Okajimas Fol. anat. jap., 43: 21-51, 1967

Light and Electron Microscopic Observations on the of the Mouse injected with dl-Thyroxine By

Tomiji Uchida

Department of Anatomy, Nagoya University School of Medicine, Nagoya, Japan (Director : Prof. Dr. Ka z u m a r o Y a m ad a)

Introduction

Probably, the first suggestion of the existence of a functional relationship between the and hypophysis was given by Niepce (1851) who described pituitary enlargement in a series of goitrous cretins. Since then numerous studies have provided detailed evidence of this relationship. The reciprocal interrelationship be- tween thyroid stimulating (TSH) secretion in the anterior hypophysis and the circulating levels of thyroid hormone has been well established, and in general, procedures which reduce effective plasma concentrations of thyroid hormone increase thyrotrophic hormone release in the anterior pituitary, whereas increased circulat- ing levels of thyroxine inhibit thyrotophic hormone secretion. This negative feedback mechanism resides both in the level of the pituitary and of a hypothalamic " TSH releasing center " (S o 1 o m on and Dowling, '60). Earlier views (Ma rin e, Rosen and Spar k, '35; Morris, '52) favored pituitary acidophile as the source of thyrotrophic hormone, but results of many recent studies implicate the basophile cell (Zeckwer, '38a and '38b; Griesbach and Purves, '45 Pur v es and Griesbac h, '46a, '46b, '51a, '51b, '51c, '57a and '57b; Goldberg and Chaikoff, '50; Salter, '50, Halmi, '50, '51, '52a , '52b, and 52c ; R ennel s, '53; Halm i and G u d e, '54 D'Angelo, '53 and '55; Knigge, '55; Elf tman, '58; Mura - s h i m a, '60 and others). Halm i ('50, '52a, '52b and '52c) using Gomori's aldehyde-fuchsin method (G o m o r i, '50) recently, differentiated clearly two functionally distinct types of basophile (beta and ) in the

21 rat pituitary, and stated beta basophiles correspond to thyrotrophic hormone secretion. P u r v es and G r i e s b a c h ('51a, '51b and '51c) moreover, applied periodic acid-Schiff's reaction to the rat pituitary which allegedly discriminates thyrotrophic and gonadotrophic hormone producing cells routinely classified as basophiles. More recently, electron microscopy has been used to study on hypophysis (W e i s s and Lansing, '53; Rinehart and Farquhar, '53; Farqu- har and Rinehart, '54a, '54b and '54c; Farquhar and Wel- lings, '57; Farquhar, '57; He dinger and Farquhar, '57; Peterson, '57; Ichikawa, '59; Yamada and Sano, '60; Barnes, '61, '62 and '63; Hymer, McShan and Christian- sen, '61; Herlant and Klastersk y, '61; Lundin and Schelin, '62; Rennels, '62 and '64; Sano, '62; Hymer and McShan, '63; Salazar, '63; Siperstein and Allison, '65; Young, Foster and Cameron, '65). Although cytological alterations in the hypophysis of hypo- and hyperthyroid animals have been extensively described by many inves- tigators, electron microscopic description of the anterior pituitary in experimental hyperthyoid animals has previously been little reported. Experimental hyperthyroidism induces a quantitative alteration of various hormonal conditions in the anterior pituitary. The present auther previously, reported on the anterior pituitary cytology of the hyperthyroid mouse made with the light (U chid a, '64) . In the present study, light and electron were employed to investigate the cytological changes in the mouse anterior pituitary following thyroxine injection.

Materials and Methods

Sixty nine young adult male DD strain mice, 51 to CO days age, were used in this study. They were divided into two groups, namely experimental group and control group. In the present study, dl- Thyroxine was dissolved in 0.02 N. sodium hydroxide made isotonic with sodium chloride and administered to the mice in the form of daily subcutaneous injection. The levels of thyroxine dosage were 0.5, 1.0, 5.0 and 10.0 pg, and the period of injection 3, 7, 14, 21 and 30 days. In the same way, control groups were injected with the same solution without thyroxine. Most animals were sacrificed 24 hours after the last injection under ether anesthesia. The pituitaries were removed immediately after death and fixed in Zenker-formalin- acetic acid fixative. The tissues were imbedded in paraffin and cut serially in the sagittal plane at 4 p. They were stained with hematoxylin-, Heidenhein's azan, periodic acid-Schiff's reaction (PAS) and Gomori's aldehyde-fuchsin. On the other hand, for electron microscopy, some of the animals were killed by decapitation. The pituitaries were dissected out quickly and fixed in buffered isotonic 1 per cent osmium tetroxide (0504) (P a la d e, '52) at ice-cold (0-4°C) temperature for 1 hour. After fixation the tissues were rinsed in distilled water, rapidly dehydrated in an ethanol series, and embedded in Epon 812 (L u f t, '61). Sections were cut with a Porter-Blume ultramicrotome, using glass knives with cutting edge angle of 45°, and mounted on mesh copper grids without a supporting film of carbon. The sections were stained by floating them face downward on a saturated solution of uranyl acetate (W a t s o n, '58) in 50 per cent ethanol for periods ranging from 30 min. to 60 min. and then were washed thoroughly in distilled water. Sections were examined and photographed with a Hitachi Hu 11A electron microscope.

Observations

The anterior pituitary of the normal mouse consisted of chromo- phobes and three types of chromophile : the acidophiles and two types of basophile, (thyrotroph) and delta cell (gonadotroph). The acidophiles (Fig. 1), by light microscopy, are more abundantly found in the lateral portion of the anterior lobe. The cells present a sharply defined cell boundary and are round or oval in shape. They contain cytoplasmic granules which are selectively stained with eosin, orange-G and azocarmin. Acidophiles vary in size, but the majority are medium in size. These medium sized cells are generally well granulated. Their nuclei are also round or oval in shape, light vesicular, and there are frequently seen cartwheel-like nuclei. Moreover, considerable numbers of these cells possess a negative image of Golgi's apparatus or perinuclear halo. The large acidophiles and small acidophiles are observed only in small numbers. Mitotic figures of acidophiles are rarely found. By electron microscopy two types of acidophile were identified. The first type cell of acidophiles (Figs. 7 and 10) is more numerous and conspicuous type. The spherical and electron dense granules of these cells are scattered randomly throughout the cytoplasm and range in size from 350 to 400 mp, although occasionally they may be as large as 500 mil. The endoplasmic reticulum of the commonly encountered form appears as a series of flattened sacs in parallel 24 Tomiji Uchida rows and is stacked to one side of the cell. But occasionally the endoplasmic reticulum is markedly enlarged. Scattered elements of the endoplasmic reticulum can be seen either around the nucleus or between the granules and the membrane. A few mitochondria and moderately developed Golgi zone are also observed. The second type cell of acidophiles (Fig. 8) is characterized by the presence of oval or irregularly shaped dense granules peculiarly concentrated near the Golgi area. Their granules range in size from 200 to 400 mp in their longest dimension, and from 100 to 200 my in their shortest. The endoplasmic reticulum is arranged as flattened parallel lamellae and stacked to one side of the cell in the vicinity of the plasma membrane. The Golgi zone is relatively small. The mitochondria are rod-shaped and possess an electron dense matrix. In both type cells, ribosomes are usually found attached to the endoplasmic reticulum membranes. The beta basophiles (Figs. 2 and 3), by light microscopy, are larger than the acidophiles, and angular or even crescent in shape. They are more numerous in the central portion of the anterior lobe, but rarely encountered. Their granules are coarser than those of the acidophiles and are stained with PAS reaction and Gomori's aldehyde-fuchsin (Fig. 3). Nuclei of the beta cells are round or oval in shape, usually smaller than those of the delta cells and contain . abundant chromatin granules. The negative image of Golgi's appa- ratus is seen indefinitely. In electron microscopic observations, these beta cells (thyrotrophs) (Figs. 9 and 10) are also polygonal in outline with a small nucleus. Their spherical secretory granules are relative- ly constant in size, ranging from 50 to 100 my, but they vary markedly in electron density. The cytoplasm of the cell is relatively devoid of fine structural detail. The fine granules of the beta cells are sparsely and randomly distributed throughout the cytoplasm. Occasionally they are particularly arranged in the periphery of the cytoplasm. The ground substance of the cytoplasm is more electron lucent than other cell types of the anterior pituitary. Few ribosomes are present, either attached to the membrane system or lying free in the cytoplasm. The Golgi's apparatus in these cells is relatively poorly developed. The endoplasmic reticulum appears as scattering of small vesicles and its development is also relatively poor. The mitochondria are small and appear rather as dense rods. The delta cells (gonadotrophs) are located mainly in the anterior portion, so-called sex-zone " and scattered in the central region of the anterior lobe. In light microscopic observations, delta cells (Fig. Light and Electron Microscopic Observations on the Anterior Pituitary 25

2) are oval or polygonal in shape and densely packed with fine, dust- like granules that are stained with aniline blue of the azan staining or PAS reaction. But these granules are not stained with aldehyde- fuchsin. They vary in size, but usually larger than those of the acidophiles. They are grouped in clusters around the portal vessels. Hyalin vacuoles are occasionally observed in the cytoplasm of the cell. Nuclei of the delta cells are round or oval in shape and contain abundant fine chromatin materials. The delta cells can be divided into two types by electron microscopy. The first type cells of the delta cell (Fig. 10) are large, usually round or oval in outline and almost invariably located adjacent to a blood vessel. Their spherical secretory granules are relatively electron lucent and range in size from 150 to 200 mp. Their granules are scattered randomly throughout the cytoplasm. Few ribosomes are present, either attached to the endoplasmic reticulum membranes or lying free in the cytoplasm. Electron dense materials are not observed within the cisternae of the endoplasmic reticulum in these cells. The mitochondria are rod-shaped with dense matrix , and relatively large. The Golgi's apparatus shows a multi-vesiculated structure and usually forms an extended half-circle in the vicinity of the nucleus. The Golgi's apparatus is well developed but the endoplasmic reticulum is poorly developed. The second type cells of the delta cell (Fig. 11) are relatively small and round or rarely polygonal in outline. Usually they are located close to a blood vessel. Their spherical granules are uniformly dense but vary in size, generally ranging from 100 to 200 m ite, and frequently are concentrated at the periphery of the cell . The nucleus is relatively large as compared with the cytoplasm . The Golgi's apparatus is moderately developed and the endoplasmic reticulum is relatively poorly developed. The mitochondria are rod-shaped with electron dense matrix. In light microscopic obsevations, chromophobe cells are small in size and contain a few granules or flocculent materials which are difficult to stain with various dyes, and they are the dominant cell types in the anterior lobe. They vary in size , but are in general about the size of acidophiles. They are irregular in shape , having poorly defined cell bounderies, and contain a light, homogeneous, nonstaining and finely granular cytoplasm . Although cells without visible granules are classed as chromophobes , by electron microscopy very few true chromophobes (Fig. 11) are seen , for most cells classed as chromophobes by light microscopy , when obserbed with electron 26 Tomiji Uchida microscope, are seen to contain variable numbers of granules of a characteristic size which would place them in either acidophile or basophile series. No granules and a few or almost no endoplasmic reticulum are seen in these true chromophobes. The nuclei are large in size in comparison with the cytoplasm. Golgi's apparatus is obscure and a few mitochondria are rarely found.

Experimental group

1) 0.5 pg daily injection group : A slight increase in number of acidophiles was observed by light microscopy. At the early stage of injection (3rd and 7th day) acidophiles with moderate granulation were frequently observed as in the controls, but in the late stage they were found somewhat less in number. Mitotic figures of acidophiles were seen as frequently as in the controls. In electron microscopic observations acidophiles did not show remarkable changes in cellular fine structure. On the other hand, the more remarkable changes occurred in the beta cells. With the Gomori's aldehyde-fuchsin staining, measurable decrease in number and size was already observed in beta cells of animals injected for 3 days. They were angular in shape and seemed to contain few aldehyde-fuchsin staining granules. With continued treatment, a more pronounced decrease in number of aldehyde- fuchsin staining beta cells was observed on the 7th day and aldehyde- fuchsin staining granules almost completely disappeared in mice which received injections for more than 14 days, whereas with the PAS staining, angular-shaped cells considered to be beta cells were still fairly present. Moreover, these angular beta cells were relatively well granulated. The negative image of the Golgi's apparatus in beta cells seemed to have increased in number at this late stage. Vacuolization was rarely found in the perinuclear region of the cytoplasm. By elccton microscopy, beta cells with a large numbcr of granules were encountered as frequently as in the controls throughout the course of this experiment. The granules of beta cells were scattered throughout the cytoplasm. The Golgi's apparatus and endoplasmic reticulum were well developed at the late stage of this experiment. Ribosomes attached to the cndoplasmic reticulum and lying free in the cytoplasm, were found more numerously. Small vesicles were frequently observed in the cytoplasm (Figs. 14 and 17). Delta cells, by light microscopy, did not show a significant Light and Electron Microscopic Observations on the Anterior Pituitary 27 change. On the 7th day, a slight decrease in number and size of delta cells was observed. During this period partially granulated and vesiculated delta cells were encountered. But from the 14th day of this experiment delta cells bogan to increase in number gradually and to approach the normal level. Remarkable changes in the cellular fine structure of delta cells (in both first and second type delta cells) were not observed by electron microscopy (Figs. 14 and 17). In this group, chromophobes slightly increased in number when compared with the normal controls. 2) 1.0 lig daily injection group : The acidophiles were not different from those of the controls at the early stage. The majority of acidophiles were moderately granulated as in the controls. At the late stage (21st and 30th day), a slight decrease in number and granules of acidophiles was observed. In electron microscopic observations, the first type acidophiles were moderately granulated (Figs. 13 and 16) and a slight increase in number of granules of the second type acidophiles was observed (Figs. 13 and 16). The beta cells were also significantly decreased in number, size and aldehyde-fuchsin positive granules. With the aldehyde-fuchsin staining, they began to show a definite decrease in number and granules from the 3rd day after administration of thyroxine. Many of them contained pale aldehyde-fuchsin staining granules, which almost completely disappeared in animals injected for over 7 days, though angular beta cells were still numerously found with other staining (PAS reaction) (Fig. 4). By electron microscopy, a slight increase in secretory granules of the beta cells was observed on the 7th and 14th days (Figs. 13, 15 and 16). The granules of the beta cells were scattered in the cytoplasm. Golgi's apparatus and endoplasmic reticulum were well developed, and occa- sionally dilated (Fig. 16). The cytoplasmic background of the beta cells was relatively dark because of increasing ribosomes (Fig. 15). With continued treatment, amount of granules of the beta cells began to increase. Oval or rod-shaped mitochondria were seen in the cytoplasm (Fig. 15). The delta cells were similar to those observed in the O. fig injection group except that the relatively well granulated delta cells were more frequently observed in the late stage of this experiment. In some delta cells with vacuoles the nuclei and negative image of Golgi's apparatus were, like the signet-ring cells, compressed into thin rims of cytoplasm at the periphery of the cells. Many nonvacuolated delta cells filled with coarse deep PAS positive granules were also present. The negative image of Golgi's apparatus of these 28 Tomiji Uchida cells was not enlarged and the nuclei were often pyknotic in appear- ance. In the fine structure of the first type delta cells significant alterations were not observed. Many of them were moderately granulated. Endoplasmic reticulum of the cells appeared as small vesicles, and occasionally they were markedly dilated like vacuoles. In general the second type delta cells were also well granulated. 3) 5.0 pg and 10.0 pg injection groups : The findings in these experimental groups were generally similar to those observed in the 1.0 pg injection group. A more prounced decrease in number and amount of granules of acidophiles was observed in the late stage. In electron microscopic observations, the majority of the first type acidophiles contained relatively few secre- tory granules and endoplasmic reticulum was frequently arranged as flattened parallel lamellae (Fig. 12). Beta cells also significantly decrease in number and granules. Aldehyde-fuchsin positive beta cells completely disappeared in animals that received injections for over 7 days. A decrease in granules of these beta cells stained with PAS reaction was seen at the late stage in these experimental groups. Beta cells, by electron microscopy, were well granulated at the early stage of these experiment. From the 14th day the granules of the cell began to decrease, with more decreases in granules observed on the 21st and 30th days after injection (Fig. 18). The cell like thyroidectomy or gonadectomy cell was rarely seen at the late stage in some cases. The endoplasmic reticulum of such cell was markedly dilated. The number of delta cells increased, but the cytoplasmic granules slightly decreased at the late stage (Figs. 5 and 6). By electron microscopy the first and second type delta cells showed a slight decrease of secretory granules.

Discussion

It is well known that each endocrine influences on the other endocrine organs, and any deficiency or excess of function of any single gland calls upon the other glands either to decrease or increase their activity, so that equilibrium may be maintained. The present work was undertaken in an attempt to study, using light and electron microscopes, the cytological changes in the anterior pituitary of experimental hyperthyroid mouse. Generally, three cell types tine- tonally have been differentiated in the anterior pituitary. Acidophile cell : Two types of acidophile have been differentiated (C le v elan d Light and Electron Microscopic Observations on the Anterior Pituitary 29 and Wolfe, '32; Wolfe, Cleveland and Campbell, '33; Dawson, '38; Dawson and Friedgood, '38; Romeis, '40; Rahn and Painter, '41; Copeland, '43; Hartmann, '44; Hartman, Fain and Wolfe,'46 ;Goldbergand Chaikoff, '52; Miller and Robbins , '55; Farquhar and Rinehart, '54a , 54b and '54c; Hedinger and Farquhar, '57; Barnes, '61, '62 and '63 ; Herlan t, '64). In the present study, two types of acidophile were not differentiated clearly by light microscopy, but by electron microscopy acidophiles could clearly be divided into two types, namely the first and second types cell ; the former is considered the site of production of , and the latter is considered to be responsible for the production of , on the basis of numerous studies (H e d i n g e r and F a r q u h a r, '57 ; Ichikawa, '59; Yamada and Sano, '60; Hymer, McShan and Christiansen, '61; Barnes, '61, '62 and '63; Hymer and McShan, '63 ; Salazar, '63; Herlant, '64). It has been known for a long time that thyroid insufficiency inhibits growth. E a r tl y and L eb1011d ('54) described that thyroxine stimulates the secretion of growth hormone and in its absence the secretion of growth hormone decreases. Changes of pituitary acido- philes in hyperthyroid animals have been reported by numerous investi- gators. Early, K o j i m a ('17) described in rat treated with thyreoidea sicca decrease of acidophiles in number in the early stage, and if thyroid feeding is prolonged the number of acidophiles again increases. M o m o se ('33) showed that with a small dose of thyroid substance acidophiles increase in number but with a large dose decrease. Neuhaus ('39) reported decrease in size and amount of granula- tion of acidophiles. Also according to Halmi ('52c) acidophiles increased in number in rat treated with a small dose of thyroxine, but with a large dose decreased in size. T a j i m a ('55) described that a-granules in acidophiles disappeared in rabbit treated with thyroid powder. In our laboratory, M u r a s h i m a ('60) described that in hyperthyroid mouse acidophiles showed hypofunction. Thus, many workers have maintained that in hyperthyroid animals, acido- philes diminish in number, size and granules except in case of a small dose. In the present study, in animals treated with a relatively small dose and for a short term, acidophiles showed almost normal or slightly hyperfunctional state. On the other hand, with a large dose and long term administration decrease in amount of granulation of acidophiles was observed, and acidophiles seemed to be in hypo- f 9nction in appearance of their fine structure. Thyroid has been 30 Tomiji Uchida considered to be associated with the activety of acidophiles, especially growth hormone secreting cells. However, if the animals are normal in thyroid function acidophiles seem to be almost normal or slight hyperfunctional with administration of a small dose of thyroxine, but a large dose of thyroxine induces decrease in number and amount of granulation and hypofunction of acidophiles. Basophile cell : Basophile cells have been divided into two cell types ; the beta cell (thyrotrophic basophile) and the delta cell (gonadotrophic basophile) using new stainings, the Gomori's aldehyde-fuchsin (H a I m i, '50) and periodic acid-Schiff reaction (C at c h i p o l e, '49 ; P u r v e s and Grieshac h, '51a, '51b and '51c ; Pears e, '50, '52a, '52b and '53 Siperstein, Nichols, Grieshach and Chaikoff, '54; son and E z r i n, '54). Moreover, recently, delta cells have been further subdivided into two cell types. P u r v e s and Grieshach ('54) divided delta cells into central gonadotrophs (LH secreting cells) and peripheral gonadotrophs (FSH secreting cells). R e n n e 1 s ('57) has also differentiated two types of gonadotrophic cell using PAS staining. By electron microscopy, Farquhar and Rinehart ('54a) in the castrated rat pituitary distinguished two types of delta cell, and they stated that one correspond to FSH cell, and the other to LH cell. Furthermore, LH secreting cells and FSH secreting cells have been identified by Barnes ('61, '62 and '63) in the mouse pituitary . In the present work, two types of delta cell were distinguished by electron microscopy. A change of pituitary basophiles in hyperthyroidism has been described by many inves- tigators. Atrophic or hypofunctional changes of basophiles have been reported by numerous authors (K iy on o, '24 ; Berblinge r, '32; Momose , '33; Connor, '37; Frank, '37 ; Neuhaus, '39; Wegelin, '38; Griesbach, Kennedy and Purves, '49). Conversely, hyperfunction of basophiles in the hyperthyroid state has been described by some workers (M o m o s e, '33 ; Cam p b e 1 1, Wolfe and Phelps, '34; Severinghaus, Smelser and C l a r k, '34 ; C o h e n, '35). However, these above described authors have not distinguished two types of basophile cell. Recently baso- philes have been divided into beta and delta cells, and Hal m i ('51) stated that administration of thyroxine to male rats failed to elicit conspicuous changes in delta cells, but a striking diminution in number of beta cells resulted. P u r v es and Griesbach ('Sic) described that with sufficient thyroxine dosage given over a long enough period G o m o r is aldehype-fuchsin positive cells completely Light and Electron Microscopic Observations on the Anterior Pituitary 31 disappeared from the rat pituitary, and they concluded that aldehyde- fuchsin stained granules are thyrotrophic hormone in a storage form, since the aldehyde-fuchsin stained granules fall to zero levels while the thyrotrophic hormone contents, though lower than normal, can still be demonstrated. Moreover, they stated that the cells classified by Halmi as delta cells include all basophiles without a high TSH content and, therefore, include all gonadotrophic basophiles and those thyrotrophic cells with low hormone content. It has widely been known that a decline in pituitary thyrotrophic potency occurs in hyperthyroid animals (H ohlweg and Junkman n, '33 ; Kuschinsky, '33; Ref erzo-Membrives, '43; Adams and Jensen, '44; Purves and Griesbach, '46b). In the present data by light microscopy, agreement with the results of these above described P u r v e s and G r i e s b a c h's study ('51c) showed defined decrease in number or complete disappearance of aldehyde-fuchsin staining beta cells in animals that received thyroxine injection. But by electron microscopy, the granules of the beta cells were observed numerously in the cytoplasm, especially in the early stage of the experiment, and even an increase in granules of beta cells was seen frequently. On the basis of these cytological changes, it can be assumed that thyroxine administration induces inhibition of thyro- trophic hormone release, and hence, the secretory granules of the beta cells are accumulated in the cell. However, these granules observed by electron microscope seemed not to correspond to the hormone itself. K n i g g e ('58) noticed that in thyroidectomized animals the disappearance of granules did not coincide with disap- pearance of the hormone, and he concluded from this fact that the granules do not correspond to the hormone itself. Adams ('46) has summarized the opinions regarding the source of thyrotrophic hormone in the anterior pituitary. Recent results of many inves- tigators tend to support the view that the basophiles (beta cells) are the source of thyrotrophic hormone secretion (S al t e r , '50 ; Goldberg and Chaikoff, '50; Halmi , '50, '51 and '52a; Purves and Griesbach, '51a, '51b and '51c; Rennels , '53; D'Angelo„55 ; Elftman,'58 ; M u r a s h i m a , '60). It is possible to conclude that beta cells including aldehyde-fuchsin negative beta cells correspond to the source of thyrotrophic hormone . On the basis of numerous investigations by castration or hormone administration, delta cells are believed to be the site of production of gonadotrophic hormone (E Ti g 1 e, '29 ; E vans and Sim p so n, '30; Wolfe , '32; Severinghaus, '32 and '39; Smith, Seve- 32 Tomiji Uchida

ringhaus and Leonard, '33; Clark, '35a and '35b). Engle ('29) and Evans and Simpson ('30) demonstrated an increase in concentration of pituitary gonadotropins in the castrated rat. Recently, H a 1 m i ('51) has stated that delta cells are cor- related with gonadotrophic function. Furthermore, P u r v e s and G r i e s b a c h ('51b and 51c) postulated that the cells classified by Halm i as delta cells include all gonadotrophic basophiles plus the thyrotrophic cells, with low hormone content, which do not stain with G o m o r is staining. Hal m i ('50, '51 and '52a) and P u r v es and Griesbach ('51a, '51b, '51c, '52, '54, '55 and '57b) have estab- lished the cellular origin of gonadotrophic hormone, while Halmi ('50) and Pur v es and Griesbach ('51a and 51b) were able to show in the rat that only category of basophiles display vacuolar hypertrophy in the castrated animals. These gonadotrophic basophiles were moreover, later divided by Purves and Griesbach ('54 and '55) into two distinct classes of cells , and they suggested that the peripheral gonadotrophic basophiles produce the follicle stimulating hormone (FSH), and the central gonadotrophic cells are responsible for luteinising hormone (LH). R e n n e 1 s ('57) supported this view regarding the source of gonadotropin. By application of electron microscope, recently Farquhar and Rinehart ('54a) in the castrated rat, distinguished two types of gonadotrophic basophile. Similarly, in the mouse, Barnes ('62 and '63) identified FSH-cell and LH-cell. Anincrease in pituitary gonadotrophic function of hyperthyroid animals has been observed by some investigators. Van Horn ('31) showed that a hyperthyroid condition in female rats increases the stimulating power of the hypophysis 19 to 63 per cent. E vans and Simpson ('30) also found an increased potency of the anterior pituitary of hyperthyroid female rats. Halmi (52) stated that delta cell percentage of hypothyroid rats treated with large doses of thyroxine is slightly elevated. Also M a q s o o d and R e i n e k e ('50) reported activation of gonadal function in the mild hyperthyroid mouse. In the present study, delta cells did not show significant changes in the early stage of the experiment, but in the late period an increase in amount of granulation of the cells was frequently observed. On the other hand, in the large dose groups (5.0 and 10.0 pg), decrease in granules of delta cells (both first and second type delta cell) was found. These results suggest that gonadotrophic function is promoted in mild hyperthyroidism, but with large dose Light and Electron Microscopic Observations on the Anterior Pituitary 33 of thyroxine is diminished. The mechanisms whereby the thyroid influences gonadal function have not been fully elucidated. C h u and Y o u ('45) described that the administration of thyroid to rabbits resulted in a lower content of pituitary FSH and an increase in LH. There is some reason to believe that thyroid hormone may influence the reproductive organs directly by metabolic conditioning of the cells involved, since thyroxine added to semen in critical amounts increased both its oxypen consumption and fertilizing capacity (Schulze and Davis, '48). : Regarding chromophobes, in our laboratory, it has been considered that chromophobes represent immature, undifferentiated cells and degranulated forms of chromophiles. S e v e r i n g h a u s ('33) had identified chromophobes as belonging to either the acidophile or basophile series by the type of Golgi's apparatus they contained. Based on his observations, Severinghaus postulated a secretory cycle for the acidophile and basophile cells as two independent cellular elements, the sequence being granules accumulation and storage and their subsequent secretion after an effective stimulus, thereby returning the cell to chromophobes or nongranular state. In the present electron microscopic observations, very few true chromo- phobes which contained few or no granules were observed. Cytolo- gical changes of pituitary chromophobes in experimental hyperthy- roidism have been described by some authors (K i y o n a r i and Nishimura, '27; Momose, '33; Connor, '37; Frank, '37). On the basis of the present results, it can be assumed that alteration of chromophobes is due to an increase or decrease of degranulated chromophiles.

Summary

The anterior pituitary of the mouse injected with various dose of thyroxine was investigated by employing histochemical procedures and electron microscope, and the following results were obtained. 1. The parenchymatous cells of the anterior pituitary of the mouse were divided by light microscopy into four cell types : acidophiles, beta basophiles, delta basophiles and chromophobes , whereas by electron microscopy, moreover, two types of acidophile : the first type acidophile (Gil or growth hormone secreting cell) and the second type acidophile (LTH or luteotrophic hormone secreting cell) and three types of basophile : the beta cell (TSH or thyrotrophic 34 Tomiji Uchida hormone secreting cell), the first type delta cell (FSH or follicle stimu- lating hormone secreting cell) and the second type delta cell (LH or luteinising hormone secreting cell) were subdivided. 2. Acidophiles, in small dose of thyroxine, did not show remark- able changes. But in large dose, a decrease in the amount of gran- ulation was observed. 3. Thyrotrophic beta cells showed remarkable changes. Aldehyde- fuchsin staining granules of the beta cells were almost completely disappeared within seven days after injection. But they were clearly recognized with PAS staining. By electron microscopy, the secretory granules of the beta cells rather slightly increased in the early stage of the experiment, but in the late stage they gradually decreased. Often pyknotic nuclei was observed, at the same time, the cytoplasm of the cell was diminished. 4. Gonadotrophic delta cells slightly increased in number and amount of granulation in relatively small dose, but in large dose of thyroxine they were fairly reduced. 5. Chromophobes did not show any marked alteration throughout all the experimental periods. In electron microscopic observations, a few true chromophobes which contained few or no granules were observed.

Acknowledgement

The author wishes to express his sincere gratitude to Prof. Dr. K. Yamada for helpful suggestions and kind guidance and critism throughout the course of this study.

References

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Explanation of Figures

Fig. 1. Anterior pituitary of a control male mouse. Acidophile cells are numerously present. They are round or oval in shape, and moderately granulated. Zenker-formrlin-acetic acid fixation. Azan stain. x 400. Fig. 2. Anterior pituitary of a control male mouse. Delta and beta basophiles are observed. Cytoplasmic granules of the delta cells are diffusely distributed throughout the cytoplasm. Zenker-formalin-acetic acid fixation. PAS reac- tion. x 1300. Fig. 3. Anterior pituitary of a control male mouse. Beta cells are angular in shape. Zenker-formalin-acid fixation. Gomori's aldehyde-fuchsin stain. x 400. Fig. 4. Anterior pituitary of a mouse injected with 1.0pg of dl-Thyroxine for 14 days. Angular beta cells are observed. Zenker-formalin-acetic acid fixation. PAS reaction. x 1300 Fig. 5. Anterior pituitary of a mouse injected with 5.0pg of dl-Thyroxine for 21 days. Partial degranulation of the delta cell is observed. Zenker-formalin-acetic acid fixation. PAS reaction. x 1300. Fig. 6. Anterior pituitary of a mouse injected with 5.0pg of dl-Thyroxine for 30 days. Decrease in amount of granulation of the delta cell is observed. Zenkkr-formalin-acetic acid fixation. PAS reaction. x1300 Fig. 7. Electron micrograph of anterior pituitary from normal male mouse. The first type acidophiles (GH secreting cells) are well granulated. The secretory granules of these cells are large, round and electron dense. x 4500 Fig. 8. Electron micrograph of anterior pituitary from normal male mouse. The second type acidophile cell (LTH secreting cell) possesses characteristic oval or irregularly shaped granules. x 6500 Fig. 9. Electron micrograph of anterior pituitary from normal male mouse. Two thyrotrophic beta cells and a second type delta cell are seen. Beta cells are angular in outline, and their secretory granules are small and variable in density. The ground substance of the cytoplasm is more electron lucent. x 4000 Fig. 10. Electron micrograph of a pituitary of control mouse. An acidophile cell of the first type, beta basophile cell and delta cell of the first type are observed. The secretory granules of the first type delta cell are relatively electron lucent. x 5200 Fig. 11. Two chromophobes and the second type delta cell from a normal male mouse. Chromophobe cell contains large nucleus and few or no granules. The second type delta cell (LH secreting cell) is relatively small cell and generally rounded in outline. The secretory granules of this cell are dense, relatively small, variable in size and frequently concentrated near the periphery of the cell. x 3500 Fig. 12. The first type and second type acidophiles, and chromophobe cell from a male mouse injected with 5.0pg of thyroxine for 21 days. The first type acidophile cell contains relatively few granules and their endoplasmic re- ticulum arranges in flattened parallel lamellae. Also the second type acidophile cell shows decrease in number of the granules. x 6000 Fig. 13. Anterior pituitary from a mouse administered with 1.0pg thyroxine for 7 days. The first and second type acidophile cell, and a beta basophile cell are observed. The secretory granules of bata cell are concentrated near the periphery of the cell, as controls. Development of Golgi's apparatus and endoplasmic reticulum is poor. x 6000 Fig. 14. Anterior pituitary from a mouse injected with 0.5pg of thyroxine for 21 days. The first type acidophile cell, beta cell and the first type delta cell are present. Two angular or irregular shaped beta cells are relatively well granulated. Acidophile cell and the first type delta cell are also well gra- nulated. x•150() Fig. 15. Anterior pituitary from a mouse received injection of 1.0pg of thyroxine for 14 days. In a beta basophile cell much ribosomes are observed. x 15000 Fig. 16. Anterior pituitary from a mouse injected with 1.0pg of thyroxine for 14 days. Two types (the first and second type) of acidophile, two types (the first and second type) of delta basophile and beta basophiles are aserved. The first type acidophile cell is moderately well granulated. The second type acidophile cell shows a slight increase in the number of secretory granules. The irregularly shaped beta cell is well granulated and their Golgi's apparatus and endoplasmic reticulum are well developed. The cytoplasmic background of the beta cell is dark, because of increasing of ribosomes. The Golgi vesicles and endoplasmic reticulum appear to dilate. The first type delta cell with irregular nucleus is also well granulated, whereas the second type delta cell shows decrease in the granules. x 6000 Fig. 17. Taken from the same section as figure 14 ; the beta granules are numerous. x 10000 Fig. 18. ' Anterior pituitary of a mouse administered with 5.Oag of thyroxine for 21 days. Slight decrease in number of granules of beta cell is observed. The Golgi complex and endoplasmic reticulum are well developed and they appear as small vacuoles. x 10000

A, : The first type acidophile cell (GH secreting cell). A 2 : The second type acidophile cell (LTH secreting cell). B : The beta basophile cell (TSH secreting cell). D1: The first type delta basophile cell (FSH secreting cell). D 2 : The second type delta basophile cell (LH secreting cell). C : The chromophobe cell. Plate I 45

Plate II 47

Plate III 49 51

Plate V