Spermatogonial stem cells and their endocrine and paracrine regulation in zebrafish

In stem cell biology, the term niche refers to anatomical and functional dimensions where stem cell populations are established and can be maintained. In these specific places, stem cells are protected from differentiating signals that otherwise might lead to their depletion, and also protected from overproliferation. To achieve this, the cells forming the niche (in the testis: Sertoli cells and interstitial cell types)integrate signals from different sources and produce regulatory output modulating stem cell behavior as necessary for sustaining tissue homeostasis. In this thesis, we studied spermatogonial stem cell (SSC) candidates, their niche, and endocrine and paracrine signals modulating SSC activity in zebrafish. In zebrafish testis, the anatomical location of SSCs was identified by the “label retaining cell” approach. Two populations of SSC candidates (“active” and “reserve”) were found surrounded by Sertoli cells near the interstitial compartment. It is assumed that the vertebrate SSC niche integrates signals from Sertoli cells, and from interstitial elements (Leydig cell, myoid, blood vessels) to regulate the SSC activity, also in the zebrafish testes. The stemness and sexual plasticity of these SSC candidates was confirmed by transplantation assays, showing colonization and donor-derived spermatogenesis as well as oogenesis in recipient testis and ovaries. To broaden our knowledge about the functional aspects of the niche (signals), SSCs were activated by treating adult males with a cytostatic agent, and gene expression studies revealed an opposing effect on two growth factors expressed in Sertoli cells, the TGFβ family member Amh (anti-Müllerian hormone), and Igf3 (insulin-like growth factor 3). Recombinant Amh and Igf3 were produced and tested on zebrafish testis explants: Amh blocked spermatogonial differentiation, decreased the proliferation of type A spermatogonia, and moreover compromised Leydig cell androgen release activated by follicle-stimulating hormone (Fsh). Igf3, on the other hand, stimulated spermatogonial proliferation and differentiation towards meiosis. Finally, this these showed that Fsh reduces Sertoli cell expression of Amh, and at the same time, stimulates Igf3, which promotes germ cell differentiation. Therefore, the main progress realized in this thesis is that the Fsh effects on spermatogenesisare shown to be mediated by growth factors, and not only by steroids, as it was believed previously. Moreover, there is very little knowledge in vertebrates in general about the local “translation” of the Fsh and androgen signals into paracrine signals/cell-cell signals that modulate germ cell behaviour. Igf3, derived from a new paralogue of the igf1gene, seems to function via an evolutionary conserved insulin/Igf signaling mechanism, while the Amh effects on SSC behavior, described for the first time in fish, should be tested in mammals (or other tetrapods), as it might be a conserved but yet undetected effect