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 undertaken 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

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,

~ 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- ~ :

* ' m:- '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. 5-24 days In the 5 day dwarf the number and size of granules in the granulated pituitary cells had increased, as compared with the newborn dwarf, and cells with large granules could be distinguished from those with smaller granules. However, it was still difficult to identify typical somatotrophs and mammotrophs (Fig. 10). Oval or rounded cells with densely stained but irregularly shaped granules were present, as were stellate cells with slightly smaller, misshapen granules. By 9 days still more cells were granulated than previously but the large granules remained atypical in size and shape. Although many cells in the 14 day dwarf pars distalis were packed with granules (Fig. 11), the distinctive features of somatotrophs and mammotrophs were still not in evidence when compared with cells in the normal 14 day pars distalis (Fig. 12). t By 19 days the dwarf pars distalis showed slight distinctions between the small, medium and larger granulated cell types (Fig. 13). Nevertheless, at this stage, as well as subsequently at 24 days, cells which resembled somatotrophs and mammotrophs were still atypical when compared with their normal counterparts (Fig. 14). In the 24 day dwarf, the granules of the somatotroph-like cells still had not attained the size and shape typical of normal somatotroph granules, and the elaborate, parallel arrays of rough endoplasmic reticulum were lacking. Moreover, the cells in the 24 day dwarf pars distalis generally gave an overall impression of being ill- defined and less healthy in appearance than the sharply differentiated cells of the normal pars distalis. 414 DORIS BURDA WILSON AND ELEANOR CHRISTENSEN ~~~ 4 ( 44

k. ,*~~ 9*,...~~~~~~~R9 ... ~,* ~Rk~ * ..

4,~~~~0 #9.

Al '~~~~~.3049'' 9. 4. 7~~~~~4' 9*

V.~ ~ ~ ~ ~ . In.S"kv 13~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.

* ~~~~~~~9' '44'9 ~~~~~~~~~~~~~~~~~~~~;9, -' 4 .~ ~ ~ ~~~~~~~~~~~~~"P:

Y.%

. 44*0

mayrepreentsomaotrophs r mamtoh ogicmlxx50 Fig. 14. ormal 19 ay pars dstalis shwing typial somatoroph (STH with wel develope Golgi~~~~Ncope3g4nog nolamcrtclm(e)x50 Somatotroph and mammotroph development 415

DISCUSSION Although light microscopic studies on. the pituitary in the dwarf mouse have described the defect as a complete lack of or deficiency in somatotrophs and thyrotrophs (Smith & MacDowell, 1930; Francis, 1944; Ortman, 1956; Elftman & Wegelius, 1959), the question arises as to whether the cells develop normally and then degenerate, or whether there is a failure to differentiate properly. The results of the current histogenetic study at the light and electron microscopic levels indicate that the developmental aspects of this pituitary mutant are complex, and that defects in the pars distalis of the dwarf can already be detected in the newborn, even though growth deficits are not obvious until about five days after birth. Light microscopy of dwarf newborn pars distalis shows cells with scanty cytoplasm, indistinct cell boundaries, and a lack of granules, whereas normal newborn pars distalis contains well-defined cells with distinct granules. However, at the electron microscopic level, granules are indeed detectable in the dwarf, although they are not as large or as numerous as in the normal newborn. Somatotrophs and mammotrophs ordinarily can be distinguished from other cell types by conventional electron microscopy on the basis of their large, dense, homogeneous granules, whereas gonadotrophs characteristically have medium sized granules, and thyrotrophs and corticotrophs have the smallest granules (Yamada & Sano, 1960; Sano, 1962; Barnes, 1963; Herlant, 1963; Yamada & Yamashita, 1967; Gomez-Dumm & Echave-Llanos, 1972). In our dwarf individuals, cells with large granules were distinguishable from those with medium and small granules by 19 days postnatally, although the large granules tended to be smaller than those in normal pituitaries. In normal mice, somatotrophs can be distinguished from mammotrophs on the basis of the regular, spherical shape of the granules as well as the extensive parallel arrays of rough endoplasmic reticulum in somatotrophs, whereas the granules in mammotrophs are irregular in shape and of varying size. Also, in contrast to the oval or rounded shape of somatotrophs, mammotrophs are often stellate and show cellular processes extending toward blood vessels. Our results show that, on the basis of these criteria, it is impossible, in dwarf mice, to distinguish somatotrophs from mammotrophs in early postnatal specimens or in three to four months old adults; instead, the dwarf pituitaries contain atypical cells which variably show combinations of these features. Thus, the dwarf defect does not seem to be attribut- able to a deficit in the number of somatotrophs, but rather to the presence ofa sub- stantial population of an ambiguous somatotroph-mammotroph cell type. In their brief reports on the dwarf adult, Rennels & McNutt (1958) and Peterson (1959) did not detect any acidophils on the basis of granule content or endoplasmic reticulum, and an electron micrograph of the adult dwarf pituitary (Kurosumi, 1974) shows that most of the cells have an atrophied appearance, with almost no granules in evidence. In contrast, our results demonstrate that granules are present in many of the dwarf cells, although it is possible that this difference may be attribut- able to variations in the age of the adults previously studied as compared with the three to four months old adults used in the current study. Also, notching of the nuclei was cited as being more prominent in dwarf adults than in normal adults (Rennels & McNutt, 1958). Although some notching was noted in our dwarf material, a morphometric study of this feature would need to be undertaken to determine if there is a quantitative basis for this observation, since the nuclei in the cells of the normal pars distalis also tend to be pleomorphic. 416 DORIS BURDA WILSON AND ELEANOR CHRISTENSEN The ambiguous cell type observed in the dwarf pars distalis resembles the 'mammosomatotroph' cell which has recently been described in human pituitary adenomas and which can produce both growth hormone and prolactin (Horvath et al. 1980). Whether or not the ambiguous somatotroph-mammotroph-like cells in our dwarf pituitaries are also capable of producing both hormones remains to be demonstrated by immunocytochemical studies. Although the light microscopic study of Francis (1944) indicated that the dwarf pituitary did not show much, if any, abnormality prior to day 6, at which time the dwarf glands became hypoplastic, our analysis of the developmental aspects of the dwarf pars distalis at the EM level shows aberrancies in the acidophil cell types as early as in the newborn; hence, the condition in older postnatal stages and in the adult does not seem to represent a dedifferentiation from the neonatal condition. Although the condition appears to worsen with developmental age, this may be due to the fact that even in normal pituitaries the cell types are not as easily distinguishable from one another during early postnatal stages as at later periods of development, and thus the differences between the dwarfs and their normal counterparts tend to become more obvious in later stages and in the adult.

SUMMARY The differentiation of somatotrophs and mammotrophs in the pars distalis of dwarf (dw/dw) and normal (+ / +; dw/+) litter mates was studied by means of electron microscopy from the day of birth to 24 days, as well as in adults at 3-4 months of age. On the basis of a combination of growth measurements, including weight, total body length, and tail length, dwarfs were identifiable with certainty as early as 5 days in litters from heterozygous matings (dw/+ x dw/+). Newborn dwarfs were obtained from homozygous matings (dw/dw x dw/dw) of adult dwarfs which had been brought to sexual maturity by hormone supplementation. The results indicate that the dwarf pituitary is already abnormal at birth. In contrast to the pars distalis in the normal newborn mouse, somatotrophs and mammotrophs cannot be distinguished from one another nor can they be distinguished as a group from other cell types in the dwarf. However, as development progresses, the dwarfs show a large population of an ambiguous somatotroph-mammotroph-like cell with fine structural characteristics of both somatotrophs and mammotrophs; these ambiguous cells also occur in the 3-4 months old adult. This research was supported by National Institutes of Health grant no. HD12308 from the National Institute of Child Health and Human Development. The authors wish to thank Pratima Ganguly and Treva Valentine for technical assistance.

REFERENCES BARNES, B. G. (1963). The fine structure of the mouse adenohypophysis in various physiological states. In Cytologie de l'Ad&nohypophyse (ed. J. Benoit & C. DaLage), pp. 91-109. Paris: CNRS. BARTKE, A. (1964). Histology of the anterior hypophysis, thyroid, and gonads of two types of dwarf mice. Anatomical Record 149, 225-236. BOHNET, H. G. & FRIESEN, H. G. (1976). Effect of prolactin and growth hormone on prolactin and LH receptors in the dwarf mouse. Journal ofReproduction and Fertility 48, 307-311. ELFTMAN, H. & WEGELIUS, 0. (1959). Anterior pituitary cytology ofthe dwarf mouse. Anatomical Record 135, 43-49. FRANCIS, T. (1944). Studies on hereditary dwarfism in mice. VI. Anatomy, histology and development of the pituitary at hereditary anterior pituitary dwarfism in mice. Acta pathologica et microbiologica scandinavica 21, 928-944. Somatotroph and mammotroph development 417 GOMEZ-DUMM, L. A. & ECHAVE-LLANOS, J. M. (1972). Further studies on the ultrastructure of the pars distalis of the male mouse hypophysis. Acta anatomica 82, 254-266. HERLANT, M. (1963). Apport de la microscopie 6lectronique a l'6tude du lobe anterieur de l'hypophyse. In Cytologie de l'Adenohypophyse (ed. J. Benoit & C. DaLage), pp. 73-94. Paris: CNRS. HORVATH, E., KOVACS, K., KILLINGER, D. W., SEmin, H. S., PLArrs, M. E., WEISS, M. S. & EZRIN, C. (1980). Mammosomatotroph cell adenoma of the human pituitary. Proceedings ofthe Electron Micro- scopy Society of America 38, 726-727. KuRosuMi, K. (1974). Adenohypophysis. In An Atlas ofElectron Micrographs. Functional Morphology of Endocrine Glands. Tokyo: Igaku Shoin Ltd. ORTMAN, R. (1956). A study of some cytochemical reactions and the hormone content of the adenohypophysis in normal and in genetic dwarf mouse. Journal ofMorphology 99, 417-431. PETERSON, R. R. (1959). Electron microscope observations on the pituitary gland of the dwarf mouse. American Journal ofAnatomy 133, 322-323. RENNELS, E. G. & McNuTr, W. (1958). The fine structure of the anterior pituitary cells of the dwarf mouse. Anatomical Record 131, 591-592. SANO, M. (1962). Further studies on the theta cell of the mouse anterior pituitary as revealed by electron microscopy, with special reference to the mode of secretion. Journal of Cell Biology 15, 85-97. SMITH, P. E. & MACDOWELL, E. C. (1930). An hereditary anterior-pituitary deficiency in the mouse. Anatomical Record 46, 249-257. WILSON, D. B. (1976). Postnatal development of the pituitary gland in Snell's dwarf (dw) mice. Anatomical Record 184, 597. WILSON, D. B. & CHRISTENSEN, E. (1980). A comparison of pituitary morphology and postnatal growth characteristics in dwarf (dw) and little (lit) mice. Anatomical Record 196, 255A. YAMADA, K. & SANO, M. (1960). Electron microscopic observations of the anterior pituitary of the mouse. Okajimasfolia anatomica japonica 34, 449475. YAMADA, K. & YAMASHrIA, K. (1967). An electron microscopic study on the possible site of production of ACTH in the anterior pituitary of mice. Zeitschriftfar Zellforschung und mikroskopische Anatomie 80, 2943.

14 '4ANA 133