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.
Supplementary Fig 1. Expression levels of gonadotropin genes and their receptors. The transcript levels of A) fshb and B) lhb were significantly upregulated in the pituitary of the cyp11a2-/- mutant adults (n=5). However, no significant changes in the expression of C) fshr and D) lhcgr were found in the testes of the cyp11a2-/- mutant adults (n=5). An unpaired t test was used for all analysis. *, p<0.05; **, p<0.005.
Supplementary Fig 2. Natural breeding behaviors are absent in cyp11a2-/- mutant zebrafish. A) cyp11a2-/- male adults lacked natural breeding ability to produce fertilised eggs when paired with wild-type females (n=10). B-C) cyp11a2-/- male adults showed impaired natural mating behavior with significantly reduced frequency and duration of intimate contacts compared to wild-type males in a 5-minute period (n=6). D) There was no difference in gonadosomatic index (GSI) between cyp11a2-/- mutant males and wild-type siblings (n=5). An unpaired t test was used for all analysis. ***, p<0.0005; ****, p<0.0001.
Supplementary Table 1. Oligonucleotide sequence of guide oligo
Supplementary Table 2. Zebrafish cyp11a2 homozygous mutants have no visual background adaption.