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
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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.
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