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Sex is determined by multiple factors derived from somatic and germ cells in vertebrates. We have identifiedamhy,dmrt1,gsdfas male andfoxl2,foxl3,cyp19a1aas female sex determination pathway genes in Nile tilapia. However, the relationship among these genes is largely unclear. Here, we found that the gonads ofdmrt1;cyp19a1adouble mutants developed as ovaries or underdeveloped testes with no germ cells irrespective of their genetic sex. In addition, the gonads ofdmrt1;cyp19a1a;cyp19a1btriple mutants still developed as ovaries. The gonads offoxl3;cyp19a1adouble mutants developed as testes, while the gonads ofdmrt1;cyp19a1a;foxl3triple mutants eventually developed as ovaries. In contrast, the gonads ofamhy;cyp19a1a,gsdf;cyp19a1a,amhy;foxl2,gsdf;foxl2double andamhy;cyp19a1a;cyp19a1b,gsdf;cyp19a1a;cyp19a1btriple mutants developed as testes with spermatogenesis via up-regulation ofdmrt1in both somatic and germ cells. The gonads ofamhy;foxl3andgsdf;foxl3double mutants developed as ovaries but with germ cells in spermatogenesis due to up-regulation ofdmrt1. Taking the respective ovary and underdeveloped testis ofdmrt1;foxl3anddmrt1;foxl2double mutants reported previously into consideration, we demonstrated that oncedmrt1mutated, the gonad could not be rescued to functional testis by mutating any female pathway gene. The sex reversal caused by mutation of male pathway genes other thandmrt1, including its upstreamamhyand downstreamgsdf, could be rescued by mutating female pathway gene. Overall, our data suggested thatdmrt1is the only male pathway gene tested indispensable for sex determination and functional testis development in tilapia. 

The impacts of androgens and glucocorticoids on spermatogenesis have intrigued scientists for decades. 11β-hydroxylase, encoded by cyp11c1 , is the key enzyme involved in the synthesis of 11-ketotestosterone and cortisol, the major androgen and glucocorticoid in fish, respectively. In the present study, a Cyp11c1 antibody was produced. Western blot and immunohistochemistry showed that Cyp11c1 was predominantly expressed in the testicular Leydig cells and head kidney interrenal cells. A mutant line of cyp11c1 was established by CRISPR/Cas9. Homozygous mutation of cyp11c1 caused a sharp decrease of serum cortisol and 11-ketotestosterone, and a delay in spermatogenesis which could be rescued by exogenous 11-ketotestosterone or testosterone, but not cortisol treatment. Intriguingly, this spermatogenesis restored spontaneously, indicating compensatory effects of other androgenic steroids. In addition, loss of Cyp11c1 led to undersized testes with a smaller efferent duct and disordered spermatogenic cysts in adult males. However, a small amount of viable sperm was produced. Taken together, our results demonstrate that cyp11c1 is important for testicular development, especially for the initiation and proper progression of spermatogenesis. 11-ketotestosterone is the most efficient androgen in tilapia.