Endocrine and paracrine regulation of spermatogonial differentiation in zebrafish (Danio rerio)

Abstract

Spermatogenesis in fish, as in other vertebrates, is a developmental process triggered by pituitary gonadotropins (Fsh and Lh) that stimulate testicular production of androgens and growth factors, thus making sperm production possible. Spermatogenesis starts with single type A undifferentiated spermatogonia (Aund). These cells can show infrequent or more frequent self-renewal proliferation, or can embark on differentiating divisions to form clones of type A differentiating spermatogonia (Adiff). The latter give rise to a large number of type B spermatogonia that subsequently enter meiosis and spermiogenesis to complete a spermatogenic wave. Studying the mechanisms controlling the transition from quiescent to more actively self-renewing and then to differentiating proliferation will allow answering questions regarding the regulation of spermatogenesis in fish, and perhaps also in other vertebrates. This thesis investigated the biological activities of Fsh, thyroid hormone (TH), and of a number of growth factors identified previously as potentially relevant for regulating Fsh-modulated spermatogonial development; many experiments used a primary tissue culture system developed for adult zebrafish testis. Recombinant zebrafish Fsh increased the number and proliferation activity of single type Aund and of clones of Adiff spermatogonia in a steroid-independent manner, while increasing Leydig cell insl3 (insulin-like peptide 3) and Sertoli cell igf3 (insulin-like growth factor 3) transcript levels but decreasing Sertoli cell amh (anti-Müllerian hormone) transcript levels. Recombinant zebrafish Igf3 promoted proliferation and differentiation of spermatogonia and stimulated the transition of spermatogonia into spermatocytes. On the other hand, recombinant zebrafish Amh compromised Igf3-induced spermatogonial proliferation and reduced the transcript levels of igf3 and insl3. The latter recruits Aund into differentiating proliferation. Furthermore, we investigated if modulators of Igf3 activity, the Igf binding proteins (Igfbps), influence TH- or Fsh-mediated effects on Igf3-stimulated spermatogonial proliferation. We found that elevating Igf3 bioactivity by blocking Igfbps further increased TH- or Fsh-induced effects in particular as regards the differentiating spermatogonial divisions, leading to a depletion of type Aund spermatogonia in favor of more differentiated generations. Finally, to study Igf3 function in vivo, zebrafish igf3 mutants were generated using the CRISPR-Cas9 approach. Initial results suggest that igf3 loss-of-function increased the proportion of type Aund spermatogonia but reduced the proportion of meiotic and post-meiotic cells. Altogether, the present thesis provides new information with respect to the question how systemic hormones (Fsh, TH) modulate the production of local signaling molecules in the testis, which then act to mediate the hormones’ effects on germ cell development. Regarding Igf3, this growth factor was the first Fsh-regulated factor of testicular origin promoting germ cell development that was characterised in vertebrates.