Isolation and culture of somatotrophs from the pituitary of the rainbow trout: immunological and physiological characterization.

The aim of the present paper was to obtain somatotroph- and gonadotroph-enriched populations from collagenase dispersed pituitaries of male rainbow trout. Inasmuch as the percentage of immunoreactive gonadotrophs and somatotrophs present in pituitaries was higher at spermiation than at the beginning of spermatogenesis, we tried such a cell separation with fish at this stage of spermatogenesis. Cells were fractionated using their differences in buoyant density with centrifugation in Percoll solutions. The use of Percoll linear gradients (1.110 to 1.027 g/ml) showed that somatotroph cells have a density of between 1.102 and 1.064 g/ml whereas gonadotrophs are spread over the range of the gradient. It was thus possible, by using linear or discontinuous Percoll gradients, to obtain 95 to 67% (mean 80%) enriched somatotropic cell fractions while no enriched gonadotropic cell fractions were collected. The fractionated cells kept their ability to be cultured and to be responsive to specific secretagogues. Somatostatine induced a 80 to 85% decrease in growth hormone release per somatotroph in the initial cell suspension as well as in the different cell fractions. On the other hand, the basal growth hormone release per cell was lower in the fractions containing cells with a density lower than 1.062 g/ml. Inversely, the gonadotrophs have a basal release per cell independent of their density, and this is also available for their responsiveness to salmon gonadotropin-releasing hormone.

Steroid activation of the brain-pituitary complex gonadotropic function in the triploid rainbow trout Oncorhynchus mykiss.

Testosterone and estradiol were implanted into triploid rainbow trout, and their effects on brain and pituitary salmon gonadotropin-releasing hormone (sGnRH) content and on gonadotropin (GtH2) levels in the pituitary and blood were studied. In the hypothalamic areas, neither steroid altered sGnRH content. In the brain tissues (ventral telencephalon and preoptic areas), testosterone (0.5 mg/kg BW) significantly increased sGnRH contents and larger doses induced a more rapid increase. Estradiol induced a similar increase which occurred later than the response to the same dose of testosterone. Both steroids also stimulated pituitary sGnRH, with estradiol being less potent. Only testosterone induced long-term increases in plasma GtH2 content. Both steroids may act on the synthesis and release of GnRH, with testosterone having a greater influence on the control of gonadotropin secretion, especially at the end of the cycle.

Effects of steroids on GTH I and GTH II secretion and pituitary concentration in the immature rainbow trout Oncorhynchus mykiss.

Using specific radio-immunoassays for rainbow trout GTH I and GTH II, the effects of testosterone and estradiol 17 beta have been studied or reinvestigated on the regulation of the secretion and the synthesis of the these two pituitary gonadotropins in the immature rainbow trout. After steroid implantation, the GTH II pituitary concentration is stimulated by testosterone and estradiol 17 beta for the entire period during which the plasma levels of these hormones are maintained to values comparable to those measured in the adult vitellogenic female rainbow trout. On the other hand, only testosterone induced a transient increase in the GTH I pituitary content 15 days after implantation, and estradiol provoked a decrease at day 30. The secretion of both GTH I and GTH II is stimulated by testosterone but not by estradiol 17 beta. Altogether, these results show that in the immature rainbow trout, testosterone preferentially modifies GTH I secretion, but not that of GTH II. They confirm that the stimulation of GTH II accumulation after testosterone or estradiol treatment would correspond to a stimulation of hormone synthesis. They evidence a differential action of both steroids on the synthesis of the two gonadotropins, especially a possible inhibition of GTH I synthesis by estradiol. They let suppose that the regulation of GTH I synthesis would involve factors other than steroids.

Gonadotropin hormone (GtH) receptors in the ovary of the brown trout Salmo trutta L. in vitro studies.

A method has been developed suitable for the study of the binding of salmon (Oncorhynchus tsawytscha) gonadotropic hormone (GtH) to brown trout (Salmo trutta) ovarian particulate fractions. Several proteolytic enzyme inhibitors were tested to preserve the binding capacity of particulate fractions (1000, 20,000, and 110,000g) from yolky oocytes. The best preservation was obtained with trypsin inhibitor. The effects on binding of the pH, time, and temperature of the incubation were investigated. Binding increased linearly with amount of particulate fraction, and is saturable. In both the 20,000 and 110,000g fractions, there are two classes of receptors: one with a high-affinity constant (3.14 and 0.95 10(10) M-1 for the 20,000 and 110,000g fractions, respectively) and a low capacity (1.76 and 0.63 fmol/g of ovary), the other with a higher-affinity constant (3.44 and 1.78 10(9) M-1) but a higher capacity (7.37 and 7.42 fmol/g of ovary). GtH binding was not affected by ovine luteinizing hormone (oHL), ovine follice-stimulating hormone (oFSH), and human chorionic gonadotropin (hCG), but was partially inhibited by 1 micrograms of bovine thyroid-stimulating hormone (bTSH) and salmon prolactin (sPRL). The results are discussed in relation to the biological specificity of fish GtH and to the unsolved problem concerning the number of gonadotropins in fish.

Type II iodothyronine deiodinase is preferentially expressed in rainbow trout (Oncorhynchus mykiss) liver and gonads.

It is well admitted that thyroid hormones (TH) play a role in the development of vertebrates. The major secretory product of the thyroid is a pro-hormone, T(4), which is activated in peripheral tissues by outer ring deiodination to T(3). We have isolated from rainbow trout testis, a full length cDNA encoding type II iodothyronine deiodinase (rtD2). The cDNA was 2410 nucleotides long and coded for a polypeptide of 264 amino acids including a selenocysteine residue. The predicted molecular weight of rtD2 was 29.3 kDa and the isoelectric point 8.71. The deduced amino acids sequence showed 80% identity with Fundulus heteroclitus D2 (fhD2) but only 68-69% identity with rat, mouse, and human D2. The 3' UTR contained a putative selenocysteine insertion sequence (SECIS) similar to that described in human cDNA. The rtD2 gene was isolated and the gene structure was similar to that described in human with two exons separated by a large intron. We studied rtD2 gene expression by Northern blot analysis using total RNA extracted from testis, ovary, and other tissues. We found a high expression of a 3 kb transcript in liver and in gonads. A lower expression was also detected in posterior kidney. In testis, rtD2 mRNA expression was dependent on spermatogenic stages: it increased at the onset of spermatogenesis. Our results show that the structural characteristics of the D2 protein and gene have been highly conserved during evolution. The rtD2 mRNA expression in the gonads suggests that rtD2 may be a key factor regulating local supply of active T(3) during rainbow trout gametogenesis.