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Cytochrome P450 side-chain cleavage enzyme, encoded by the  CYP11A1  gene, catalyzes the first and rate-limiting step of steroid hormone biosynthesis. Previous morpholino-knockdown studies in zebrafish suggested  cyp11a2  is a functional equivalent of human  CYP11A1  and is essential for interrenal steroidogenesis in zebrafish larvae. The role of Cyp11a2 in adult zebrafish, particularly in gonadal steroidogenesis, remains elusive. To explore the role of Cyp11a2 in adults, we developed zebrafish mutant lines by creating deletions in  cyp11a2  using the CRISPR/Cas9 genomic engineering approach. Homozygous  cyp11a2  mutant zebrafish larvae showed an upregulation of the hypothalamic–pituitary–interrenal axis. Furthermore, these Cyp11a2-deficient zebrafish demonstrated profound glucocorticoid and androgen deficiencies.  Cyp11a2  homozygotes only developed into males with feminized secondary sex characteristics. Adult  cyp11a2 −/−   mutant fish showed a lack of natural breeding behaviors. Histological characterization revealed disorganized testicular structure and significantly decreased numbers of mature spermatozoa. These findings are further supported by the downregulation of the expression of several pro-male genes in the testes of  cyp11a2  homozygous zebrafish, including  sox9a ,  dmrt1  and  amh . Moreover, the spermatogonia markers  nanos2  and  piwil1  were upregulated, while the spermatocytes marker  sycp3  and spermatids marker  odf3b  were downregulated in the testes of  cyp11a2  homozygous mutants. Our expression analysis is consistent with our histological studies, suggesting that spermatogonia are the predominant cell types in the testes of  cyp11a2  homozygous mutants. Our work thus demonstrates the crucial role of Cyp11a2 in interrenal and gonadal steroidogenesis in zebrafish larvae and adults.

The P450 side-chain cleavage enzyme Cyp11a2 facilitates steroidogenesis in zebrafish