Hormonal regulation of spermatogenesis in zebrafish

Abstract

Across vertebrates, spermatogenesis is under the endocrine control of two hormones, follicle-stimulating hormone (FSH) and androgens; the testicular production and secretion of the latter are controlled by luteinizing hormone. In fish, also the strong steroidogenic potency of Fsh should be taken into consideration indicating that a large part of Fsh action in fish spermatogenesis is mediated by androgens. In the studies encompassing this thesis, we aimed at elucidating the mechanisms, by which FSH and especially androgens regulate adult spermatogenesis with particular interest for the spermatogonial phase. Zebrafish was chosen as the experimental animal model. First, our studies identified 11-ketotestosterone (11-KT) as the main end product of the androgen synthesis pathway in zebrafish testis. Together with testosterone, 11-KT binds to and activates the zebrafish Ar at physiologically relevant concentrations. Subsequently, our developed experimental models revealed that all stages of spermatogenesis are dependent of androgens, and that the spermatogonial phase is particularly sensitive. In an in vitro model, an organ culture system for adult zebrafish testis, complete spermatogenesis was supported in the presence of 11-KT. Even more, a higher proportion of type A spermatogonia proliferated and differentiated to commit to the transformation process towards becoming spermatozoa. In contrast, in the absence of 11-KT spermatogenesis in the same testis culture system became depleted. This relates very well to the spontaneous and rapid downfall of the steroidogenic system under basal culture conditions. An in vivo model for oestrogen-induced androgen insufficiency revealed a marked inhibition of spermatogonial proliferation and differentiation, and germ cell depletion became more prominent with their progressive differentiation. The mechanism behind the inhibition of spermatogenesis in this in vivo model is negative oestrogenic feedback on the brain-pituitary system causing a reduction of expression of factors responsible for androgen synthesis, ultimately leading to reduced testicular androgen production. Sertoli cells express functional receptors for both FSH and androgens, in contrast to germ cells. We showed that zebrafish Sertoli cells contacting type A spermatogonia express high levels of ar transcript. It is thought that Sertoli cells mediate androgen or Fsh action by regulating the secretion of paracrine factors, which in turn influence spermatogonial differentiation. We showed that the gonad-specific fish representative of such a group of paracrine factors, the insulin-like growth factors (i.e. igf3), was highly expressed by zebrafish Sertoli cells contacting type A spermatogonia. Furthermore, its mRNA expression was positively modulated by both androgens as well as Fsh, for the latter independent of its steroidogenic potential. Studies on the action of a commercially available recombinant fish Igf protein (i.e. Igf1) on zebrafish spermatogenesis in vitro revealed a stimulatory effect on the proliferation of spermatogonia and their entrance into meiosis, and demonstrated the ability to enhance 11-KT-stimulated spermatogenesis. The present thesis expanded the knowledge on the sites of action and the mechanisms of endocrine control of adult fish spermatogenesis. In addition, novel experimental models to investigate the endocrine control of adult spermatogenesis were developed. Conclusively, zebrafish were proven to be an exciting animal model to investigate endocrine control mechanisms of the spermatogonial phase