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International audience; For continued studies of GnRH receptor regulation in the winter flounder, we have developed an in vitro system consisting of cultured pituitary cells dissociated by collagenase. Using immunocytochemical staining methods for gonadotropin, growth hormone, and prolactin, these cell types were represented at the levels of 25, 20, and 19.5% of total pituitary cell population, respectively. Receptors for GnRH were characterized in intact monolayered attached pituitary cells, maintained in RPMI culture medium. The cell GnRH receptor characteristics were compared with those previously described using pituitary homogenates. The cells were capable of binding GnRH in a similar manner on Day 2 or Day 3 of culture, indicating the integrity of GnRH receptors. The specificity of binding was demonstrated since only high doses of cold GnRHa competed with 125I-GnRHa uptake, different peptides being without effect. The specific binding is saturable and the data suggest the presence of a single class of high-affinity (apparent Ka = 1.50 x 10(9) M-1), high-capacity sites (binding capacity = 25.03 fmol/2.5 x 10(5) cells or 242.23 x 10(3) sites/gonadotroph) which is in accordance with the characteristics of GnRH receptors present in homogenates of pooled male and female pituitary glands. All these observations suggest that such an in vitro pituitary cell system would be appropriate for studying GnRH receptor characteristics under different physiological conditions.

Using primary cultures of whole dispersed pituitary cells collected from rainbow trout at different stages of spermatogenesis, basal and GnRH-induced GtH release and cell GtH content were studied in control and steroid-pretreated cultures. Steroid pretreatments were performed for 3 days with 11-ketotestosterone (11KT) and 17 alpha-hydroxy,20 beta-dihydroprogesterone (17 alpha 20 beta p) at levels corresponding to those circulating at the time of spermiation (50 and 20 ng/ml, respectively). In control cultures, basal GtH release and cell GtH content increased with the stage of spermatogenesis in a characteristic pattern as predicted from in vivo results concerning plasma and pituitary GtH contents. The pituitary response to salmon GnRH (sGnRH) also varied as indicated by the decrease in the minimal effective dose of GnRH able to induce a significant GtH release with the advancement of spermatogenesis: 10(-7) M at the spermatocyte stage, 10(-9) M at prespermiation and spermiation. Steroid pretreatments were shown to have a direct effect on pituitary gonadotrophs and particularly on pituitary response to sGnRH, depending on the stage at which they are applied. At the beginning of spermatogenesis both of them induced an increase in GtH release and at prespermiation they have a slight negative effect, significant only with 17 alpha 20 beta P. At spermiation they have no effect except for 17 alpha 20 beta P which increased the response to 10(-8) M of sGnRH. Results are discussed in relation to hormonel changes (gonadotropin and steroid) observed by different authors during the sexual cycle.

International audience; Growth hormone (GH) polypeptide was purified from pituitary glands of the gilthead sea bream (Sparus aurata) by a two-step procedure involving gel filtration on Sephadex G-100 and reverse-phase high-performance liquid chromatography (rpHPLC). At each stage of purification, fractions were monitored by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and by immunoblotting using anti-bonito GH antiserum. The molecular weight of the sea bream GH was estimated by SDS-PAGE to be 21 kDa when electrophoresed in the absence of p-mercaptoethanol (nonreduced conditions) and 22 kDa when electrophoresed under reduced conditions (in the presence of 1% beta-mercaptoethanol). Pituitary RNA was used to direct cell-free translation. When specific immunoisolation from S-35-labeled proteins was conducted, using antisera against Sparus or tilapia GH, a larger prehormone was immunoprecipitated. The size of the pre-GH was estimated to be 27-28 kDa under reduced conditions and 26-27 kDa under nonreduced conditions, in agreement with the calculated molecular weight of Sparus pre-GH of 26,296 based on the deduced amino acid sequence of Sparus GH cDNA. The specificity of the immunoprecipitation reaction was demonstrated by the ability of recombinant tilapia GH to compete with the radioactively labeled translation product. No such competition was found after the addition of BSA. Our results demonstrate that the sea bream GH is similar in its size to other purified fish GHs and provide direct evidence for the synthesis of GH as a prepeptide, thus supporting the conclusions presented earlier by GH cDNA cloning.

International audience; Juvenile rainbow trout received gonadotropic hormone (GtH) treatment to investigate the response of the immature gonad and its effect on the onset of rising pituitary GtH levels, an early sign of initiation of sexual maturation in the teleost fishes. Brief exposure to GtH evoked increases in plasma androgens and significantly increased pituitary GtH in males; the accumulation of pituitary GtH was not observed in female trout which showed significant but smaller increases in plasma androgen resulting from GtH administration. There was no evidence of gonadal negative feedback in intact immature fish since pituitary GtH and plasma androgens remained at basal levels in castrate juvenile trout. Since the castrate male trout failed to respond to GtH injections, the data suggest that the immature gonad produces a steroid capable of triggering pituitary GtH production by hypothalmo-pituitary feedback in males.