Cellular changes in the of the mouse following Pituitrin-injection

By

Masao Sano

Department of Anatomy, Nagoya University School of Medicine, Nagoya, Japan.

(Director : Prof. Dr. K. Y a ma da)

Introduction It has been recognized that the anterior and posterior lobes of the pituitary are clearly bordered by connective tissues morphologically, and that these two lobes have not neural but humoral connection through a pituitary portal system (Popa and Fielding, '30; Wislocki and King, '36 ; 0 hf uj i, '53). On the other hand, it has also been believed that hormone(s) is secreted by the of the posterior lobe. Recently, however, B a r gmann ('49) and co-workers postulated that the hormone is produced by certain nerve cells in the , and that the posterior pituitary plays the role of storage and release of the hormone. Unrelated to the site of production of posterior pituitary hormone, it is presumable that the hormone acts directly on the anterior pituitary by the general blood- circulation or the portal vessels, or indirectly through the other organs. By what mechanism does the posterior pituitary hormone influence the anterior pituitary ? What histological changes are revealed in the anterior lobe then? These problems deserve much interest, but only a few studies, regarding these, have been made so far. Ito ('53) reported that an intimate relationship exists between posterior pituitary hormone and basophile cells of the anterior lobe. Also it has been shown that the hormones of the posterior pituitary and the have antagonistic actions on sodium and water excretion under various experimental conditions (Winter and In gram, '43; Little et al., '47; Sartorius and Roberts, '49 and others). It has been, similarly, pointed out that large doses of pitressin stimu- 248 M. Sano late the release of adrenocorticotrophic hormone followed by activation of the adrenal cortex as judged by an ascorbic acid depletion test in rats (Nagareda and Gaunt, '51; Stutinsky, Schneider and Denoyelle, '52 ; Arimu r a, '55). In these reports, however, there are no descriptions of the morphological changes in the anterior pitui- tary. For the purpose of clarifying the influence that the posterior pitu- itary hormone exerts on the anterior lobe, the hypophyses obtained from mice injected with this hormone were examined, in the present study. On the other hand, it is generally believed that the posterior pituitary hormone consists of three elements (pitocin, oxytocin and adiuretin) which have influences on their target organs respectively. It has not yet been established, however, whether these three functions depend on a single substance or on separate ones. Although purifica- tion methods for the hormone have been studied for a long time, isola- tion of these substances in separate forms has not been successful. Accordingly, a posterior pituitary preparation containing all three elements was used in the present experiment.

Materials and Methods Adult mice of both sexes ranging from 15 to 25 gm. body weight and ages of from 60 to 140 days were used. They were divided into two groups (Pituitrin-injection and saline solution-injection groups). In the Pituitrin-injection group, the total of 36 mice were divided into three groups of 12 animals each, of large dosage-injection group (A-group), medium dosage-injection group (B-group) and small dosage- injection group (C-group). All were injected intramuscularly with " Pituitrin " prepared by the Institute of Dai Nippon Zohki Co. The animals of A-, B- and C-groups were injected respectively with 1000 mU. (0.1 cc), 100 mU. (0.01 cc) and 10 mU. (0.001 cc) of the hormone everyday. According to the number of injection, the animals of each group were designated as A-1, A-3, A-5, A-10 ; B-1, B-3, B-5, B-10; and C-1, C-3, C-5, C-10 (Table 1). Eight mice of the saline solution-injection group were injected with 0.1 cc of 0.85 per cent physiological saline solution once a day for 1, 3, 5 and 10 days. All mice of both Pituitrin-injection and saline solution-injection groups were sacrificed on the day following the last injection under Cellular changes in the anterior pituitary of the mouse 249

Table 1.

chloroform anesthesia: At that time the body weights were examined (Table 2). In addition to these two groups (Pituitrin-injection and saline solution-injection groups), 16 adult mice were observed without any treatment as normal controls. The majority of pituitary glands removed were fixed in Zenker- formol-acetic acid (Zenker's stock solution 20 cc, formalin 0.5 cc and glacial acetic acid 1 cc), embedded in paraffin and sectioned at 4 microns serially in a sagittal plane. These sections were stained by hematoxylin- eosin stain and by Heidenhain's azan stain modified by Mallory . Some of the pituitary glands were fixed in sublimate-formal-acetic acid (saturated sublimate solution 20 cc, formalin 2 cc and glacial acetic acid 0.5 cc), and were stained by periodic acid Schiff method modified by Coleman ('38) and by Gomori's aldehyde-fuchsin stain ('50a).

Results

Normal control grout The anterior pituitary of the mouse consists of parenchyma and interstitial tissue. The former occupies the greater part of the anterior pituitary, while the latter is composed of blood capillaries and sinusoids 250 M. Sano with a small quantity of connective tissue. These blood capillaries and sinuspids, in which the blood corpuscles are abundant, are distributed as a network. They, moreover, are abundant in the anterior portion of the anterior pituitary. The cells of the parenchyma are arranged in irregularly anastomos- ing cords, supported by a very delicate network of reticular connective tissue fibers. As a rule, the cells comprised in the cords do not lie in direct contact with the capillaries, but are separated from them by a small amount of connective tissue. A typical arrangement of the cells in the cords is consistently absent in the regions adjacent to the inferior border of the residual cleft. The cells of the pars anterior connect with those of the without a distinct boundary existing between them. Rarely, the so-called pituitary cysts are observed in the anterior pituitary. They are almost always present in the transitional zone where the pars anterior and become continuous. As in the anterior of all other mammals studied, that of the mouse is generally considered to be composed of three fundamental types (, basophile and chromophobe cells). The chromophobes are most numerous, the acidophiles few, and the basophiles least of all.

I. Acidophile cell The acidophile cell is the most easily recognized cell type in the pituitarygland, because of a large number of distinct, highly acido- philic granules in its cytoplasm. In number it usually lies between the other two types. Acidophiles are not equally distributed through- out the lobe, but are considerably more abundant in the lateral ex- tremities than in the middle of the gland. The acidophile cells can be divided into three varieties, namely small, medium size and large acidophile cells, since the acidophiles are variable in size. 1) The small acidophile cell (Fig. 1). The cells outlined clearly are round or oval in form. Their cyto- plasmic granules stain with azocarmine or eosin, and are densely distributed throughout the cytoplasm. The round or oval nuclei being centrally situated in the cytoplasm in general, contain a single, large acidophilic nucleolus and show a coarse chromatin network. Consequently, they usually have a dark cast, and look like the spots of a die. Cellular changes in the anterior pituitary of the mouse 251 2) The medium size acidophile cell (Fig. 1). The cells accounting for the greatest number of these three acido- phile cell types, are generally round or oval in shape, but are frequently polygonal or elongated, especially when lying in groups along a blood capillary. The nuclei of the cells are round or oval in form, and are generally situated eccentrically in the cytoplasm. They have a prominent acido- philic nucleolus and contain dust-like to coarse chromatin particles. The majority of these nuclei are light vesicular when compared with that of the small acidophile cell. The chromatin network of the cells is relatively rough and often arranged in a " cartwheel-like " fashion. The nuclear membrane .of the cells is strained in general, but some- times crenated or depressed ones are observed. The cytoplasmic granules of the cells which are distributed slightly coarser than those of the small acidophile cell, are somewhat fine, brilliantly acidophilic and spherical. These granules are scattered throughout the cytoplasm in general, but they are often found more abundantly in the peripheral portion than in the perinuclear region in the cytoplasm. Frequently near the nucleus, there is a " negative image " of the Golgi-apparatus, in which corpuscles faintly blue with aniline blue are observed. A pen-nuclear " halo " devoid of cytoplasmic granules is seen repeatedly. 3) The large acidophile cell (Fig. 2). The large acidophile cell, the fewest of the three types of acido- philes, is similar to the medium size acidophile cell in general features except for some respects as follows : 1) Since the chromatin network of the cells is considerably coarse, the nucleus is lighter than that of the medium size cell. 2) The cytoplasmic granules of the cells are more sparse.'3) The majority of the cells indicate a prominent nega- tive image of the Golgi-apparatus and a pen-nuclear halo. In addition to these three types described above, pycnotic forms are rarely observed in the anterior pituitaries of the normal mice. These cells have irregularly shaped cytoplasm staining very deeply red- dish with eosin or azocarmine, and have pycnotic nuclei. Sometimes mitotic figures of acidophile cells appear in the anterior pituitaries of the normal mature mice. 252 M. Sano

IL Basophile cell Although the basophile cell exceeds the other cells in size, the number of these cells in the pituitary gland is considerably less than the others. The basophiles are obviously concentrated in the area adjacent to the intermediate lobe and especially at the anterior edge in the so-called " sex zone," and they are also numerous in the central portion of the lobe.. The basophile cell will be divided into two varieties for descrip- tive purposes. Later in the article of discussion, it will be shown that these two types represent different responses in the present experiment. 1) The first type of basophile cell (Figs. 3 and 4). The first type cell of basophiles is not evenly distributed through- out the anterior lobe. The junctional zone of the pars anterior with the pars tuberalis is composed almost entirely of this cell, and they are also localized at the lower portion of the anterior pituitary and the upper region of the anterior lobe adjacent to the residual cleft. The cells are large, oval or polygonal and show a relatively indis- tinct cell boundary. Their cytoplasmic granules staining pale blue with aniline blue are very fine, and are diffusely distributed throughout the cytoplasm. These granules react positively with periodic acid Schiff reagent, but do not stain with Gomori's aldehyde-fuchsin. The nucleus of the cell is round or oval in shape, and only rarely is indented. It usually shows a light cast, because it contains a single large, acidophilic nucleolus and its abundant chromatin particles are dust-like to granular. 2) The second type of basophile cell (Fig. 5). These cells being relatively scarce in number are dispersed in the central area of the anterior lobe. The large and angular cells are delinea- ted by a distinct cell boundary and contain dark blue and coarse granules densely. These granules stain purple red with Gomori's aldehyde-fuchsin, and also stain pink by periodic acid Schiff method. Although the nuclei of these cells are generally oval or elongated, sometimes they are very irregularly shaped being bibbed, indented and wrinkled. The nuclei are usually smaller than those of the first type. These nuclei contain not so abundant chromatin particles which vary from granular to coarse clumps and are provided with an acidophilic nucleolus: Accordingly the nuclei, as a rule, show a dark cast. Frequently the basophile cells of these two types contain one, two or more pale blue hyaline vacuoles in the cytoplasm. These vacuoles Cellularchanges in the anteriorpituitary of the mouse 253 are usually smallest near the center of the cell and increase in size as they approach the cell borders. Mitotic figures of basophile cells are almost never observed in mature mice.

III. The chromophobe cell (Figs. 6 and 7) is most numerous of 'the three cell types of the anterior pituitary. They occupy the central region and the upper part of the anterior lobe adjacent to the residual cleft. In some chromophobes (Fig. 6) the cell boundary is indistinct and, since so little cytoplasm is present, a group of such cells has the appearance of a compact aggregate of nuclei. The whole mass resembles a syncytium and corresponds to " Kernhaufen." The round or oval, dark nucleus with one or two intensely staining nucleoli is surrounded by a slender margin of cytoplasm. Chromophobes containing an elongated sponge-gourd-shaped nucleus and very little cytoplasm are rarely observed. For descriptive purposes, it will be designated as " sponge-gourd-shaped cell " (Fig. 7), because it appears frequently in the present experiment. Chromophobes with a somewhat large amount of cytoplasm are also found, and frequently show indistinct granular or flocculent struc- tures staining faintly with eosin in the cytoplasm. These cells show obscure cell boundaries and vary in size. In some of these cells, the round nuclei are smaller than the others, and they have relatively abundant, coarse chromatin particles. From this, the nuclei of the cells resemble that of the small acidophile cell. In the others, the large nuclei are oval, and they show a light vesicular cast. It may be conceivable, as will be described later, that these chromophobe cells can be interpreted as a phase in the transition between chromophobes and the two types of chromophiles (Fig. 6). Certain peculiar chromophobes (Fig. 7) are also sometimes observed. In female mice, however, they are more numerous than in males. Their nuclei are round or oval in form and contain relatively abundant, coarse chromatin particles, and indicate similar features to that of the medium size acidophile cell. In the peripheral zone of these cells many filaments, of a cap-like structure, are found. These elements stain with hematoxylin or azocarmine. Mitoses of chromophobes are rarely observed. 254 M. Sano

The Pituitrin-injection group I. The large dosage-injection group (A-group) a) The acidophile cell. It appears that the acidophiles are fewer . in number than those of the normal controls. Moreover, the longer the term of injection, the greater is the decrease in number of these acidophiles. Although the nuclei are generally round or oval in shape, in A-3, A-5 and A-10 it is observed that irregularly shaped nuclei, indented or wrinkled, become somewhat numerous when compared with those in the normal (Fig. 8). In the greater number of acidophiles the nuclei are vesicular, whereas dark nuclei occur increasingly with the number of injection, and in A-10 the majority of acidophiles contain dark nuclei. Acidophiles with a pycnotic nucleus are slightly numerous in A-1 and A-10 (Fig. 9). Cells with cartwheel-like nuclei show a decrease in number gradually with the number of injection. Mitotic figures are very scarce in all serial sections. The acidophiles with a negative image of the Goigi-apparatus or a pen-nuclear halo appear only in a small number in A-3 and A-5, and these cells are scarcely observed in A-10. But the large acidophile cells appearing somewhat numerous in A-3 and A-5, possess the image and halo in general. b) The basophile cell. The number of basophiles is least in A-3, more numerous in A-1 and A-5, and most numerous in A-10. In A-10, it is as numerous as in the normal. This numerical change is due principally to the first type cell. The nuclei have usually a dark cast, and irregularly shaped ones show an increase in number according to progress of the experi- ment (Fig. 10). Cells with pycnotic nuclei are observed somewhat numerously when compared with the normal. Except for A-1, basophiles with hyaline vacuoles in the cytoplasm (Fig. 11), are found in less numbers than those in the normal. Mitosis of basophile cell does not occur during the experiment. c) The chromophobe cell. It is observed that the number of chromophobes increases with the frequency of injection. Although the majority of chromophobes contain light vesicular nuclei, dark nuclei appear with considerable abundance during the second half of experi- ment. In the same manner, the sponge-gourd-shaped cell (Fig. 6) mentioned above shows an increase in number during the second half. Cellular changes in the anterior pituitary of the mouse 255 IL The medium dosage-injection group (B-group) a) The acidophile cell. Acidophiles are generally fewer in number than in the normal, while they are more numerous than in the A- group. It is observed that the small acidophile cell is numerous in B-1, and the large one abundant in B-5. Dark or irregularly shaped nuclei show an increase in number with the length of experiment. On the contrary, cells with cartwheel-like nuclei decrease in number gradually. It is observed, moreover, that acidophile cells with a bibbed nucleus appear in relatively large numbers in B-3 and B-10. In B-10 mitotic figures of acidophiles are found more numerously than in the normal, while they do not appear so frequently in B-1, B-3 and B-5. The acidophile cell with a negative image of the Golgi-apparatus appears only in a small number in this group. In addition the cell with a pen-nuclear halo is almost not observed. b) The basophile cell. The appearance of basophiles shows a diametrically opposite relationship to that in A-group. Namely the number of basophiles is most numerous in B-3, less so in B-1 and B-5, and least in B-10. Furthermore, in B-1.0 the number of basophiles are slightly less than in the normal. This numerical change is due principally to the first type of basophiles as in the A-group. Dark or irregularly shaped nuclei are usually less than those of A-group, and cells with pycnotic nuclei are rarely observed. Cells with cytoplasmic hyaline vacuoles appear less than in the normal. Mitosis of basophile cell is scarcely observed. c) The chromophobe cell. In B-10 the number of chromophobes is slightly numerous when compared with the normal. Irregularly shaped or sponge-gourd-shaped nuclei are somewhat numerous when compared with those in the normal, but they appear less than in the A-group. Mitotic figures are sometimes observed in B-10.

III. The small dosage-injection group (C-group) a) The acidophile cell. In C-1, C-3 and C-5 acidophiles is more numerous than those in the normal controls, while in C-10 they are somewhat less. The small acidophile cell is observed somewhat abun- dantly in C-1, and the large one in C-5 and C-10. In C-group, there appear, though not so. conspicuous, the nuclear changes described in A- and B-groups. The cells with a negative image of the Golgi-apparatus or a pen-nuclear halo are somewhat less than in the normal. The cells with a few hyaline vacuoles in the 256 M. Sano cytoplasm are abundantly observed in C-5. Mitotic figures of acido- philes are generally less seen than in the normal. b) The basophile cell. In C-3 and C-5 basophiles are slightly nuemrous, while in C-1 and C-10, they are as many as in the normal. Irregularly shaped nuclei show a slight increase in number. c) The chromophabe cell. Sponge-gourd-shaped nuclei are slightly numerous in C-10. Mitotic figures appear somewhat abundantly in C-5 and C-10.

The saline solution.injection grout , The acidophiles and chromophobes do not show any cellular changes. On the other hand, the number of basophiles indicate a slight increase at the beginning of experiment, and this change depends upon the first type of basophiles as in the Pituitrin-injection group.

Discussion The acidophile cell The anterior pituitary has been a subject of histological interest for a long time. This interest has been directed largely toward a study of distinct cell types. At first, Flesch (1884) divided the cells of the anterior pituitary into two types (chromophobes and chromo- philes) with Weigert's chrome-hematoxylinlack in horse, dog and human pituitaries. Similarly Dostoiewsky (1884 and 1886), Lothringer and Rogowitsch (1889) described these two types in several species. Schiinemann (1892) further divided the chromophiles into acidophiles and cyanophiles employing Balmer's alaunhematoxylin and eosin stain. Subsequently, many investigators have published various classifications and nomenclatures for the anterior pituitary cells in many species (Thom,'01; Erdheim,'03;Scaffidi,'04; Gemelli,'07; Löwenstein, '07; Trautmann , '09; Erdheim and Stumme, '09; Stendell, '13; Biedl, '13; Kraus, '14 and '26; Maurer and Lewis, '22; Collin, '24a and '24b; Bailey and Davidoff , '25; Wolfe, Cleveland and Campbell, '33; Romeis, '40; Goldberg and Chaikoff, '52). It is, however, believed today that two chromophile types together with the chromophobes remain as the well-established cell types of the normal gland. From a histophysiological standpoint, Saint Remy (1892) re- ported that these cell types (acidophiles, basophiles and chromophobes) Cellularchanges in the anteriorpituitary of the mouse 257 of the anterior pituitary represent merely various phases of secretory activity. Similarly Benda ('00), St endell ('14), Collin ('24a and '24b), Fr a n c k ('35a, '35b and '35c), S u s man ('35) and others arrived at the same results. Subsequently, however, Benda ('32) abandoned this unitalistic concept. Gemelli ('07) reported that acidophiles can be transformed to basophiles through a transitional form, while chromo- phobes have no connection with the cycle of the chromophiles. On the other hand, Trautmann ('09) Kraus ('14), Urasov ('27), Severinghaus ('32, '33, '35, '37 and '39) and Yamada ('34, '37a, '37b and '43) postulated a dualism. Namely, there is no transition between acidophiles and basophiles, and the two chromophile types originate separately from chromophobes. Kraus ('14) observed the transitional cells between chromophobes and two types of chromophiles.. Ur a so v ('27) differentiated acidophies and basophiles by morphological differ- ences between their respective Golgi-apparatus in mice. At well ('29) also referred to this difference in the cat. Although he differentiated the Golgi-apparatus of acidophiles from those of the other two types, he could not demonstrate striking morphological differences between the Golgi-apparatus of .chromophobes and that of basophiles in this species. Se veringhaus ('32) mentioned that in the rat, the Golgi-apparatus of the basophile cell has a definite and constant structure different entirely from that of the acidophile cell. In chromophobes, moreover, he discovered two distinct types of the Golgi-apparatus corresponding respectively to the basophilic and acidophilic types ('33). This is strong evidence in support of the theory that chromophobic cells are pro- genitors of distinct and divergent chromophilic cell types, and that no transition between basophiles and acidophiles is possible. He also pointed out that chromophiles revert to their. chromophobic forms from a study of the pituitaries of castrated animals. In the study of normal human anterior hypophysis, he reported that the nuclear differences in chromophiles seem to be definitely correlated with the chromophilic chromophobic cycle ('35). Against the authors mentioned above, Bied1 ('22) and Rome is ('40) supported a pluralism. B ie d 1 ('22) postulated that " ungranu- lierte Hauptzellen " are neither the starting nor the end-point of a secretory cycle, but are merely the mother cells from which " granu- lierte Zellen " differentiate. He also reported that three cell types (" Hauptzellen," " Eosinophilen " and " Basophilen ") of the anterior pituitary have independent functions, and that they originate from a " Starnmform." Rome is ('40) has denied unitalism from a study of 258 M. Sano human pituitaries. At the same time, he reported that his " undiffe- renzierte Zellen (Stammzellen)" grow into alpha, beta, gamma and delta cells, while an is differentiated from the gamma cell. These 5 cell classes have been said to have independent functions respectively. In the present data the number of acidophiles shows a decrease according to the increase in Pituitrin dosage, while chromophobes increase gradually. It is probable that the chromophobes increasing in the Pituitrin-injection group are cells transformed from acidophiles, since the mitotic figures of chromophobes are observed only rarely in this group. Severinghaus ('33) has previously divided chromophobes into acidophilic and basophilic chromophobes by differences in their Golgi-apparatus. It cannot be concluded whether the chromophobes increasing in the present experiment are identical or not with his acidophilic chromophobes, since the present sections were not fixed in osmic acid such as in his procedure. However, it can be presumed that the majority of these chromophobes correspond" to his acidophilic chromophobes. In the hypophysectomized tadpole intraperitoneal injection of an anterior lobe extract elaborated the growth (Smith and Smith, '23a). Evans and Long ('21a and '21b) have shown that the rate of growth in animals injected intraperitoneally with an anterior lobe extract, is greater than in the controls, wheareas hypophysis-fed animals and non-treated controls showed neither signficant differences in growth nor in the age of maturity. Similarly, Hoskins and Hoskins ('20) have observed that feeding hypophysis to hypophysectomized larvae stimulates growth. Putnam, Benedict and Teel ('29) have shown that a dog which received daily for fourteen months sterile aqueous extracts of the anterior lobe of cow pituitaries grew to almost twice the weight of its littermate control. Also, they emphasized that the condition produced appears to merit the designation of experimental acromegaly. These works show that a growth promoting element exists in the anterior pituitary. Smith and Smith ('23b) have reported that hypophysectomized tadpoles receiving the outer portion of bovine pituitary, which contains many acidophiles, grow unusually large, and that those injected with the central part grow more slowly than those receiving the outer portion. It was discovered that hereditary dwarfism in the mouse is associated with the absence of acidophiles in the anterior pituitary (Smith and McDowell, '30). Kraus ('26) found hyperplasia of the Cellularchanges in the anteriorpituitary of the mouse 259 hypophysis with hypertrophy of acidophile cells in acromegaly cases. A histological study on 89 cases of human pituitary adenomas (Bailey and Davidoff, '25) indicated that 35 of 89 cases manifested a syndrome of acromegaly at the same time, these cases showed a conspicuous increase in number of alpha cells microscopically. They reported also that 54 cases which did not manifest an acromegalic symptom indicated chromophobic adenomas. On the other hand, Poindecker ('13) found that both hypophysis-hyperplasia and hypophyseal adenomas which are observed in acromegaly cases depend not only on the increase in number of acidophiles, but also on the increase in chromophobic element. From these facts, it is generally believed that growth promoting hor- mone is secreted by acidophile cells in the anterior pituitary. The present results regarding acidophiles are as follows 1) Acido- philes are reduced in number in accordance with the increase in Pituitrin- dosage injected. 2) Cells with dark or pycnotic nuclei are increased gradually. 3) The cells with a negative image of the Golgi-apparatus or a pen-nuclear halo decrease in number. From these facts, it can be presumed that acidophiles fall into hypofunction as a result of Pituitrin- administration. Moreover, in A-10 injected with the largest dose of Pituitrin, the average value of increased body weight during the ex- periment was —0.75gm. (Table 2). This supports the view that the acidophiles are the site of production of growth promoting hormone. It is not clear whether these changes taking place in the acidophiles depend on a direct action of Pituitrin or on an indirect one. Selye ('37 and '39) reported that an adrenocorticotrophic hormone demand inhibits the secretion of growth promoting hormone. It was, moreover, indicated that ACTH inhibits body growth in normal rats (Evans, Simpson and Li, '43). These changes of acidophiles in the present experiment, may depend on an indirect action through ACTH, since it is conceivable that such large dosage Pituitrin-injection is followed by a release of ACTH. In A-3 and A-5, the large acidophiles appear somewhat numerous when compared with the normal. It is conceivable that these cells are in an active phase functionally, since they have acidophilic granules to a moderate degree, a negative image of the Golgi apparatus and a peri- nuclear halo. It may be presumed that the great occurrence of large acidophiles represents emergency measures against the general hypo-, function of acidophiles. 260 M. Sano The basophile cell In 1940, Romeis published the results of an extensive study of the anterior pituitary of several species. Employing the kresofuchsin- a.zan stain of Berblinger and Bur gdorf ('35), he differentiated 5 cell types and, following a suggestion by Bailey and Davidoff ('25), designated them alpha through epsilon. Although Romeis stated that these 5 cells could be differentiated in the anterior pituitary, later investigators were unable to differentiate clearly the beta and delta cells of the rat by his kresazan method (Brolin, '45; Goldberg and Chaikoff, '50; Gomori,'50b). Recently, however, Gomor i ('50a) has shown that a new elastic stain, aldehyde- fuchsin, in combination with a modified azan technique, permits a clear-cut distinction between two types of basophiles in rat pituitary. Subsequently, this has been confirmed by Pur v es and Griesbach ('51c) and Haim i ('50 and '52) in rat and mouse. The description of a new technique (M c Ma flu s, '46) of staining with periodic acid followed by Schiff's reagent has permitted the demonstration of glycoprotein in histological sections. This technique was elaborated by Hotchkiss ('48) and has been applied with con- spicuous success by Catchpole ('49) in a study of the hormone content of rat pituitary. Using frozen dried material, Catchpole was able to demonstrate in cyanophile cells an accumulation of granules which gave a positive periodic acid Schiff reaction (PAS) for glyco- protein. Pear se ('49, '50, '52a, '52b and '53) employed the McManus- Hotchkiss technique to human and animal anterior pituitaries and has shown PAS positive granules in basophile cells. Purves and Griesbach ('51a) have stated that two types of glycoprotein containing cells are distinguishable. According to them, one of the two is an oval or round cell staining intensely by PAS and localized at the lower surface of the anterior pituitary and the upper surface adjacent to the pars intermedia, while the second type of glycoprotein containing cells is polyhedral in shape and occupies the central region of the anterior lobe. Moreover, they emphasized that so-called " basophiles " (the cells which stain blue with aniline blue by azan technique) are glyco- protein producing cells. Previously, Reese, Koneff and Wainm an ('43) pointed out the distinction between these two types of basophile cells as seen by the Mallorylazan type of staining. Purves and Griesbach ('51a) also reported that the aniline blue of the azan method stains the same elements demonstrated by the PAS method and that all the cytological Cellularchanges in the anterior pituitaryof the mouse 261 features and functional responses described in their experiments on gonadal and disturbances for the two types of cells are obser- vable by the Mallory or azan techniques. According to the present observations, it is possible to differentiate two types of basophiles in the anterior pituitary of the mouse by azan stain, and they were designated as the first and second types. In addi- tion, referring to Gomor is aldehyde-fuchsin stain and PAS method, it can be acknowledged that the first type corresponds to Romeis's and the second type to the , in several respects as follows : the appearance, distribution, form, stainability of granules and structure of nuclei. From cytophysiological view points, Halmi ('50) has concluded that the delta cells are the most likely source of FSH and thyrotrophin, while the possible riire of the beta cells in the formation of ACTH requires further investigation. Subsequently, however, he has suggested that the beta and delta cells are correlated respectively with thyro- trophic and gonadotrophic function ('51). On the other hand, Pu ryes and Griesbach ('51b and '51c) have considered that the Gomori positive granules are thyrotrophic hormone in a storange form, and that " gonadotrophs" is the site of production of gonadotrophic hor- mone. They postulated that the cells classified by Halmi as delta cell include all gonadotrophic basophiles plus those thyrotrophic cells with low hormone content which are unstained by the Gomori stain. The present data showed significant changes occurring in the basophile cells of the mouse anterior pituitary. In the A-group, the number of basophiles decreases markedly during the first half of experiment. In the B-group, on the contrary, the basophiles show a distinct increase in number during the same period. Moreover, in the C-group, slight increase in number of basophiles is observed only in the middle. It is interesting that the number of basophile cells re- turned to normal level in these three groups at the end of the experi- ment. These changes are due principally to the first type, and the second type cells are scarcely concerned. On the other hand, with the increase in dosage of Pituitrin, the dark or irregularly shaped nuclei of both types of basophiles increase gradually. But it is not generally observed that these cells show marked degenerative signs. Referring to the opinion of Pu ryes and Griesbach, the present observations seem to indicate that Pituitrin-injection has an action on their " gonadotrophs ". In the present results, however, the first type basophile cell, even if not conspicuous, showed an increase in number 262 M. Sano in the saline solution-injectiongroup too. Since it is unreasonable that the saline solution has an influence directly upon the , and since the changes taking place in the first type basophile cell appear rapidly after the Pituitrin-injection,it is presumablethat these changesin the first type cell are not pituitary histologicalmanifestations followingthe indirect action of the saline solutionor the Pituitrin on the anterior pituitary through the gonads, but to their direct effect on the anterior lobe. Selye ('37) previously demonstrated•that the administration of noxioussubstances resulted in adrenal hypertrophyin normal animals but not in hypophysectomizedones. He also postulated that exposure to stress resulted in the release of ACTHby the anterior pituitary gland. Subsequently,many investigatorshave proved this phenomenon. In addition, it has been shown that the hormonesof the posterior pituitary and the adrenal cortex have antagonisticactions on sodium and water excretion under various experimental conditions (Winter and Ingram, '43; Little et al., '47; Sartoriu s and Roberts, '49 and others). It has been, similarly,pointed out that smallcloses (5 mil) of pitressin did not stimulate the adrenal cortex as judged by the ascorbicacid depletiontest in rats, whereaslarger dose(100 or 400mU.) did so (Nagareda and Gaunt, '51). Stutinsky, Schneider and Den oyelle ('52)have also reportedthat vasopressinresulted in a signi- ficant depletionof ascorbicacid of the adrenal cortex in rats. Jailer ('50)has been indicated that the administrationof 100mU. pituitrin has no effect on the adrenal ascorbicacid contentin 3-4 day old rats, while newbornrats reveala sensitivepituitary-adrenocortical response by the administrationof an ACTH preparation(M o on, '40 ; Jailer, '50). In the presentexperiment, the Pituitrin dosageinjected is very large whencompared with the bodyweight of the mouse. It is therefore, ade- quatelypresumable that the Pituitrin willwork on the anteriorpituitary by way of stress, and will be followedby the release of ACTH. Accordingto the contributionsof numerousworkers, it has been established that ACTH is produced only by the anterior pituitary. Althoughmany investigatorshave attempted to ascribethe production of ACTHto one type of the anteriorpituitary cells, it has not yet been conclusivelyestablished. Further, some studies ascribe its secretion to acidophilesand some to basophiles. Classical studies ascribed the secretion of ACTH to basophiles. In pathologicmaterials, Kraus ('26 and '27) and Kraus and Tr aube ('28) reportedthat the numberof basophilesshows a marked diminution Cellularchanges in the anteriorpituitary of the mouse 263 on the of the pituitary gland following death from Addison's disease. Moreover, in the autopsy of patients from Addison's disease, Crooke and Russell ('35). observed that cliromOphobe cells are in- creased in number and acidophile cells reduced, and that constant fea- tures are the extreme reduction of basophiles and the presence of a series of abnormal basophile transitional cells. It has also been reported that Cushing's syndrome is accompanied with basophilic ade- nomas and adrenocortical hyperplasia in general (Cushing, '32a, '32b and '33). In addition, Cr o o k e ('35) has postulated that the 'hyaline changes observed in the basophiles of cases of Cushing's •syndrome, are not degenerative processes, but show a functional phase of the basophiles. These facts suggest that the basophiles secrete ACTH. Baillif ('38) stated that hypophyses of albino rats exposed to cold are characterized by a series of changes interpreted as phases of hypersecretion. Namely, in the anterior lobe there is a prompt and severe degranulation of alpha cells ; this process is preceded by the formation of small vacuoles ; beta cells are found to increase in size, to acquire vacuolated cytoplasm, and to discharge these vacuoles; exposure to severe stress results in profuse cellular degeneration followed by colloid formation. Reese, Koneff and A kim o to ('39) in a study of the anterior pituitary glands of male rats showing acute symptom after double adrenal ablation, found a diminution in number and size of acidophiles, correlated with progressive loss of granular material and regressive changes in the Golgi-apparatus. They observed, moreover, that the basophiles of their rats are also obviously diminished in number and size with cytological changes depending upon severity of symptoms and length of post-operative period. These two reports suggest that ACTH is correlated with both acidophiles and basophiles. Heinbecker and Rolf ('44), in a study of hypophysial aci- dophile cell and sensitivity, attributed adrenocorticotrophic secretion to the acidophile' cell. According to Finerty and Br iseno ('39), an increase in number of pituitary acidophile cells has been observed in rats 15 days after unilateral adrenalectomy, at w-hich time compensatory adrenal hypertrophy is evident. From this they concluded that the increased adrenocorticotrophic activity which' follows reduction in circulating adrenal cortical hormone (s) is mediated by an increase in number and activity of the acidophile cells of the hypophysis , and that the source of ACTH is the acidophiles. Several authors advocated that in the rat the percentage of anterior pituitery basophiles cannot be correlated with increased or decreased demand for endo- 264 M. Sano genous ACTH (Finerty and Briseno, '49; Halmi and Bogdanove, '51a and '5113; Halmi and Barker , '52; Golden and Bondy, '52; Finerty, Hess and Binhammer, '52). Finerty, Hess and Binhammer ('52) also suggested that pituitary cytological changes associated with ACTH secretion following severe burns involve only acidophile cells. In their study, however, it was pointed out that no significant change in the percentage of acidophile cells has been observed in an acute severe stress of scalding. Assuming that ACTH is secreted by acidophiles, the acidophiles must show a marked degra- nulation or diminution in the anterior pituitary of the rat exposed to severe stress, since ACTH demand must increase rapidly as a result of this stress especially at the early stage after treatment. It may be inadequate to admit the opinion of Finerty and co- workers, since the present data indicate that the number of acidophiles decreases in accordance with the increase of Pituitrin-dosage, and since it is likely that these acidophiles fall into hypofunction in general. As regards the secretion of ACTH, Gemzell, VanDyke, Tobias and Evans ('51) have shown an immediate and progressive increase in ACTH secretion, following adrenalectomy which may possibly be considered to be a severe, prolonged form of stress. A similar, transient pituitary ACTH depletion has been demonstrated by Sayers and Cheng ('49) following adrenalectomy. They observed that during the first 24 hours after adrenalectomy, there is a drop in pituitary ACTH content. According to the results of an extensive study of the general adaptation syndrome by Selye, the whole reaction of adaptation consists of three stages; stage of alarm, stage of resistance and stage of exhaustion. In the stage of alarm, pituitary ACTH content showed a marked reduction (Sayers and Cheng, '49; Gemzell et al., '51). It was proved that there is an increased pituitary ACTH content (Gemzell et al., '51) and an augmentation of basophile cell counts (Selye, '36; o k a, '37) in the stage of resistance. Moreover, it is probable that the histological changes described above in the autopsy of patients with Addison's disease (Kraus, '27; Kraus and Traub e, '28; Crooke and Russell, '35) correspond to the stage of exhaustion. D'Angelo, Gordon and Charipper ('48) found the increased percentage of basophiles concomitant with adrenal hypertrophy during inanition in guinea-pigs. From this fact, they postulated that these results are interpreted to mean that cortical hypertrophy in the starving guinea-pig results from augmentation of ACTH secretion by the basop- hiles of the anterior, pituitary. Similarly, Zeck wer ('38), Meligren Cellular changes in the anterior pituitary of the mouse 265

('48), Golden, Bondy and Sheldon ('50), Chiti and Zoinolli ('52) and Zondek ('53) ascribed ACTH secretion to basophiles. Marshall ('51) indicated that basophiles must contain ACTH, in a study of locali- zation of ACTH with histochemical and immunochemical methods. According to the present results, in the A-group the number of the first type basophiles shows a marked decrease in the first half of the experiment. It is presumable that this stadium corresponds to Selye's alarm stage, since it can be thought that the animals of this group have been exposed to a severe stress. Sayer s and C h e n g ('49), moreover, observed that the pituitary ACTH content shows a conspi- cuous decrease at this stage. The basophiles returned to normal number in A-10 of the present data. It is conceivable that this stadium coincides with a transitional period up to the stage of resistance. On the other hand, in the B-group the basophiles appear more numerous than in the normal controls in the first half of experiment. It may be accepted that the stage of alarm progresses rapidly and the stage of resistance comes in the early stages, and that ACTH release is in a low degree in comparison with its formation, since the stress which the animals of 13-group have received is somewhat milder than that of the A-group. It was also observed that numerous basophiles appear in this resistant stage (Selye, '35; Oka, '37). In the C-group and saline solution-jnjection group, the increase in number of basophiles is more slight than in the A- and B-groups, and this may depends on the fact that the stress is more weak in these groups than in the others. Furthermore, in the present observations, these changes in basophiles are due principally to the first type cell. From these results, the first type basophiles may be more closely connected with rise and fall of ACTH demand than in any other cell types of the anterior pituitary. Golden, Bondy and Sheldon ('50) found, in man after ACTH therapy, an increase in the total number of basophiles, Crooke's hyaline cytoplasmic changes in these cells and the basophilic stippling of many chromophobes. In this study they supposed that these changes reflect the storage of endogenous ACTH following stimulation of the adrenal cortex by the therapeutic administration of this hormone. Subsequently, however, Golden and Bondy ('52) have reported that the percentage of anterior pituitary basophiles and the presence of Crooke's change cannot be correlated with increased or decreased demand for endogenous ACTH. Ha1mi and Bog danove ('51a) reported that neither the granulation of acidophiles nor that of beta cells can be the morphological equivalent of ACTH storage. 266 M. Sano They also concluded that adenohypophysial ACTH is not associated with the delta granules, since no parallelism was observed between the ACTH content and the amount of delta granules in rat pituitaries ('51b), It was shown that there was no histological change which could have been correlated with diminished ACTH output in the pituitaries of cortisone treated rats (Hal mi and Barker, '52). How- ever, it stands to reason that the amount of beta- or delta-granules is not correlated with the pituitary ACTH content, since (1), the granules of basophile cells are not a definite hormone itself but presumably a precursor of the hormone; since (2), the number of basophiles or the state of basophilic granules at a certain moment is controlled by two factors, i. e. release and formation of the hormone (Sayers and Cheng, '49); and since (3), the pituitary -ACTH content which can be appre- ciated by a bioassay method should include the hormone present not only intracellularly but also extracellularly. According to Purves and Griesbach ('51a), it would seem unlikely that any other non-glycoprotein hormone will be produced by PAS positive cells. The three pituitary hormones containing sugar in significant amounts are follicle stimulating, luteinizing and thyrotrophic hormones (Li and Evans, '48). Although many investigators have worked on the purification of ACTH for a long time, its chemical structure has not yet been conclusively established (Co llip, Anderson and Thomson, '33; Lyons, '37; Li, Simpson and Evans, '42; Li, Evans and Simpson, '43; Sayers, White and Long, '43; Burns et al., '49). For this reason, this problem will not be discussed. It is generally believed that there is an intimate relationship between the anterior pituitary and the gonads (Heller and Nelson, '48). It was established that castration is followed by augmented production of pituitary gonadotrophins, as reflected in the increase in number of pituitary basophiles and in an augmented gonadotrophic potency of the ,gland (Smith, Severinghaus and Leonard, '33; Hayashi, '55). On the other hand, the adrenal cortex is also closely connected with the gonads through ACTH. Shumacker and Fir or ('34) have reported that the hypophyses of adrenalectomized rats show a decrease in gonadostimulating power. On the other hand, S z ego and White ('51) reported that previous castration in mice or rats inhibits significantly pituitary-adrenocortical response in inanition. Furthermore, it has been confirmed that the inanition is effective as a stress in augmenting pituitary-adrenocortical activity (White and Dougherty, '47; D' Angelo, Gordon and Cellular changes in the anterior pituitary of the mouse 267 Charipper, '48 ; Szego and White, '51). These facts suggest that there is a mutual interference between the gonadotrophic potency and adrenocorticotropic power of the anterior pituitary. Se 1ye ('36, '37 , '39 and '40) postulated that the atrophy of the sex organs is a part of the " general adaptation syndrome," and that in cases of emergency, the pituitary tends to produce more adrenotropic and less gonadotropic hormone than under normal conditions. He interpreted this fact that, under such conditions, an abundant supply of a life-maintaining principle of the adrenal cortex is a more imminent necessity than the preservation of normal sex function. Se lye and Co Ilip ('36) expressed the opinion that under the influence of damaging agents, there may be a " shift phenomenon " in the hormone production of the pituitary, since the same agents which cause atrophy of the sexual glands and anestrus in the female, usually also elicit enlargement of the adrenal cortex and inhibition of growth. Mellgr en ('48) and D'Angelo, Gordon and Char ipp e r ('48) also supported this shift phenomenon. Although the determination of site of ACTH production requires further studies, from the present data, it may be assumed that besides gonadotrophin production, the first type basophile cell is correlated intimately with adronocorticotrophin production or release. In the present results the basophiles show various cytological changes, and their number are variable in experimental conditions. In spite of the fact, mitotic figures of basophiles cannot be observed. Moreover, in chromophobes there are the cells conceivable as a • tran- sitional form to basophiles. For this reason it can be presumed that the basophiles and a part of the chromophobes are mutually trans- formable as described in the article of chromophobe cell.

The chromophobe cell The cells of the anterior pituitary were divided into chromophile and chromophobe cells by F les c h (1884). Since then the chromophobe cells have been named " Hauptzelle," " Chromophobe," " Kernhaufen " ungranulierte Zelle " etc ," . by various authors. In the present paper , the chromophobes are classified as follows : ungranulated, granulated and peculiar chromophobe cells. 1) The ungranulated chromophobe cell. The cytoplasm of this chromophobe cell is scanty and difficult to stain. The nucleus of the cell is small, round or oval and has little coarse chromatin particles and an acidophilic nucleolus. The nucleus shows the features of the spots of a die. It is without doubt that these chromophobes are identical 268 M. Sano • with Rogowitsch's " Kernhaufen " or Romeis's " undifferenzierte Zellen." In addition to this chromophobe cell, the sponge-gourd-shaped cell is observed numerously in the Pituitrin-injection group. It will be accepted from the character of the nucleus, that this cell is immature and undifferentiated. The significance of their occurrence in the present experiment is not clear. 2) The granulated chromophobe cell. These chromophobes contain a small quantity of cytoplasm. Their cytoplasm show a few granular or flocculent structures, and are difficult to stain with common stain- ing methods. It is possible to divide some of these cells into two types by their nuclear characters. One of them is smaller. Their nuclei resemble that of the acidophile cell, since they contain abundant coarse chromatin particles and a prominent acidophilic nucleolus. The other is larger. Its oval nucleus shows a delicate network of fine chromatin granules and an acidophilic nucleolus. The nucleus is similar to that of basophiles. It is conceivable that these two types of granulated chromophobe cells are either the undifferentiated cell from which acidophile and basophile cells originate, or the differentiated cell (" gamma-Zelle " proposed by Romeis). I think that these chromophobes are not identical with Romeis's " gamma-Zelle," but with the undifferentiated cells, since these cells contain only a few graunles in the cytoplasm and indistinct cell boundaries, and since, as described above, there are transitional forms between chromophobes and the two types of chromophiles. It is probable, moreover, that these two types of granulated chromophobe cells correspond to Severinghaus's acidophilic and basophilic chrome- phobe cells. 3) The peculiar chromophobe cell. This chromophobe cell contains a cap-like structure surrounding the cytoplasm. The structure consists of many filaments staining with hematoxylin or azocarmine, and it is generally believed that these filaments represent ribonucleic acid (Desciin, '40 ; Aboling, '52 and others). These chromophobes are few in male mice, but are observed in more numbers in the female. In the present experiment their appearance was of the same degree as in the normal. Although it was reported that these chromophobes reveal an increase in number in (Desclin, '40 ; K at o, '56) and after castration (Yamada, Kato and Sano, '56), regarding the significance of their appearance in the present experiment, further studies are required. Cellularchanges in the anterior pituitaryof the mouse 269

Summary A histological examination was made on the hypophyses of adult mice injected with various dosages of Pituitrin. 1. Acidophiles became reduced gradually in number in accordance with the increase in dose of Pituitrin. 2. Basophiles showed a decrease in number in the large dosage injection group (daily dose 1000 mU. of Pituitrin), and an increase in number in the medium dosage injection group (daily dose 100 mU. of Pituitrin). In these groups the number of basophile cells returned to normal at the end of the experiment. In the small dosage injection group (daily dose 10 mU. of Pituitrin), there were no significant changes in the number of basophiles. These numerical changes in the basophiles were due principally to the first type cell. From this, it may be assumed that besides gonadotrophin production, the first type basophile cell is connected intimately with adrenocorticotrophin pro- duction or release. 3. Chromophobes revealed an increase in number with increased dosage of Pituitrin, and the sponge-gourd-shaped chromophobe cell was observed frequently in the Pituitrin-injection group. 4. In spite of these numerical changes in the secretory cells of the anterior pituitary under various experimental conditions, mitotic figures of these cells did not appear so markedly, though transitional forms between chromophobes and two type chromophiles were seen. From this fact, it can be accepted that the chromophobes and the two type chromophiles are mutually transformable. I wish to thank Prof. Dr. K. Yamada for his constant guidance and encouragement throughout this work.

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Explanations of Figures As Small acidophile cell Am Medium size acidophile cell Al Large acidophile cell B1 The first type basophile cell B2 The second type basophile cell

. 276 M. Sano

Ca The chromophobe cell interpreted as a phase of transition between chromophobe and acidophile cells Cb The chromophobe cell interpreted as a phase of transition between chromophobe and basophile cells Cp Peculiar chromophobe cell Cs Sponge-gourd-shaped chromophobe cell Cu Undifferentiated chromophobe cell

Plate 1. Fig. 1. Small acidophile cells with round cytoplasm and a centrally situated round nucleus are observed. Many medium size acidophile cells reveal oval or elonga- ted cytoplasm and provide an eccentrically situated light vesicular nucleus. From a 60-day-old female mouse of normal control group. Fixation: Zenker-formol-acetic acid. Azan stain. x1300. Fig. 2. There are observed three large-acidophile cells showing a light vesicular nucleus, a perinudear halo and a negative image of the Golgi-apparatus. From a female mouse of A-3 group animals. Fixation: Zenker-formol-acetic acid. Hematoxylin-eosin stain. x1500. Fig. 3. The first type basophile cell of round and polygonal form. Their nuclei show a light cast. Their granules staining pale blue with aniline blue are very fine, and are diffusely distributed throughout the cytoplasm . From a 130-day-old male mouse of normal control group. Fixation: Sublimate-formol-acetic acid. Azan stain. x1500. Fig. 4. The figure shows the junctional zone of the pars anterior with the pars tuberalis, and many the first type basophile cells. From a 130-day-old male mouse of normal control group. Fixation: Sublimate-formol-acetic acid. Azan stain. x 1300.

Plate 2. Fig. 5. The second type basophile cells are angular in form and contain dense dark blue, coarse granules. Their nuclei show a very dark cast. From a 130-day-old female mouse of normal control group. Fixation : Sublimate-formol-acetic acid. Azan stain. x 1500. Fig. 6. Two sponge-gourd-shaped cells and several undifferentiated chromophobe cells are seen. There are, moreover, chromophobe cells interpreted as a phase of tran- sition betWeen chromophobe and chromophile cells. From a male mouse of A-10 group animals. Fixattion: Zenker-formol-acetic acid. Hematoxylin-eosin stain. x 1500. Fig. 7. There are seen two peculiar chromophobe cells. From a 110-day-old fem- ale -mouse of normal control group. Fixation: Sublimate-formol-acetic acid. Azan stain. x1500. Fig. 8. Acidophile cells containing irregularly shaped nuclei, indented or wrinkled, are seen. From a female mouse of A-5 group animals. Fixation: Zenker-formol-acetic acid. Hematoxylin-eosin stain. x 1600.

Plate 3. Fig. 9. Several acidophile cells With pycnotic nuclei are observed. From a male mouse of A-10 group animals. Fixation : Zenker-formol-acetic acid. Hematcrxylin-eosin stain. x1300. Fig. 10. There appear irregularly shaped nuclei of the first type basophile cells. Cellular changes in the anterior pituitary of the mouse 277

From a male mouse of A-10 group animals. Fixation : Zenker-forinol-acetic acid. Azan stain. x 1400. Fig. 11. Vacuolated the first type basophile cel!s are seen. These cells, in all Pitui- trin-injection groups except for A-1 group animals, are found in less numbers than in normal control animals. From a female mouse of A-1 group animals. Fixation : Zenker- formol-acetic acid. Azan stain. x 1100. M. Sano Plate I

Fig. 1. Fig. 2.

Fig. 3. Fig. 4. Cellular changes in the anterior pituitary of the mouse Plate II

Fig. 5. Fig, 6.

Fig. 7. Fig. S. M. Sano Plate III

Fig. 9. Fig. 10.

Fig. 11.