Distinct and Cooperative Roles of and in Self-Renewal and Differentiation of Male Germ Cells in Zebrafish.

Spermatogenesis is a fundamental process in male reproductive biology and depends on precise balance between self-renewal and differentiation of male germ cells. However, the regulative factors for controlling the balance are poorly understood. In this study, we examined the roles of and in male germ cell development by generating their mutants with Crispr/Cas9 technology in zebrafish. mutant zebrafish displayed a female-biased sex ratio, and both male and female mutants developed hypertrophic gonads due to uncontrolled proliferation and impaired differentiation of germ cells. A large number of proliferating spermatogonium-like cells were observed within testicular lobules of the -mutated testes, and they were demonstrated to be both Vasa- and PH3-positive. Moreover, the average number of Sycp3- and Vasa-positive cells in the mutants was significantly lower than in wild-type testes, suggesting a severely impaired differentiation of male germ cells. Conversely, all the -mutated testes displayed severe testicular developmental defects and gradual loss of all Vasa-positive germ cells by inhibiting their self-renewal and inducing apoptosis. In addition, several germ cell and Sertoli cell marker genes were significantly downregulated, whereas a prominent increase of Insl3-positive Leydig cells was revealed by immunohistochemical analysis in the disorganized -mutated testes. Our data suggest that might act as a guardian to control the balance between proliferation and differentiation of male germ cells, whereas might be required for the maintenance, self-renewal, and differentiation of male germ cells. Significantly, this study unravels novel functions of gene in fish.