Okajimas Fol. anat. jap., 43: 21-51, 1967 Light and Electron Microscopic Observations on the Anterior Pituitary 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 thyroid 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 hormone (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 cell 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 staining method (G o m o r i, '50) recently, differentiated clearly two functionally distinct types of basophile (beta and delta cell) 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 microscope (U chid a, '64) . In the present study, light and electron microscopes 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-eosin, 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, beta cell (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.