Cell Tissue Res. 202, 399406 (1979)
Cell and Tissue Research 9 by Springer-Verlag 1979
Morphological Changes of Pituitary Gonadotrophs and Thyrotrophs Following Treatment with LH-RH or TRH in vitro* Masataka Shiino Department of Anatomy, The University of Texas Health ScienceCenter at San Antonio, San Antonio, Texas, USA
Summary. To investigate the relationship between L H - R H and T R H and the formation of castration cells and thyroidectomy cells, pituitary glands of 14-day old female rats were cultured with L H - R H or T R H for 10 days. Observed in these glands were external and internal zones, the former containing active, healthy appearing cells, and the latter consisting of m a n y degenerative and necrotic cells. G o n a d o t r o p h s and thyrotrophs were readily demonstrated in the external zone of the organs by immunocytochemistry. Hypertrophic gonadotrophs (castration cells) and thyrotrophs (thyroidectomy cells) were observed only in the external zone by electron microscopy. Neither the typical signet-ring gonadotrophs nor intracisternal granules of thyroidectomy cells were seen in the present study. However, the hypertrophic gonadotrophs or thyrotrophs were similar to castration cells or thyroidectomy cells observed in the pituitary gland following castration or thyroidectomy. The results indicate that L H - R H and T R H directly stimulate the secretion of hormones and alter the morphological features of pituitary target cells. Key words: Castration cells - Thyroidectomy cells - L H R H - T R H - Organ culture. It is generally accepted that the secretion of gonadotrophins and thyrotrophins, as well as the other hormones of the anterior pituitary gland, is under hypothalamic control, e.g., via releasing hormones. Luteinizing hormone-releasing hormone ( L H - R H ) has been demonstrated by Schally and his collaborators to stimulate the secretion of not only L H but also F S H in vivo and in vitro (Schally et al., 1972; Arimura et al., 1972). Schally and Redding (1967) and Bowers et al. (1970) demonstrated that thyrotrophin-releasing hormone (TRH) stimulates the release of thyrotrophin both in vivo and in vitro. Send offprint requests to: Dr. Masataka Shiino, Department of Anatomy, The University of Texas
Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78284, U.S.A. * Supported by USPHS Grant HD 11826. The author wishesto expresshis thanks to NIAMDD for providing pituitary hormones
0302-766X/79/0202/0399/$02.40
400
M. Shiino
By e l e c t r o n - m i c r o s c o p i c a l m e t h o d s Shiino et al. (1972) d e m o n s t r a t e d t h a t the e x t r u s i o n o f secretory granules f r o m L H g o n a d o t r o p h s c o u l d be o b s e r v e d within 2.5 m i n after the injection o f pure, n a t u r a l p o r c i n e L H - R H in persistent-estrous rats. D r a w i n g f r o m the a b o v e e x p e r i m e n t a l results, Z a m b r a n o et al. (1974) expected to find m o r p h o l o g i c a l changes associated with anterior pituitary cells following their i n c u b a t i o n with L H - R H in o r g a n culture. They r e p o r t e d t h a t the F S H cells, as d e s c r i b e d by K u r o s u m i (1968), c h a n g e d into typical p o s t - c a s t r a t i o n signet-ring cells when glands f r o m rats were i n c u b a t e d for 3 a n d 6 h with L H - R H ( o r g a n culture). A f t e r a n t e r i o r p i t u i t a r y glands were i n c u b a t e d for 24 h with T R H , C u e r d o R o c h a a n d Z a m b r a n o (1974) r e p o r t e d the a p p e a r a n c e in t h y r o t r o p h s o f i n t r a c i s t e r n a l m a t e r i a l similar to t h a t o b s e r v e d in t h y r o i d e c t o m y cells as well as m a s s i v e exocytosis. H o w e v e r , they d i d n o t observe typical t h y r o i d e c t o m y cells. Shiino a n d Rennels (1975) stated t h a t e x o g e n o u s T R H s t i m u l a t e d t h y r o t r o p h s o f a n t e r i o r p i t u i t a r y glands t r a n s p l a n t e d u n d e r the kidney capsule o f the hyp o p h y s e c t o m i z e d rats. A r i m u r a et al. (1976) d e m o n s t r a t e d t h a t the a p p e a r a n c e o f c a s t r a t i o n cells or h y p e r t r o p h i c g o n a d o t r o p h s following g o n a d e c t o m y was p r e v e n t e d by injection o f a n t i - L H - R H . This m a y indicate t h a t c a s t r a t i o n cells occur due to the elevated secretion o f h y p o t h a l a m i c L H - R H triggered by g o n a d e c t o m y . I n fact, N a l l a r a n d M c C a n n ( | 965) f o u n d t h a t p l a s m a levels o f L H - R H activity o f h y p o p h y s e c t o m i z e d rats are higher t h a n those o f n o r m a l rats. H o w e v e r , unlike Z a m b r a n o et al. (1974), we h a v e n o t o b s e r v e d c a s t r a t i o n cells after such s h o r t - t e r m o r g a n culture with L H R H as 3 or 6 h. In a d d i t i o n , we can occasionally observe signet-ring cells similar to c a s t r a t i o n cells even in a n t e r i o r p i t u i t a r y glands o f n o r m a l intact rats. In the study p r e s e n t e d herein, the a u t h o r e m p l o y e d i m m a t u r e rats for the p r o l o n g e d culture o f a n t e r i o r p i t u i t a r y glands with L H - R H o r T R H to ascertain w h e t h e r o r n o t the m o r p h o l o g i c a l changes in g o n a d o t r o p h s or t h y r o t r o p h s are due solely to elevated levels o f L H - R H or T R H . It has been r e p o r t e d t h a t the p h y s i o l o g i c a l level o f L H - R H or T R H is a b o u t 10-11 M in n o r m a l rats ( C h i a p p a et al., 1977). C o n s e q u e n t l y the a u t h o r used 10-7 a n d 1 0 - 6 M o f L H - R H o r T R H for the o r g a n - c u l t u r e experiments.
Materials and Methods Fourteen-day old female rats (Sprague-Dawley) were used. The animals were kept under air conditioning and light control (0600 to 2000 h), and fed Purina Laboratory Chow and tap water ad libitum. Anterior pituitary glands were removed from 30 rats and were placed in Hank's solution and cut into 1 mm s pieces with sterilized razor blades. These were placed on filter-paperrafts over stainless steel ring bases (1 cm diameter) in Petri dishes (Falcon plastic) containing 1 ml of Ham's FI0 medium supplemented with 5% horse serum, 10% fetal bovine serum, streptomycin (50~tm/ml), penicillin (50 iu/ml) and fungisone (0.5 ~tm/ml) for 10 days. Each dish contained 5-8 explants. The dishes were maintained in a humidified atmosphere of 56 CO 2 and 95 % air at 37~C. The culture medium was changed every 24 h. Five experimental groups, consisting of two dishes each in which pituitary tissue was obtained from 5 female rats, were used. The glands were incubated in: 1) Ham's F10 medium as a control, 2) Ham's F10 medium with 10 - 7 M LH-RH, 3) 10 - 6 M LH-RH, 4) 10-7 M TRH, or 5) with 10-6M TRH. For immunocytochemical examination, the tissues were fixed in Zamboni's solution and embedded in paraffin. Five-~tm sections of all paraffin blocks were prepared. The sections were stained by the
Castration Cells and Thyroidectomy Cells
401
technique of Sternberger et al. (1970). Antiserum to adrenocorticotrophin (anti-ACTH) was provided by Dr. E.P. Bowie, but antisera to the following hormones were prepared by the author using rabbits" ovine growth hormone (NIH-GH-S12), ovine PRL (NIH-P-S 12) and ovine TSH (NIH-TSH-S7). Specificityof these antisera was established by using standard immunological techniques. In addition, immunocytochemical specificitywas confirmed by the loss of specificreaction of pituitary ceils by the absorption of each antiserum with its homologous antigen (1 mg/ml). For electron-microscopic observations the tissues were fixed in a mixture of equal parts of ice-cold 2 % paraformaldehyde prepared in cacodylate buffer (0.2 M, pH 7.4) and2 % osmic acid. Four hours after fixation the tissues were placed in ice-cold 0.5 % uranyl acetate overnight, and they were then dehydrated with alcohol and acetone. The tissues were embedded in Spurr's plastic mixture (Spurr, 1969). The sections were stained with lead citrate and observed with a Siemens 101 electron microscope.
Results
Control The organ cultures o f anterior pituitary glands revealed the presence o f two clearly defined zones: 1) an extensive internal zone with m a n y small-sized and some necrotic cells, and 2) a relatively thin external zone with healthy-appearing cells. Cells reactive to antisera for each o f the hormones used in this investigation were recognized in the external zone a n d some were identified in the internal zone o f the organ. Cells that reacted to these antisera were generally scarce and reactions were weak. By electron m i c r o s c o p y it was observed that most o f the cells h a d a decreased n u m b e r o f secretory granules a n d that the cells o f the internal zone were atrophic. They contained n u m e r o u s lipid bodies and lysosomes, characteristic o f cells in organ culture. M a n y cytoplasmic projections were seen on the surface cells o f the external zone (Fig. 5).
Culture with T R H All five o f the pituitary h o r m o n e s were recognized in the cultured organs by i m m u n o c y t o c h e m i c a l methods. Prolactin cells were observed in the internal zone o f the o r g a n but were scarce in the external zone. A C T H cells, which reacted only weakly to the antiserum, were observed only in small numbers in the external zone. L H cells were scarce and small. However, the cells that c o r r e s p o n d i m m u n o cytochemically to T S H cells were clearly observed as hypertrophic cells in the external zone (Figs. 1 and 2). These cells contained well-developed endoplasmic reticula (ER), but only a few secretory granules and lipid bodies (Fig. 7). In the internal zone, there were m a n y cells that reacted weakly with the antiserum against TSH, but all o f them were atrophic or necrotic (Fig. 6). The two different concentrations o f T R H (10-7 and 10 -6 M) did not produce any clear differences in the organ cultures.
402
M. Shiino
Fig. 1. Distribution of TSH cells in the pituitary organ culture revealed by immunocytochemistry. TSHcontaining cells are distributed in both the external and internal zones, but hypertrophic thyrotrophs are seen in the external zone. x 48. (TRH-treated culture group) Fig. 2. Higher magnification of Fig. 1. Immunocytochemically positive cells by anti-TSH serum are revealed mostly as large polygonal cells in the external zone. • 768 Fig. 3. Distribution of gonadotrophs in the pituitary organ culture revealed by immunocytochemistry. M a n y LH-containing cells are observed in the external zone and some weakly stained cells are seen in the internal zone of the organ. • 48. (LH-RH-treated culture group) Fig. 4. High magnification of Fig. 3. The cells are round or oval in shape. The stained cells are larger than most of the unstained cells. • 768
Castration Cells and Thyroidectomy Cells
403
Fig. 5. A n example o f n u m e r o u s cytoplasmic projections seen on the surface o f the external zone of the organ. The projections m a y serve to determine whether the organ culture performed well or not. • 12,240. (Control culture group) Fig. 6. M a n y atrophic and degenerating cells (/9) are observed in the internal zone of the organ. Arrows indicate lipid bodies, x 3,655. (Control group)
404
M. Shiino
Fig. 7. An example of hypertrophic (thyroidectomy) cell following the culture with TRH. Prominent dilated endoplasmic reticulum is observed, x 6,120 Fig. 8. An example of hypertrophic gonadotrophs following the incubation with LH-RH. The hypertrophic cells contain a well developed Golgi zone and rough surfaced endoplasmic reticulum. One hypertrophic gonadotroph contains a lot of lipid. These cells correspond to castration cells in vivo. x 3,655
Castration Ceils and ThyroidectomyCells
405
Culture with L H - R H Localization and cytological features of GH, PRL and A C T H cells were similar to those of cells cultured in Ham's F 10 medium supplemented with TRH. Some TSH cells were scattered in the explants but only a few were seen in the external zone. G H cells were clearly recognized in the external zone of the explants but were very scarce in the internal zone. Gonadotrophs were observed in both zones but were more prominent in the external zone (Fig. 3). When the tissues were observed with the electron microscope, many hypertrophic gonadotrophs containing well-developed Golgi zones, dilated ER and many lipid bodies in the cytoplasm were observed (Fig. 8).
Discussion
Zambrano et al. (1974) employed L H - R H (about 2 • 10 - 4 M) and Cuerdo-Rocha and Zambrano (1974) used 10 - 4 M T R H with in vitro systems to induce castration and thyroidectomy cells, respectively. Since it is known that the circulating blood contains about 10-11 M L H - R H (Chiappa et al., 1977), the concentration of LHR H which Zambrano and his collaborators employed in their culture is very high. Generally, it is known that a high concentration of hormone(s) may inhibit the release of hormone(s) from the target cells. Shiino et al. (1979) reported that the most effective level of L H - R H for stimulation of release of gonatropin in vitro was 10- 7 to 10- 6 M. The concentration of L H - R H employed by Zambrano et al. (1974) seems therefore to be quite high for use in culture. Furthermore, in our preliminary studies, pituitary cells in organ culture with L H - R H for 3-6 h did not reveal dramatic morphological changes in gonadotrophs except for extrusion of granules. It is not of rare occurrence that the adenohypophysis of adult male rats contains signet-ring gonadotrophs even without ablation of the gonads (Shiino and Rennels, 1973). It is for this reason that immature female rats for organ culture in this investigation. When pituitary glands are transplanted under the kidney capsule many prolactin cells remain active (Rennels, 1962), probably because of weak influence of the prolactin inhibitory factor (PIF) from the hypothalamus of the gland. However, prolactin cells were not well developed in the explants in the present experiment. This may be due to: 1) the presence of immature or underdeveloped prolactin cells in the anterior pituitary glands of the 14-day old rats employed, or 2) an incubation period that was not sufficient to promote development of the prolactin cells in the organ culture. The addition of L H - R H into the culture medium resulted in hypertrophic pituitary cells that reacted specifically with antiserum to LH. These hypertrophic gonadotrophs were very similar to the gonadotrophs observed following the ablation of the gonads. This indicates that hypei?trophic gonadotrophs or castration cells develop as a result of persistent stimulation of the gonadotrophs by considerably elevated levels of LH-RH. These morphological changes, however, may not be acute but rather may occur gradually when gonadotrophs are subjected to continuous stimulation with LH-RH. Similarly, the morphological features of
406
M. Shiino
thyrotrophs were altered within 10 days by incubation with TRH. We assume that thyroidectomy cells occurred owing to the constant stimulation by levels of TRH that were higher than those of normal blood. These results are in good agreement with our previous results with rats bearing autografts of pituitary glands that were treated with TRH (Shiino and Rennels, 1975). It is concluded that LH-RH and TRH stimulated directly the secretion of their respective hormones and concomitantly change the morphological features of the pituitary target cells. References Arimura, A., Debeljuk, L., Schally, A.V.: Stimulation of FSH release in vivo by prolonged infusion of synthetic LH-RH. Endocrinology 91, 529 532 (1972) Arimura, A., Shiino, M., DeLa Cruz, K.D., Rennels, E.G., Schally, A.V.: Effect of active and passive immunization with luteinizing hormone and follicle stimulating hormone-releasing hormone on serum luteinizing hormone and follicle stimulating hormone levels and the ultrastructure of the pituitary gonadotrophs in castrated male rat. Endocrinology 99, 291-303 (1976) Bowers, C.Y., Schally, A.V., Enzman, F., Baler, J., Folkers, K.: Porcine thyrotropin releasing hormone is (pyro)glu-his-pro(NH2). Endocrinology 86, 1143-1153 (1970) Chiappa, S.A., Fink, G., Sherwood, N.M.: Immunoreactive luteinizing hormone releasing factor (LRF) in pituitary stalk plasma from female rats: Effects of stimulating diencephalon, hypocampus and amygdala. J. Physiol. 267, 625-640 (1977) Cuerdo-Rocha, S., Zambrano, D.: The action of protein synthesis inhibitors and thyrotrophin releasing factor on the ultrastructure of rat thyrotrophs. J. Ultrastruct. Res. 48, 1-16 (1974) Kurosumi, K.: Functional classification of cell types of the anterior pituitary glands accomplished by electron microscopy. Arch. Histol. Jpn. 29, 329 362 (1968) Nailer, R., McCann, S.M.: Luteinizing hormone-releasing activity in plasma of hypophysectomized rats. Endocrinology 76, 272-275 (1965) Rennels, E.G.: An electron microscope study of pituitary autograft cells in the rat. Endocrinology 71, 713-722 (1962) Schally, A.V., Redding, T.W.: In vitro studies with thyrotrophin releasing factor. Proc. Soc. Exp. Biol. Med. 126, 320-325 (1967) Schally, A.v., Redding, T.W., Matsuo, H., Arimura, A.: Stimulation of FSH and LH release in vitro by natural and synthetic LH and FSH releasing hormone. Endocrinology 90, 1561-1568 (1972) Shiino, M., Rennels, E.G.: Ultrastructural observations of gonadotrophin release in rats treated neonatally with testosterone. Rep. Biol. Med. 31, 215-228 (1973) Shiino, M., Rennels, E.G.: Effects of TRH on pituitary grafts in the rat. Electron Microscopic Concepts of Secretion: Ultrastructure of Endocrine and Reproductive Organs (ed. M. Hess) pp. 271-286, New York: John Wiley & Sons, 1975 Shiino, M., Arimura, A., Schally, A.V., Rennels, E.G.: Ultrastructural observations of granule extrusion from rat anterior pituitary cells after injection of LH-releasing hormone. Z. Zellforsch. 128, 152-161 (1972) Shiino, M., Maruyama, T., Tanaka, T.: Gonadotroph-rich cell line derived from pituitary clonal cells (2A8) grafted under the kidney capsule. Cell Tissue Res. (In Press) 1979 Spurr, A.R.: A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 31-43 (1969) Sternberger, L.A., Hardy, P.H., Jr., Cuculis, J.J., Meyer, H.G.: The unlabeled antibody enzyme method of immunohistochemistry: Preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antiperoxidase) and its use in identification of spirochetes. J. Histochem. Cytochem. 18, 315-333 (1970) Zambrano, D., Cuerdo-Rocha, S., Bergman, I.: Ultrastructure of rat pituitary gonadotrophs following incubation of the gland with synthetic LH-RH. Cell Tissue Res. 150, 179-192 (1974) Accepted August 1, 1979
Cell and Tissue Research 9 by Springer-Verlag 1979
Morphological Changes of Pituitary Gonadotrophs and Thyrotrophs Following Treatment with LH-RH or TRH in vitro* Masataka Shiino Department of Anatomy, The University of Texas Health ScienceCenter at San Antonio, San Antonio, Texas, USA
Summary. To investigate the relationship between L H - R H and T R H and the formation of castration cells and thyroidectomy cells, pituitary glands of 14-day old female rats were cultured with L H - R H or T R H for 10 days. Observed in these glands were external and internal zones, the former containing active, healthy appearing cells, and the latter consisting of m a n y degenerative and necrotic cells. G o n a d o t r o p h s and thyrotrophs were readily demonstrated in the external zone of the organs by immunocytochemistry. Hypertrophic gonadotrophs (castration cells) and thyrotrophs (thyroidectomy cells) were observed only in the external zone by electron microscopy. Neither the typical signet-ring gonadotrophs nor intracisternal granules of thyroidectomy cells were seen in the present study. However, the hypertrophic gonadotrophs or thyrotrophs were similar to castration cells or thyroidectomy cells observed in the pituitary gland following castration or thyroidectomy. The results indicate that L H - R H and T R H directly stimulate the secretion of hormones and alter the morphological features of pituitary target cells. Key words: Castration cells - Thyroidectomy cells - L H R H - T R H - Organ culture. It is generally accepted that the secretion of gonadotrophins and thyrotrophins, as well as the other hormones of the anterior pituitary gland, is under hypothalamic control, e.g., via releasing hormones. Luteinizing hormone-releasing hormone ( L H - R H ) has been demonstrated by Schally and his collaborators to stimulate the secretion of not only L H but also F S H in vivo and in vitro (Schally et al., 1972; Arimura et al., 1972). Schally and Redding (1967) and Bowers et al. (1970) demonstrated that thyrotrophin-releasing hormone (TRH) stimulates the release of thyrotrophin both in vivo and in vitro. Send offprint requests to: Dr. Masataka Shiino, Department of Anatomy, The University of Texas
Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78284, U.S.A. * Supported by USPHS Grant HD 11826. The author wishesto expresshis thanks to NIAMDD for providing pituitary hormones
0302-766X/79/0202/0399/$02.40
400
M. Shiino
By e l e c t r o n - m i c r o s c o p i c a l m e t h o d s Shiino et al. (1972) d e m o n s t r a t e d t h a t the e x t r u s i o n o f secretory granules f r o m L H g o n a d o t r o p h s c o u l d be o b s e r v e d within 2.5 m i n after the injection o f pure, n a t u r a l p o r c i n e L H - R H in persistent-estrous rats. D r a w i n g f r o m the a b o v e e x p e r i m e n t a l results, Z a m b r a n o et al. (1974) expected to find m o r p h o l o g i c a l changes associated with anterior pituitary cells following their i n c u b a t i o n with L H - R H in o r g a n culture. They r e p o r t e d t h a t the F S H cells, as d e s c r i b e d by K u r o s u m i (1968), c h a n g e d into typical p o s t - c a s t r a t i o n signet-ring cells when glands f r o m rats were i n c u b a t e d for 3 a n d 6 h with L H - R H ( o r g a n culture). A f t e r a n t e r i o r p i t u i t a r y glands were i n c u b a t e d for 24 h with T R H , C u e r d o R o c h a a n d Z a m b r a n o (1974) r e p o r t e d the a p p e a r a n c e in t h y r o t r o p h s o f i n t r a c i s t e r n a l m a t e r i a l similar to t h a t o b s e r v e d in t h y r o i d e c t o m y cells as well as m a s s i v e exocytosis. H o w e v e r , they d i d n o t observe typical t h y r o i d e c t o m y cells. Shiino a n d Rennels (1975) stated t h a t e x o g e n o u s T R H s t i m u l a t e d t h y r o t r o p h s o f a n t e r i o r p i t u i t a r y glands t r a n s p l a n t e d u n d e r the kidney capsule o f the hyp o p h y s e c t o m i z e d rats. A r i m u r a et al. (1976) d e m o n s t r a t e d t h a t the a p p e a r a n c e o f c a s t r a t i o n cells or h y p e r t r o p h i c g o n a d o t r o p h s following g o n a d e c t o m y was p r e v e n t e d by injection o f a n t i - L H - R H . This m a y indicate t h a t c a s t r a t i o n cells occur due to the elevated secretion o f h y p o t h a l a m i c L H - R H triggered by g o n a d e c t o m y . I n fact, N a l l a r a n d M c C a n n ( | 965) f o u n d t h a t p l a s m a levels o f L H - R H activity o f h y p o p h y s e c t o m i z e d rats are higher t h a n those o f n o r m a l rats. H o w e v e r , unlike Z a m b r a n o et al. (1974), we h a v e n o t o b s e r v e d c a s t r a t i o n cells after such s h o r t - t e r m o r g a n culture with L H R H as 3 or 6 h. In a d d i t i o n , we can occasionally observe signet-ring cells similar to c a s t r a t i o n cells even in a n t e r i o r p i t u i t a r y glands o f n o r m a l intact rats. In the study p r e s e n t e d herein, the a u t h o r e m p l o y e d i m m a t u r e rats for the p r o l o n g e d culture o f a n t e r i o r p i t u i t a r y glands with L H - R H o r T R H to ascertain w h e t h e r o r n o t the m o r p h o l o g i c a l changes in g o n a d o t r o p h s or t h y r o t r o p h s are due solely to elevated levels o f L H - R H or T R H . It has been r e p o r t e d t h a t the p h y s i o l o g i c a l level o f L H - R H or T R H is a b o u t 10-11 M in n o r m a l rats ( C h i a p p a et al., 1977). C o n s e q u e n t l y the a u t h o r used 10-7 a n d 1 0 - 6 M o f L H - R H o r T R H for the o r g a n - c u l t u r e experiments.
Materials and Methods Fourteen-day old female rats (Sprague-Dawley) were used. The animals were kept under air conditioning and light control (0600 to 2000 h), and fed Purina Laboratory Chow and tap water ad libitum. Anterior pituitary glands were removed from 30 rats and were placed in Hank's solution and cut into 1 mm s pieces with sterilized razor blades. These were placed on filter-paperrafts over stainless steel ring bases (1 cm diameter) in Petri dishes (Falcon plastic) containing 1 ml of Ham's FI0 medium supplemented with 5% horse serum, 10% fetal bovine serum, streptomycin (50~tm/ml), penicillin (50 iu/ml) and fungisone (0.5 ~tm/ml) for 10 days. Each dish contained 5-8 explants. The dishes were maintained in a humidified atmosphere of 56 CO 2 and 95 % air at 37~C. The culture medium was changed every 24 h. Five experimental groups, consisting of two dishes each in which pituitary tissue was obtained from 5 female rats, were used. The glands were incubated in: 1) Ham's F10 medium as a control, 2) Ham's F10 medium with 10 - 7 M LH-RH, 3) 10 - 6 M LH-RH, 4) 10-7 M TRH, or 5) with 10-6M TRH. For immunocytochemical examination, the tissues were fixed in Zamboni's solution and embedded in paraffin. Five-~tm sections of all paraffin blocks were prepared. The sections were stained by the
Castration Cells and Thyroidectomy Cells
401
technique of Sternberger et al. (1970). Antiserum to adrenocorticotrophin (anti-ACTH) was provided by Dr. E.P. Bowie, but antisera to the following hormones were prepared by the author using rabbits" ovine growth hormone (NIH-GH-S12), ovine PRL (NIH-P-S 12) and ovine TSH (NIH-TSH-S7). Specificityof these antisera was established by using standard immunological techniques. In addition, immunocytochemical specificitywas confirmed by the loss of specificreaction of pituitary ceils by the absorption of each antiserum with its homologous antigen (1 mg/ml). For electron-microscopic observations the tissues were fixed in a mixture of equal parts of ice-cold 2 % paraformaldehyde prepared in cacodylate buffer (0.2 M, pH 7.4) and2 % osmic acid. Four hours after fixation the tissues were placed in ice-cold 0.5 % uranyl acetate overnight, and they were then dehydrated with alcohol and acetone. The tissues were embedded in Spurr's plastic mixture (Spurr, 1969). The sections were stained with lead citrate and observed with a Siemens 101 electron microscope.
Results
Control The organ cultures o f anterior pituitary glands revealed the presence o f two clearly defined zones: 1) an extensive internal zone with m a n y small-sized and some necrotic cells, and 2) a relatively thin external zone with healthy-appearing cells. Cells reactive to antisera for each o f the hormones used in this investigation were recognized in the external zone a n d some were identified in the internal zone o f the organ. Cells that reacted to these antisera were generally scarce and reactions were weak. By electron m i c r o s c o p y it was observed that most o f the cells h a d a decreased n u m b e r o f secretory granules a n d that the cells o f the internal zone were atrophic. They contained n u m e r o u s lipid bodies and lysosomes, characteristic o f cells in organ culture. M a n y cytoplasmic projections were seen on the surface cells o f the external zone (Fig. 5).
Culture with T R H All five o f the pituitary h o r m o n e s were recognized in the cultured organs by i m m u n o c y t o c h e m i c a l methods. Prolactin cells were observed in the internal zone o f the o r g a n but were scarce in the external zone. A C T H cells, which reacted only weakly to the antiserum, were observed only in small numbers in the external zone. L H cells were scarce and small. However, the cells that c o r r e s p o n d i m m u n o cytochemically to T S H cells were clearly observed as hypertrophic cells in the external zone (Figs. 1 and 2). These cells contained well-developed endoplasmic reticula (ER), but only a few secretory granules and lipid bodies (Fig. 7). In the internal zone, there were m a n y cells that reacted weakly with the antiserum against TSH, but all o f them were atrophic or necrotic (Fig. 6). The two different concentrations o f T R H (10-7 and 10 -6 M) did not produce any clear differences in the organ cultures.
402
M. Shiino
Fig. 1. Distribution of TSH cells in the pituitary organ culture revealed by immunocytochemistry. TSHcontaining cells are distributed in both the external and internal zones, but hypertrophic thyrotrophs are seen in the external zone. x 48. (TRH-treated culture group) Fig. 2. Higher magnification of Fig. 1. Immunocytochemically positive cells by anti-TSH serum are revealed mostly as large polygonal cells in the external zone. • 768 Fig. 3. Distribution of gonadotrophs in the pituitary organ culture revealed by immunocytochemistry. M a n y LH-containing cells are observed in the external zone and some weakly stained cells are seen in the internal zone of the organ. • 48. (LH-RH-treated culture group) Fig. 4. High magnification of Fig. 3. The cells are round or oval in shape. The stained cells are larger than most of the unstained cells. • 768
Castration Cells and Thyroidectomy Cells
403
Fig. 5. A n example o f n u m e r o u s cytoplasmic projections seen on the surface o f the external zone of the organ. The projections m a y serve to determine whether the organ culture performed well or not. • 12,240. (Control culture group) Fig. 6. M a n y atrophic and degenerating cells (/9) are observed in the internal zone of the organ. Arrows indicate lipid bodies, x 3,655. (Control group)
404
M. Shiino
Fig. 7. An example of hypertrophic (thyroidectomy) cell following the culture with TRH. Prominent dilated endoplasmic reticulum is observed, x 6,120 Fig. 8. An example of hypertrophic gonadotrophs following the incubation with LH-RH. The hypertrophic cells contain a well developed Golgi zone and rough surfaced endoplasmic reticulum. One hypertrophic gonadotroph contains a lot of lipid. These cells correspond to castration cells in vivo. x 3,655
Castration Ceils and ThyroidectomyCells
405
Culture with L H - R H Localization and cytological features of GH, PRL and A C T H cells were similar to those of cells cultured in Ham's F 10 medium supplemented with TRH. Some TSH cells were scattered in the explants but only a few were seen in the external zone. G H cells were clearly recognized in the external zone of the explants but were very scarce in the internal zone. Gonadotrophs were observed in both zones but were more prominent in the external zone (Fig. 3). When the tissues were observed with the electron microscope, many hypertrophic gonadotrophs containing well-developed Golgi zones, dilated ER and many lipid bodies in the cytoplasm were observed (Fig. 8).
Discussion
Zambrano et al. (1974) employed L H - R H (about 2 • 10 - 4 M) and Cuerdo-Rocha and Zambrano (1974) used 10 - 4 M T R H with in vitro systems to induce castration and thyroidectomy cells, respectively. Since it is known that the circulating blood contains about 10-11 M L H - R H (Chiappa et al., 1977), the concentration of LHR H which Zambrano and his collaborators employed in their culture is very high. Generally, it is known that a high concentration of hormone(s) may inhibit the release of hormone(s) from the target cells. Shiino et al. (1979) reported that the most effective level of L H - R H for stimulation of release of gonatropin in vitro was 10- 7 to 10- 6 M. The concentration of L H - R H employed by Zambrano et al. (1974) seems therefore to be quite high for use in culture. Furthermore, in our preliminary studies, pituitary cells in organ culture with L H - R H for 3-6 h did not reveal dramatic morphological changes in gonadotrophs except for extrusion of granules. It is not of rare occurrence that the adenohypophysis of adult male rats contains signet-ring gonadotrophs even without ablation of the gonads (Shiino and Rennels, 1973). It is for this reason that immature female rats for organ culture in this investigation. When pituitary glands are transplanted under the kidney capsule many prolactin cells remain active (Rennels, 1962), probably because of weak influence of the prolactin inhibitory factor (PIF) from the hypothalamus of the gland. However, prolactin cells were not well developed in the explants in the present experiment. This may be due to: 1) the presence of immature or underdeveloped prolactin cells in the anterior pituitary glands of the 14-day old rats employed, or 2) an incubation period that was not sufficient to promote development of the prolactin cells in the organ culture. The addition of L H - R H into the culture medium resulted in hypertrophic pituitary cells that reacted specifically with antiserum to LH. These hypertrophic gonadotrophs were very similar to the gonadotrophs observed following the ablation of the gonads. This indicates that hypei?trophic gonadotrophs or castration cells develop as a result of persistent stimulation of the gonadotrophs by considerably elevated levels of LH-RH. These morphological changes, however, may not be acute but rather may occur gradually when gonadotrophs are subjected to continuous stimulation with LH-RH. Similarly, the morphological features of
406
M. Shiino
thyrotrophs were altered within 10 days by incubation with TRH. We assume that thyroidectomy cells occurred owing to the constant stimulation by levels of TRH that were higher than those of normal blood. These results are in good agreement with our previous results with rats bearing autografts of pituitary glands that were treated with TRH (Shiino and Rennels, 1975). It is concluded that LH-RH and TRH stimulated directly the secretion of their respective hormones and concomitantly change the morphological features of the pituitary target cells. References Arimura, A., Debeljuk, L., Schally, A.V.: Stimulation of FSH release in vivo by prolonged infusion of synthetic LH-RH. Endocrinology 91, 529 532 (1972) Arimura, A., Shiino, M., DeLa Cruz, K.D., Rennels, E.G., Schally, A.V.: Effect of active and passive immunization with luteinizing hormone and follicle stimulating hormone-releasing hormone on serum luteinizing hormone and follicle stimulating hormone levels and the ultrastructure of the pituitary gonadotrophs in castrated male rat. Endocrinology 99, 291-303 (1976) Bowers, C.Y., Schally, A.V., Enzman, F., Baler, J., Folkers, K.: Porcine thyrotropin releasing hormone is (pyro)glu-his-pro(NH2). Endocrinology 86, 1143-1153 (1970) Chiappa, S.A., Fink, G., Sherwood, N.M.: Immunoreactive luteinizing hormone releasing factor (LRF) in pituitary stalk plasma from female rats: Effects of stimulating diencephalon, hypocampus and amygdala. J. Physiol. 267, 625-640 (1977) Cuerdo-Rocha, S., Zambrano, D.: The action of protein synthesis inhibitors and thyrotrophin releasing factor on the ultrastructure of rat thyrotrophs. J. Ultrastruct. Res. 48, 1-16 (1974) Kurosumi, K.: Functional classification of cell types of the anterior pituitary glands accomplished by electron microscopy. Arch. Histol. Jpn. 29, 329 362 (1968) Nailer, R., McCann, S.M.: Luteinizing hormone-releasing activity in plasma of hypophysectomized rats. Endocrinology 76, 272-275 (1965) Rennels, E.G.: An electron microscope study of pituitary autograft cells in the rat. Endocrinology 71, 713-722 (1962) Schally, A.V., Redding, T.W.: In vitro studies with thyrotrophin releasing factor. Proc. Soc. Exp. Biol. Med. 126, 320-325 (1967) Schally, A.v., Redding, T.W., Matsuo, H., Arimura, A.: Stimulation of FSH and LH release in vitro by natural and synthetic LH and FSH releasing hormone. Endocrinology 90, 1561-1568 (1972) Shiino, M., Rennels, E.G.: Ultrastructural observations of gonadotrophin release in rats treated neonatally with testosterone. Rep. Biol. Med. 31, 215-228 (1973) Shiino, M., Rennels, E.G.: Effects of TRH on pituitary grafts in the rat. Electron Microscopic Concepts of Secretion: Ultrastructure of Endocrine and Reproductive Organs (ed. M. Hess) pp. 271-286, New York: John Wiley & Sons, 1975 Shiino, M., Arimura, A., Schally, A.V., Rennels, E.G.: Ultrastructural observations of granule extrusion from rat anterior pituitary cells after injection of LH-releasing hormone. Z. Zellforsch. 128, 152-161 (1972) Shiino, M., Maruyama, T., Tanaka, T.: Gonadotroph-rich cell line derived from pituitary clonal cells (2A8) grafted under the kidney capsule. Cell Tissue Res. (In Press) 1979 Spurr, A.R.: A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 31-43 (1969) Sternberger, L.A., Hardy, P.H., Jr., Cuculis, J.J., Meyer, H.G.: The unlabeled antibody enzyme method of immunohistochemistry: Preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antiperoxidase) and its use in identification of spirochetes. J. Histochem. Cytochem. 18, 315-333 (1970) Zambrano, D., Cuerdo-Rocha, S., Bergman, I.: Ultrastructure of rat pituitary gonadotrophs following incubation of the gland with synthetic LH-RH. Cell Tissue Res. 150, 179-192 (1974) Accepted August 1, 1979
