Fsh stimulates Leydig cell Wnt5a production, enriching zebrafish type A spermatogonia

Follicle-stimulating hormone (Fsh) modulates vertebrate spermatogenesis by regulating somatic cell functions in the testis. We have found previously that zebrafish Fsh stimulated the differentiating proliferation of type A undifferentiated spermatogonia (A und ) in an androgen-independent manner by regulating the production of growth factors and other signaling molecules in both Sertoli (SCs) and Leydig cells (LCs). For example, Fsh triggered the release of Igf3 that subsequently activated β-catenin signaling to promote the differentiating proliferation of A und . In the present study, we report that Fsh moreover uses the non-canonical Wnt pathway to promote the proliferation and accumulation of A und . Initially, we found that the stimulatory effect of Fsh on the proliferation activity of A und was further strengthened when β-catenin signaling was inhibited, resulting in an accumulation of A und . We then showed that this Fsh-induced accumulation of A und was associated with increased transcript levels of the non-canonical Wnt ligand, wnt5a. In situ hybridization of insl3 mRNA, a gene expressed in LCs, combined with Wnt5a immunocytochemistry identified LCs as the cellular source of Wnt5a in the adult zebrafish testis. Addition of an antagonist of Wnt5a to incubations with Fsh decreased both the proliferation activity and the relative section area occupied by A und , while an agonist of Wnt5a increased these same parameters for A und . Taken together, our data suggest that Fsh triggered LCs to release Wnt5a, which then promoted the proliferation and accumulation of A und . Hence, Fsh uses non-canonical Wnt signaling to ensure the production of A und , while also triggering β-catenin signaling via Igf3 to ensure spermatogonial differentiation.