Cell Tissue Res (1992) 267:t69-183
Cell e Tissue Research 9 Springer-Verlag 1992
Differentiation of the melanotrophic cells of rat pituitary primordium in organotypic culture in defined medium P. Vuillez, F. Ren6, M. Plante, C. Hindelang, M.J. Klein, J.M. F61ix, and M.E. Stoeckel Laboratoire de Physiologic,URA CNRS no 1446, 21, Rue Descartes, F-67084 Strasbourg, France ReceivedJuly 5, 1991 / AcceptedAugust 26, 1991
Summary. Organotypic cultures, in defined medium, of pituitary primordia obtained from 15-day-old rat fetuses were performed in order to study the in vitro differentiation of melanotrophic cells. The morphological and ultrastructural features of the transplants resembled those of the gland developing in vivo. In situ hybridization on semi-thin sections, using a 3SS-labelled oligonucleotide probe, revealed pro-opiomelanocortin-mRNA-containing cells on the first day of culture in the anterior lobe and after 2-3 days in the intermediate lobe. Immunoperoxidase labelling of adjacent sections showed that the same cells reacted with antibodies against c~-melanocyte-stimulating hormone (c~MSH), 73MSH and adrenocorticotropic hormone in both lobes. The pro-opiomelanocortin-mRNA-containing cells formed progressively conspicuous areas in the intermediate lobe, which was almost uniformly labelled after 6 days. In the anterior lobe, these cells remained scattered in small cell groups, and colloidal gold immunolabelling showed the progressive disappearance of c~MSH labelling from the secretory vesicles in cells exhibiting morphological features of adult corticotrophic cells. Both the c~MSH content of the explants and eMSH release into the culture medium increased with time. Treatment with the dopamine agonist bromocriptine induced a strong dose-dependent decrease in c~MSH secretion, which was significant after 3 days in culture, indicating that dopamine D2 receptors are able to regulate hormonal release of melanotrophic cells at early stages. This system constitutes a suitable model for further studies of factors controlling cell differentiation and cellular interactions involved in histogenesis. Key words: Fetal intermediate lobe Tissue culture Immunocytochemistry - In situ hybridization - Dopamine - Rat (Wistar)
Cell differentiation and organogenesis are complex processes in vertebrate pituitary glands. Whereas the neural Offprint requests to : P. Vuillez
lobe (NL) is derived from the floor of the diencephalon, the anterior and intermediate lobes (AL and IL, respectively) arise from the hypophyseal placode, which is presumably derived from the anterolateral neural ridge (Couly and Le Douarin 1985) through Rathke's pouch formation. Differentiation results in phenotypically distinct cell types: thyreotrophs, somatotrophs, lactotrophs, gonadotrophs and corticotrophs in the AL, and melanotrophs in the IL. The temporal differentiation of the different AL cell types in situ has been extensively studied in rats and mice: Dupouy and Dubois (1975), Chatelain et al. (1979), Watanabe and Daikoku (1979), Begeot et al. (1981), Gash et al. (1982) and, more recently, Nemesk~ri et al. (1988). Furthermore, searches for factors and/or tissue relationships inducing differentiation have been carried out using in vitro experiments: Ferrand and Nanot (1968), Nemesk~ri et al. (1976), Daikoku et al. (1982), Watanabe (1982), Begeot et al. (1982, 1987), Schechter et al. (1985), Nemesk6ri and Halfisz (1989). Recently, a pituitary-specific transcription factor (Pit-1 or GHF-I) able to activate both rat prolactin and growth hormone gene promoters has been identified as a homeobox-gene-derived protein (Ingraham et al. 1988; Bodner et al. 1988) that specifies the somatotroph and lactotroph phenotypes (Simmons et al. 1990). With respect to the IL and the melanotrophs, the available results concern mainly the in situ ontogenesis of the expression of the pro-opiomelanocortin (POMC) gene, and the biosynthesis and release of the main related peptides. In adult pituitary, POMC is a pro-hormone expressed in the corticotrophs and the melanotrophs, although the peptide formed, the regulation of POMC gene expression, and the biosynthesis and release of the related peptides are different (for reviews, see Smith and Funder 1988; Autelitano etal. 1989). In the melanotrophs, the main synthesized and released peptides are ~-melanocyte-stimulating hormone (c~MSH) and acetylated/~-endorphin. POMC gene expression and eMSH secretion are negatively regulated by dopamine (DA), via a D2 receptor and by 7-aminobutyric acid (GABA) (see Eberle 1988); both transmitters are co-localized in the axons innervating the IL (Vuillez et al. 1987). In the rat, as pointed out by Pintar and Lugo (1987) and Hin-
170 d e l a n g et al. (1990), the o n s e t o f synthesis o f P O M C m R N A in the I L occurs a t e m b r y o n i c stage 1 5 - I 6 d a y s ( E l 5-16), a n d P O M C m R N A t r a n s l a t i o n a n d p r o - h o r m o n e p r o c e s s i n g s t a r t w i t h o u t d e l a y as i n d i c a t e d b y i m munocytochemical data and radioimmunoassay (Chatelain et al. 1976; C h a t e l a i n a n d D u p o u y 1981 ; S c h w a r t z b e r g a n d N a k a n e 1982; D a i k o k u et al. 1983; K h a c h a t u r i a n et al. 1983; N e m e s k ~ r i et al, 1988). A l t h o u g h p u t a tive f u n c t i o n a l D A D z r e c e p t o r s h a v e been d e t e c t e d in the I L d u r i n g o n t o g e n e s i s as e a r l y as E17 (Sales et al. 1989), p h y s i o l o g i c a l d o p a m i n e r g i c i n h i b i t o r y c o n t r o l only o p e r a t e s a t p o s t - n a t a l d a y 5 ( P N 5 ) o n e M S H release ( D a v i s et al. 1984) a n d on P O M C gene e x p r e s s i o n ( H i n d e l a n g et al. 1990). In vitro d e v e l o p m e n t o f the I L a n d d i f f e r e n t i a t i o n o f the m e l a n o t r o p h s a r e o n l y d e a l t with in the u l t r a s t r u c t u r a l s t u d y o f W a t a n a b e et al. (1973). The present paper mainly concerns IL organogenesis a n d the d i f f e r e n t i a t i o n o f m e l a n o t r o p h s o f the p i t u i t a r y p r i m o r d i u m in o r g a n o t y p i c c u l t u r e in d e f i n e d m e d i u m , e x a m i n e d b y c o m b i n i n g in situ h y b r i d i z a t i o n a n d imm u n o c y t o c h e m i c a l techniques. O b s e r v a t i o n s o n the dev e l o p m e n t o f the o t h e r p i t u i t a r y lobes in the s a m e cultures a r e also r e p o r t e d .
Materials and methods
Animals Female Wistar rats were singly housed in a temperature-controlled and light-cycled (14 h light, 10 h dark) animal room. All received standard food and water ad libitum. The time of conception was assessed from vaginal smears carried out on the morning after overnight mating. The sperm-positive day was designated as embryonic day zero (E0). Pituitary primordia were explanted at E15. Under deep barbiturate anaesthesia, tile embryos were removed by caesarean section, decapitated, and the pituitary primordium and the diencephalon were exposed. Under a dissecting microscope, the pituitary primordium was carefully separated from the roof of the oral cavity and severed from the diencephalon by a section close to the infundibular recess. The isolated explants thus included Rathke's pouch, its associated mesenchyme and the extension of the putative hypothalamus corresponding roughly to the NL,
Organ culture Pituitary primordia were each placed on a cellulose nitrate membrane filter (0.45 gm, Whatman, Maidstone, UK) supported by a stainless steel grid. Incubation was carried out in a cellular plastic culture-dish (35 mm diameter, Costar, Cambridge, Mass., USA) containing 1 ml medium, consisting of RPMI 1640 supplemented with glucose (final concentration 6 g/l), albumin (2.5 g/l), ascorbic acid (0.02 g/l), insulin, progesterone, transferrin, putrescin and selenium (according to Bottenstein and Sato 1979), streptomycin (0.05 g/l), fungizone (0.125 g/l), penicillin (50.000 U/l), and gentamicin (0.05 g/l). During incubation, the gas phase consisted of 5% CO2 and 95% air. The culture medium was first changed on the day following initiation of the culture and then every 3 days.
Electron microscopy The explants were fixed in 5% gtutaraldehyde in 0.1 M phosphate buffer pH 7.4 (PB), for 1 h, and postfixed for I h with 1% OsO4
in PB. After dehydration in graded alcohols, they were conventionally embedded in an Araldite-Epon mixture. Semi-thin sections for light-microscope controls were stained with 1% toluidine blue in 5% sodium borate. Thin sections, contrasted with uranyl acetate and lead citrate, were examined on a Jeol 100 CX electron microscope. For post-embedding immunogold labelling, the explants were fixed for 2 h in 0.1 M PB containing 4% paraformaldehyde and 0.25% glutaraldehyde and embedded in Lowicryl according to Valentino et al. (1985). For the demonstration of exocytotic figures, cultures were incubated for 20 rain in the presence of 1.5% tannic acid in the culture medium prior to fixation (Buma et al./984).
In situ hybridization POMC mRNA was detected using a synthetic single-stranded oligodeoxynucleotide probe labelled with 35S deoxyadenosine triphosphate (specific activity 400~5000 Ci/mmole, NEN, Dupont de Nemours, USA), purified and tested for specificity as previously reported (Hindelang et al. 1990). Hybridization was carried out on the whole explant, and the probe was detected by autoradiography on semi-thin sections according to Guitteny et al. (1988) with slight modifications. Briefly, the explants were fixed for 30 min in a mixture containing 4% paraformaldehyde and 0.25% glutaraldehyde in PB. The explants were then rinsed 3 • 5 min in PB, acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine, 0.9% NaC1, pH 8, for 10 rain, and washed 3 x 10 rain in 2 x SSC (1 • SSC= 0.15 M NaC1, 0.015 M sodium citrate). They were incubated overnight at 35~ C in 100 gl hybridization buffer containing 25 ng/ml probe, washed for 3 x 10 min in 2 x SSC at room temperature, then for 30 min in 0.5 x SSC at 50~ C, and post-fixed for 30 rain in the paraformaldehyde/glutaraldehyde mixture. After a brief rinse in PB, the explants were dehydrated and embedded in the AralditeEpon mixture. Semi-thin sections (1.5 gm) were mounted on gelatin-coated slides. They were first exposed for 24-48 h to fiMax hyper-film (Amersham, UK) and were then dipped into K5 emulsion (Ilford, France) diluted 1/2 and exposed for 4-8 days. Film and emulsion developments were performed as previoulsy described (Hindelang et al. 1990). Alternate sections were used for immunocytochemical studies.
Immunocytochemistry For light microscopy, the conventional indirect immunoperoxidase method was applied using antisera raised in the rabbit, and peroxidase-labelled anti-rabbit Fab (1/200, Biosys, Compi6gne, France) was revealed with diaminobenzidine tetrahydrochloride as already described (Schimchowitsch et al. 1983). Semi-thin sections adjacent >
Fig. 1. l-day culture. Anterior lobe (AL). Presence of dense secretory vesicles in a cell extension at the periphery of the transplant. Basement membrane at upper left (arrow). • 18 500. Bar." 0.5 gm Fig. 2. 5-day culture. Distinct cell types, as indicated by the size of the dense secretory vesicles, are intermingled in the AL. x 6500. Bar: 2 pin Fig. 3. 9-day culture. Intermediate lobe (IL). Melanotrophic cells contain dense secretory vesicles disposed along the ceil borders. Note the tight interdigitations of the plasma membranes (arrows). x 7600. Bar: 2 pm Fig. 4a, b. 9-day culture, IL. Incubation in the presence of tannic acid prior to fixation reveals several secretory vesicles undergoing exocytosis. In b, several compound exocytotic figures occur in two adjacent cells, x 25000. Bar: 0.5 I-tm
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173 to those used for autoradiography and semi-thin sections from explants similarly fixed, but not hybridized, were processed after removal of the embedding medium with sodium methoxide. The antisera used were raised against synthetic ~MSH (1/2000; the late G. Schmitt, Strasbourg, France), y3MSH (1/1000; H. Vaudry Rouen, France) and 1-39 adrenocorticotropic hormone (ACTH; 1/1000; H. Vaudry, Rouen, France), prolactin (1/300; the late M.P. Dnbois, Nouzilly, France), luteinizing hormone (LH; 1/1000; M. Poissonnier, Gif Sur Yvette, France) and GABA (1/5000; Immunotech, Marseille, France). Anti-gtial fibrillary acidic protein (GFAP; 1/100; Dako, Copenhagen, Denmark) and anti-laminin (1/1000; M. Vigny, Paris, France) antisera were applied to cultures fixed in the same fixative, and whole-mounted after clearing in xylene. [mmunostaining specificity for peptides was tested by incubating the diluted antisera for 1 h at 37~ C in the presence of 10 gg/ml of the corresponding antigen, or for GABA, GFAP and laminin, by replacing the antiserum by pre-immune serum. Although the high specificity of the anti-c~MSH and 7aMSH versus other POMCderived peptides had already been established in radio-immunoassay (see Stoeckel et al. 1985), complementary tests were performed to check possible cross-reactivity of the eMSH antibody with ACTH in the culture sections and in sections from pituitary glands of adult rats, fixed and embedded under the same conditions. The diluted antiserum was therefore incubated in the presence of 0.05, 0.1, 0.5, 1, 5 and 10 Ixg/ml synthetic c~MSH or ACTH before immunostaining. The ACTH antiserum was preincubated with the same concentrations of ACTH, c~MSH and CLIP (corticotrophin-like intermediate lobe peptide, 14-39 ACTH). For electron microscopy, c~MSH and 73MSH immunoreactivities were detected on thin sections from the Lowicryl embedded explants by using the immunogold procedure with 5- or 15-nm anti-rabbit-IgG-coated gold particles (Janssen, Beerse) and double labelling was performed using a recto-verso method, as already described (Vuillez et al. 1987). The characteristics of immunogold labelling with both antibodies have already been reported (Stoeckel et al. 1985). In the present study, the specificity tests were performed on the explant sections, by preincubating the antibodies with 1 and 10 gg/ml of the homologous peptide, and, in the case of the anti-eMSH antibody, with 1, 5, and 10 ~tg/ml ACTH.
Radioimmunoassay of ~ M S H Explants were extracted by sonication in ice-cold 1% acetic acid and 20 gg/ml aprotinin. The supernatants were separated from the cell debris by centrifugation at 10000 x g for 10 rain. The culture medium was adjusted to give final concentrations of 1% acetic acid and 20 gg/ml aprotinin. All samples were kept at - 2 0 ~ until radioimmunoassay. Both explant extracts and media were assayed for eMSH as described by Schmitt et al. (1979). The antibody used was from Dr. A.N. Eberle (Basel, Switzerland); raised
Fig. 5. 9-day culture, IL. Folliculo-stellate cells, linked by apical junctions, project microvilli into the lumen of a follicle, x 10500. Bar: 1 gm Fig. 6. 14-day culture, AL. Isolated connective-tissue space lined with a basement membrane encloses a cell exhibiting morphological features of a fibroblast, x 4700. Bar: 2 gm Fig. 7. 9-day culture. Connective tissue layer containing collagen bundles separates the intermediate (left) and the neural (right) lobes, x 8000. Bar." 1 gm Fig. 8. 14-day culture. Note contact between intermediate (right) and neural (left) lobes. Loosely arranged collagen fibrils persist between the cells (arrows). Pituicyte (P), containing lipid inclusions, intermingled with melanotrophic cells. Note the decreased electron density of the secretory vesicles in the melanotrophic cells (compare with Fig. 3). x 4700. Bar. 2 gm
against synthetic c~MSH, it recognizes the free C-terminal part of the desacetylated (ACTH 1-13), and acetylated and diacetylated eMSH on a nearly equimolecular basis; it also gives a very low cross-reaction with ACTH (molar cross-reactivity < 0.1%) (Foll6nius et al. 1985), and no detectable reaction with higher molecular forms of ACTH peptides (personal observations). The c~MSH content was separately measured for each explant. The expression of the c~MSH content per explant reflects the variations of both the cell numbers producing c~MSH and hormone content, independent of the variations in other cell-type differentiation and/or proliferation. The eMSH degradation rates in the medium were estimated by eMSH measurements in the medium at various stages of culture, immediately or after an additional 6-h incubation at 37~ C without explant.
Protein measurement The determination of the whole protein value of the explants was performed by the technique of Lowry et al. (1951) using a microassay procedure with bovine albumin as a standard.
Statistical analysis Data in figures are means_+ SEM. Statistical differences between data were analysed using Student's t-test (F test).
Chemicals and drugs These were purchased fiom Gibco-BRL (Cergy Pontoise, France) in the case of RPMI 1640, albumin, streptomycin, fungizone, penicillin, and gentamicin; from Jacques Boy laboratory (Reims, France) in the case of transferrin ; from Le Brun laboratory (Paris, France) as regards haloperidol; from Sigma (La Verpilli6re, France) as regards triethanolamine, insulin, progesterone, putrescin, aprotinin, 2-bromo-c~-ergocryptine, and selenium; from Fluka (Buchs, Switzerland) with respect to glutaraldehyde; and from Kodak (Touzart et Matignon, France) as regards paraformaldehyde.
Results
Morphofunctional aspects of Rathke's pouch differentiation The explanted primordia rapidly flattened and exhibited a r o u n d e d shape, ~ 2 m m in d i a m e t e r a n d J 00 g m thick, after 9 d a y s in culture. U p to 5 days, the persistence o f the R a t h k e ' s p o u c h l u m e n e n a b l e d the i d e n t i f i c a t i o n o f the p r i m o r d i a o f the A L , the I L a n d the t u b e r a l lobe on the t o l u i d i n e b l u e - s t a i n e d s e m i - t h i n sections. In the h o r i z o n t a l section p l a n e , the A L a p p e a r e d as a large cell m a s s f r o m w h i c h the connective spaces, w h i c h were d e v e l o p i n g in the p r i m o r d i u m a r o u n d the p e n e t r a t i n g vessels at the time o f e x p l a n t a t i o n , h a d d i s a p p e a r e d (Fig. 9). A t t w e l l ' s recess was still r e c o g n i z a b l e at 4 d a y s o f culture, b e t w e e n the A L a n d the t u b e r a l l o b e p r i m o r dia. T h e IL, s e p a r a t e d f r o m the A L b y the R a t h k e ' s p o u c h lumen, was f o r m e d b y a thick c u r v e d cell m a s s t h a t r a p i d l y lost its p r i m i t i v e p a l i s a d i c a r r a n g e m e n t . M i totic cells were r e g u l a r l y set o u t a l o n g the l u m e n o f R a t h k e ' s p o u c h , w h i c h c o n t a i n e d cell d e b r i s a n d large
174 cells with vacuolated cytoplasm. In the early stages, no marked differences in cell shape and basophilia were obvious between the IL and the AL primordia. The NL, deeply engulfed in the IL, consisted of a uniform population of cells slightly larger and less basophilic than the adenohypophysial cells (Fig. 9). In several explants, the NL was in continuity with hypothalamic fragments of various sizes, the presence of which had no obvious effects on the development of the pituitary explants. In the later stages, the partial disappearance of Rathke's pouch and Attwell's recess made histological identification of the adenohypophysial lobes more difficult.
Electron microscopy After 1 day of culture, cells containing secretory vesicles (SV) could be detected at the periphery of the explants in the putative AL (Fig. 1). The number of the SV-containing cells rapidly increased. After 5 days, several cell types, as indicated by the size of their SV, were present in the AL (Fig. 2). Small SV occurred regularly in cells containing abundant glycogen particles, and obviously belonging to the pars tuberalis. In the IL, the melanotrophic cells contained SV of 100-120 nm in diameter, set out along the cell borders. The conspicuous development of interdigitations of the plasma membranes was a striking feature of these cells (Fig. 3). At 9 days, most adenohypophysial cells devoid of SV could be classified as folliculo-stellate cells. They were linked together by well-developed junctional systems in contact with follicles of various sizes (Fig. 5). No colloid was, however, detected in either follicles or Rathke's pouch lumen, at any stage. Both glandular and follicular cells were often seen undergoing mitosis. A few typical fibroblasts were found in apparently isolated, tiny connective spaces, containing small collagen bundles, and lined with a basement membrane (Fig. 6). Basement membranes were sometimes interposed between glandular cells; they did not continuously border cell cords as in the AL in situ. No such images were observed in the IL primordia. A thin connective tissue layer most often separated the IL and NL primordia (Fig. 7). In later stages (9 and 14 days), this barrier frequently disappeared and the melanotrophic cells were in direct contact and even intermingled with the pituicytes. The latter were characterized by frequent lipid inclusions (Fig. 8). In the samples incubated in the presence of tannic acid prior to fixation, exocytotic figures were frequently observed in the IL primordium (Fig. 4) but rarely in the AL. After bromocriptine treatment (10-7 tool/l), the exocytotic figures were less frequently observed in the IL primordium.
Immunocytochemistry The specificity controls of the antibodies performed by preabsorption with the homologous antigen resulted, in all cases, in an absence of immunostaining. This was also the case when the primary antibody was replaced
by pre-immune rabbit serum. Tests concerning the antic~MSH and the anti-ACTH antibodies were performed in pituitaries of adult rats, where c~MSH is known to predominate in the IL and ACTH in the AL (Smith and Funder 1988). The anti-~MSH antibody strongly labelled the melanotrophic cells of the IL, whereas the corticotrophic cells in the AL were faintly labelled. In the IL, ~MSH labelling was partly inhibited at 0.1 pg/ml eMSH and completely abolished by 10 gg/ml eMSH; in contrast, eMSH labelling was unchanged by ACTH at concentrations up to 5 pg/ml. In the AL, the faint ~MSH labelling was abolished in the presence of 0.01 gg/ ml c~MSH, and 0.5 gg/ml of ACTH, the lowest concentration tested. The anti-ACTH antibody equally labelled the corticotrophic AL cells and the melanotrophic IL cells. This labelling decreased when antiserum treated with 0.5 pg/ml ACTH was used and was abolished in the presence of 5 gg/ml ACTH. It was decreased but not abolished when the antibody was preincubated in the presence of 10 l,tg/ml CLIP or ~MSH. Taken together, these tests indicate the specificities of the anti-c~MSH and anti-ACTH antibodies, whith only faint cross-reactivities for the related POMC-derived peptides. In the explants, eMSH, 73MSH and ACTH immunoreactivities could be detected on semi-thin sections in a few cells scattered in the AL primordium from the first day of culture. In the IL, c~MSH-immunoreactive (ir) cells occurred after 2 days in some explants and in all of them after 3 days of culture. After 4 days, eMSH-ir cells formed conspicuous areas in the IL, whereas such cells remained regularly scattered in small clusters in the AL primordium (Fig. 11 a). In these preparations, eMSH labelling was totally abolished in the presence of 5 gg/ml eMSH, still strongly inhibited at 0.5 gg, and unaltered by 0.05 gg/ml eMSH; it remained unchanged in the presence of 5 gg/ml ACTH. In all cases, inhibition of the reaction was similar in the AL and IL primordia. ACTH immunolabelling showed the same distribution as ~MSH labelling (Fig. 11 b), and adjacent sections demonstrated that the same cells reacted with the antibodies raised against the POMC-derived peptides. ACTH immunoreaction was abolished in the presence of 5 pg/ml ACTH, but persisted in the presence of the same concentration of CLIP or c~MSH. The ir cells had roughly the same shape and size in the two lobes, and showed a characteristic distribution of labelling along the cell borders (Fig. 14a, b). In the later stages (9 and 14 days), the cells of the IL progressively filled up with ir material (Fig. 14c). In the AL, most of the cells exhibited labelling of the periphery as in the early stages, whereas some others appeared more densely packed with ir material (Fig. 14d). Electron-microscopic immunogold labelling showed uniform labelling of the SV of the IL primordia cells with both anti-73MSH and anti-eMSH antibodies (Fig. 15 a). These antibodies similarly labelled stellateshaped cells scattered throughout the AL, as shown by double-labelled preparations (Fig. 15 b). Gold particles were concentrated on dense SV, ~ 120 140 nm in diameter, regularly set out under the plasma membrane, and occasionally accumulated in a cell extension. In some
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Fig. 9. 3-day culture. Toluidine-blue staining of a semi-thin section in the horizontal plane reveals the organization of the pituitary primordium: the well-defined, though collapsed, hypophysial cleft separates the A L from the IL; a hypothalamic fragment (H) is attached to the NL, x 75. Bar: 200 gm Fig. 10. 3-day culture. Autoradiographic detection of in situ hybridized POMC mRNA on a semi-thin section shows labelling concenA L cells, the a n t i - ~ M S H a n t i b o d y less intensely labelled the SV, some o f which were completely free f r o m gold particles (Fig. 16b, b o t t o m cell). The variations in labelling intensities o f the SV in the same cell were particularly obvious in cells with dense granules at their periph-
trated in the IL primordium; labelling occurs in small patches in the A L and is absent from the NL. • 85. Bar: 200 ~tm Fig. 11 a, b. 4-day culture. Immunoperoxidase labelling for ~MSH
(a) and ACTH (b) shows similar labelling with both antibodies in the AL, whereas the IL reacts less intensely for ACTH than the AL. x 64. Bar: 200 gm
eries, resembling adult corticotrophic cells. In such cells, there was, however, no obvious relationship between labelling intensity and electron density o f the SV. In other cells, with m o r e a b u n d a n t SV that were diffusely scattered t h r o u g h the cytoplasm, almost all SV were h o m o -
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Figs. 12, 13. Examples of 6-day and 14-day cultures. Adjacent semithin sections processed for autoradiographic detection of in situ hybridized POMC mRNA (a), immunoperoxidase labelling of c~MSH (b), and prolactin (e). a Hybridization signals are strongly increased at the 14-day stage, although in this example, AL label-
ling is decreased with both in situ hybridization and ~MSH immunocytochemistry (b). e Prolactin cells are, at first, concentrated in the central area at 6 days, but are scattered throughout the transplant at 14 days. The intense reaction on the cells in the b o t t o m area of the AL in Fig. 13b and e is non-specific, x 55. B a r . 200 gm
geneously ~ M S H - i r (Fig. 16b, top cell). The anti-73MSH a n t i b o d y u n i f o r m l y labelled all SV (Fig. 16a). Such discrepancies between ~ M S H - a n d 73MSI-I i m m u n o r e a c t i vities o c c u r r e d m o r e frequently in the later stages. Specificity tests s h o w e d inhibition o f labelling with the antic~MSH a n t i b o d y in the presence o f 1 gg/ml e M S H , al-
t h o u g h it was n o t abolished by 5 gg/ml A C T H . The specificity o f c~MSH i m m u n o g o l d labelling was further s u p p o r t e d by absence o f labelling o f the SV in the corticotrophic cells o f adult rat pituitaries. These d a t a c o m plete previous results using the same antibodies (Stoeckel et al. 1985). I m m u n o l a b e l l i n g with the anti-73MSH anti-
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Fig. 14a-f. Detail of immunoperoxidase localization of eMSH in the IL (a, e) and the AL (b, d) at 3 days (a, b) and 9 days (e, d), and of prolactin (e) and LH (f) at 6 days of culture. At 3 days, eMSH immunoreactivity lines the cell borders and is less intense in the IL than in the AL cells (a, b). At 9 days, the labelled cells
of the IL are tightly packed and immunoreactive material tends to fill the cytoplasm (e). Note the heterogeneity of the labelling intensity in the AL (d). Localization of prolactin immunolabelling is concentrated in a juxtanuclear area (e), whereas LH labelling is distributed throughout the cytoplasm (f). x 450. Bar: 25 gm
b o d y was abolished in the presence of 10 gg/ml h o m o l o gous peptide. At 6 days, prolactin-ir (Figs. 13 c, 14 e) and L H - i r cells (Fig. 14f) were clearly detected in the AL. Prolactincontaining cells exhibited striking, intense granular labelling in the juxta-nuclear zone, contrasting with the m a r g i n a l labelling o f the A C T H - i r / M S H - i r cells. L a m i n i n i m m u n o s t a i n i n g on whole cultures (at 6 and
9 days) showed a discontinuous a r r a n g e m e n t o f the basem e n t m e m b r a n e s t h r o u g h o u t the A L and their absence f r o m the I L (Fig. 17), as previously seen in the electronm i c r o s c o p e preparations. T h e a n t i b o d y against G F A P intensely labelled pituicytes in the N L and, m o r e weakly, some cells in the A L (Fig. 18), as in the pituitary gland in situ (see Allaerts et al. 1990). In the adjacent h y p o t h a l a m i c areas, n u m e r o u s cells
Fig. 15a, b. 13-day culture. Electron-microscopic detection of c~MSH and 73MSH by immunogold double-labelling (eMSH with 5-nm particles; ?3MSH with 15-nm particles) in the IL (a) and the AL (b). In both locations, most secretory vesicles are doublelabelled, x 42300. Bar." 0.5 ~,m
Fig. 16a, b. 13-day culture, AL. Immunogold detection of y3MSH (a) shows labelling of all secretory vesicles; in b, c~MSH labelling occurs on almost all secretory vesicles in the cell at the top, whereas few secretory vesicles are labelled in the bottom cell. The dotted line indicates the position of the separating plasma membranes between these two cells, x 32200. Bar: 0.5 gm
179
Fig. 17. 9-day culture. Whole-mount laminin-immunoperoxidase labelling reveals different distribution patterns of basement membranes in the three lobes. Discontinuous stellate-shaped figures are scattered throughout the AL; labelled structures are almost absent from the IL; a delicate network is labelled in the neural lobe (NL). x 250. Bar: 50 p.m
Table 1. aMSH content of pituitary primordia and secretions at different stages of organotypic culture. Secretion was measured as the content of eMSH in the medium, divided by the culture time in days. Results are means of the contents or secretions from separate pituitary primordia. Number of cases in brackets
Fig. 18. 9-day culture. Whole-mount GFAP-immunoperoxidase shows intensely labelled cells in the NL primordium and in the adjacent hypothalamic area (top). Weakly labelled cells are scattered in the AL primordium, x 60. Bar: 200 gm
~MSH content Stages (days)
1
3
6
9
14
eMSH fmol_+ SEM
140_+20 (4)
1414• (6)
5245+_723 (6)
11269+_740 (6)
32718_+2518 (6)
Stages (days)
1-3
3-6
6-9
9 12
1~14
eMSH fmol/day _+SEM
41.5+_2.8 (26)
317.6+_22 (20)
1079+_57 (13)
1226_+125 (8)
1100+_133 (8)
~MSH secretion
were labelled w i t h the a n t i b o d y a g a i n s t G A B A . But no ir a x o n s c o u l d be d e t e c t e d in either a d e n o h y p o p h y s e a l lobes at a n y stage e x a m i n e d .
In situ hybridization W h e n cultures were initiated, a w e a k signal for P O M C m R N A - c o n t a i n i n g cells was o b s e r v e d in a few cells, in
o n l y s o m e R a t h k e ' s p o u c h explants. A f t e r I d a y o f culture, P O M C - m R N A - c o n t a i n i n g cells were s c a t t e r e d in the A L . I m m u n o c y t o c h e m i s t r y a n d in situ h y b r i d i z a t i o n on serial sections i n d i c a t e d t h a t their n u m b e r was g r e a t e r t h a n the A C T H - i r / c ~ M S H - i r cells. P O M C - p o s i t i v e cells a p p e a r e d after 2 d a y s in the I L , w h i c h was well d e l i m i t e d a n d labelled at 3 d a y s (Fig. 10). A t l a t e r stages, the intensity o f cell labelling a n d the
180 Table 2. Effects of bromocriptine on the e M S H content of pituitary primordia and secretions during the first 9 days of culture. Bromocriptine (10-7 tool/l) was added after one day in culture. Results are means of contents or secretions from separate pituitary primorStages (days)
dia. Media contained 0.001% ethanol for dissolving bromocriptine. Secretion was measured as indicated in Table 1. Number of cases in brackets
c~MSH content fmol__ SEM 3
c~MSH secretion fmol/day__ SEM
6
9
1-3
3-6
6-9
Control
1314 • (6)
62
5330 +474 (6)
10780 • 1090 (5)
4/.8 +_2.7 (25)
337 +25.9 (12)
l / 9 4 • 109 (13)
Bromocriptine 10- 7 tool/1
1720 • 242 (6)
8702 • 874 (7)
13 460 • 5110 (5)
25.6 • 1.9 (27)
116.3 • 14.6 (13)
307 • (14)
medium also increased with time (Table 1). The low levels found during the first days of culture were not the result of a higher eMSH degradation in the culture media, since evaluation of the degradation rates indicated no statistical differences with culture time (not shown). Treatment by the DA agonist bromocriptine induced a strong decrease in eMSH secretion, which was significant (P
Cell e Tissue Research 9 Springer-Verlag 1992
Differentiation of the melanotrophic cells of rat pituitary primordium in organotypic culture in defined medium P. Vuillez, F. Ren6, M. Plante, C. Hindelang, M.J. Klein, J.M. F61ix, and M.E. Stoeckel Laboratoire de Physiologic,URA CNRS no 1446, 21, Rue Descartes, F-67084 Strasbourg, France ReceivedJuly 5, 1991 / AcceptedAugust 26, 1991
Summary. Organotypic cultures, in defined medium, of pituitary primordia obtained from 15-day-old rat fetuses were performed in order to study the in vitro differentiation of melanotrophic cells. The morphological and ultrastructural features of the transplants resembled those of the gland developing in vivo. In situ hybridization on semi-thin sections, using a 3SS-labelled oligonucleotide probe, revealed pro-opiomelanocortin-mRNA-containing cells on the first day of culture in the anterior lobe and after 2-3 days in the intermediate lobe. Immunoperoxidase labelling of adjacent sections showed that the same cells reacted with antibodies against c~-melanocyte-stimulating hormone (c~MSH), 73MSH and adrenocorticotropic hormone in both lobes. The pro-opiomelanocortin-mRNA-containing cells formed progressively conspicuous areas in the intermediate lobe, which was almost uniformly labelled after 6 days. In the anterior lobe, these cells remained scattered in small cell groups, and colloidal gold immunolabelling showed the progressive disappearance of c~MSH labelling from the secretory vesicles in cells exhibiting morphological features of adult corticotrophic cells. Both the c~MSH content of the explants and eMSH release into the culture medium increased with time. Treatment with the dopamine agonist bromocriptine induced a strong dose-dependent decrease in c~MSH secretion, which was significant after 3 days in culture, indicating that dopamine D2 receptors are able to regulate hormonal release of melanotrophic cells at early stages. This system constitutes a suitable model for further studies of factors controlling cell differentiation and cellular interactions involved in histogenesis. Key words: Fetal intermediate lobe Tissue culture Immunocytochemistry - In situ hybridization - Dopamine - Rat (Wistar)
Cell differentiation and organogenesis are complex processes in vertebrate pituitary glands. Whereas the neural Offprint requests to : P. Vuillez
lobe (NL) is derived from the floor of the diencephalon, the anterior and intermediate lobes (AL and IL, respectively) arise from the hypophyseal placode, which is presumably derived from the anterolateral neural ridge (Couly and Le Douarin 1985) through Rathke's pouch formation. Differentiation results in phenotypically distinct cell types: thyreotrophs, somatotrophs, lactotrophs, gonadotrophs and corticotrophs in the AL, and melanotrophs in the IL. The temporal differentiation of the different AL cell types in situ has been extensively studied in rats and mice: Dupouy and Dubois (1975), Chatelain et al. (1979), Watanabe and Daikoku (1979), Begeot et al. (1981), Gash et al. (1982) and, more recently, Nemesk~ri et al. (1988). Furthermore, searches for factors and/or tissue relationships inducing differentiation have been carried out using in vitro experiments: Ferrand and Nanot (1968), Nemesk~ri et al. (1976), Daikoku et al. (1982), Watanabe (1982), Begeot et al. (1982, 1987), Schechter et al. (1985), Nemesk6ri and Halfisz (1989). Recently, a pituitary-specific transcription factor (Pit-1 or GHF-I) able to activate both rat prolactin and growth hormone gene promoters has been identified as a homeobox-gene-derived protein (Ingraham et al. 1988; Bodner et al. 1988) that specifies the somatotroph and lactotroph phenotypes (Simmons et al. 1990). With respect to the IL and the melanotrophs, the available results concern mainly the in situ ontogenesis of the expression of the pro-opiomelanocortin (POMC) gene, and the biosynthesis and release of the main related peptides. In adult pituitary, POMC is a pro-hormone expressed in the corticotrophs and the melanotrophs, although the peptide formed, the regulation of POMC gene expression, and the biosynthesis and release of the related peptides are different (for reviews, see Smith and Funder 1988; Autelitano etal. 1989). In the melanotrophs, the main synthesized and released peptides are ~-melanocyte-stimulating hormone (c~MSH) and acetylated/~-endorphin. POMC gene expression and eMSH secretion are negatively regulated by dopamine (DA), via a D2 receptor and by 7-aminobutyric acid (GABA) (see Eberle 1988); both transmitters are co-localized in the axons innervating the IL (Vuillez et al. 1987). In the rat, as pointed out by Pintar and Lugo (1987) and Hin-
170 d e l a n g et al. (1990), the o n s e t o f synthesis o f P O M C m R N A in the I L occurs a t e m b r y o n i c stage 1 5 - I 6 d a y s ( E l 5-16), a n d P O M C m R N A t r a n s l a t i o n a n d p r o - h o r m o n e p r o c e s s i n g s t a r t w i t h o u t d e l a y as i n d i c a t e d b y i m munocytochemical data and radioimmunoassay (Chatelain et al. 1976; C h a t e l a i n a n d D u p o u y 1981 ; S c h w a r t z b e r g a n d N a k a n e 1982; D a i k o k u et al. 1983; K h a c h a t u r i a n et al. 1983; N e m e s k ~ r i et al, 1988). A l t h o u g h p u t a tive f u n c t i o n a l D A D z r e c e p t o r s h a v e been d e t e c t e d in the I L d u r i n g o n t o g e n e s i s as e a r l y as E17 (Sales et al. 1989), p h y s i o l o g i c a l d o p a m i n e r g i c i n h i b i t o r y c o n t r o l only o p e r a t e s a t p o s t - n a t a l d a y 5 ( P N 5 ) o n e M S H release ( D a v i s et al. 1984) a n d on P O M C gene e x p r e s s i o n ( H i n d e l a n g et al. 1990). In vitro d e v e l o p m e n t o f the I L a n d d i f f e r e n t i a t i o n o f the m e l a n o t r o p h s a r e o n l y d e a l t with in the u l t r a s t r u c t u r a l s t u d y o f W a t a n a b e et al. (1973). The present paper mainly concerns IL organogenesis a n d the d i f f e r e n t i a t i o n o f m e l a n o t r o p h s o f the p i t u i t a r y p r i m o r d i u m in o r g a n o t y p i c c u l t u r e in d e f i n e d m e d i u m , e x a m i n e d b y c o m b i n i n g in situ h y b r i d i z a t i o n a n d imm u n o c y t o c h e m i c a l techniques. O b s e r v a t i o n s o n the dev e l o p m e n t o f the o t h e r p i t u i t a r y lobes in the s a m e cultures a r e also r e p o r t e d .
Materials and methods
Animals Female Wistar rats were singly housed in a temperature-controlled and light-cycled (14 h light, 10 h dark) animal room. All received standard food and water ad libitum. The time of conception was assessed from vaginal smears carried out on the morning after overnight mating. The sperm-positive day was designated as embryonic day zero (E0). Pituitary primordia were explanted at E15. Under deep barbiturate anaesthesia, tile embryos were removed by caesarean section, decapitated, and the pituitary primordium and the diencephalon were exposed. Under a dissecting microscope, the pituitary primordium was carefully separated from the roof of the oral cavity and severed from the diencephalon by a section close to the infundibular recess. The isolated explants thus included Rathke's pouch, its associated mesenchyme and the extension of the putative hypothalamus corresponding roughly to the NL,
Organ culture Pituitary primordia were each placed on a cellulose nitrate membrane filter (0.45 gm, Whatman, Maidstone, UK) supported by a stainless steel grid. Incubation was carried out in a cellular plastic culture-dish (35 mm diameter, Costar, Cambridge, Mass., USA) containing 1 ml medium, consisting of RPMI 1640 supplemented with glucose (final concentration 6 g/l), albumin (2.5 g/l), ascorbic acid (0.02 g/l), insulin, progesterone, transferrin, putrescin and selenium (according to Bottenstein and Sato 1979), streptomycin (0.05 g/l), fungizone (0.125 g/l), penicillin (50.000 U/l), and gentamicin (0.05 g/l). During incubation, the gas phase consisted of 5% CO2 and 95% air. The culture medium was first changed on the day following initiation of the culture and then every 3 days.
Electron microscopy The explants were fixed in 5% gtutaraldehyde in 0.1 M phosphate buffer pH 7.4 (PB), for 1 h, and postfixed for I h with 1% OsO4
in PB. After dehydration in graded alcohols, they were conventionally embedded in an Araldite-Epon mixture. Semi-thin sections for light-microscope controls were stained with 1% toluidine blue in 5% sodium borate. Thin sections, contrasted with uranyl acetate and lead citrate, were examined on a Jeol 100 CX electron microscope. For post-embedding immunogold labelling, the explants were fixed for 2 h in 0.1 M PB containing 4% paraformaldehyde and 0.25% glutaraldehyde and embedded in Lowicryl according to Valentino et al. (1985). For the demonstration of exocytotic figures, cultures were incubated for 20 rain in the presence of 1.5% tannic acid in the culture medium prior to fixation (Buma et al./984).
In situ hybridization POMC mRNA was detected using a synthetic single-stranded oligodeoxynucleotide probe labelled with 35S deoxyadenosine triphosphate (specific activity 400~5000 Ci/mmole, NEN, Dupont de Nemours, USA), purified and tested for specificity as previously reported (Hindelang et al. 1990). Hybridization was carried out on the whole explant, and the probe was detected by autoradiography on semi-thin sections according to Guitteny et al. (1988) with slight modifications. Briefly, the explants were fixed for 30 min in a mixture containing 4% paraformaldehyde and 0.25% glutaraldehyde in PB. The explants were then rinsed 3 • 5 min in PB, acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine, 0.9% NaC1, pH 8, for 10 rain, and washed 3 x 10 rain in 2 x SSC (1 • SSC= 0.15 M NaC1, 0.015 M sodium citrate). They were incubated overnight at 35~ C in 100 gl hybridization buffer containing 25 ng/ml probe, washed for 3 x 10 min in 2 x SSC at room temperature, then for 30 min in 0.5 x SSC at 50~ C, and post-fixed for 30 rain in the paraformaldehyde/glutaraldehyde mixture. After a brief rinse in PB, the explants were dehydrated and embedded in the AralditeEpon mixture. Semi-thin sections (1.5 gm) were mounted on gelatin-coated slides. They were first exposed for 24-48 h to fiMax hyper-film (Amersham, UK) and were then dipped into K5 emulsion (Ilford, France) diluted 1/2 and exposed for 4-8 days. Film and emulsion developments were performed as previoulsy described (Hindelang et al. 1990). Alternate sections were used for immunocytochemical studies.
Immunocytochemistry For light microscopy, the conventional indirect immunoperoxidase method was applied using antisera raised in the rabbit, and peroxidase-labelled anti-rabbit Fab (1/200, Biosys, Compi6gne, France) was revealed with diaminobenzidine tetrahydrochloride as already described (Schimchowitsch et al. 1983). Semi-thin sections adjacent >
Fig. 1. l-day culture. Anterior lobe (AL). Presence of dense secretory vesicles in a cell extension at the periphery of the transplant. Basement membrane at upper left (arrow). • 18 500. Bar." 0.5 gm Fig. 2. 5-day culture. Distinct cell types, as indicated by the size of the dense secretory vesicles, are intermingled in the AL. x 6500. Bar: 2 pin Fig. 3. 9-day culture. Intermediate lobe (IL). Melanotrophic cells contain dense secretory vesicles disposed along the ceil borders. Note the tight interdigitations of the plasma membranes (arrows). x 7600. Bar: 2 pm Fig. 4a, b. 9-day culture, IL. Incubation in the presence of tannic acid prior to fixation reveals several secretory vesicles undergoing exocytosis. In b, several compound exocytotic figures occur in two adjacent cells, x 25000. Bar: 0.5 I-tm
171
172
173 to those used for autoradiography and semi-thin sections from explants similarly fixed, but not hybridized, were processed after removal of the embedding medium with sodium methoxide. The antisera used were raised against synthetic ~MSH (1/2000; the late G. Schmitt, Strasbourg, France), y3MSH (1/1000; H. Vaudry Rouen, France) and 1-39 adrenocorticotropic hormone (ACTH; 1/1000; H. Vaudry, Rouen, France), prolactin (1/300; the late M.P. Dnbois, Nouzilly, France), luteinizing hormone (LH; 1/1000; M. Poissonnier, Gif Sur Yvette, France) and GABA (1/5000; Immunotech, Marseille, France). Anti-gtial fibrillary acidic protein (GFAP; 1/100; Dako, Copenhagen, Denmark) and anti-laminin (1/1000; M. Vigny, Paris, France) antisera were applied to cultures fixed in the same fixative, and whole-mounted after clearing in xylene. [mmunostaining specificity for peptides was tested by incubating the diluted antisera for 1 h at 37~ C in the presence of 10 gg/ml of the corresponding antigen, or for GABA, GFAP and laminin, by replacing the antiserum by pre-immune serum. Although the high specificity of the anti-c~MSH and 7aMSH versus other POMCderived peptides had already been established in radio-immunoassay (see Stoeckel et al. 1985), complementary tests were performed to check possible cross-reactivity of the eMSH antibody with ACTH in the culture sections and in sections from pituitary glands of adult rats, fixed and embedded under the same conditions. The diluted antiserum was therefore incubated in the presence of 0.05, 0.1, 0.5, 1, 5 and 10 Ixg/ml synthetic c~MSH or ACTH before immunostaining. The ACTH antiserum was preincubated with the same concentrations of ACTH, c~MSH and CLIP (corticotrophin-like intermediate lobe peptide, 14-39 ACTH). For electron microscopy, c~MSH and 73MSH immunoreactivities were detected on thin sections from the Lowicryl embedded explants by using the immunogold procedure with 5- or 15-nm anti-rabbit-IgG-coated gold particles (Janssen, Beerse) and double labelling was performed using a recto-verso method, as already described (Vuillez et al. 1987). The characteristics of immunogold labelling with both antibodies have already been reported (Stoeckel et al. 1985). In the present study, the specificity tests were performed on the explant sections, by preincubating the antibodies with 1 and 10 gg/ml of the homologous peptide, and, in the case of the anti-eMSH antibody, with 1, 5, and 10 ~tg/ml ACTH.
Radioimmunoassay of ~ M S H Explants were extracted by sonication in ice-cold 1% acetic acid and 20 gg/ml aprotinin. The supernatants were separated from the cell debris by centrifugation at 10000 x g for 10 rain. The culture medium was adjusted to give final concentrations of 1% acetic acid and 20 gg/ml aprotinin. All samples were kept at - 2 0 ~ until radioimmunoassay. Both explant extracts and media were assayed for eMSH as described by Schmitt et al. (1979). The antibody used was from Dr. A.N. Eberle (Basel, Switzerland); raised
Fig. 5. 9-day culture, IL. Folliculo-stellate cells, linked by apical junctions, project microvilli into the lumen of a follicle, x 10500. Bar: 1 gm Fig. 6. 14-day culture, AL. Isolated connective-tissue space lined with a basement membrane encloses a cell exhibiting morphological features of a fibroblast, x 4700. Bar: 2 gm Fig. 7. 9-day culture. Connective tissue layer containing collagen bundles separates the intermediate (left) and the neural (right) lobes, x 8000. Bar." 1 gm Fig. 8. 14-day culture. Note contact between intermediate (right) and neural (left) lobes. Loosely arranged collagen fibrils persist between the cells (arrows). Pituicyte (P), containing lipid inclusions, intermingled with melanotrophic cells. Note the decreased electron density of the secretory vesicles in the melanotrophic cells (compare with Fig. 3). x 4700. Bar. 2 gm
against synthetic c~MSH, it recognizes the free C-terminal part of the desacetylated (ACTH 1-13), and acetylated and diacetylated eMSH on a nearly equimolecular basis; it also gives a very low cross-reaction with ACTH (molar cross-reactivity < 0.1%) (Foll6nius et al. 1985), and no detectable reaction with higher molecular forms of ACTH peptides (personal observations). The c~MSH content was separately measured for each explant. The expression of the c~MSH content per explant reflects the variations of both the cell numbers producing c~MSH and hormone content, independent of the variations in other cell-type differentiation and/or proliferation. The eMSH degradation rates in the medium were estimated by eMSH measurements in the medium at various stages of culture, immediately or after an additional 6-h incubation at 37~ C without explant.
Protein measurement The determination of the whole protein value of the explants was performed by the technique of Lowry et al. (1951) using a microassay procedure with bovine albumin as a standard.
Statistical analysis Data in figures are means_+ SEM. Statistical differences between data were analysed using Student's t-test (F test).
Chemicals and drugs These were purchased fiom Gibco-BRL (Cergy Pontoise, France) in the case of RPMI 1640, albumin, streptomycin, fungizone, penicillin, and gentamicin; from Jacques Boy laboratory (Reims, France) in the case of transferrin ; from Le Brun laboratory (Paris, France) as regards haloperidol; from Sigma (La Verpilli6re, France) as regards triethanolamine, insulin, progesterone, putrescin, aprotinin, 2-bromo-c~-ergocryptine, and selenium; from Fluka (Buchs, Switzerland) with respect to glutaraldehyde; and from Kodak (Touzart et Matignon, France) as regards paraformaldehyde.
Results
Morphofunctional aspects of Rathke's pouch differentiation The explanted primordia rapidly flattened and exhibited a r o u n d e d shape, ~ 2 m m in d i a m e t e r a n d J 00 g m thick, after 9 d a y s in culture. U p to 5 days, the persistence o f the R a t h k e ' s p o u c h l u m e n e n a b l e d the i d e n t i f i c a t i o n o f the p r i m o r d i a o f the A L , the I L a n d the t u b e r a l lobe on the t o l u i d i n e b l u e - s t a i n e d s e m i - t h i n sections. In the h o r i z o n t a l section p l a n e , the A L a p p e a r e d as a large cell m a s s f r o m w h i c h the connective spaces, w h i c h were d e v e l o p i n g in the p r i m o r d i u m a r o u n d the p e n e t r a t i n g vessels at the time o f e x p l a n t a t i o n , h a d d i s a p p e a r e d (Fig. 9). A t t w e l l ' s recess was still r e c o g n i z a b l e at 4 d a y s o f culture, b e t w e e n the A L a n d the t u b e r a l l o b e p r i m o r dia. T h e IL, s e p a r a t e d f r o m the A L b y the R a t h k e ' s p o u c h lumen, was f o r m e d b y a thick c u r v e d cell m a s s t h a t r a p i d l y lost its p r i m i t i v e p a l i s a d i c a r r a n g e m e n t . M i totic cells were r e g u l a r l y set o u t a l o n g the l u m e n o f R a t h k e ' s p o u c h , w h i c h c o n t a i n e d cell d e b r i s a n d large
174 cells with vacuolated cytoplasm. In the early stages, no marked differences in cell shape and basophilia were obvious between the IL and the AL primordia. The NL, deeply engulfed in the IL, consisted of a uniform population of cells slightly larger and less basophilic than the adenohypophysial cells (Fig. 9). In several explants, the NL was in continuity with hypothalamic fragments of various sizes, the presence of which had no obvious effects on the development of the pituitary explants. In the later stages, the partial disappearance of Rathke's pouch and Attwell's recess made histological identification of the adenohypophysial lobes more difficult.
Electron microscopy After 1 day of culture, cells containing secretory vesicles (SV) could be detected at the periphery of the explants in the putative AL (Fig. 1). The number of the SV-containing cells rapidly increased. After 5 days, several cell types, as indicated by the size of their SV, were present in the AL (Fig. 2). Small SV occurred regularly in cells containing abundant glycogen particles, and obviously belonging to the pars tuberalis. In the IL, the melanotrophic cells contained SV of 100-120 nm in diameter, set out along the cell borders. The conspicuous development of interdigitations of the plasma membranes was a striking feature of these cells (Fig. 3). At 9 days, most adenohypophysial cells devoid of SV could be classified as folliculo-stellate cells. They were linked together by well-developed junctional systems in contact with follicles of various sizes (Fig. 5). No colloid was, however, detected in either follicles or Rathke's pouch lumen, at any stage. Both glandular and follicular cells were often seen undergoing mitosis. A few typical fibroblasts were found in apparently isolated, tiny connective spaces, containing small collagen bundles, and lined with a basement membrane (Fig. 6). Basement membranes were sometimes interposed between glandular cells; they did not continuously border cell cords as in the AL in situ. No such images were observed in the IL primordia. A thin connective tissue layer most often separated the IL and NL primordia (Fig. 7). In later stages (9 and 14 days), this barrier frequently disappeared and the melanotrophic cells were in direct contact and even intermingled with the pituicytes. The latter were characterized by frequent lipid inclusions (Fig. 8). In the samples incubated in the presence of tannic acid prior to fixation, exocytotic figures were frequently observed in the IL primordium (Fig. 4) but rarely in the AL. After bromocriptine treatment (10-7 tool/l), the exocytotic figures were less frequently observed in the IL primordium.
Immunocytochemistry The specificity controls of the antibodies performed by preabsorption with the homologous antigen resulted, in all cases, in an absence of immunostaining. This was also the case when the primary antibody was replaced
by pre-immune rabbit serum. Tests concerning the antic~MSH and the anti-ACTH antibodies were performed in pituitaries of adult rats, where c~MSH is known to predominate in the IL and ACTH in the AL (Smith and Funder 1988). The anti-~MSH antibody strongly labelled the melanotrophic cells of the IL, whereas the corticotrophic cells in the AL were faintly labelled. In the IL, ~MSH labelling was partly inhibited at 0.1 pg/ml eMSH and completely abolished by 10 gg/ml eMSH; in contrast, eMSH labelling was unchanged by ACTH at concentrations up to 5 pg/ml. In the AL, the faint ~MSH labelling was abolished in the presence of 0.01 gg/ ml c~MSH, and 0.5 gg/ml of ACTH, the lowest concentration tested. The anti-ACTH antibody equally labelled the corticotrophic AL cells and the melanotrophic IL cells. This labelling decreased when antiserum treated with 0.5 pg/ml ACTH was used and was abolished in the presence of 5 gg/ml ACTH. It was decreased but not abolished when the antibody was preincubated in the presence of 10 l,tg/ml CLIP or ~MSH. Taken together, these tests indicate the specificities of the anti-c~MSH and anti-ACTH antibodies, whith only faint cross-reactivities for the related POMC-derived peptides. In the explants, eMSH, 73MSH and ACTH immunoreactivities could be detected on semi-thin sections in a few cells scattered in the AL primordium from the first day of culture. In the IL, c~MSH-immunoreactive (ir) cells occurred after 2 days in some explants and in all of them after 3 days of culture. After 4 days, eMSH-ir cells formed conspicuous areas in the IL, whereas such cells remained regularly scattered in small clusters in the AL primordium (Fig. 11 a). In these preparations, eMSH labelling was totally abolished in the presence of 5 gg/ml eMSH, still strongly inhibited at 0.5 gg, and unaltered by 0.05 gg/ml eMSH; it remained unchanged in the presence of 5 gg/ml ACTH. In all cases, inhibition of the reaction was similar in the AL and IL primordia. ACTH immunolabelling showed the same distribution as ~MSH labelling (Fig. 11 b), and adjacent sections demonstrated that the same cells reacted with the antibodies raised against the POMC-derived peptides. ACTH immunoreaction was abolished in the presence of 5 pg/ml ACTH, but persisted in the presence of the same concentration of CLIP or c~MSH. The ir cells had roughly the same shape and size in the two lobes, and showed a characteristic distribution of labelling along the cell borders (Fig. 14a, b). In the later stages (9 and 14 days), the cells of the IL progressively filled up with ir material (Fig. 14c). In the AL, most of the cells exhibited labelling of the periphery as in the early stages, whereas some others appeared more densely packed with ir material (Fig. 14d). Electron-microscopic immunogold labelling showed uniform labelling of the SV of the IL primordia cells with both anti-73MSH and anti-eMSH antibodies (Fig. 15 a). These antibodies similarly labelled stellateshaped cells scattered throughout the AL, as shown by double-labelled preparations (Fig. 15 b). Gold particles were concentrated on dense SV, ~ 120 140 nm in diameter, regularly set out under the plasma membrane, and occasionally accumulated in a cell extension. In some
175
Fig. 9. 3-day culture. Toluidine-blue staining of a semi-thin section in the horizontal plane reveals the organization of the pituitary primordium: the well-defined, though collapsed, hypophysial cleft separates the A L from the IL; a hypothalamic fragment (H) is attached to the NL, x 75. Bar: 200 gm Fig. 10. 3-day culture. Autoradiographic detection of in situ hybridized POMC mRNA on a semi-thin section shows labelling concenA L cells, the a n t i - ~ M S H a n t i b o d y less intensely labelled the SV, some o f which were completely free f r o m gold particles (Fig. 16b, b o t t o m cell). The variations in labelling intensities o f the SV in the same cell were particularly obvious in cells with dense granules at their periph-
trated in the IL primordium; labelling occurs in small patches in the A L and is absent from the NL. • 85. Bar: 200 ~tm Fig. 11 a, b. 4-day culture. Immunoperoxidase labelling for ~MSH
(a) and ACTH (b) shows similar labelling with both antibodies in the AL, whereas the IL reacts less intensely for ACTH than the AL. x 64. Bar: 200 gm
eries, resembling adult corticotrophic cells. In such cells, there was, however, no obvious relationship between labelling intensity and electron density o f the SV. In other cells, with m o r e a b u n d a n t SV that were diffusely scattered t h r o u g h the cytoplasm, almost all SV were h o m o -
176
Figs. 12, 13. Examples of 6-day and 14-day cultures. Adjacent semithin sections processed for autoradiographic detection of in situ hybridized POMC mRNA (a), immunoperoxidase labelling of c~MSH (b), and prolactin (e). a Hybridization signals are strongly increased at the 14-day stage, although in this example, AL label-
ling is decreased with both in situ hybridization and ~MSH immunocytochemistry (b). e Prolactin cells are, at first, concentrated in the central area at 6 days, but are scattered throughout the transplant at 14 days. The intense reaction on the cells in the b o t t o m area of the AL in Fig. 13b and e is non-specific, x 55. B a r . 200 gm
geneously ~ M S H - i r (Fig. 16b, top cell). The anti-73MSH a n t i b o d y u n i f o r m l y labelled all SV (Fig. 16a). Such discrepancies between ~ M S H - a n d 73MSI-I i m m u n o r e a c t i vities o c c u r r e d m o r e frequently in the later stages. Specificity tests s h o w e d inhibition o f labelling with the antic~MSH a n t i b o d y in the presence o f 1 gg/ml e M S H , al-
t h o u g h it was n o t abolished by 5 gg/ml A C T H . The specificity o f c~MSH i m m u n o g o l d labelling was further s u p p o r t e d by absence o f labelling o f the SV in the corticotrophic cells o f adult rat pituitaries. These d a t a c o m plete previous results using the same antibodies (Stoeckel et al. 1985). I m m u n o l a b e l l i n g with the anti-73MSH anti-
177
Fig. 14a-f. Detail of immunoperoxidase localization of eMSH in the IL (a, e) and the AL (b, d) at 3 days (a, b) and 9 days (e, d), and of prolactin (e) and LH (f) at 6 days of culture. At 3 days, eMSH immunoreactivity lines the cell borders and is less intense in the IL than in the AL cells (a, b). At 9 days, the labelled cells
of the IL are tightly packed and immunoreactive material tends to fill the cytoplasm (e). Note the heterogeneity of the labelling intensity in the AL (d). Localization of prolactin immunolabelling is concentrated in a juxtanuclear area (e), whereas LH labelling is distributed throughout the cytoplasm (f). x 450. Bar: 25 gm
b o d y was abolished in the presence of 10 gg/ml h o m o l o gous peptide. At 6 days, prolactin-ir (Figs. 13 c, 14 e) and L H - i r cells (Fig. 14f) were clearly detected in the AL. Prolactincontaining cells exhibited striking, intense granular labelling in the juxta-nuclear zone, contrasting with the m a r g i n a l labelling o f the A C T H - i r / M S H - i r cells. L a m i n i n i m m u n o s t a i n i n g on whole cultures (at 6 and
9 days) showed a discontinuous a r r a n g e m e n t o f the basem e n t m e m b r a n e s t h r o u g h o u t the A L and their absence f r o m the I L (Fig. 17), as previously seen in the electronm i c r o s c o p e preparations. T h e a n t i b o d y against G F A P intensely labelled pituicytes in the N L and, m o r e weakly, some cells in the A L (Fig. 18), as in the pituitary gland in situ (see Allaerts et al. 1990). In the adjacent h y p o t h a l a m i c areas, n u m e r o u s cells
Fig. 15a, b. 13-day culture. Electron-microscopic detection of c~MSH and 73MSH by immunogold double-labelling (eMSH with 5-nm particles; ?3MSH with 15-nm particles) in the IL (a) and the AL (b). In both locations, most secretory vesicles are doublelabelled, x 42300. Bar." 0.5 ~,m
Fig. 16a, b. 13-day culture, AL. Immunogold detection of y3MSH (a) shows labelling of all secretory vesicles; in b, c~MSH labelling occurs on almost all secretory vesicles in the cell at the top, whereas few secretory vesicles are labelled in the bottom cell. The dotted line indicates the position of the separating plasma membranes between these two cells, x 32200. Bar: 0.5 gm
179
Fig. 17. 9-day culture. Whole-mount laminin-immunoperoxidase labelling reveals different distribution patterns of basement membranes in the three lobes. Discontinuous stellate-shaped figures are scattered throughout the AL; labelled structures are almost absent from the IL; a delicate network is labelled in the neural lobe (NL). x 250. Bar: 50 p.m
Table 1. aMSH content of pituitary primordia and secretions at different stages of organotypic culture. Secretion was measured as the content of eMSH in the medium, divided by the culture time in days. Results are means of the contents or secretions from separate pituitary primordia. Number of cases in brackets
Fig. 18. 9-day culture. Whole-mount GFAP-immunoperoxidase shows intensely labelled cells in the NL primordium and in the adjacent hypothalamic area (top). Weakly labelled cells are scattered in the AL primordium, x 60. Bar: 200 gm
~MSH content Stages (days)
1
3
6
9
14
eMSH fmol_+ SEM
140_+20 (4)
1414• (6)
5245+_723 (6)
11269+_740 (6)
32718_+2518 (6)
Stages (days)
1-3
3-6
6-9
9 12
1~14
eMSH fmol/day _+SEM
41.5+_2.8 (26)
317.6+_22 (20)
1079+_57 (13)
1226_+125 (8)
1100+_133 (8)
~MSH secretion
were labelled w i t h the a n t i b o d y a g a i n s t G A B A . But no ir a x o n s c o u l d be d e t e c t e d in either a d e n o h y p o p h y s e a l lobes at a n y stage e x a m i n e d .
In situ hybridization W h e n cultures were initiated, a w e a k signal for P O M C m R N A - c o n t a i n i n g cells was o b s e r v e d in a few cells, in
o n l y s o m e R a t h k e ' s p o u c h explants. A f t e r I d a y o f culture, P O M C - m R N A - c o n t a i n i n g cells were s c a t t e r e d in the A L . I m m u n o c y t o c h e m i s t r y a n d in situ h y b r i d i z a t i o n on serial sections i n d i c a t e d t h a t their n u m b e r was g r e a t e r t h a n the A C T H - i r / c ~ M S H - i r cells. P O M C - p o s i t i v e cells a p p e a r e d after 2 d a y s in the I L , w h i c h was well d e l i m i t e d a n d labelled at 3 d a y s (Fig. 10). A t l a t e r stages, the intensity o f cell labelling a n d the
180 Table 2. Effects of bromocriptine on the e M S H content of pituitary primordia and secretions during the first 9 days of culture. Bromocriptine (10-7 tool/l) was added after one day in culture. Results are means of contents or secretions from separate pituitary primorStages (days)
dia. Media contained 0.001% ethanol for dissolving bromocriptine. Secretion was measured as indicated in Table 1. Number of cases in brackets
c~MSH content fmol__ SEM 3
c~MSH secretion fmol/day__ SEM
6
9
1-3
3-6
6-9
Control
1314 • (6)
62
5330 +474 (6)
10780 • 1090 (5)
4/.8 +_2.7 (25)
337 +25.9 (12)
l / 9 4 • 109 (13)
Bromocriptine 10- 7 tool/1
1720 • 242 (6)
8702 • 874 (7)
13 460 • 5110 (5)
25.6 • 1.9 (27)
116.3 • 14.6 (13)
307 • (14)
medium also increased with time (Table 1). The low levels found during the first days of culture were not the result of a higher eMSH degradation in the culture media, since evaluation of the degradation rates indicated no statistical differences with culture time (not shown). Treatment by the DA agonist bromocriptine induced a strong decrease in eMSH secretion, which was significant (P
