Fine Structure of Somatotrophs and Mammotrophs

J. Anat. (1981), 133, 3, pp. 407-417 407 With 14 figures Printed in Great Britain Fine structure of somatotrophs and mammotrophs during development of the dwarf (dw) mutant mouse DORIS BURDA WILSON AND ELEANOR CHRISTENSEN Division ofAnatomy, M-004, Department of Surgery, University of California, San Diego, La Jolla, California 92093 (Accepted 4 November 1980) INTRODUCTION Numerous light microscopic studies on the adult adenohypophysis of Snell's dwarf (dw) mutant mouse have shown that in the homozygotes (dw/dw) there is a lack of the typical acidophils (somatotrophs and mammotrophs) seen in normal (+ / +; dw/+) adult mice, as well as a deficiency of thyrotrophs (Smith & Mac- Dowell, 1930; Francis, 1944; Ortman, 1956; Elftman & Wegelius, 1959). Sparse information is available on the fine structure of the adenohypophysis in the adult dwarf mouse (Rennels & McNutt, 1958; Peterson, 1959; Kurosumi, 1974), and only light microscopic studies have been made of the immature dwarf adenohypophysis during postnatal development (Francis, 1944; Wilson, 1976). Whether the cellular defect represents a failure of cytodifferentiation or a regression of normally differentiated cells is, however, a fundamental question. In our colony, we have been able to detect dwarf individuals as early as five days postnatally by a combination of criteria, including lower body weights, shorter tails, and shorter body lengths (Wilson & Christensen, 1980). In view of this relatively early manifestation of dwarfism, the current electron microscopic study was under- taken in order to analyze the fine structural organization of somatotrophs as they differentiate and mature in the dwarf mouse from the time of birth until 24 days of age. Observations on the differentiation of mammotrophs in the dwarf are also included because of their various structural and tinctorial similarities to somatotrophs, as well as the known growth-promoting effects of prolactin (Bohnet & Friesen, 1976). Although dwarf individuals cannot be distinguished from normal litter mates in newborn litters from heterozygous matings, we were able to obtain known dwarf newborns by mating dwarf adults which were supplemented with hormones in order to induce sexual maturity (Bartke, 1964). MATERIALS AND METHODS Dwarf heterozygotes (dw/+) maintained on a diet of Purina mouse chow in a temperature controlled room with a light-dark schedule (14 hours light-10 hours dark) were mated, and the date of birth was designated as day 0 (newborn). Pitui- taries were removed from male dwarfs (dw/dw) and normal (+/+, dw/+) litter mates on days 5, 9, 14, 19 and 24, and from male adults at 3 to 4 months of age. In order to obtain newborn dwarf pituitaries, litters composed entirely of dwarfs were obtained from matings of adult dwarf homozygotes which had received hor- 408 DORIS BURDA WILSON AND ELEANOR CHRISTENSEN F AL it.~~~~~iSNoqdAK: j , jma I 3L4f ..* X: j:s.' WF r; ... .. *.....* ^:f.e'.v V4,ewJ- UW '. V 7 4) Fig. 1. Light micrograph of normal adult pars distalis. Dark areas in cytoplasm (arrows) represent concentrations of granules. x 480. Fig. 2. Electron micrograph of normal adult pars distalis showing a somatotroph (STH) and mammotroph (LTH). x 5600. Somatotroph and mammotroph development 409 mone supplementation as follows: Thyroxine (DL-thyroxine, sodium salt) in solution was administered by intraperitoneal injections to male and female dwarfs three times weekly for eight weeks. Male dwarfs 1-2 months of age and female dwarfs 2-3 months of age received 1.5 ,ug thyroxine in 0 07 ml volume for two weeks, 30 1ug thyroxine in 0-14 ml volume for two weeks, and 455,ag thyroxine in 0-20 ml for the last four weeks. After eight weeks of thyroxine supplementation, dwarfs were mated. To maintain pregnancy, a female with a vaginal plug was given prolactin supplementation for eight days beginning on the day that the plug was seen. Daily injections of at least 125 ,g of prolactin in 0-2 ml volume were given at 9.00 am ± 30 minutes. Pituitaries were removed on the day of birth (day 0, newborn). Normal newborn pituitaries were removed from litters obtained from + /+ x ?/ + matings. The tissues were fixed in a fresh solution of 3 % glutaraldehyde in 0 1 M sodium phosphate buffer, pH 7-4, at 4 °C for a minimum of six hours. The specimens were rinsed three times in phosphate buffer and post-fixed in I % osmium tetroxide in 0ff1 M phosphate buffer for one hour at room temperature. Tissues were then dehydrated in graded alcohols and propylene oxide, embedded in fresh Epon- Araldite mixture and then polymerized at 60 'C. Thick sections (1-2 ,um) obtained for orientation and stained with 1 % methylene blue-azure ii solution were mounted on slides. In order to maintain consistency in our samples, thin sections were confined to the lateral portions of the pars distalis and were cut with a diamond knife, mounted on naked copper grids, stained with uranyl acetate and lead citrate, and then observed with a Zeiss 9S-2 electron microscope at direct magnifications up to 28000. RESULTS In the following account, the term 'normal' represents + / + or dw/ + individuals; 'dwarf' designates dw/dw individuals. Adult Thick sections (1-2,m) of pars distalis of normal adult mice at 3-4 months of age showed numerous plump granulated cells (Fig. 1). At the electron microscopic (EM) level the somatotrophs were easily identified by their rounded or oval shape and large (300-400 nm) densely stained spherical granules (Fig. 2). Mammotrophs were stellate in shape, with cellular processes often extending toward blood vessels. Although their granules showed a homogeneous density similar to that of the somatotroph granules, mammotrophs were characterized by irregularly shaped granules, of various sizes, which were rarely larger than 600 nm (Fig. 2). In the pars distalis of dwarf adult mice, light micrographs showed a multitude of small and apparently non-granulated cells which appeared to be shrunken and poorly defined (Fig. 3). Electron micrographs revealed that although many of the cells appeared atrophied or poorly differentiated, they did indeed contain granules, some of which showed the same homogeneous density as in normal somatotrophs and mammotrophs (Fig. 4). However, although the granules in these cells were larger than the medium granules and the small granules in the other cell types, the granules tended to be smaller than those in normal somatotrophs. Moreover, the granules in these cells were often irregular and elliptical in shape and bore some resemblance to those in normal mammotrophs. However, despite their mammotroph-like characteristics, these cells resembled somatotrophs in terms of their 410 DORIS BURDA WILSON AND ELEANOR CHRISTENSEN 'sEr_~~~~~~~~~~~~~~~~~~~~~~~r 9-, *.'*. * ..*,**~0 .5', .~~~~~~~.41, %*O~*, ~ *9 . At&A v,U 7 k~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~P Vj1 Fig. 3. Light micrograph of dwarf adult pars distalis. Note shrunken cells with poorly defined boundaries and an apparent lack of granules in cytoplasm. x 480. Fig. 4. Electron micrograph of dwarf adult pars distalis. Some cells (+) with large granules may represent atypical somatotrophs or mammotrophs. x 5600. STH <~~~~ - ~ ~ ~ ~ -4 V* ** A 4 p~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~4 ~~~ ~~~IN-4 It, .4, '9 ~ N. ,-. 19* ~ ~ 41 *~~~~~~~,0 4V,7 "J.* V Fig5 igtmcrgrph f oralnewor prs isals Nteditict rauls (rrwsWi ctpamX75 Fig. 6. Electron micrograph of normal newborn pars distalis showing a somatotroph (STH). x 5600. Fig. 7. A mammotroph-like cell (LTH) in normal newborn pars distalis. x 5600. Fig. 8. Light micrograph of dwarf newborn pars distalis. Note indistinct cell boundaries and apparent lack of granules in cytoplasm. x 750. Fig. 9. Electron micrograph of dwarf newborn pars distalis. Note small, sparsely distributed granules. x 5600. 412 DORIS BURDA WILSON AND ELEANOR CHRISTENSEN Tji, t >^ ~~~~~~~~~~~~~~~--' 4x ....f W -tttR ':6.V* -W.' 2.j..~ I 4 I I0- ~ : I * ' m:- 'p.- . 'p 1 it Fig. 10. Dwarf 5 day pars distalis. Note increase in the size of granules as compared with the newborn, but the cell types are ambiguous. x 5600. Fig. 11. Dwarf 14 day pars distalis. Granules have increased in number, but cell types remain ambiguous. x 5600. Fig. 12. Normal 14 day pars distalis showing a typical somatotroph (STH). x 5600. Somatotroph and mammotroph development 413 round to oval shape. Although they often exhibited a Golgi complex, the rough endoplasmic reticulum did not appear to be as highly developed or as prominent as in normal somatotrophs, where it was often arranged in parallel, concentric stacks (Fig. 2). Thus, in adult dwarfs it was difficult to identify or distinguish between mammotrophs and somatotrophs on the basis of typical fine structural criteria. Newborn At birth, the normal pars distalis was characterized by a mixture of granulated and non-granulated cells. At the light microscopic level, the granules were not as densely packed as in the adult, and were thus more distinct (Fig. 5). At the electron microscopic level, somatotrophs with large dense spherical granules were common and often showed a well developed rough endoplasmic reticulum (Fig. 6). An occasional cell resembling a mammotroph could also be found (Fig. 7). The dwarf newborn pars distalis already showed differences from the normal, even at the light microscopic level (Fig. 8). The cell boundaries tended to be indistinct, and granules were not in evidence. However, at the EM level, sparsely granulated cells could be seen (Fig. 9). The granules were not as large as those in normal somatotrophs and mammotroph-like cells, and there were only slight differences in granule size among all the cells. Hence, at this age it was not easy to distinguish somatotrophs and mammotrophs as a group apart from other cell types with smaller granules. Many of the cells, however, did show a rounded or oval shape with some of the concentric arrays of rough endoplasmic reticulum which are characteristic of somatotrophs.

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