Regulação funcional dos testículos de Zebrafish (Danio rerio) adultos através do T3 E IGF3

Abstract

The regulation of early stages of spermatogenesis in adult fish is a process that is not yet well understood, in particular the aspects related to the proliferation of Sertoli cells and self-renewal or differentiation of spermatogonial stem cells (SSC). In this context, the present study investigated the potentially direct effects of triiodothyronine (T3) in the testes of adult zebrafish, as well as the involvement and biological activity of the growth factor insulin-like 3 (Igf3/igf3) in the T3-mediated effects. It is known in the literature that thyroid hormones participate in regulating growth and homeostatic processes in vertebrates, including development and adult functioning of the reproductive system. Here we report a new stimulatory role of thyroid hormone on the proliferation of Sertoli cells and single, type A undifferentiated spermatogonia (Aund) in adult zebrafish testes. A direct role for T3 in zebrafish testis is suggested by in situ hybridization studies (ISH), which localized thyroid receptor alpha (thr) in Sertoli cells and the thyroid receptor beta (thr) mRNA in Sertoli andLeydig cells. Using a primary zebrafish testis tissue culture system, the effect of T3 on steroid release, spermatogenesis and the expression of selected genes was evaluated. Basal steroid release and Leydigcell gene expression did not change in response to T3. However, in the presence of follicle-stimulating hormone (Fsh), T3 potentiated gonadotropin-stimulated androgen release as well as androgenreceptor (ar) and 17-hydroxylase/17,20 lyase (cyp17a1) gene expression. Moreover, T3 alone stimulated the proliferation of both Sertoli cells and Aund, potentially resulting in newly formed spermatogenic cysts. Additional tissue culture studies demonstrated that Igf3, a new, gonad-specific member of the insulin-like growth factor family, mediated the stimulatory effect of T3 on the proliferation of Aund and Sertoli cells. Finally, T3 induced changes in connexin 43 mRNA levels in the testis, a known T3-responsive gene. Taken together, our studies suggest that T3 expands the population of Sertoli cells and Aund involving Igf signalling, and potentiates gonadotropin-stimulated testicular androgen production as well as androgen sensitivity. In another aspect of this work, we also investigate the regulation and the potential functions of Igf3/ifg3 in the zebrafish testis. It is known that in vertebrates the IGF system plays an important role in the cell survival, migration, proliferation and differentiation. Thus, we have shown that under the influence of Fsh, in an androgen-independent manner, stimulated zebrafish spermatogenesis to progress from the proliferation of spermatogonial of type A until the formation of haploid germ cells. In addition, we demonstrated that Fsh stimulated the expression of Igf3/igf3 in adult zebrafish testes,involving the activation of transcription factors Creb (cAMP response element binding protein) and ATF-1 (cAMP-dependent transcription factor 1), present in the promoter region of the igf3 gene, as demonstrated by transfection studies in HEK 293T cells. Using immunocytochemistry and ISH studies, Igf3 was localised in Sertoli cells contacting spermatogonia type A, suggesting that Igf3 effects can mainly be expected on this type of germ cells. Testicular explants incubated with rzf Igf3 increased the expression of differentiation (dazl) and mitosis (piwil2) related genes and also stimulated the proliferation of Aund, which was dramatically reduced in the presence of the IGF -1R kinase inhibitor, NVP-AEW541. This inhibitor was also able to significantly reduce Fsh-stimulated spermatogonial proliferation, suggesting that even without the participation of androgens, this gonadotropin can stimulate spermatogenesis in adult zebrafish via Igf3. In summary, the results ofthis study provide important and novel information to better understand the regulation of testicular functions in adult zebrafish .