1 Reproductive Biology Group, Division Developmental Biology, Department of Biology, Institute of Biodynamics and Biocomplexity, Faculty of Science, University of Utrecht, NL-3584 CH Utrecht, The Netherlands
2 Reproduction and Developmental Biology Group, Institute of Marine Research, Nordnes, Bergen, Norway
Correspondence should be addressed to R W Schulz: firstname.lastname@example.org
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 (Aund) 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 Aund. In the present study, we report that Fsh moreover uses the non-canonical Wnt pathway to promote the proliferation and accumulation of Aund. Initially, we found that the stimulatory effect of Fsh on the proliferation activity of Aund was further strengthened when β-catenin signaling was inhibited, resulting in an accumulation of Aund. We then showed that this Fsh-induced accumulation of Aund 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 Aund, while an agonist of Wnt5a increased these same parameters for Aund. Taken together, our data suggest that Fsh triggered LCs to release Wnt5a, which then promoted the proliferation and accumulation of Aund. Hence, Fsh uses non-canonical Wnt signaling to ensure the production of Aund, while also triggering β-catenin signaling via Igf3 to ensure spermatogonial differentiation.
Supplemental figure 1. (A-E) Immunocytochemical detection of BrdU in sections of zebrafish testis incubated in the presence of 100 ng/mL Fsh (A) or 100 ng/mL Fsh and XAV939 (10 µM) (B) or 100 ng/mL Fsh, 10 µM XAV939 and 50 µM IWP-12 (C) or 100 ng/mL Fsh and 50 µM IWP-12 (D) or under basal condition, presence of 10 µM XAV939 or 50 µM IWP-12 (E) for 5 days, showing BrdU-positive (+) and BrdU-negative (-) Aund and Adiff spermatogonia. (K-I) Whole-mount co-localization of Wnt5a by immunocytochemistry and insl3 mRNA by in situ hybridization in Leydig cells of adult zebrafish testis.