Immunocytochemistry of gonadotropic cells in the pituitary of Pomadasys jubelini (teleost fish)

GENERAL

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ENDOCRINOLOGY

79, 439-445 (1990)

lmmunocytochemistry of Gonadotropic Cells in the Pituitary of Pomadasys jubelini (Teleost Fish) A. FANTODJI,*

E. FOLLENIUS,?

AND A. S.N’DIAYE*

*Dkpartemen? de Biologie et de Physiologie animale, Facultk des Sciences et techniques C6te d’lvoire, West Africa, and fLaboratoire de Cytologie Animale, UniversitC Louis VUniversitC, 67000 Strasbourg, France

22 BP.582, Abidjan, Pasteur, 12 Rue de

Accepted October 30, 1989 An antibody specific for the p subunit of carp gonadotropin was applied to pituitary sections from Pomadasys jubelini in order to characterize and localize the gonadotropic cells by immunocytological means. It reveals the gonadotropic zone lying mainly at the periphery of the proximal pars distalis with lateral extensions around the pars intermedia. The separation between the pars proximalis and the pars intermedia is irregular. Short finger-like extensions of the gonadotropic zone protrude more or less into the pars intermedia. The immunocytological results corroborate previous tentative identifications of the gonadotropic cells by classical cytological methods and suggest that the gonadotropic hormone produced is immunologically related with several previously studied fish gonadotropins. 6 19!B Academic Press, Inc.

In recent years, immunochemical techniques have greatly enhanced our potential for investigating the reproductive physiology of fishes. Earlier immunocytological and immunological studies (for review see Follenius et al., 1978) had shown that the antibodies against mammalian hormones also cross-reacted with the corresponding hormones of nonmammalian vertebrates and might therefore be useful for detecting homologous substances in fish endocrine glands, especially in the pituitary. The gonadotropin-producing cells in the teleost pituitary have been localized mostly in hatchery-reared species (Cyprinus carpio: Billard et al., 1971; Salmo furio: Olivereau and Nagahama, 1983; Oreochromis sp.: Bogomolnaya-Bass and Yaron, 1987, 1988) or in aquarium-reared ones (Mollienisiu lutipinnu: Goos et al., 1976; Xiphophorus muculutus: Schreibman and MargolisKazan, 1979; Curussius uurutus: Dubourg et al., 1985). Relatively few marine (Dicentrurchus lubrux: Cambre et al., 1986; Spurus uurutu: Mancera et al., 1989) or am-

phibiotic

species (Sulmo sulur: Ekengren et al., 1978; Anguilla unguillu: Olivereau and

Nagahama, 1983) have been studied so far with immunocytological techniques. The present study was a first attempt to localize and characterize the gonadotropic cells of Pomudusys jubelini. The distribution area of this amphibiotic species extends along the west African coast from Mauritania to Angola (Fischer et al., 1981). In C&e d’Ivoire, P. jubelini first lives for 2 to 3 years in the lagoon where the first spawning takes place before the migration to the sea. The adults living in the sea migrate to the estuaries for spawning (Fantodji, 1987). The knowledge of its reproductive physiology may help improve the management of this species, which has an interesting local economic potential. MATERIALS

AND METHODS

Male and female P. jubelini at different stages of sexual maturity caught in the sea and the Abidjan Lagoon were used in this study.

439 0016~6480/9O $1.50 Copyright All rights

0 1990 by Academic Press. Inc. of reproduction in any form reserved.

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Sag&al and parasagittal pituitary gland sections (5 pm), fixed in sublimated Bouin-Hollande solution and embedded in para&, were put on slides in groups of six to eight. They were exposed to a specific immunocytochemical reaction as described by Nakane (1970). The primary antigen-antibody reaction was revealed with a peroxidase-labeled second antibody. The antigonadotropin antibody had been prepared from rabbit by Burzawa-Gerard and Kerdelhue (1978) from puritied carp pituitary gonadotropic hormone (BurzawaGerard, 1971). Only an antibody against the 8 subunit of the gonadotropic hormone of carp (ISBcGTH) was used. After deparaftining and bleaching, the sections were rinsed three times for 5 min in Mayer’s buffer diluted l/5 in distilled water. After drying, they were covered with a specific antibody at a final dilution of l/2000 in Mayer’s buffer and incubated for 24 to 36 hr at 4” in a humid chamber. After three washes in Mayer’s buffer and short drying, they were incubated again at room temperature for 1 h in a peroxidase-labeled sheep anti-rabbit immunoglobulin (Inst. Past. 75011). After three rinses with Mayer’s buffer the peroxidase was revealed with the method of Graham and Karnovsky (1966). The sections were transferred to a diaminobenzidin solution (3-3’diaminobenzidine tetrahydrochloride, Sigma) in Tris buffer (0.5 M, pH 7.6) in the presence of hydrogen peroxide. The reaction, in the dark, took about 10 min. The sections were again washed three times in Mayer’s buffer before being colored either with Evan’s blue (l/10,000) or with an eosine solution (l/100) in distilled water. They were dehydrated with alcohol and toluene and mounted with Canada balsam to obtain permanent preparations. The specificity of the antibody was checked by incubating reference sections with either the labeled second antibody alone or using a first antibody which had been previously neutralized with 10 mg of antigen/ml of antibody. No immunocytochemical reaction was observed in either case.

RESULTS Incubating pituitary gland sections with the anti-p subunit of carp gonadotropin re-

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veals strongly immunoreactive cells in the proximal pars distalis (PPD). On sagittal sections they form a band in the medioventral part of the gland (Figs. 1 and 2). The more laterally situated sections reveal that, in this species, the gonadotropic cells of the pituitary form a thin sheet covering the proximal pars distalis and extending caudally around part of the pars intermedia as finger-like processes which, on some sections, are seen as immunoreactive islets. No immunoreactive cells were observed in the pars intermedia proper (Fig. 4) nor in the rostra1 pars distalis (RPD). In the peripheral proximal pars distalis, a great majority of cells display a strong immunoreaction (Figs. 3-5). Their whole cytoplasm displays a strong immunoreaction which is indicative of a heavy load of secretory products. With the techniques applied, no further details could be defined. Only a few islets or bands of cells are not immunoreactive. They probably correspond to the thyrotropic cells (Fig. 5) or to gonadotropic cells devoid of secretory products. Staining some of the sections close to those exposed to immunocytochemistry with Schiff’s periodic acid and with Herlant’s tetrachrome shows that the immunoreactive cells are PAS +, They also stain with Alcian and Alizarin blue. Comparing the tetrachrome-stained preparations and the immunocytological ones shows that the immunoreaction belongs to the large Alcian blue cells, which, as shown earlier (Fantodji and N’Diaye, 1983), constitute the ventral pars proximalis of the pituitary.

FIGS. 1 AND 2. Topographical arrangement of the GTH cells, as demonstrated by immunocytology. They form a band in the medioventral proximal pars distalis. Some GTH cell islets are present at the periphery of the gland, around the pars intermedia (PI). The rostral pars distalis (RPD), the pars nervosa (PN), and the pars intermedia contain no gonadotropic cells. x42. FIG. 3. The gonadotropin in the gonadotropic cells shows immunoreaction with the antibody against the B subunit of carp gonadotropin. x340. FIG. 4. The antibody shows selectively the gonadotropin content in the gonadotropic cells at the periphery of the pituitary. The nearby melanotropic cells of the pars intermedia are devoid of immunoreactive material. X 340. FIG. 5. Presumed thyrotropic cells, with no immunoreaction, form some bands or islets in the gonadotropic zone (heavy arrow). x425.

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The high specificity of the anti-c l3 subunit gonadotropin immunoreaction (see Discussion) clearly identifies these cells as undeniable producers of gonadotropic hormone . DISCUSSION In our previous histological and cytological studies (Fantodji and N’Diaye, 1983; Fantodji, 1987), seven different cell types were tentatively identified in the pituitary gland of P. jubelini. The techniques used to localize these cell types were not specific enough to demonstrate the hormones secreted by each. Therefore, more productspecific immunocytochemical methods were called for. The general topographical distribution of the gonadotropic cells of P. jubelini is similar to that described recently in two related perciform species: Dicentrarchus labrax (Cambre et al., 1986) and Sparus aurata (Mancera et al., 1989). In all these species, the gonadotropic zone is not strictly confined to the periphery of the PPD but also covers the pars intermedia superficially. In some previous studies, the antibodies available to reveal and measure pituitary hormones of fish were not always specific enough. Schreibman and Margolis-Kazan (1979) pointed out that both glycoprotidic hormones, the gonado- and thyrotropins of Xiphophorus maculatus, are immunoreactive with antibodies against whole trout, carp, or salmon gonadotropins. Olivereau and Nagahama (1983), using an antibody to whole salmon gonadotropin, obtained similar results on the pituitaries of Sarotherodon mosambicus, Carassius auratus, Anguilla anguilla, Salmo trutta fario, and Sarpa salpa. The same lack of specificity

was also observed with antibodies against the cx subunit of carp gonadotropin (Schreibman and Margolis-Kazan, 1979). The thyrotrops of several teleost species have been specifically identified immunocytologically with an antibody against the h

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l3 TSH subunit (Ueda et al., 1983; Van Putten et al., 1983). For this reason, a more specific antibody was applied to selectively characterize the gonadotropic hormone without interference with the thyrotropic hormone. As shown earlier (Fontaine and Burzawa-Gerard, 1977; Schreibman and Margolis-Kazan, 1979), only the anti-l3 subunit antibody specifically reveals the gonadotropic hormone. The selectivity and the specificity of the antibody we applied, as established in these studies, allows us to conclude that only the gonadotropic hormone was revealed. There are three types of cells in the PPD of P. jubelini pituitary gland (Fantodji and N’Diaye, 1983). From the precise localization of the somatotropic cells, with histochemical techniques, and from that of the gonadotropic cells by immunocytochemistry, we can deduce where the thyrotropic cells may be. Several radioimmunological studies (Burzawa-Gerard et al., 1980; BurzawaGerard, 1982) have established the immunological relatedness between the p subunits of cGTH and of bovine luteinizing hormone (LH). Cross-reactions between antibodies against mammalian gonadotropins and the corresponding fish antigens have also provided a tool to visualize the presence of gonadotropins in specific cell types of the fish pituitary. An ovine LH antibody cross-reacts with the pituitary gonadotropin of salmon (McKeown and Van Overbeeke, 1971). Tests with an antimammalian follicle-stimulating hormone (FSH) antibody did not reveal FSH-like hormones in the same species, but anti-h FSH revealed the gonadotrops of Xiphophorus (Margolis-Kazan and Schreibman, 1981). It is known that the p subunit of fish gonadotropin shows a greater similitude with the p subunit of LH than with the l3 subunit of mammalian FSH (Jolles et al., 1977). The production of FSH-like gonadotropin in the fish pituitary is still controver-

GONADOTROPIC

sial (for review see Fontaine and Dufour, 1987 and Itoh et al., 1988). The fish gonadotropins probably differ from those of mammals as shown earlier by Burzawa-Gerard and Fontaine (1972) and Fontaine and Burzawa-Gerard (1978), and they may even differ from one group of teleosts to the other (Bye et al., 1980). An antibody against GTH from the Atlantic croaker does not cross-react with carp or salmon GTH but does react with pituitary extracts from striped bass (Morone saxatilis) and to a lesser extent with extracts from several other species (Copeland and Thomas, 1987, 1989). The reasons for this heterogeneity of fish gonadotropins are still being investigated (Ando and Ishi, 1988). For the moment, only limited information on the amino acid sequence of fish gonadotropin subunits is available. Few sequence data are available: a partial sequence of carp GTHa and p (Jolles et al., 1977) and a complete sequence of GTH l3 from Onchorhynchus tschuwytschu (Trinh et al., 1986) and 0. ketu (Itoh er al., 1988) are known. The p subunit of the chinook salmon has already been cloned (Hew, 1989). Recent successful attempts to purify gonadotropins of Chunnu punctutus and Cutlu cutlu have shown that only a single gonadotropin having two dissimilar subunits (CYMW:16-18,000; p MW: 2627,000) exists in these species (Banerjee et al., 1989). Further studies are needed to characterize more closely the gonadotropins of Pomudusys jubelini and to establish whether the immunoreaction registered with an anti-c$GTH reveals the GTH-2 isoform as it does in the carp (Van der Kraak et al., 1987). All this information about the biochemical and immunological characteristics of the fish gonadotropins strongly supports our immunocytochemical identification of gonadotropins in the corresponding pituitary cells. There are at least some common immunological determinants in the go-

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nadotropins of several species investigated with the antibody used in this study (Dicentrurchus lubrux: CambrC et al., 1986; Spurus auruta: Mancera et al., 1989; Carassius uurutus: Dubourg et al., 1983, but biochemical information is needed to formally establish this. ACKNOWLEDGMENT We are indebted to Dr. Burzawa-Gerard erous gift of the anti-cpGTH.

for the gen-

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