a MPi > > > > > a< ^ ■<* tH O o O o o o o (0 43 J3 XI X! ^ X JD bD th CO lO ^ s o3 i^ 133 o3 § o^Si « " ^ J2, (D a> CD CD OJ ID hJ cols- 1—1 "O ^ •" ro O) (D 9? 9. , ^ >> o J3 -'^ 1q fH o ^ H life sl-j^ 1^ T— 1 c^ » I-H (M t^ 1-H IN CO CO -*^ CO CO 4J CO 94 PIGMENTARY GROWTH AFTER ABLATION OF In order to determine accurately the diminution in the adrenal cortex in the albino and its increase in the thyroidectomized tadpole, this substance was accurately drawn with the camera at a magnification of 300 diameters in three specimens, a 67-mm. albino, a 56-mm. thyroidless tadpole, and a 42-mm. normal animal. The drawings were transferred to a wax plate of pro- portionate thickness and the weight of the models of the cortex TABLE 9 Table giving the weight of wax models {XSOO) of cortex of left adrenal (frog tadpoles) SPECIMEN Weight of model in grams Individual specimen number Type Length' Age, dated from opera- tive stage (days) Diet 36 Albino 67 -/-/0.2 228 Anterior lobe May 6 to July 18; then liver 144.43 41 Normal 42 -/-/12 228 Liver 382.75 t 2 Thyroidec- tomized 56 -/-/2.0 228 Anterior lobe 759.60 pll Partial albino 41 -/-/16 286 Same as spec. 36 360.15 '■ The length of the component parts of the tadpole is given in the following order: Total /Hind/Fore Body /Tail/legs /legs thus secured for these three specimens (table 9). The cortical substance of one adrenal — the left in each case — was treated in this way The albino, although 11 mm. longer than the thy- roidectomized tadpole, and 25 mm. longer than the normal speci- men, had but 38 per cent, of the cortical tissue presented by the normal and 19 per cent, of that of the thyroidectomized speci- men. It would thus appear to be established that the cortical tissue is greatly diminished in the albino as compared with THE PARS BUCCALIS OF THE HYPOPHYSIS 95 the normal or thyroidectomized tadpole. On the other hand, the cortical adrenal tissue of the thyroidectomized tadpole exceeded by twice that of the normal. Although the thyroid- less animal is somewhat larger than the normal (11 mm.), yet the discrepancy in the size of the adrenals is out of proportion to the difference in the size of the specimens, and it thus seems certain that the cortical tissue is hypertrophied in the thjrroid- less larva as compared to the normal. Changes as striking but of a different nature are evident between the adrenal medulla of a normal or thyroidectomized tadpole and a hypophysectomized tadpole. In the normally pigmented larva the medullary cells present a diverse appear- ance, since certain cells are deeply stained and the cytoplasm limited by a definite cell membrane, while the opposite extreme is seen in certain other cells whose cytoplasm is reticular or even vacuolated and but slightly tinged by the dye, the cell membrane not being distinguishable (fig. 40). Between these two extremes all gradations can be found, a condition suggestive of the various secretory. states exhibited by the mammalian chromaffin tissue. In the albino, on the other hand, the cells are of one type; their cell boundaries are distinct and their cytoplasm moderately and uniformly stained (fig. 41). Moreover, the cells are uniformly larger than those either of the normal or thyroidectomized tad- pole as shown by table 8. Not only are the chromaffin cells larger in the albino, but their relation to each other and to the surrounding tissue is pecuHar to these animals. They closely approximate each other and the adjacent cortical tissue, spaces between or around them being seldom evident. In the normal or thyroidectomized tad- pole, on the other hand, an interval frequently separates these cells from each other and from the cortical tissue. This con- dition, which simulates shrinkage, was at first believed to be an artifact. Its constant occurrence in the normal and absence in the albino with identical technique suggests two possible causative factors. Either these spaces exist during life or the physical constitution of the cells of the normal and thyroidless larvae is such that shrinkage inevitably occurs during the manipu- 96 PIGMENTARY GROWTH AFTER ABLATION OF lations involved in the technical treatment. Be the explanation as it may, it is certain that in the preparations the chromaffin tissue of the normal animal almost invariably does not fill the intracortical space accorded it. A duplication in wax of the volume of the medulla (x 187.5) has been made for each of the three types of animal. In the normal and thyroidectomized animals the total intracortical medullary space has been included. It is evident, then, that if rABLE 10 Tabk giving the weight of wax models {X187.S) of adrenal medulla (frog tadpoles) SPECIMEN Weight of model in grams Individual specimen number Type Lengthi Age, dated from opera- tive stage (days) Diet 34 Albino 54 19/35/0.2 217 Anterior lobe May 6 to July 18; then liver 27.32 39 Normal 39 ■ 15/24/4.5 217 Liver 14.93 t 3 Thyroidec- tomized 55 19/36/1.0 217 Anterior lobe 24.46 ' The length of the component parts of the tadpole is given in the following order: Total /Hind/Fore Body /Tail/legs /legs only the actual chromaffin tissue exclusive of the surrounding spaces had been drawn, an undertaking too laborious and uncer- tain to be considered, the mass of the model in the normal and thyroidless larvae would have been considerably reduced. From the included table it will be seen that the volume of the medulla while not seriously out of proportion to the size of the specimens would appear to be increased in both the albino and the thyroid- less larvae (table 10). Attention has already been repeatedly called to the incom- pletely hypophysectomized tadpole, the so-called 'partial' albino, THE PARS BUCCALIS OF THE HYPOPHYSIS 97 and their characteristics described. Especial interest is associ- ated with these specimens in the study of the adrenal, since the pigmentation in Addison's disease is usually referred to a de- rangement of the adrenal. Both whole mounts and sections, however, reveal the fact that neither the adrenal cortex nor medulla suffers a serious disturbance in the 'partial' albino as compared with the normal animal. To test more exactly the amount of cortex in one such specimen, it was reproduced in wax (x300), as has been previously explained. These findings show that the cortical tissue is present in relatively as great an amount as in the normal tadpole (table 9). Apparently, then, as in the case of the thjrroid, a relatively small amount of hypophysial tissue is sufficient to give rise to a normal adrenal. The epithelial bodies The epithelial bodies (Maurer) might be suspected of par- ticipating in the general endocrine upset experienced by the tadpole suffering from pituitary deficiency. Such, indeed, ap- pears to be the case, although these bodies do not suffer as do the thyroids, adrenal cortex, or neural hypophysis. Models of these glands made from four normal and four albinous tadpoles reveal in many cases a profound diminution in the albino, al- though in other cases this decrease does not transcend the limits of variation of the individual bodies in the normal. When we take cognizance of the total amount of tissue, however, there can be no question but that it is profoundly diminished in the albino (table 11). There appears to be no serious structural abnormalities in these bodies. Thus in their reaction to epi- thehal hjrpophysectomy these bodies align themselves with all the other endocrine organs thus far examined (thyroids, adrenal cortex, neural h3rpophysis) save one, the adrenal medulla. An opposite response — one of increasing size — is evoked in the epithelial bodies (Allen) by thyroidectomy. In this opposed response in thjToidectomy they thus align themselves with the enlargement which the pituitary (Allen, Hoskins) and adrenal cortex (Smith) enjoy. MEMOIR NO. 11. 98 PIGMENTARY GROWTH AFTER ABLATION OF X t-H CO 10 CO 05 r^ rt 50 CO »o »o ■^ CO 4^ o Eh S t>^ CO 00 oi •* d CO IN (N CO l-H T-H T-H M S i> TJH CO CO 00 CO rH CD i "^ 1-H 00 l-H 1> IN CO i-H ai 43 60 "^ Tti ■* TJH tH CO ■* (N ^ tH 5 O H-l S '-< CO rH CO CO 00 lO ►^ ,4a I I> 05 00 >o (N OS ij H M H 3 60 00 06 C3 d CO CO 10 •* H p^ l-H 2 .a. 01 CO t>. »o (N CO s 00 03 CD 02 (N i-H l> S ■* ■* tH ,— ( CO >o (N « 1— t ,— I M II S s 10 CO m I— 1 00 CO to ?5 (N 05 00 CO u ^f 4^ 1 1 ^ -9. t g 1 .2 c, s -+J ■ -H -4^ 3 3 a -' 1^ 3 3 3 cl ^ < <^ •^(io) S fa »_ "S ® C8 m" tH 1-H rH -* i-H l-H 1— 1 t-H l-H IN o> 05 Age, from tive (d ,—) T-H tH i-H i-H 1— I ,—1 rH 10 T)H T-H 1-1 CO CO T-H ,-H 1 d 5i CO d d d ?2 ^^ 22-^ os:^ 1- e?. co^ ^i -> J^ -*^ tA ^ 1 TtH r u3 t:r -cH t^ ^ 1 ira f 10 p Ph t— 1 l-H CO 60 bD bD M bD a) Cu -2 S •"°-S 2^ 2^ ^■^ ^^-^^ fe< '-k' TV. -igv -;-'•'"-' ■~fe>^a»tATK :i 'f'3.,-crr7//~ 110 PLATE 4 EXPLANATION OF FIGURES 19 The pigmentary system" of a 'light and heat' adapted 38/6.0 normal frog tadpole which exhibited a contraction of the epidermal melanophores under the influence of this stimulus. Fixed 5}/^ months after the operative stage by dropping into Helly's fluid. Liver diet. X226. 20 The pigmentary system of a 'light and heat' adapted 46/0.5 albino frog tadpole. Fixed 5J^ months after epithelial hypophysectomy by dropping into Helly's fluid. Liver diet. X226. 21 The pigmentary system of a 46/0.1 albino frog tadpole, supplied with a continuous diet of posterior lobe, 'standard' environment. Fixed by dropping into Helly's fluid, 4}/^ months after epithelial hypophysectomy. X226. 120 20 >-». ^ yi'^xt-^' 121 PI.ATE 5 EXPLANATION Or FIGURES 22 The pigmentary system of a 46/0.1 albino frog tadpole, 'light and heat' adapted. This tadpole was supplied with a posterior-lobe diet for 4M months. Prior to fixation it had been on a Uver diet for one week. Fixed 43^ months after epithelial hypophysectomy. X226. 23 The pigmentary system of a 43/0.1 albino frog tadpole, 'light and heat' adapted. This tadpole was supplied with a posterior-lobe diet for 3}4 months. It had been on a liver diet for 5 weeks prior to fixation. Fixed by dropping into Helly's fluid 4}^ months after epithelial hypophysectomy. X226. 122 iM.A'i'h; ■(■■ ■ ■: ' ^ [ ' ■'■■ \ > t V \^ 2i: ■i^-;«!''"v''"~^' 3-:. \ \ \ " . ^ N \ ~v. ■ -- \ - — ?s...^.-.f - ■ ^ —-'5^ 73> 25 ..>-* — -.i-aMi jii::-^'^"^- g z^.-^'^. ~ , ■*• ^T-A^ .*^ "TZ^^ r-J-^-^fli^"^ y 123 PLATE 6 EXPLANATION OP PIGUEES 24 A diagram of the ventral view of the brain of a 45/6.0 frog tadpole to show the position of the hypophysis. 25 A model of the infundibular process and hypophysial components of a 38/3.5 normal frog tadpole (specimen 15), fixed 63 days after the operative stage. Liver diet. The caudal end of the infundibular process faces the top of the page, a, dorsal, 6, ventral, c, median sagittal, views. X89. 124 PLATE 125 PLATE 7 EXPLANATION OF FIGURES 26 A model of the infundibular process and hypophysial components of a 43/0.1 albino frog tadpole (specimen 18), fixed 101 days after epithelial hypophysectomy. Liver diet. The caudal end of the infundibular process faces the top of the page. a, dorsal, b, ventral, c, median sagittal, views. X89. 27 A model of the infundibular process and hypophysial components of a 36/4.2 'partial' albino frog tadpole (specimen, p 1), fixed 66 days after the operative stage. Liver diet. The caudal end of the infundibular process faces the top of the page, o, dorsal, 6, ventral, c, median sagittal, views. X89. 126 PLATE 7 p.tl'. \,p.f. 0''--p>^ ^*5^.-.>> ,s — 26 /^ t.p.t: tpc'P zi 127 PLATE 8 EXPLANATION OF FIGURES 28 A median sagittal section through the infundibular process and the hypo- physial components of a 38/3.5 normal frog tadpole. Liver diet. Fixed 63 days after the operative stage. X227. 29 A median sagittal section through the infundibular process and the hypo- physial components of a 43/0.1 albino frog tadpole (specimen 16). Liver diet. Fixed 101 days after epithelial hypophysectomy. X227. 30 A median sagittal section through the infundibular process and the hypo- physial components of a 36/4.2 'partial' albino frog tadpole (specimen p 1). Liver diet. Fixed 66 days after the attempted epithelial hypophysectomy. X227. 128 28 p.tv. ji / \ / is^iir S " - P/: n pn „ >^# &(,'? « ** •,;. ^ P CP- X .&^ / 7^^ «;-*> 29 ^ 30 MEMOIR NO. 11. 129 PLATE 9 EXPLANATION OP FIGURES 31 (a) Ventral and (b) median views of a model of the left thyroid of a 38/3.5 normal frog tadpole (specimen 15). Liver diet. Fixed 63 days after the operative stage. X89. 32 (a) Ventral and (b) median views of a model of the left thyroid of a 43/0.1 albino frog tadpole (specimen 18). Liver diet. Fixed 101 days after epithelial hypophysectomy. X89. 130 ^' ij? ym '^i.1 52 131 PLATE 10 EXPLANATION OP FIGURES 33 A cross-section through the largest portion of the left thyroid of a 40/5.0 normal frog tadpole. Liver diet. Fixed 64 days after the operative stage. X227. 34 A cross-section through the largest portion of the left thyroid of a 40/1.5 albino frog tadpole. Liver diet. Fixed 64 days after epithelial hypophysectomy. X227. 35 The follicle, shown by an arrow, in figure 33. X765. 36 The follicle, shown by an arrow, in figure 34. X765. 132 I'l.ATIO 10 sf%»/* / s-t- vs- ~j^ ,-«(^^ ^ #. ^ 5^'^ .'^ e *4 'j3t #*^ 33 j-**^^ ' 34 4;' 35 i:i:j PLATE 11 EXPLANATION OF PIGUKES 37 The ventral surface of the dorsal abdominal wall of a 44/9.0 frog tadpole to show the position of the adrenals. 38 The adrenal cortex of a 43/8.0 normal frog tadpole. Drawn from a whole mount. Osmium-bicromate fixation. The specimen was killed 221 days after the operative stage. Liver diet. X24. 39 The adrenal cortex of a 55/0.2 albinous frog tadpole. Same fixation, age, and diet as above. X24. 134 i'i,A'ri': II f^'" £•/■<*- ^■'H^ W^^ #--*^-, 0** %^ 39 \k 0/ ly.i PLATE 12 EXPLANATION OF FIGURES 40 A small portion of the adrenal of a 48/9.0 normal frog tadpole. Liver diet. Fixed in osmium-biehromate 195 days after the operative stage. Babes' safranin stain. Cortex black, medulla and nuclei red. XV33. 41 A small portion of the adrenal of a 54/0.2 albino frog tadpole. Same age, diet, fixation, and stain as above. X733. 136 PLATE 12 40 41 137 138 < -*»»- r,-^j?:^?i-p.^ja..i 139 o X 140 ** * i \\-y 141 1— I CO PS P 1^ O o < 142 ^•'TS ;■> J7> i « f 143 1? o ■< ■< X T3 a ,0 O =3 « s M °5 J- ft g §■- ■3 CI £ o - X — o '-3 p- 1 03 -a o > > S D- o Xi a, §2 cS o m a P< ■a " ■13 ft C3 e =3 e !0' bC ft O * .S ■* "3 .2 .3 m o -e j3 ji ft o "2 2 § O 05 o -P ^ g O §1 144 s f .-•"S. "rl- ^%.„.- 145 PLATE 17 EXPLANATION OP FIGUltKS 55. Photographs of a normal and an albinous frog tadpole to show the xantho- leuoophores. Photographed May 26th. The same specimens as shown in figure 44. X7. 56. An enlargement of a portion of the dorsal body surface of the specimens shown in figure 55. 146 PI,ATE 17 ■ V '«l>,'^T^^^€' wmw 56 I 55 147 PLATE 18 EXPLANATION OF PIGTTRES •57. An albinous and a normal frog tadpole showing reciprocal sldn exchanges. Taksn four hours after the skin exchange was effected. Age of specimens 70 days, dated from the operative stage. X5, 58. Enlargements of the skin exchanges and surrounding region of the specimens shown in figure 57. 148 PLATE 1» 58 57 149 PLATE 19 EXPLANATION OF FIGOKES 59. The mesonephroi and adrenal bodies, fixed in the osmium-bichromate solu- tion, (a) 'Partial' albino young adult frog (specimen p. 7). (b) Normal young adult frog (specimen 33). (c) Albinous frog larva. Age of specimens 356 days. Specimens a and b are shown in figure 53. 60. An albinous (a) and a thyroidles.? (b) frog tadpole photographed to show the fat-bodies. Taken immediately after death. These specimens had been subjected to inanition for six weeks prior to death. Age 335 days. X2. 150 PLATE 10 b 59 60 151 AMERICAN ANATOMICAL MEMOIRS No. 1. The Anatomy and Development of the Systemic Lymphatic Vessels in the Domestic Cat, by George S. Huntington, Professor of Anatomy, Columbia University, New York City, states the various theories held in regard to lymphatic development in general and then presents the result of six years' careful investigation on mammalian lymphatic development. Part I deals with the development of the systemic lymphatic vessels in their relation to the blood vascular system. Part II deals with the development of the preazygos and azygos seg- ments of the thoracic duct. 175 pages of tejct, 8 text figures (two in color), 254 photomicro- graphs and 21 colored plates. Sent post paid to any country for $4.00. 1911. No. 2. Contribution to the Study of the Hypophysis Cerebri with Especial Refer- ence to its Comparative Histology, by Frederick Tilney. Associate in Anatomy, Columbia University, New York City. Part I contains a historical review of the literature. Part II deals with the comparative histology of the pituitarj' gland and gives a report of six hypophy- sectomies performed upon cats. 72 pages of text, 2 text figures, 60 photomicrographs and plates. Sent post paid to any country for S1.50. 1911. No. 3. Early Stages of Vasculogenesis in the Cat fFelis Domestica) with Especial Reference to the Mesenchymal Origin of Endothelium, by H. Von Schulte, Depart- ment of Anatomy, Columbia University, New York City. 90 pages of text and 33 figures, of which 14 are in colors. Sent post paid to any country for $1.50. 1914. No. 4. The Development of the Lymphatic System in Fishes, with Especial Refer- ence to its Development in the Trout, by C. F. W. McClure, Department of Comparative Anatomy, Princeton University. 140 pages, 41 figures, 11 of which are in colors. Sent post paid to any country for $2.50. 1915. No. 5. The Development of the Albino Rat, Mus Norvegicus Albinus: I. From the pronuclear stage to the stage of mesoderm anlage; end of the first to the end of the ninth day: II. Abnormal ova; end of the first to the end of the ninlh day; by G. Carl Huber, Department of Anatomy, University of Michigan, and the Division of Embryology, Wistar Institute of Anatomy and Biology, Philadelphia. 142 pages of text and 42 figures from drawings by the author. Sent post paid to any country for $2.50. 1915. No. 6. The Rat, compiled and edited by Henry H. Donaldson. Reference tables and data for the Albino Rat (Mus Norvegicus Albinus, and the Norway Rat (Mus Norvegicus), 280 pages. Sent post paid to any country for $3.00. 1915. No. 7. An Experimental Analysis of the Origin of Blood and Vascular Endothe- lium: I. The origin of blood and vascular endothelium in embryos without a circulation of the blood and in the normal embryo (forty-nine figures); II. A study of wandering mesenchymal cells on the Uving yolk-sac and their development products; chromatophores, vascular en- dothelium and blood cells (thirty-five figures); by Charles R. Stockard, Department of Anat- omy, Cornell University Medical School, New York City. 174 pages. Sent post paid to any country for $2.50 1915. No. 8. On the Behavior of Bufo and Rana toward Colloidal Dyes of the Acid Azo Group (trypan blue and dye No. 161). I. With reference to the portal of entry of the dye, and the causes which underlie the initiation of the process by which colloidal dye particles are stored in the cytoplasm of certain typical cells of the embryo, and II. With reference to the development of the lymphatic system; by Charles F. W. McClure, Laboratory of Comparative Anatomy, Princeton University. 64 pages. Sent post paid to any country for $1.25. 1918. No. 9. The Morphology and Evolutional Significance of the Pineal Body: being Part I of a contribution to the study of the epiphysis cerebri with an interpretation of the mor- phological, physiological and clinical evidence; by Frederick Tilney, M.D., Ph.D., Professor of Neurology, Columbia University, N. Y., and Luther F. Warren, A.B., M.D., Professor of Medi- cine, Long Island College Hospital, N. Y. 258 pages and 97 figures. Sent post paid to any country for $3.00. 1919. ^''Vr^^^'{;';i,ll<'i^^;"!/Ms^Hf}|^^^^^^^^^^