Growth hormone (GH) and gonadotropin subunit gene expression and pituitary and plasma changes during spermatogenesis and oogenesis in rainbow trout (Oncorhynchus mykiss).

In order to evaluate potential interactions between somatotropic and gonadotropic axes in rainbow trout (Oncorhynchus mykiss), changes in pituitary content of the specific messenger RNA of growth hormone (GH) and gonadotropin (GTH) alpha- and beta-subunits were studied during gametogenesis with respect to pituitary and plasma hormone concentrations. Quantitative analyses of mRNA and hormones were performed by dot blot hybridization and homologous RIA on individual fish according to stage of spermatogenesis and oogenesis. All transcripts were detectable in 9-month-old immature fish. GH, GTH IIbeta, and GTH alpha increased moderately throughout most of gametogenesis and then more dramatically at spermiation and during the periovulatory period. GTH Ibeta mRNA increased first from stage I to V in males and more abruptly at spermiation, while in females GTH Ibeta transcripts increased first during early vitellogenesis and again around ovulation. Pituitary GH absolute content (microgram/pituitary, not normalized with body weight) increased slowly during gametogenesis and more abruptly in males during spermiation. In the pituitary of previtellogenic females and immature males, GTH I beta peptide contents were 80- to 500-fold higher than GTH II beta peptide contents. GTH I contents rose regularly during the initial phases of vitellogenesis and spermatogenesis and then more abruptly in the final stages of gonadal maturation, while GTH II contents show a dramatic elevation during final oocyte growth and maturation, in postovulated females, and during spermiogenesis and spermiation in males. Blood plasma GTH II concentrations were undetectable in most gonadal stages, but were elevated during spermiogenesis and spermiation and during oocyte maturation and postovulation. In contrast, plasma GTH I was already high ( approximately 2 ng/ml) in fish with immature gonads, significantly increased at the beginning of spermatogonial proliferation, and then increased again between stages III and VI to reach maximal levels ( approximately 9 ng/ml) toward the end of sperm cell differentiation, but decreased at spermiation. In females, plasma GTH I rose strongly for the first time up to early exogenous vitellogenesis, decreased during most exogenous vitellogenesis, and increased again around ovulation. Our data revealed that patterns of relative abundance of GTH Ibeta mRNA and pituitary and plasma GTH I were similar, but not the GTH II patterns, suggesting differential regulation between these two hormones at the transcriptional and posttranscriptional levels. Pituitary and plasma GH changes could not be related to sexual maturation, and only a weak relationship was observed between GH and gonadotropin patterns, demonstrating that no simple connection exists between somatotropic and gonadotropic axes at the pituitary level during gametogenesis.

Insulin-like growth factor (IGF-I) mRNA and IGF-I receptor in trout testis and in isolated spermatogenic and Sertoli cells.

Few data exist concerning the occurrence and potential role of an insulin-like growth factor (IGF) system in fish gonads. Using Northern and slot blot hybridization with a specific salmon IGF-I cDNA, we confirmed that IGF-I transcription occurs in trout testis. Testicular IGF-I mRNA abundance may be increased by long-term GH treatment in juvenile fish, while shorter treatment with growth hormone (GH) or a gonadotropin (GTH-II) in maturing males had no statistically significant effect. Radiolabelled recombinant human IGF-I binds with high affinity to crude trout testis preparation, to cultured isolated testicular cells, and to a membrane fraction of these cells (Ka = 0.2 to 0.7 x 10(10) M-1; Bmax = 10 to 20 fmol/10(7) cells, and 68 fmol/mg protein of membrane). The binding site was identified as type 1 IGF receptor by its binding specificity (IGF-I > IGF-II >>> insulin) and the molecular size of its alpha-subunit labelled with 125I-IGF-I (M(r)125-140 kDa). 125I-IGF-II also bound to the type 1 receptor whereas IGF-II/ mannose 6 phosphate receptors could not be detected. Separation of isolated testicular cells by Percoll gradient and centrifugal elutriation provided populations enriched in different types of intratubular cells. IGF-I mRNA (detected by reverse transcription + polymerase chain reaction [PCR]) and IGF-I receptors (measured by competitive binding) were observed to a greater extent in Sertoli cell-enriched populations and in spermatogonia with primary spermatocytes. Therefore, IGF-I is a potential paracrine/autocrine regulator inside the spermatogenic compartment and appears as a possible mediator of GH action at the gonadal level in fish.

Evidence for binding and action of growth hormone in trout testis.

Growth hormone (GH) binding to testis tissue and GH action on trout testicular cells were studied in vitro. Labeled salmon GH (sGH) was able to bind to a trout testis membrane preparation. Binding sites showed high affinity (Ka = 1-2 x 10(9) M-1) and low capacity (11 fmol/g fresh tissue) for 125I-sGH. Salmon GH and bovine GH, but not salmon gonadotropin, could compete with 125I-sGH for site occupancy. The binding characteristics were similar to those of trout liver GH receptors that we previously described. Salmon GH (0.1 and 1 microgram/ml) and bovine GH (10 micrograms/ml) could modulate steroidogenesis in cultured testicular cells: 17 alpha-hydroxy, 20 beta-dihydroprogesterone (17 alpha 20 beta OHP) accumulation in culture medium was stimulated by GH addition, and this effect increased with duration of culture and/or stimulation; 11-ketotestosterone accumulation tended to be inhibited in the presence of GH at the beginning of culture. These effects were dependent on GH concentration and were observed both in the absence and presence of gonadotropin. The amplitude of the sGH effect varied between experiments, probably according to the physiological state of the cells used. In vivo, GH and 17 alpha 20 beta OHP plasma levels increased at the beginning of spermiation (sperm production) and decreased at the end of spermiation. This relationship suggests that, at the end of the reproductive cycle, high GH levels are associated with the production of 17 alpha 20 beta OHP, a progestin necessary for efficient spawning in this species. We conclude that GH may play a role in testicular physiology, at least at certain stages of spermatogenesis.