Follicle-stimulating hormone (Fsh) is a major regulator of spermatogenesis, targeting somatic cell functions in the testes. We reported previously that zebrafish Fsh promoted the differentiation of type A undifferentiated spermatogonia (A_und) by stimulating the production of factors that advance germ cell differentiation, such as androgens, insulin-like peptide 3 (Insl3) and insulin-like growth factor 3 (Igf3). In addition, Fsh also modulated the transcript levels of several other genes, including some belonging to the Wnt signaling pathway. Here, we evaluated if and how Fsh utilizes part of the canonical Wnt pathway to regulate the development of spermatogonia. We quantified the proliferation activity and relative section areas occupied by A_und and type A differentiating (A_diff) spermatogonia and we analyzed the expression of selected genes in response to recombinant proteins and pharmacological inhibitors. We found that from the three downstream mediators of Fsh activity we examined, Igf3, but not 11-ketotestosterone or Insl3, modulated the transcript levels of two β-catenin sensitive genes (cyclinD1 and axin2). Using a zebrafish β-catenin signaling reporter line, we showed that Igf3 activated β-catenin signaling in type A spermatogonia and that this activation did not depend on the release of Wnt ligands. Pharmacological inhibition of the β-catenin or of the phosphoinositide 3-kinase (PI3K) pathways revealed that Igf3 activated β-catenin signaling in a manner involving PI3K to promote the differentiation of A_und to A_diff spermatogonia. This mechanism represents an intriguing example for a pituitary hormone like Fsh using Igf signaling to recruit the evolutionary conserved, local β-catenin signaling pathway to regulate spermatogenesis.

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.

The IGF-binding proteins (IGFBPs) play a dual role in the regulation of the activity and bioavailability of IGFs in different tissues. Diverse evidence has shown that IGFBPs can inhibit and/or potentiate IGF actions. In this study, igfbp1, 2, 3, 4, 5, and 6 were isolated in the fine flounder, a flat fish species that shows slow growth and inherent Gh resistance in muscle. Subsequently, the expression of all igfbps was assessed in the skeletal muscle of flounder that underwent different nutritional statuses. igfbp1 was not expressed in muscle during any of the nutritional conditions, whereas igfbp3 and igfbp5 were the lowest and the highest igfbps expressed respectively. A dynamic expression pattern was found in all the igfbps expressed in skeletal muscle, which depended on the nutritional status and sampling period. During the fasting period, igfbp2, 4, and 5 were downregulated, whereas igfbp3 was upregulated during part of the fasting period. The restoration of food modulated the expression of the igfbps dynamically, showing significant changes during both the long- and short-term refeeding. igfbp3 and igfbp6 were downregulated during short-term refeeding, whereas igfbp5 was upregulated, and igfbp2 and igfbp4 remained stable. During long-term refeeding, the expression of igfbp2, 4, 5, and 6 increased, while igfbp3 remained unchanged. In conclusion, this study shows for the first time the isolation of all igfbps in a single fish species, in addition to describing a dynamic nutritional and time-dependent response in the expression of igfbps in the skeletal muscle of a nonmammalian species.

Changes in zebrafish testicular gene expression induced by follicle-stimulating hormone (Fsh) or anti-Mullerian hormone (Amh) suggested that Amh inhibition and Fsh stimulation of spermatogenesis involved up and downregulation, respectively, of prostaglandin (PG) signaling. We found that Sertoli cells contacting type A undifferentiated (A_und) and differentiating (A_diff) spermatogonia expressed a key enzyme of PG production (Ptgs2); previous work showed that Sertoli cells contacting A_diff and B spermatogonia and spermatocytes showed ptges3b expression, an enzyme catalyzing PGE₂ production. In primary testis tissue cultures, PGE₂, but not PGD₂ or PGF_2α, reduced the mitotic activity of A_diff and their development into B spermatogonia. Vice versa, inhibiting PG production increased the mitotic activity of A_diff and B spermatogonia. Studies with pharmacological PG receptor antagonists suggest that an Ep4 receptor mediates the inhibitory effects on the development of spermatogonia, and cell-sorting experiments indicated this receptor is expressed mainly by testicular somatic cells. Combined inhibition of PG and steroid production moreover reduced the mitotic activity of A_und spermatogonia and led to their partial depletion, suggesting that androgens (and/or other testicular steroids), supported by PGE₂, otherwise prevent depletion of A_und. Androgens also decreased testicular PGE₂ production, increased the transcript levels of the enzyme-catabolizing PGs and decreased PGE₂ receptor ptger4b transcript levels. Also Fsh potentially reduced, independent of androgens, PGE₂ production by decreasing ptges3b transcript levels. Taken together, our results indicate that PGE₂, via Ep4 receptors, favors self-renewal in conjunction with androgens and, independent of Fsh and androgens, inhibits differentiating divisions of spermatogonia.