Interplay between Fsh, Igf and Wnt signaling in the regulation of spermatogonial development in the zebrafish Danio rerio

Abstract

Spermatogenesis relies on the activation of a small population of undifferentiated germ cells called spermatogonial stem cells (SSCs). SSCs reside in a specific microenvironment, called “niche”, formed by somatic cells such as Sertoli, Leydig and myoid cells as well as the signaling molecules secreted by these cells. These signaling molecules can modulate the proliferation and differentiation behavior of the SSCs. Thus, SSCs can either be quiescent, proliferate to self-renew, thereby producing more SSCs, or undergo a differentiating proliferation to eventually produce spermatozoa. A balanced self-renewal/differentiation activity is critical for maintaining testis tissue homeostasis. The pituitary follicle-stimulating hormone (Fsh) is a major regulator of vertebrate spermatogenesis, targeting somatic cell functions in the testes. The present thesis explored mechanisms used by Fsh to regulate adult fish spermatogenesis, with a strong emphasis but not exclusively focusing on the signaling system used by the Fsh-regulated growth factor Igf3. Igf-binding proteins (Igfbps) were shown to modulate the biological activity of Igf3. The intracellular signaling system used by Igf3 in germ cells was identified as depending on b-catenin signaling, and finally, the function of an Fsh-regulated, non-canonical Wnt ligand in regulating spermatogonial fate was detected. Igf bioactivity is modulated by a family of 9 Igfbps in zebrafish. The first results showed that pharmacological inhibition of the Igfbps promoted differentiating division of type A spermatogonia, suggesting that Igfbps protect these cells from excessive, Igf3-mediated differentiation. Further studies showed that Fsh is a regulator of Igfbp transcript levels, which also responded to factors downstream of Fsh (androgens and Igf3). Five of the nine Igfbps responded to the factors investigated by up- or down-regulation. Down-regulation occurred faster than up-regulation, which lagged for at least 3 days. Finally, functional studies involving pharmacological blocking or de-blocking of Igfbps allowed concluding that three Igfbps inhibited while two others supported Igf3-stimulated differentiation of spermatogonia. Large-scale gene expression studies on salmon and zebrafish testis tissue suggested that Fsh action involved the Wnt signaling system. Igf can activate b-catenin-dependent signaling. Indeed, experiments with different inhibitors revealed that Fsh-triggered Igf3 release activated the β-catenin signaling pathway in a Wnt ligand-independent manner in zebrafish type A spermatogonia to induce their differentiation. Finally, this thesis also showed that Fsh used the non-canonical Wnt pathway to promote self-renewal of type A spermatogonia by increasing the synthesis and release of Wnt5a by Leydig cells. We conclude that Fsh stimulates the proliferation and differentiation activity of type A spermatogonia in a balanced manner by regulating self-renewal via non-canonical Wnt (Wnt5a) signaling and differentiation by activating β-catenin-dependent (Igf3) signaling.