Cloning and sequencing of the gilthead sea bream estrogen receptor cDNA.

We report here the complete nucleotide sequence of a cDNA clone containing the full-coding sequence of the Sparus aurata estrogen receptor (ER) isolated from an expression library prepared from gilthead sea bream liver poly A+ RNA. The library was screened using a single strand rainbow trout ER cDNA probe, corresponding to the C-D domain. The cDNA sequence containing an insert of 2369 nucleotides was found to encode a protein of 579 amino acids. The 5'- and 3'-untranslated regions of the message are 186 and 392 nucleotides long, respectively. The gilthead sea bream ER shows the higher homology with the ER of another perciform, Chrysophrys major (93%), moderate to high homology with Oreocromis aureus (78%) medaka (77%) and rainbow trout (70.7%) ERs and lower homology with japanese eel (45%), amphibian (47%), avian (48.5%) and mammalian (47-47.5%) ERs. The sequence homologies and phylogenetic analysis of the various ERs suggest that gilthead sea bream ER should be considered as a ER alpha-like.

Insulin-like growth factor-I stimulates gonadotrophin production from eel pituitary cells: a possible metabolic signal for induction of puberty.

Insulin-like growth factor (IGF)-I has been suggested as a potential signal linking growth and puberty in mammals. Using the juvenile European eel as a model, we employed a long-term, serum-free primary culture of pituitary cells to study the direct effect of IGF-I on gonadotrophin (GtH-II=LH) production. IGF-I increased both cell content and release of GtH-II in a time- and dose-dependent manner. IGF-I and IGF-II had similar potencies but insulin was 100-fold less effective, suggesting the implication of an IGF type 1 receptor. Other growth and metabolic factors, such as basic fibroblast growth factor and thyroid hormones, had no effect on GtH-II production. IGF-I did not significantly increase the number of GtH-II immunoreactive cells, indicating that its stimulatory effect on GtH-II production does not result from gonadotroph proliferation. Comparison of IGF-I and somatostatin (SRIH-14) effects showed that both factors inhibited growth hormone (GH) release but only IGF-I stimulated GtH-II production by eel pituitary cells. This indicates that the effect of IGF-I on gonadotrophs is not mediated by the reduction of GH released by somatotrophs into the culture medium. This study demonstrates a specific stimulatory effect of IGF-I on eel GtH-II production, played out directly at the pituitary level. These data obtained in a primitive teleost suggest that the role of IGF-I as a link between body growth and puberty may have been established early in the evolution of vertebrates.

Metabolic studies on eel (Anguilla anguilla L.) hepatocytes in primary culture: effect of 17 beta-estradiol and growth hormone.

Previous studies demonstrated that native and recombinant growth hormone from mammalian and fish species potentiate the estrogenic induction of vitellogenin synthesis by cultured eel hepatocytes. In the present study, the metabolic competence (respiratory activity and estradiol catabolism) of cultured hepatocytes and their functional capacity to synthesize a specific protein, vitellogenin, in the presence of estradiol and/or bovine growth hormone was investigated. In addition, we examined the possible role of insulin-like growth factors as mediators of growth hormone. Hepatocytes retain a high level of metabolic activity under the primary culture conditions applied. Estradiol has a half life of several hours in the hepatocyte culture, and is metabolized into conjugated forms. Estradiol and/or growth hormone had no effects on respiratory activity of the cultured hepatocytes. Moreover, the estradiol catabolic parameters were not affected by growth hormone. Finally, human and trout recombinant insulin-like growth factors do not potentiate vitellogenin synthesis induced by estradiol.

Potentiating effect of growth hormone on vitellogenin synthesis induced by 17 beta-estradiol in primary culture of female silver eel (Anguilla anguilla L.) hepatocytes.

Previous in vivo experiments have indicated a potentiating role of growth hormone (GH) during experimentally induced vitellogenesis by 17-beta-estradiol (E2) in the female silver eel (Anguilla anguilla L.). To investigate whether GH has direct hepatic actions, the effects of hypophysial-purified and recombinant QH on vitellogenin (Vg) synthesis in response to E2 were tested on primary cultures of hepatocytes. Hepatocytes were prepared from control or E2-primed eels. Addition of E2 alone into the culture medium induced both Vg synthesis and secretion in a dose- and time-related fashion. Bovine growth hormone (bGH) alone had no effect on the induction of Vg synthesis or secretion. Bovine GH enhanced the in vitro effects of F2 on both Vg synthesis and secretion, an effect attenuated by an in vivo E2 priming which was dose-dependent with an ED(50) of 5 ng/ml. To investigate the specificity of GH action, purified eel and salmon GH and salmon, trout, and tilapia prolactins (PRL), as well as recombinant trout and tilapia GH, were tested, and the responses were compared to bGH. Purified salmon and homologous eel GH potentiated the vitellogenic response to F2. Recombinant GH were highly efficacious, excluding the presence of active contaminants in the potentiating effect of GH preparations. The potentiating effect of recombinant trout GH on the vitellogenic response was reduced at high doses (above 20 ng/ml), suggesting a down-regulation of GH binding sites by GH itself. Salmon PRL has minimal activity, but not trout and tilapia PRL, indicating that PRL is not an important potentiating factor on Vg synthesis in our model. It is concluded that GH acts directly on the liver to potentiate E2 induction of eel hepatic Vg synthesis. The potentiating effect of GH appears to be time- and dose-dependent and modulated as a function of hormonal status (E2 priming) of the eel.

Synthesis of vitellogenin by eel (Anguilla anguilla L.) hepatocytes in primary culture: requirement of 17 beta-estradiol-priming.

Control and 17 beta-estradiol-primed eels were used to investigate the hormonal requirement of vitellogenesis in an immature fish, the eel. A primary culture of isolated liver cells from female silver eels was developed. The hepatocytes were maintained as monolayers on poly-L-lysine coated dishes for up to 12 days in a defined medium alone or supplemented with 17 beta-estradiol (E2, from 10(-8) to 10(-5) M). The amounts of vitellogenin (Vg) in the cells and secreted into the medium were measured at 2-day intervals using a homologous vitellogenin ELISA. Different E2-priming conditions were determined before hepatocyte isolation (one injection of 250 micrograms of E2 21 days, 17 days, or 24 hr). The vitellogenic response of hepatocytes to E2 stimulation was studied in relation to the duration of the E2-priming. After 8 days of culture, when hepatocytes from control eels were used, Vg was undetectable both in cells and in culture media, even if the culture was performed in the presence of E2 10(-5) M. However, Vg was detectable both in cells and in culture media of hepatocytes from E2-primed eels. If the priming was performed 24 hr before the culture, the Vg synthesis significantly increased (P < 0.001) in the presence of E2 10(-5) M after 10 days of culture but remained low. When the culture was performed 17 or 21 days after the priming, the level of the vitellogenic response was higher than after a short priming. In particular, with hepatocytes from 21-day E2-primed eel, the concentration of secreted Vg was 1.5 times higher than in control dishes (P < 0.01), in the presence of E2 10(-8) M after 12 days of culture. Higher doses of E2 (10(-5) M) increased Vg 2.7-fold over control values (P < 0.01) after 4 days of culture. In control dishes, cultured without steroid, the amounts of secreted and intracellular Vg remained unchanged over 12 days of culture (respectively, 72.8 +/- 2.7 ng/10(6) cells/48 hr and 28.7 +/- 2.7 ng/10(6) cells). These results show that cultured hepatocytes retain their functional capacity by synthesizing a specific protein, Vg, in the presence of E2 and there are dose- and time-related effects of E2 on in vitro Vg synthesis. The induction of hepatic vitellogenesis in vitro requires a preliminary in vivo E2-priming.

[Implication of growth hormone in experimental induction of vitellogenesis by estradiol-17 beta in female silver eel (Anguilla anguilla L.)]. / Implication de l'hormone de croissance au cours de l'induction expérimentale de la vitellogenèse par l'oestradiol-17 beta chez l'anguille argentée femelle (Anguilla anguilla L.).

The effect of different preparations of growth hormone (GH) was assayed with 17 beta-estradiol on vitellogenesis in hypophysectomized or normal female silver eels. Vitellogenin (Vg) plasma levels were taken as the index of hepatic vitellogenesis. The E2 doses were chosen to give the same pattern for the plasma Vg level as in the controls. They decreased or remained undetectable in hypophysectomized or normal animals. GH also failed to induce alone a significant modification. When E2 was injected together with a GH, plasma Vg levels were 5.13 +/- 1.30 times higher with salmon GH in hypophysectomized eels and 2.01 +/- 0.25 times higher with bovine GH in normal eels. GH is shown to enhance the effects of E2 on hepatic vitellogenesis induction in a teleost.

Effects of 17 beta-estradiol and carp gonadotropin on vitellogenesis in normal and hypophysectomized European silver female eel (Anguilla anguilla L.) employing a homologous radioimmunoassay for vitellogenin.

The European silver eels are at the early stages of vitellogenesis before the marine reproductive migration. Vitellogenesis was induced by 17 beta-estradiol (E2) alone and by a purified carp gonadotropin (cGTH). We studied and compared their effects on plasma vitellogenin (Vg) levels and ovarian yolk contents in female normal (N) and hypophysectomized (H) eels for both treatments. To this purpose an homologous radioimmunoassay (RIA) was established. Eel Vg was purified to homogeneity on 0.1% SDS-Electrophoresis. Native Vg has a molecular weight of 340 +/- 15 kilodalton (kDa) and was partially separated into subunits. The RIA was established with a sensitivity of 1.1 ng and was specific for eel Vg. In control (N and H) silver eels, plasma Vg levels were 0.04 +/- 0.02 microgram/ml and unchanged throughout the experiment. Similarly, yolk was indetectable in control ovarian extracts. E2 treatment increased plasma Vg levels proportionally with time to 783.4 +/- 130.7 micrograms/ml in N eels. The same profile was seen in H eels but terminally the mean value was 36.7 times lower than in N eels (P less than 0.01). Yolk at 0.005 microgram/g in N eels was indetectable in H eels. cGTH treatment gave a biphasic kinetic change: plasma Vg increased within 12 days, peaked at 93.6 +/- 13.0 micrograms/ml at 20 to 24 days, and stabilized to decrease at 40.2 +/- 7.5 micrograms/ml. The gonadosomatic index (GSI) increased alongside the yolk content (980.4 +/- 153.1 micrograms Vg/g). The kinetic profile for H eels was different: a peak was not apparent, rather there was a delayed increase, and at 67 days levels were still 8.23 times lower than in N eels (P less than 0.01). The GSI increased as the yolk content to 202.7 +/- 64.8 micrograms Vg/g ovary showing an ovarian incorporation of Vg in H eels.

Purification of gonadotropic hormone from the pituitary of the African catfish, Clarias gariepinus (Burchell), and the development of a homologous radioimmunoassay.

Glycoprotein gonadotropic hormone (GTH) was purified from 2000 pituitaries of male and female African catfish, Clarias gariepinus. The first step was chromatography on concanavalin A-Sepharose followed by filtration on Ultrogel Aca 54, chromatography on DEAE-cellulose, and filtration on Ultrogel Aca 54, respectively. Finally, the purified fractions were analyzed by polyacrylamide gel electrophoresis. The gonadotropic activity in the different fractions was characterized using two tests: the radioimmunoassay for carp gonadotropin-beta subunit was used to quantify the immunoreactive GTH and a cAMP accumulation test was applied to measure the GTH biological activity. The purified glycoprotein GTH was used to raise antibodies and to develop a radioimmunoassay. This resulted in an assay with a variation between assays of approximately 4%, a precision of 4-8%, and an accuracy of 4-8%. GTH levels can be measured over a range of 0.8 to 12.5 ng/ml.

Light and electron microscopic identification of gonadotrophic cells in the pituitary gland of the goldfish by means of immunocytochemistry.

Immunocytochemical techniques were used at the light and electron microscopical levels in order to localize and to characterize the gonadotrophs in the goldfish pituitary gland by means of antibodies to carp gonadotrophin (c-GTH) or its subunit (c-GTH beta). At the light microscopical level antibodies to c-GTH reacted weakly with cells located in the rostral pars distalis (RPD) and strongly with cells of the proximal pars distalis (PPD). The labeling was restricted to the proximal pars distalis when antibodies to c-GTH beta were employed. The PAP and colloidal-gold postembedding procedures demonstrated that two cell types of the PPD react with both immune sera. These cells correspond to the so-called globular and nonglobular basophils of the goldfish pituitary. The labeling was located over the small secretory granules and the large globules. A relationship was noted between the intensity of the labeling and the electron density of the globules.

Effect of carp gonadotropin (cGTH) and a fraction unabsorbed on concanavalin A-Sepharose obtained from cGTH on vitellogenesis in the hypophysectomized marine teleost Gobius niger.

To investigate whether a differential potency on vitellogenesis exists between the carp gonadotropin (cGTH) and fraction I-cGTH (proteins from cGTH unbound on concanavalin A-Sepharose, which represent 5% of cGTH in weight), hypophysectomized Gobius niger were treated with the two hormonal preparations at the same level. Vitellogenesis was checked for synthesis of vitellogenin and yolk incorporation in the ovary by means of immunological studies and histological techniques (light and electron microscopy). In addition, increased synthesis of vitellogenin was induced by injection of estradiol 17 beta together with each gonadotropin to assess the action of the two hormonal preparations on vitellogenin incorporation. Oogenesis was enhanced by cGTH and fraction I-cGTH, and at the same dose levels both treatments produced a similar pattern of stimulation of vitellogenesis. Vitellogenin was found in all the blood samples of animals treated by the hormones (cGTH and fraction I-cGTH) alone. Vesicles of pinocytosis were detected by electron microscopy up to Stage IIIa of oogenesis. When a high synthesis of vitellogenin was induced by exogeneous estradiol 17 beta injections, the two gonadotropic preparations had similar effects in yolk incorporation. cGTH was not less potent than fraction I-cGTH in these processes even though the cGTH preparation contains only 5% of fraction I-cGTH. The contamination of cGTH by a small amount of material unbound on concanavalin cannot be solely responsible for the vitellogenic activity of cGTH which consists of 95% glycoproteins.

Isolation of gonadotrops from the pituitary of the African catfish, Clarias lazera. Morphological and physiological characterization of the purified cells.

Dispersed pituitary cells from male African catfish, Clarias lazera, were fractionated in a density gradient of Percoll. Five fractions were isolated, consisting of about 6, 19, 39, 95 and 83% gonadotrops, respectively. The gonadotrops were identified by their ultrastructural characteristics, by immunocytochemistry, and by measuring their hormone content. After one day in culture, in each fraction the secretion of gonadotropin could be stimulated by a luteinizing hormone-releasing hormone analogue, indicating that the cells had retained their functional integrity. Since the regulatory mechanisms of different cell types from the pituitary have some similarity, purification of the gonadotrops provides a model to study the regulation of gonadotropin secretion.

Pituitary gonadotropic hormone from a chondrostean fish, starred sturgeon (Acipenser stellatus Pall.) IV. Differences in biological action of isoforms and absence of sex-specific forms.

The specific activities of four subfractions of the starred sturgeon (Acipenser stellatus Pall.) pituitary gonadotropin (aci-GTH-A, -B, -C, -D) were compared using several test systems. The ratios of two of them (A and D) that contained the isoforms differing by the values of isoelectric point were shown to be different, especially when in vitro oocyte maturation and spermiation tests were used. These differences were not due to the presence in one of these preparations of the component affecting spermiation rather than oocyte maturation. The qualitative differences in the biological action of aci-GTH-A and -D were also revealed by the comparison of dose-response curves using toad oocyte in vitro maturation. Finally, as the spectra of aci-GTH isoforms were practically identical when female or male individual pituitary extracts were submitted to isoelectric focusing, we concluded that the structural and functional heterogeneity of aci-GTH was related neither to sex nor to genetic intrapopulation polymorphism.

Pituitary gonadotropic hormone from a chondrostean fish, starred sturgeon (Acipenser stellatus Pall.) III. Polymorphism.

Four biologically active fractions of gonadotropic hormone (aci-GTH-A, -B, -C, -D) were isolated and purified from acetonized pituitaries of the starred sturgeon (Acipenser stellatus Pall.). Their separation was achieved by DEAE-cellulose chromatography. Disc-electrophoresis and especially isoelectric focusing in polyacrylamide gel showed that each fraction contained several components. Not less than 15 different components as a whole with isoelectric points ranging from 4.5 to 7.0 could be counted in four aci-GTH preparations. All these components were active in toad oocyte maturation test. Only two of four preparations (aci-GTH-A and -D) were practically free of common components. All aci-GTH preparations were shown to be homogeneous and identical by molecular weight, sedimentation coefficient, sialic acid content, and some immunological properties. N-terminal amino acid analysis revealed tyrosine and leucine in all aci-GTH preparations, with the only exception of aci-GTH-D that contained an additional polypeptide with N-terminal glycine. No differences in the spectra of aci-GTH isoforms were found when pituitary extract, newly purified or 3 years older hormone preparations were submitted to isoelectric focusing.

Zoological origin of gonadotropin subunits and association kinetics.

Mammalian luteinizing hormone (LH) is an association of two dissimilar subunits, alpha and beta. In vitro studies, mainly using difference spectrophotometry, had shown that this phenomenon was slow, especially at low temperatures. If the situation was the same in poikiloterms, it would probably make gonadotropin (GTH) synthesis difficult for these animals in a cold environment. We have found that the formation of a teleost gonadotropin is in fact strikingly more rapid and less thermodependent than formation of mammalian LH. Also, studies with a fish-mammal hybrid molecule have allowed us to estimate the respective influence of the alpha and beta subunits in determining these differences.

[Speed of in vitro reassociation of gonadotropin subunits in a teleost fish, the carp (Cyprinus carpio L.)]. / Rapidité de la réassociation in vitro des sousunités de gonadotropine chez un Poisson Téléostéen, la Carpe (Cyprinus carpio L.).

Purified gonadotropin subunits of a teleost Fish, the Carp, reassociated in vitro, according to a second order rate reaction. The rate constant was temperature dependent. Compared to ovine LH, the Q10 was about three times less and the rate constant much greater (160 times for instance at 20 degrees C). The possible physiological significance of these differences is underlined.

The evolution of gonadotropins: some molecular data concerning a non-mammalian pituitary gonadotropin, the hormone from a teleost fish (Cyprinus carpio L.).

The amino acid and sugar compositions as well as long N-terminal sequences and the C-terminal amino acids of the two subunits of carp gonadotropin, SU I and SU II, were determined. An important homology was demonstrated between SU I and alpha-subunits and between SU II and beta-subunits of mammalian gonadotropins. Moreover SU II was more closely related to the beta-subunit of LH than to the beta-subunit of FSH.