Distinct and Cooperative Roles of amh and dmrt1 in Self-Renewal and Differentiation of Male Germ Cells in Zebrafish

Abstract

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.

The roles of Amh in zebrafish gonad development and sex determination

“…4B, F, J). Averaging results from females homozygous for the amh -10 and amh -26 298 alleles, the gonadal-somatic index (GSI, (gonad weight / body weight)*100) of amh mutants was 299 about 2.6-fold larger than their respective wild-type siblings, confirming prior results in zebrafish 300 and mouse (DURLINGER et al 1999;LIN et al 2017). Adult ovaries in 8mpf amh -26 zebrafish mutants 301 lacked oocytes that had matured beyond stage III ( Fig.…”

“…We conclude that amh mutants are slow to adapt a male 981 phenotype, and many never do, leading to a female-biased sex ratio among amh mutants. 982 Ovaries in mature adult amh mutant females were swollen by immature oocytes to nearly three 983 times the size of ovaries in wild-type siblings, confirming previous results (LIN et al 2017). The , showing that in zebrafish, Amh helps slow gonad growth both in males and in females.…”

“…S2). This Results showed that amh activity promotes, but is not essential for, male development in 938 zebrafish because homozygous amh mutants were only about 20% as likely to develop into males 939 as their wild-type siblings (see also (LIN et al 2017)). In addition, most mature adult male mutant 940 gonads we examined contained a few early stage oocytes while at the same age, wild-type siblings .…”

Loss ofdmrt1restores female fates in the absence ofcyp19a1abut notrbpms2

“…In mammals, Dmrt1 acts by directly activating male determining genes and simultaneously suppressing female determining genes [2,16]. Recent studies verified that dmrt1 is expressed in both the germline and somatic gonad in zebrafish and as in mammals, is required for male differentiation, as mutants disrupting Dmrt1 cause female-biased sex ratios [11,17].…”

“…In mammals, Dmrt1 acts by directly activating male determining genes and simultaneously suppressing female determining genes [2,16]. Recent studies verified that dmrt1 is expressed in both the germline and somatic gonad in zebrafish and as in mammals, is required for male differentiation, as mutants disrupting Dmrt1 cause female-biased sex ratios [11,17].Aromatase, encoded by cyp19, required for the final steps of estrogen synthesis [18,19], is a critical factor for sex differentiation in fish [10, 20]. Zebrafish have two copies of cyp19 (ovarian cyp19a1a and brain cyp19a1b) and, in contrast to dmrt1, knockout of the ovarian aromatase cyp19a1a results in an all-male phenotype that is characterized by early transition to male gonad features with an apparent absence of a bipotential phase of development [21].Bone morphogenetic protein 15 (Bmp15) is a signaling molecule expressed primarily by the oocyte in mice and zebrafish [20,22,23].…”