Cell Tiss. Res. 169, 335-344 (1976)
Cell and Tissue Research 9 by Springer-Verlag 1976
Hypothalamus and Cytodifferentiation of the Foetal Pituitary Gland Study in vivo A. Chatelain*, M. P. Dubois**, and J. P. Dupouy* * Laboratoire de PhysiologieAnimale, Facult~ des Sciences,80039 Amiens Cedex, France ** INRA, Centre de Recherchesde Tours, Nouzilly B.P. 1, 37380 Monnaie, France
Summary. To investigate whether the hypothalamus is involved in the cytodifferentiation of the anterior pituitary gland, rat foetuses were encephalectomized in utero on day 16 of pregnancy. Pituitary sections from encephalectomized and normal littermate foetuses were studied on day 21 with the immunofluorescence technique using antibodies anti ~-MSH, anti fl-MSH, anti ~-(17-39) A C T H and anti fl-(1-24) ACTH. On day 16, only the anti fi-MSH revealed a few cells in the pars distalis but not in the pars intermedia. On the other hand, on day 21, the pituitary cells reacting with antibodies anti a-MSH, anti fi-MSH and anti e - ( 1 7 - 3 9 ) A C T H were as numerous in the encephalectomized foetuses as in the normal littermate foetuses. The cells revealed with the antibody anti fl-(1-24) A C T H were less numerous and less fluorescent in the pars distalis and intermedia of the hypophysis of the encephalectomized foetuses. On day 21, the adrenals of the encephalectomized foetuses were atrophied in comparison with those of the normal littermate foetuses but they were larger than on day 16. These data suggest that the cytodifferentiation of the corticotroph and melanotroph cells of the hypophysis occurs without the influence of the hypothalamus which is necessary for the normal release of ACTH. Key words: Adenohypophysis -- Rat foetus -- Cytoimmunology: MSH, A C T H -- Hypothalamus -- Cytodifferentiation. Introduction Previously reported data have shown that the hypothalamus of the rat is involved in the control of A C T H release in late pregnancy. The ablation of the hypothalamus 9 of the foetus by total removal of the brain, leaving the hypophysis in situ (encephalectomy), induced atrophy of the adrenals (Jost, 1966; Jost et al., 1966; D u p o u y and Jost, 1970; Fujita et al., 1970; Jost et al., 1970; Dupouy, 1971; Cohen et al., Send offprint requests to: Dr. J.P. Dupouy, Laboratoire de PhysiologieAnimale, Facult~ des Sciences, 80039 Amiens Cedex, France
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1971; Dupouy and Jost, 1972; Mitskevich and Rumyantseva, 1972; Dupouy et al., 1973; Eguchi et al., 1973)and decrease of adrenocortical activity (Dupouy and Jost, 1970; Dupouy et al., 1973). The corticostimulating activity of the hypophysis of the encephalectomized foetuses could be initiated by an implant of or an extract from an adult rat hypothalamus, by an extract from the hypothalamus of a rat foetus or by purified CRF (Jost et al., 1966; Jost et al., 1970). Moreover, the hypothalamus of the rat foetus contained some CRF-like activity (Hiroshige and Sato, 1971 ; Dupouy and Jost, 1972; Dupouy, 1975). The presence of the entire hypothalamus was necessary for the response of the foetal hypophysis and adrenals to the stress produced by formalin injection (Cohen et al., 1968) or ether inhalation (Cohen and Negellen, 1974; NegellenPerchellet and Cohen, 1975). This raises some questions regarding the cytodifferentiation of the foetal pituitary gland. Is the hypothalamus implicated in the differentiation ofcorticotroph cells as it is involved in the control of ACTH release? It is worth noting that the first ACTH cells appeared in the posterior half of the hypophysis, in a region distant from the hypothalamus (Dupouy and Dubois, 1975). If this part did not receive a blood supply from the portal system, this reported observation could suggest that the hypothalamus does not influence the differentiation of the ACTH cells. Moreover, some signs of cytodifferentiation, such as secretory granules, appeared in the hypophyseal anlagen when they were taken on days 12, 14 or 15 of pregnancy and put in tissue culture for 9, 6 or 5 days (Watanabe et al., 1973). The first ACTH cells appeared on day 17 of pregnancy in the pars distalis of the hypophysis and only on day 18 in the pars intermedia as revealed by the immunofluorescence reaction with anti/~-(1-24) ACTH (Dupouy, 1974; Dupouy and Dubois, 1975). Other investigators noted corticotroph cells earlier, on day 16 (Setalo and Nakane, 1972)./%MSH cells were observed on day 16 in the pars anterior and on day 17 in the pars intermedia, whereas ~-MSH cells appeared only on day 18 in the pars intermedia (Dupouy, 1974; Dupouy and Dubois, 1975). The purpose of the present work was to investigate whether the hypothalamus was involved in the cytodifferentiation of the corticotroph and melanotroph cells in the pituitary gland. Accordingly, these cells were made visible by the immunofluorescence technique, on day 21 of pregnancy, in normal rat foetuses as well as in littermate foetuses, encephalectomized on day 16. The data reported here have been previously summarized (Begeot et al., 1975).
Material and Methods A. Foetal Age Determination: Experiments were performed on pregnant rats of the Wistar strain, bred in the laboratory. One male and several females were caged overnight from 18:00 to 08:00. The pregnant rats were screened by palpation 14 days later. Precise foetal age was expressed in days and hours after fertilization, which was assumed to occur at 01:00 (Jost and Picon, 1970). B. Foetal Surgery: Pregnant rats were laparotomized on day 16 of pregnancy under ether anesthesia in order to encephalectomize 1 to 2 foetuses in every litter. The encephalectomy was performed using the previously described technique (Jost, 1966; Jost et al., 1966).
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C. Maternal and Foetal Sacrifices: On day 21, the pregnant rats were killed rapidly by a blow on the head and bled from the carotid artery. The normal and encephalectomized foetuses were decapitated and weighed. Foetal adrenals were excised and weighed. Some foetuses from intact mothers were killed on day 16 or on day 21.
D. CytoimmunologicaI Study: 1. Specific antibodies were prepared in the rabbit using synthetic bovine /~-(1-24) ACTH, ~-(17 39) ACTH, ~-MSH and /3-MSH (Dubois, 1971, 1972). Immunofluorescence reactions were performed using the indirect method. 2. The pituitary glands of 15 normal foetuses and 16 foetuses encephalectomized on day 16 and sacrificed on day 21 were fixed for 24 h with Stieve's fixative (saturated HgC12:75 ml; neutral formaline: 20 ml; glacial acetic acid: 5 ml) and embedded in paraffin. Pituitary sections 7 p.m thick were deparaffinized, rehydrated and washed in 0.1 M veronal buffer, pH 7.3, then exposed for 30 minutes to the appropriate antibody diluted 20 to 40 times with buffer containing proteins (egg albumin, HSA or BSA 10/00). The fixation of the specific antibodies on cell receptors was made conspicuous using anti-rabbit sheep 7 globulins, conjugated with fluorescein isothiocyanate (Institut Pasteur, Paris). The treated sections were stained with 1 : 10,000 Evans Blue. The sections were mounted in a glycerol mixture (9 parts glycerol; 1 part 0.1 M veronal buffer, pH 8.6) and examined with an adapted microscope (BG 12 activating filter and K 530 stoppage barrier filter). 3. The abundance of the reactive cells and the fluorescence intensity were directly noted. In order to attain information about the abundance of the immunoreactive cells, the pituitary sections were arbitrarily classified in four categories: sections without revealed cells, - - ; sections with very few § numerous + + or very numerous + + + fluorescent cells. In order to facilitate the comparison between experimental groups, the percentage of sections in each category was determined. With respect to the intensity of the fluorescence, the sections were also classed in four categories: no fluorescence - - , weak +, high + + or intense + + + fluorescence.
Results
A. Adrenal Growth in the Foetus
The adrenals of normal foetuses from intact mothers grew rapidly between day 16 and day 21 of pregnancy (p < 0.001) (Table 1). The adrenal growth was not impaired by maternal laparotomy on day 16; the adrenals of normal foetuses from Table 1. Body and adrenal weight of normal and encephalectomized foetuses Mothers
Foetuses
Age (days)
Number of cases
Body weight headless (g)
Adrenal weight (mg)
Intact
Normal
16 21
22 35
0.37 - 0.07 3.51 +0.12
0.48 + 0.08 2.59 -+0.11
Laparotomized
Normal
21
15
3.57 + 0.29
2.38 + 0.24
Encephalectomized
21
15
2.66 -+ 0.28
1.43 --- 0.18
Mean values -+ confidence interval (p -- 0.05) The mean values of the encephalectomized foetuses were compared with those of normal foetuses (** * highly significant difference, p ( 0.001)
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laparotomized mothers were, on day 21, as heavy as those of the foetuses from intact mothers (no significant difference, p > 0.05) (Table 1). On the contrary, the adrenals of the encephalectomized foetuses were atrophied in comparison with those of normal littermates (p < 0.001) but larger (p < 0.001) than those of normal foetuses, on day 16 (Table 1). The adrenals grew slightly between the time ofencephalectomy and the time of sacrifice.
B. Cytoimmunological Study The antiserum against e-(17-39)ACTH revealed corticotroph cells in the pars distalis from day 17 and in the pars intermedia from day 18 of pregnancy (Table 2). When the encephalectomy was performed, on day 16, the antisera against e-MSH, c~-(17-39) ACTH and fl-(1-24) ACTH did not reveal immunoreactive cells in the pars distalis or in the pars intermedia (Table 2). However, already by day 16 a very small number of melanotrophs was shown with the fl-MSH antibody in the pars distalis (Table 2). As previously reported, e-MSH cells were never revealed by the antibody anti c~-MSH in the pars distalis of the foetal adenohypophysis, from day 15 to day 21 (Table 2). Yet, in the present observations a very small number of melanotrophs was shown on day 21 of pregnancy with this c~-MSH antibody in the pars distalis of the encephalectomized foetuses and of normal littermate foetuses (Fig. 1) but not in the pars distalis of the mothers. However, the fluorescence of these melanotrophs was weak in the pars distalis and intense in the pars intermedia (Fig. 2).
T a b l e 2. O n t o g e n e s i s o f the m e l a n o t r o p h a n d c o r t i c o t r o p h cells in the h y p o p h y s i s o f the foetal r a t Immunoreactive cells r e v e a l e d by a n t i s e r u m against:
F o e t a l age (days)
15
16
17
18
19
20
21
+ + +
+ + +
Pars distalis e-MSH" fl-MSH" e-(17-39)ACTH fl-(1-24) A C T H a
----
. + ---
.
. + + §
. + + +
. + + +
.
Pars intermedia c~-MSH"
--
fl-MSH" c~-(17-39) A C T H fl-(1-24) A C T H "
----
a -
-
----
--
+
+
+
+
+ ---
+ + +
+ + +
+ + +
+ + +
a c c o r d i n g to D u p o u y a n d D u b o i s (1975) n o r e a c t i v e cells r e v e a l e d § r e a c t i v e cells r e v e a l e d
339
Hypothalamusand Cytodifferentiationof the FoetalPituitaryGland ~,~/istar
1 6 . 2 1 d.
./. Pars distalis
y. Pars i n t e r m e d i a
100
100.
*~MSH 5 0 ~
5 4.
100.
I00.
50
0
§
"IF
§247
100.
100.
100
100.
.,~17-39ACTH
i9 1-24ACTH
§
§247
"H-§
0
~
-
§
4.§
+4.4.
-
+
§247
§247
-
§
§
§247
Fig. 1. Abundanceof the cells. [] Normal foetuses. 9 Encephalectomizedfoetuses
The encephalectomy of the foetuses on day 16 did not prevent the cytodifferentiation of the corticotrophs and melanotrophs in the pituitary gland (Figs. 3-7). On day 21, the pituitary cells reacting with the antibodies anti c~-MSH (Fig. 3), anti fl-MSH (Figs. 4, 5) and anti ct-(17-39) ACTH (Fig. 6) were as numerous in the encephalectomized foetuses as in the normal littermates (Fig. 1). Yet, the cells reacting with the antibody anti ~-(1-24) ACTH were fewer in the two parts of the pituitary of the encephalectomized foetuses (Fig. 7) than in those of normal foetuses (Fig. 1). A weakening of the fluorescence intensity of the corticotrophs revealed by the antiserum anti fl-(1-24) ACTH was observed in the encephalectomized foetuses in comparison with the controls (Fig. 2). No appreciable difference was noted between encephalectomized and normal foetuses when the other antibodies were used (Fig. 2).
Discussion
The cytodifferentiation of the rat adenohypophysis occurs without hypothalamic influence, as the pituitary gland of 21 day-old encephalectomized foetuses showed
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W i s tar"
Pars distalis
16 - 21 d
Pars intermedia
100
100
*~ M S H
4"
lOO
1oo
50
5
4.
4.4-
§247
100.
-
4-
§
+4.4"
-
4-
4-4-
4-4-§
4-
4-§
4.
4.#.
100
17-39 ACTH
§
+4-
lOO.
4-4-+
1oo
1-24 ACTH
4-
+4-
Fig. 2. Fluorescence intensity. [] Normal foetuses. 9 Encephalectomized foetuses
Fig. 3. Hypophysis of an 21 day-old encephalectomized foetus. The melanotroph cells revealed with an anti ~-MSH antibody are numerous in the pars intermedia and very scarce in the pars distalis. • 120
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Fig. 4. Hypophysis of a 21 day-old encephalectomized foetus. Melanotroph cells revealed with an anti fl-MSH antibody, x 120
Fig. 5. Hypophysis of a 21 day-old normal foetus. Melanotroph cells revealed with an anti fl-MSH antibody, x 120
Fig. 6. Hypophysis of a 21 day-old encephalectomized foetus. Corticotroph cells revealed with an anti ~-(17-39)ACTH antibody, x 120
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Fig. 7. Hypophysis of a 21 day-old encephalectomized foetus. Corticotroph cells revealed with an anti fl-(1 24)ACTH antibody. • 120
not only ACTH and MSH cells but also LH and TSH cells (Dupouy et al., unpublished data). The differentiation of corticotrophs, gonadotrophs, thyrotrophs and somatotrophs was reported in similarly performed experiments (Watanabe and Daikoku, 1975; and personal communication). Furthermore, these cells were demonstrated in vitro after Rathke's pouch was isolated on day 12 and cultivated for 9 days (Watanabe and Daikoku, 1975; and personal communication). Then the secretory granules observed in the cultivated hypophyseal anlagen (Watanabe et al., 1973) could be correlated with pituitary hormones. In the absence of hypothalamic tissue, the pituitary gland of the encephalectomized foetuses showed some corticostimulating activity; indeed, the adrenals of these foetuses were heavier on day 21 than those of normal foetuses on day 16. Nevertheless, the corticostimulating activity of the hypophysis was weak in comparison with that of normal littermates. Similar results were reported when foetuses were encephalectomized on day 17 of pregnancy and killed on day 19 (Cohen et al., 1971). In the pituitary gland of the anencephalic human foetus or newborn, corticomelanotroph cells could be revealed by using specific antibodies, but these cells were smaller and less numerous than those seen in the hypophysis of the normal newborn (Begeot et al., 1975; Dubois et al., 1975). These results could be related with the adrenal atrophy observed in the anencephalic foetuses from the 20th week, while the adrenal weight of normal foetuses increased from 1 to 7 g during the second part of pregnancy (Jost et al., 1970). These observations suggest that the human foetal hypothalamus, like that of the rat foetus, is not necessary for the cytodifferentiation of the anterior pituitary gland. In contrast, in these two species, the corticostimulating activity of the hypophysis was reduced in the absence of the hypothalamus. In other vertebrate species such as the chick embryo, the cytodifferentiation and the functional differentiation of the pituitary takes place in the absence of any hypothalamic influence (Le Douarin et al., 1967; Le Douarin and Ferrand, 1968; Ferrand, 1970, 1971, 1972a, b). Moreover, ACTH cells developed only when
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R a t h k e ' s p o u c h a n d its s u r r o u n d i n g m e s e n c h y m e i s o l a t e d f r o m t h e h y p o t h a l a m u s w e r e g r o w n t o g e t h e r in c h o r i o a l l a n t o i c m e m b r a n e f o r 12 d a y s ( F e r r a n d et al., 1974). T h e s e e x p e r i m e n t s s u g g e s t t h a t t h e c e p h a l i c m e s e n c h y m e c o u l d p l a y a d i r e c t r o l e in t h e d e v e l o p m e n t o f t h e p i t u i t a r y p r i m o r d i u m . W h e t h e r this p r e m i s e o f a n i n f l u e n c e o f m e s e n c h y m e h o l d s t r u e f o r t h e rat o r h u m a n h y p o p h y s i s r e m a i n s to b e e l u c i d a t e d .
Acknowledgements: The authors would like to express their gratitude to Mrs. N. Chatelain for the correction of the English text, and to Mr. J.B. Zazac for excellent technical assistance.
References Begeot, M., Chatelain, A., Dubois, M.P., Dubois, P.M., Dupouy, J.P.: Influence de l'hypothalamus sur les cellules corticotropes et m61anotropes de l'hypophyse foetale. Etude chez le Rat et l'homme. J. Physiol. (Paris) 70, 25B (1975) Cohen, A., Dupouy, J.P., Jost, A.: Influence de l'hypothalamus sur l'activit6 corticostimulante de l'hypophyse foetale du Rat au cours de la gestation. C.R. Acad. Sci. (Paris) 273, 883 886 (1971) Cohen, A., Negellen, E.: Changes in adrenal glands during stress in fetal rats. Gen. comp. Endocr. 22, 400-401 (1974) Cohen, A., Pernot, J.C., Jost, A.: R61e de l'hypothalamus dans la r6ponse des surr6nales du foetus de Rat/t une agression par le formol. C.R. Soc. Biol. (Paris) 162, 2070-2073 (1968) Dubois, M.P.: Les cellules corticotropes de l'hypophyse des bovins, ovins et porcins. Ann. Biol. anim. 11, 589-624 (1971) Dubois, M.P.: Localisation cytologique par immunofluorescence des s~cr6tions corticotropes, ct et fl m61anotropes au niveau de l'ad6nohypophyse des bovins, ovins et porcins. Z. Zellforsch. 125, 200-209 (1972) Dubois, P.M., Berthenod, M., Gilly, R., Dubois, M.P.: Etude des activit6s cortico-m61anotropes dans l'antbhypophyse d'un anenc6phale et d'un nouveau-n6 normal. Arch. fran~. P6diat. 32, 647-654 (1975) Dupouy, J.P.: R6ponse du complexe hypothalamo-hypophysaire du foetus de Rat/t un blocage de la biosynth+se de corticost6roides par la m6topirone. Influence du cortisol. C.R. Acad. Sci. (Paris) 273, 962-965 (1971) Dupouy, J.P.: La fonction corticostimulante de l'hypophyse foetale du rat; ontogen6se des cellules corticotropes et 6volution de l'activit6 corticosurr6nalienne. J. Physiol. (Paris) 69, 194A (1974) Dupouy, J.P.: C.R.F activity in fetal rat hypothalamus in late pregnancy. Neuroendocrinology 19, 303-313 (1975) Dupouy, J.P., Dubois, M.P.: Ontogenesis of the ct-MSH, fl-MSH and ACTH ceils in the foetal hypophysis of the rat. Correlation with the growth of the adrenals and adrenocortical activity. Cell Tiss. Res. 161, 373-384 (1975) Dupouy, J.P., Coffigny, H., Jost, A.: Action de l'hypophyse et de l'hypothalamus sur la teneur en corticost6rone et l'ultrastructure de la surr6nale foetale du rat. 5e colloque de Neuroendocrinologie. Marseille, Sept. 1973 Dupouy, J.P., Jost, A.: Activit6 corticotrope de l'hypophyse foetale du rat: influence de l'hypothalamus et des corticost6roides. C.R. Soc. Biol. (Paris) 164, 2422-2427 (1970) Dupouy, J.P., Jost, A.: Hypothalamus et fonction corticostimulante de l'hypophyse foetale du Rat. Lille m6d. XVII, 10, 1395 (1972) Eguchi, Y., Hirai, O., Morikawa, Y., Hashimoto, Y.: Critical time in the hypothalamic control of the pituitary-adrenal system in fetal rats: observations in fetuses subjected to hypervitaminosis A and hypothalamic destruction. Endocrinology 93, 1 11 (1973) Ferrand, R.: Diff6renciation de tissu ad6nohypophysaire ~t partir de la poche de Rathke greff6e chez des embryons de Poulet priv6s d'hypothalamus. C.R. Acad. Sci. (Paris) 170, 1480-1481, (1970) Ferrand, R. : Activit6 thyr6otrope du tissu ad6no-hypophysaire diffbrenci6 en greffe chorio-allantoidienne chez l'embryon de Poulet. C. R. Soc. Biol. (Paris) 165, 1976 1978 (1971) Ferrand, R.: Etude exp6rimentale des facteurs de la diff6renciation histologique et physiologique de l'ad6nohypophyse chez l'embryon d'oiseau. Th6se Sciences Naturelles. Nantes (1972 a)
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Ferrand, R.: Etude exp6rimentale des facteurs de la diff6renciation cytologique de l'ad6nohypophyse chez l'embryon de Poulet. Arch. Biol. (Li6ge) 83, 297-371 (1972b) Ferrand, R., Pearse, A.G.E., Polak, J.M., Le Douarin, N.M.: Immunohistochemical studies on the development of avian embryo pituitary corticotrophs under normal and experimental conditions. Histochemistry 38, 133-141 (1974) Fujita, T., Eguchi, Y., Morikawa, Y., Hashimoto, Y.: Hypothalamic hypophysial adrenal and thyroid systems: observations in fetal rats subjected to hypothalamic destruction, brain compression and hypervitaminosis A. Anat. Rec. 166, 659-671 (1970) Hiroshige, T., Sato, T.: Changes in hypothalamic content of corticotropin-releasing activity following stress during neonatal maturation in the rat. Neuroendocrinology 7, 257-270 (1971) Jost, A.: Anterior pituitary function in foetal life. In: The pituitary gland (G.W. Harris and B.T. Donovan, eds.), Vol. 2, pp. 299-323 (1966) Jost, A., Dupouy, J.P., Geloso-Meyer, A. : Hypothalamo-hypophyseal relationships in the fetus. In: "The hypothalamus", ed. by Martini, L., Motta, M., Fraschini, F., pp. 605-615 (1970) 3ost, A., Dupouy, J.P., Monchamp, A.: Fonction corticotrope de l'hypophyse et hypothalamus chez le foetus de rat. C.R. Acad. Sci. (Paris)262, 147-150 (1966) Jost, A., Picon, L.: Hormonal control of fetal development and metabolism. In: Advances in metabolic disorders 4, 123-184 (1970) Le Douarin, G., Ferrand, R.: Diff6renciation fonctionnelle de l'6bauche 6pith61iale de l'ad6nohypophyse isol6e du plancher enc6phalique: activit6 thyr6otrope. C.R. Acad. Sci. (Paris) 266, 697-699 (1968) Le Douarin, N., Ferrand, R., Le Douarin, G.: La diff6renciation de l'6bauche 6pith61iale de l'hypophyse s6par6e du plancher enc6phalique et plac6e dans des m6senchymes h6t6rologues. C.R. Acad. Sci. (Paris) 64, 3027-3029 (1967) Mitskevich, M.S., Rumyantseva, O.N.: A possible role of the hypothalamus in the control of adrenocortical and thyroid functions during fetal life. Ontogenesis 3, 376-384 (1972) Negellen-Perchellet, E., Cohen, A.: Effect of ether inhalation by adrenalectomized pregnant rats on the adrenal corticost6rone concentration in normal, decapitated and encephalectomized fetuses. Neuroendocrinology 17, 225-235 (1975) Setalo, G., Nakane, P.K.: Studies on the functional differentiation of cells in fetal anterior pituitary glands of rats with peroxidase-labeled antibody method. Anat. Rec. 172, 403-404 (1972) Watanabe, Y.G., Daikoku, S. : Immunocytological study on the cytodifferentiation of rat anterior pituitary in vivo and in vitro. 10th Int. Cong. Anat. Tokyo, p. 275 (1975) Watanabe, Y.G., Matsumura, H., Daikoku, S.: Electron microscopic study of rat pituitary primordium in organ culture. Z. Zellforsch. 146, 453-461 (1973)
Received December 19, 1975 / in final form March 8, 1976
Cell and Tissue Research 9 by Springer-Verlag 1976
Hypothalamus and Cytodifferentiation of the Foetal Pituitary Gland Study in vivo A. Chatelain*, M. P. Dubois**, and J. P. Dupouy* * Laboratoire de PhysiologieAnimale, Facult~ des Sciences,80039 Amiens Cedex, France ** INRA, Centre de Recherchesde Tours, Nouzilly B.P. 1, 37380 Monnaie, France
Summary. To investigate whether the hypothalamus is involved in the cytodifferentiation of the anterior pituitary gland, rat foetuses were encephalectomized in utero on day 16 of pregnancy. Pituitary sections from encephalectomized and normal littermate foetuses were studied on day 21 with the immunofluorescence technique using antibodies anti ~-MSH, anti fl-MSH, anti ~-(17-39) A C T H and anti fl-(1-24) ACTH. On day 16, only the anti fi-MSH revealed a few cells in the pars distalis but not in the pars intermedia. On the other hand, on day 21, the pituitary cells reacting with antibodies anti a-MSH, anti fi-MSH and anti e - ( 1 7 - 3 9 ) A C T H were as numerous in the encephalectomized foetuses as in the normal littermate foetuses. The cells revealed with the antibody anti fl-(1-24) A C T H were less numerous and less fluorescent in the pars distalis and intermedia of the hypophysis of the encephalectomized foetuses. On day 21, the adrenals of the encephalectomized foetuses were atrophied in comparison with those of the normal littermate foetuses but they were larger than on day 16. These data suggest that the cytodifferentiation of the corticotroph and melanotroph cells of the hypophysis occurs without the influence of the hypothalamus which is necessary for the normal release of ACTH. Key words: Adenohypophysis -- Rat foetus -- Cytoimmunology: MSH, A C T H -- Hypothalamus -- Cytodifferentiation. Introduction Previously reported data have shown that the hypothalamus of the rat is involved in the control of A C T H release in late pregnancy. The ablation of the hypothalamus 9 of the foetus by total removal of the brain, leaving the hypophysis in situ (encephalectomy), induced atrophy of the adrenals (Jost, 1966; Jost et al., 1966; D u p o u y and Jost, 1970; Fujita et al., 1970; Jost et al., 1970; Dupouy, 1971; Cohen et al., Send offprint requests to: Dr. J.P. Dupouy, Laboratoire de PhysiologieAnimale, Facult~ des Sciences, 80039 Amiens Cedex, France
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1971; Dupouy and Jost, 1972; Mitskevich and Rumyantseva, 1972; Dupouy et al., 1973; Eguchi et al., 1973)and decrease of adrenocortical activity (Dupouy and Jost, 1970; Dupouy et al., 1973). The corticostimulating activity of the hypophysis of the encephalectomized foetuses could be initiated by an implant of or an extract from an adult rat hypothalamus, by an extract from the hypothalamus of a rat foetus or by purified CRF (Jost et al., 1966; Jost et al., 1970). Moreover, the hypothalamus of the rat foetus contained some CRF-like activity (Hiroshige and Sato, 1971 ; Dupouy and Jost, 1972; Dupouy, 1975). The presence of the entire hypothalamus was necessary for the response of the foetal hypophysis and adrenals to the stress produced by formalin injection (Cohen et al., 1968) or ether inhalation (Cohen and Negellen, 1974; NegellenPerchellet and Cohen, 1975). This raises some questions regarding the cytodifferentiation of the foetal pituitary gland. Is the hypothalamus implicated in the differentiation ofcorticotroph cells as it is involved in the control of ACTH release? It is worth noting that the first ACTH cells appeared in the posterior half of the hypophysis, in a region distant from the hypothalamus (Dupouy and Dubois, 1975). If this part did not receive a blood supply from the portal system, this reported observation could suggest that the hypothalamus does not influence the differentiation of the ACTH cells. Moreover, some signs of cytodifferentiation, such as secretory granules, appeared in the hypophyseal anlagen when they were taken on days 12, 14 or 15 of pregnancy and put in tissue culture for 9, 6 or 5 days (Watanabe et al., 1973). The first ACTH cells appeared on day 17 of pregnancy in the pars distalis of the hypophysis and only on day 18 in the pars intermedia as revealed by the immunofluorescence reaction with anti/~-(1-24) ACTH (Dupouy, 1974; Dupouy and Dubois, 1975). Other investigators noted corticotroph cells earlier, on day 16 (Setalo and Nakane, 1972)./%MSH cells were observed on day 16 in the pars anterior and on day 17 in the pars intermedia, whereas ~-MSH cells appeared only on day 18 in the pars intermedia (Dupouy, 1974; Dupouy and Dubois, 1975). The purpose of the present work was to investigate whether the hypothalamus was involved in the cytodifferentiation of the corticotroph and melanotroph cells in the pituitary gland. Accordingly, these cells were made visible by the immunofluorescence technique, on day 21 of pregnancy, in normal rat foetuses as well as in littermate foetuses, encephalectomized on day 16. The data reported here have been previously summarized (Begeot et al., 1975).
Material and Methods A. Foetal Age Determination: Experiments were performed on pregnant rats of the Wistar strain, bred in the laboratory. One male and several females were caged overnight from 18:00 to 08:00. The pregnant rats were screened by palpation 14 days later. Precise foetal age was expressed in days and hours after fertilization, which was assumed to occur at 01:00 (Jost and Picon, 1970). B. Foetal Surgery: Pregnant rats were laparotomized on day 16 of pregnancy under ether anesthesia in order to encephalectomize 1 to 2 foetuses in every litter. The encephalectomy was performed using the previously described technique (Jost, 1966; Jost et al., 1966).
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C. Maternal and Foetal Sacrifices: On day 21, the pregnant rats were killed rapidly by a blow on the head and bled from the carotid artery. The normal and encephalectomized foetuses were decapitated and weighed. Foetal adrenals were excised and weighed. Some foetuses from intact mothers were killed on day 16 or on day 21.
D. CytoimmunologicaI Study: 1. Specific antibodies were prepared in the rabbit using synthetic bovine /~-(1-24) ACTH, ~-(17 39) ACTH, ~-MSH and /3-MSH (Dubois, 1971, 1972). Immunofluorescence reactions were performed using the indirect method. 2. The pituitary glands of 15 normal foetuses and 16 foetuses encephalectomized on day 16 and sacrificed on day 21 were fixed for 24 h with Stieve's fixative (saturated HgC12:75 ml; neutral formaline: 20 ml; glacial acetic acid: 5 ml) and embedded in paraffin. Pituitary sections 7 p.m thick were deparaffinized, rehydrated and washed in 0.1 M veronal buffer, pH 7.3, then exposed for 30 minutes to the appropriate antibody diluted 20 to 40 times with buffer containing proteins (egg albumin, HSA or BSA 10/00). The fixation of the specific antibodies on cell receptors was made conspicuous using anti-rabbit sheep 7 globulins, conjugated with fluorescein isothiocyanate (Institut Pasteur, Paris). The treated sections were stained with 1 : 10,000 Evans Blue. The sections were mounted in a glycerol mixture (9 parts glycerol; 1 part 0.1 M veronal buffer, pH 8.6) and examined with an adapted microscope (BG 12 activating filter and K 530 stoppage barrier filter). 3. The abundance of the reactive cells and the fluorescence intensity were directly noted. In order to attain information about the abundance of the immunoreactive cells, the pituitary sections were arbitrarily classified in four categories: sections without revealed cells, - - ; sections with very few § numerous + + or very numerous + + + fluorescent cells. In order to facilitate the comparison between experimental groups, the percentage of sections in each category was determined. With respect to the intensity of the fluorescence, the sections were also classed in four categories: no fluorescence - - , weak +, high + + or intense + + + fluorescence.
Results
A. Adrenal Growth in the Foetus
The adrenals of normal foetuses from intact mothers grew rapidly between day 16 and day 21 of pregnancy (p < 0.001) (Table 1). The adrenal growth was not impaired by maternal laparotomy on day 16; the adrenals of normal foetuses from Table 1. Body and adrenal weight of normal and encephalectomized foetuses Mothers
Foetuses
Age (days)
Number of cases
Body weight headless (g)
Adrenal weight (mg)
Intact
Normal
16 21
22 35
0.37 - 0.07 3.51 +0.12
0.48 + 0.08 2.59 -+0.11
Laparotomized
Normal
21
15
3.57 + 0.29
2.38 + 0.24
Encephalectomized
21
15
2.66 -+ 0.28
1.43 --- 0.18
Mean values -+ confidence interval (p -- 0.05) The mean values of the encephalectomized foetuses were compared with those of normal foetuses (** * highly significant difference, p ( 0.001)
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laparotomized mothers were, on day 21, as heavy as those of the foetuses from intact mothers (no significant difference, p > 0.05) (Table 1). On the contrary, the adrenals of the encephalectomized foetuses were atrophied in comparison with those of normal littermates (p < 0.001) but larger (p < 0.001) than those of normal foetuses, on day 16 (Table 1). The adrenals grew slightly between the time ofencephalectomy and the time of sacrifice.
B. Cytoimmunological Study The antiserum against e-(17-39)ACTH revealed corticotroph cells in the pars distalis from day 17 and in the pars intermedia from day 18 of pregnancy (Table 2). When the encephalectomy was performed, on day 16, the antisera against e-MSH, c~-(17-39) ACTH and fl-(1-24) ACTH did not reveal immunoreactive cells in the pars distalis or in the pars intermedia (Table 2). However, already by day 16 a very small number of melanotrophs was shown with the fl-MSH antibody in the pars distalis (Table 2). As previously reported, e-MSH cells were never revealed by the antibody anti c~-MSH in the pars distalis of the foetal adenohypophysis, from day 15 to day 21 (Table 2). Yet, in the present observations a very small number of melanotrophs was shown on day 21 of pregnancy with this c~-MSH antibody in the pars distalis of the encephalectomized foetuses and of normal littermate foetuses (Fig. 1) but not in the pars distalis of the mothers. However, the fluorescence of these melanotrophs was weak in the pars distalis and intense in the pars intermedia (Fig. 2).
T a b l e 2. O n t o g e n e s i s o f the m e l a n o t r o p h a n d c o r t i c o t r o p h cells in the h y p o p h y s i s o f the foetal r a t Immunoreactive cells r e v e a l e d by a n t i s e r u m against:
F o e t a l age (days)
15
16
17
18
19
20
21
+ + +
+ + +
Pars distalis e-MSH" fl-MSH" e-(17-39)ACTH fl-(1-24) A C T H a
----
. + ---
.
. + + §
. + + +
. + + +
.
Pars intermedia c~-MSH"
--
fl-MSH" c~-(17-39) A C T H fl-(1-24) A C T H "
----
a -
-
----
--
+
+
+
+
+ ---
+ + +
+ + +
+ + +
+ + +
a c c o r d i n g to D u p o u y a n d D u b o i s (1975) n o r e a c t i v e cells r e v e a l e d § r e a c t i v e cells r e v e a l e d
339
Hypothalamusand Cytodifferentiationof the FoetalPituitaryGland ~,~/istar
1 6 . 2 1 d.
./. Pars distalis
y. Pars i n t e r m e d i a
100
100.
*~MSH 5 0 ~
5 4.
100.
I00.
50
0
§
"IF
§247
100.
100.
100
100.
.,~17-39ACTH
i9 1-24ACTH
§
§247
"H-§
0
~
-
§
4.§
+4.4.
-
+
§247
§247
-
§
§
§247
Fig. 1. Abundanceof the cells. [] Normal foetuses. 9 Encephalectomizedfoetuses
The encephalectomy of the foetuses on day 16 did not prevent the cytodifferentiation of the corticotrophs and melanotrophs in the pituitary gland (Figs. 3-7). On day 21, the pituitary cells reacting with the antibodies anti c~-MSH (Fig. 3), anti fl-MSH (Figs. 4, 5) and anti ct-(17-39) ACTH (Fig. 6) were as numerous in the encephalectomized foetuses as in the normal littermates (Fig. 1). Yet, the cells reacting with the antibody anti ~-(1-24) ACTH were fewer in the two parts of the pituitary of the encephalectomized foetuses (Fig. 7) than in those of normal foetuses (Fig. 1). A weakening of the fluorescence intensity of the corticotrophs revealed by the antiserum anti fl-(1-24) ACTH was observed in the encephalectomized foetuses in comparison with the controls (Fig. 2). No appreciable difference was noted between encephalectomized and normal foetuses when the other antibodies were used (Fig. 2).
Discussion
The cytodifferentiation of the rat adenohypophysis occurs without hypothalamic influence, as the pituitary gland of 21 day-old encephalectomized foetuses showed
340
A. Chatelain et al.
W i s tar"
Pars distalis
16 - 21 d
Pars intermedia
100
100
*~ M S H
4"
lOO
1oo
50
5
4.
4.4-
§247
100.
-
4-
§
+4.4"
-
4-
4-4-
4-4-§
4-
4-§
4.
4.#.
100
17-39 ACTH
§
+4-
lOO.
4-4-+
1oo
1-24 ACTH
4-
+4-
Fig. 2. Fluorescence intensity. [] Normal foetuses. 9 Encephalectomized foetuses
Fig. 3. Hypophysis of an 21 day-old encephalectomized foetus. The melanotroph cells revealed with an anti ~-MSH antibody are numerous in the pars intermedia and very scarce in the pars distalis. • 120
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Fig. 4. Hypophysis of a 21 day-old encephalectomized foetus. Melanotroph cells revealed with an anti fl-MSH antibody, x 120
Fig. 5. Hypophysis of a 21 day-old normal foetus. Melanotroph cells revealed with an anti fl-MSH antibody, x 120
Fig. 6. Hypophysis of a 21 day-old encephalectomized foetus. Corticotroph cells revealed with an anti ~-(17-39)ACTH antibody, x 120
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A. Chatelain et al.
Fig. 7. Hypophysis of a 21 day-old encephalectomized foetus. Corticotroph cells revealed with an anti fl-(1 24)ACTH antibody. • 120
not only ACTH and MSH cells but also LH and TSH cells (Dupouy et al., unpublished data). The differentiation of corticotrophs, gonadotrophs, thyrotrophs and somatotrophs was reported in similarly performed experiments (Watanabe and Daikoku, 1975; and personal communication). Furthermore, these cells were demonstrated in vitro after Rathke's pouch was isolated on day 12 and cultivated for 9 days (Watanabe and Daikoku, 1975; and personal communication). Then the secretory granules observed in the cultivated hypophyseal anlagen (Watanabe et al., 1973) could be correlated with pituitary hormones. In the absence of hypothalamic tissue, the pituitary gland of the encephalectomized foetuses showed some corticostimulating activity; indeed, the adrenals of these foetuses were heavier on day 21 than those of normal foetuses on day 16. Nevertheless, the corticostimulating activity of the hypophysis was weak in comparison with that of normal littermates. Similar results were reported when foetuses were encephalectomized on day 17 of pregnancy and killed on day 19 (Cohen et al., 1971). In the pituitary gland of the anencephalic human foetus or newborn, corticomelanotroph cells could be revealed by using specific antibodies, but these cells were smaller and less numerous than those seen in the hypophysis of the normal newborn (Begeot et al., 1975; Dubois et al., 1975). These results could be related with the adrenal atrophy observed in the anencephalic foetuses from the 20th week, while the adrenal weight of normal foetuses increased from 1 to 7 g during the second part of pregnancy (Jost et al., 1970). These observations suggest that the human foetal hypothalamus, like that of the rat foetus, is not necessary for the cytodifferentiation of the anterior pituitary gland. In contrast, in these two species, the corticostimulating activity of the hypophysis was reduced in the absence of the hypothalamus. In other vertebrate species such as the chick embryo, the cytodifferentiation and the functional differentiation of the pituitary takes place in the absence of any hypothalamic influence (Le Douarin et al., 1967; Le Douarin and Ferrand, 1968; Ferrand, 1970, 1971, 1972a, b). Moreover, ACTH cells developed only when
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R a t h k e ' s p o u c h a n d its s u r r o u n d i n g m e s e n c h y m e i s o l a t e d f r o m t h e h y p o t h a l a m u s w e r e g r o w n t o g e t h e r in c h o r i o a l l a n t o i c m e m b r a n e f o r 12 d a y s ( F e r r a n d et al., 1974). T h e s e e x p e r i m e n t s s u g g e s t t h a t t h e c e p h a l i c m e s e n c h y m e c o u l d p l a y a d i r e c t r o l e in t h e d e v e l o p m e n t o f t h e p i t u i t a r y p r i m o r d i u m . W h e t h e r this p r e m i s e o f a n i n f l u e n c e o f m e s e n c h y m e h o l d s t r u e f o r t h e rat o r h u m a n h y p o p h y s i s r e m a i n s to b e e l u c i d a t e d .
Acknowledgements: The authors would like to express their gratitude to Mrs. N. Chatelain for the correction of the English text, and to Mr. J.B. Zazac for excellent technical assistance.
References Begeot, M., Chatelain, A., Dubois, M.P., Dubois, P.M., Dupouy, J.P.: Influence de l'hypothalamus sur les cellules corticotropes et m61anotropes de l'hypophyse foetale. Etude chez le Rat et l'homme. J. Physiol. (Paris) 70, 25B (1975) Cohen, A., Dupouy, J.P., Jost, A.: Influence de l'hypothalamus sur l'activit6 corticostimulante de l'hypophyse foetale du Rat au cours de la gestation. C.R. Acad. Sci. (Paris) 273, 883 886 (1971) Cohen, A., Negellen, E.: Changes in adrenal glands during stress in fetal rats. Gen. comp. Endocr. 22, 400-401 (1974) Cohen, A., Pernot, J.C., Jost, A.: R61e de l'hypothalamus dans la r6ponse des surr6nales du foetus de Rat/t une agression par le formol. C.R. Soc. Biol. (Paris) 162, 2070-2073 (1968) Dubois, M.P.: Les cellules corticotropes de l'hypophyse des bovins, ovins et porcins. Ann. Biol. anim. 11, 589-624 (1971) Dubois, M.P.: Localisation cytologique par immunofluorescence des s~cr6tions corticotropes, ct et fl m61anotropes au niveau de l'ad6nohypophyse des bovins, ovins et porcins. Z. Zellforsch. 125, 200-209 (1972) Dubois, P.M., Berthenod, M., Gilly, R., Dubois, M.P.: Etude des activit6s cortico-m61anotropes dans l'antbhypophyse d'un anenc6phale et d'un nouveau-n6 normal. Arch. fran~. P6diat. 32, 647-654 (1975) Dupouy, J.P.: R6ponse du complexe hypothalamo-hypophysaire du foetus de Rat/t un blocage de la biosynth+se de corticost6roides par la m6topirone. Influence du cortisol. C.R. Acad. Sci. (Paris) 273, 962-965 (1971) Dupouy, J.P.: La fonction corticostimulante de l'hypophyse foetale du rat; ontogen6se des cellules corticotropes et 6volution de l'activit6 corticosurr6nalienne. J. Physiol. (Paris) 69, 194A (1974) Dupouy, J.P.: C.R.F activity in fetal rat hypothalamus in late pregnancy. Neuroendocrinology 19, 303-313 (1975) Dupouy, J.P., Dubois, M.P.: Ontogenesis of the ct-MSH, fl-MSH and ACTH ceils in the foetal hypophysis of the rat. Correlation with the growth of the adrenals and adrenocortical activity. Cell Tiss. Res. 161, 373-384 (1975) Dupouy, J.P., Coffigny, H., Jost, A.: Action de l'hypophyse et de l'hypothalamus sur la teneur en corticost6rone et l'ultrastructure de la surr6nale foetale du rat. 5e colloque de Neuroendocrinologie. Marseille, Sept. 1973 Dupouy, J.P., Jost, A.: Activit6 corticotrope de l'hypophyse foetale du rat: influence de l'hypothalamus et des corticost6roides. C.R. Soc. Biol. (Paris) 164, 2422-2427 (1970) Dupouy, J.P., Jost, A.: Hypothalamus et fonction corticostimulante de l'hypophyse foetale du Rat. Lille m6d. XVII, 10, 1395 (1972) Eguchi, Y., Hirai, O., Morikawa, Y., Hashimoto, Y.: Critical time in the hypothalamic control of the pituitary-adrenal system in fetal rats: observations in fetuses subjected to hypervitaminosis A and hypothalamic destruction. Endocrinology 93, 1 11 (1973) Ferrand, R.: Diff6renciation de tissu ad6nohypophysaire ~t partir de la poche de Rathke greff6e chez des embryons de Poulet priv6s d'hypothalamus. C.R. Acad. Sci. (Paris) 170, 1480-1481, (1970) Ferrand, R. : Activit6 thyr6otrope du tissu ad6no-hypophysaire diffbrenci6 en greffe chorio-allantoidienne chez l'embryon de Poulet. C. R. Soc. Biol. (Paris) 165, 1976 1978 (1971) Ferrand, R.: Etude exp6rimentale des facteurs de la diff6renciation histologique et physiologique de l'ad6nohypophyse chez l'embryon d'oiseau. Th6se Sciences Naturelles. Nantes (1972 a)
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Ferrand, R.: Etude exp6rimentale des facteurs de la diff6renciation cytologique de l'ad6nohypophyse chez l'embryon de Poulet. Arch. Biol. (Li6ge) 83, 297-371 (1972b) Ferrand, R., Pearse, A.G.E., Polak, J.M., Le Douarin, N.M.: Immunohistochemical studies on the development of avian embryo pituitary corticotrophs under normal and experimental conditions. Histochemistry 38, 133-141 (1974) Fujita, T., Eguchi, Y., Morikawa, Y., Hashimoto, Y.: Hypothalamic hypophysial adrenal and thyroid systems: observations in fetal rats subjected to hypothalamic destruction, brain compression and hypervitaminosis A. Anat. Rec. 166, 659-671 (1970) Hiroshige, T., Sato, T.: Changes in hypothalamic content of corticotropin-releasing activity following stress during neonatal maturation in the rat. Neuroendocrinology 7, 257-270 (1971) Jost, A.: Anterior pituitary function in foetal life. In: The pituitary gland (G.W. Harris and B.T. Donovan, eds.), Vol. 2, pp. 299-323 (1966) Jost, A., Dupouy, J.P., Geloso-Meyer, A. : Hypothalamo-hypophyseal relationships in the fetus. In: "The hypothalamus", ed. by Martini, L., Motta, M., Fraschini, F., pp. 605-615 (1970) 3ost, A., Dupouy, J.P., Monchamp, A.: Fonction corticotrope de l'hypophyse et hypothalamus chez le foetus de rat. C.R. Acad. Sci. (Paris)262, 147-150 (1966) Jost, A., Picon, L.: Hormonal control of fetal development and metabolism. In: Advances in metabolic disorders 4, 123-184 (1970) Le Douarin, G., Ferrand, R.: Diff6renciation fonctionnelle de l'6bauche 6pith61iale de l'ad6nohypophyse isol6e du plancher enc6phalique: activit6 thyr6otrope. C.R. Acad. Sci. (Paris) 266, 697-699 (1968) Le Douarin, N., Ferrand, R., Le Douarin, G.: La diff6renciation de l'6bauche 6pith61iale de l'hypophyse s6par6e du plancher enc6phalique et plac6e dans des m6senchymes h6t6rologues. C.R. Acad. Sci. (Paris) 64, 3027-3029 (1967) Mitskevich, M.S., Rumyantseva, O.N.: A possible role of the hypothalamus in the control of adrenocortical and thyroid functions during fetal life. Ontogenesis 3, 376-384 (1972) Negellen-Perchellet, E., Cohen, A.: Effect of ether inhalation by adrenalectomized pregnant rats on the adrenal corticost6rone concentration in normal, decapitated and encephalectomized fetuses. Neuroendocrinology 17, 225-235 (1975) Setalo, G., Nakane, P.K.: Studies on the functional differentiation of cells in fetal anterior pituitary glands of rats with peroxidase-labeled antibody method. Anat. Rec. 172, 403-404 (1972) Watanabe, Y.G., Daikoku, S. : Immunocytological study on the cytodifferentiation of rat anterior pituitary in vivo and in vitro. 10th Int. Cong. Anat. Tokyo, p. 275 (1975) Watanabe, Y.G., Matsumura, H., Daikoku, S.: Electron microscopic study of rat pituitary primordium in organ culture. Z. Zellforsch. 146, 453-461 (1973)
Received December 19, 1975 / in final form March 8, 1976
