Functional Analysis Of Amh And Dmrt1 In Germ Cell Development And Sex Differentiation Of Zebrafish

Gametogenesis is a fundamental process in reproductive biology and depends on precise balance between selfrenewal and differentiation of male germ cells.However,the regulative factors for controlling the balance are poorly understood.amh and dmrt1 are essential for the gonadal development and male sex determination in mammals.In this study,we examined the roles of amh and dmrt1 in germ cell development and sex differentiation by generating their mutants with CRISPR/Cas9 technology in zebrafish.We found all homozygous amh or dmrt1 mutant zebrafish displayed a femalebiased sex ratio,indicating that both amh and dmrt1 are required for normal sex ratios in zebrafish.All the male and female amh mutants developed hypertrophic gonads due to uncontrolled proliferation and impaired differentiation of germ cells.In male zebrafish,a large number of proliferating spermatogonium-like cells were observed within testicular lobules of the amh-mutated testes,and they were demonstrated to be both Vasa-and PH3-positive.Further study revealed that these accumulated and disorganized spermatogonium-like cells were nanos2-positive spermatogonia.Moreover,the average number of Sycp3-and Vasa-positive cells in the amh mutants was significantly lower than in wild-type testes,suggesting a severely impaired differentiation of male germ cells.Compared with the WT control,only a few single Leydig cells among the disorganized germ cells were detected in amh mutants,suggesting that Leydig cell differentiation failed in amh-deficient testes.In amhmutated testes,the m RNA levels of six spermatogonium marker genes and four Sertoli cell marker genes were significantly upregulated,whereas four Leydig cell marker genes were downregulated.These results were consistent with our histological observation that spermatogonia had excessive proliferation and spermatogenic cell differentiation was severely impaired.In amh mutant female zebrafish,homozygous females had similar phenotypes to homozygous males,including enlarged abdomen and hypertrophic ovary.The hypertrophic ovaries of homozygous females were filled with lots of primary growth oocytes and the oocyte maturation was arrested at an early stage,indicating that loss of amh function resulted in an excessive proliferation of germ cells and a profound oocyte maturation defect.We further confirmed that these accumulated germ cells are premeiotic oogonia by RNA in situ hybridization with a premeiotic germ cell marker nanos2.Taken together,amh deficiency causes overproliferation and impaired differentiation of germ cells.Thus,amh is essential for regulating germ cell differentiation in both male and female zebrafish.Conversely,all the dmrt1-mutated testes displayed a thin thread-like testicular structure and all the homozygous males were infertile.Our histological results showed that dmrt1 mutant testes were completely devoid of normal testicular structure and lost all Vasa-positive germ cells,with only disorganized somatic cells at 43 dpf,revealing that dmrt1 deletion impairs male germ cell development and testicular lobule formation.Further,we found a significant decrease of PH3-positive proliferating cells and the gradual loss of Sycp3 expression in the dmrt1 mutant testes.And there is an abnormal increase of cell apoptosis in dmrt1 mutant testes.These apoptotic cells were identified as germ cells and Sertoli cells in mutant testes.Therefore,these results indicate that dmrt1 is required for proliferation and maintenance of early male germ cells.In addition,the expression of several germ cell and Sertoli cell marker genes were significantly downregulated or completely inhibited at later stages,whereas Leydig cell marker genes were upregulated.The expression changes of three kinds of genes were consistent with our histological results that germ cells and Sertoli cells were absent or impaired,whereas numerous Insl3-positive Leydig cells existed in residual testes of the dmrt1 mutants.Therefore,their expression changes suggest that dmrt1 should be required for the maintenance of both germline genes and Sertoli cell genes during testis development.Additionally,we found that dmrt1 deletion lead to complete inhibition of amh expression in the examined testes,and the expression of dmrt1 was significantly upregulated in amh-mutated testes.However,the homozygous dmrt1 mutant females were fertile and exhibited the same normal ovarian development as WT females,demonstrating that dmrt1 is dispensable for the zebrafish ovarian development.In conclusion,our data suggest that amh might act as a guardian to control the balance between proliferation and differentiation of male germ cells,whereas dmrt1 might be required for the maintenance,self-renewal,and differentiation of male germ cells.Therefore,amh and dmrt1 cooperatively regulate self-renewal and differentiation of male germ cells in zebrafish.Significantly,this study unravels novel functions of amh gene in fish.