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 described the divergent functions of the two cyp11a paralogs in zebrafish. Cyp11a1 has been suggested to be required for early development, whereas cyp11a2 is a functional equivalent of human CYP11A1 and is essential for the initiation and maintenance of zebrafish interrenal steroidogenesis. To establish a model system with glucocorticoid and sex steroid deficiency, we developed zebrafish mutant lines by creating deletions in cyp11a2 gene using CRISPR/Cas9 genomic engineering approach. Homozygous mutant zebrafish larvae showed an upregulation of the hypothalamic-pituitary-interrenal (HPI) axis and interrenal hyperplasia. Furthermore, Cyp11a2-deficient zebrafish had a steroid profile with decreased glucocorticoid and 11-Ketotestosterone, the key active androgen in zebrafish. Downregulation of the glucocorticoid-responsive genes fkbp5 and pck1 and the androgen-responsive gene cyp2k22 indicated systemic combined steroid hormone deficiencies. Cyp11a2 homozygotes only developed into males with feminized secondary sex characteristics. Adult cyp11a2 null-allele mutant fish showed lack of natural breeding behavior and reduced sperm concentration, suggesting reproductive impairment in adult fish. Histological characterization of the testes revealed disorganized testicular structure and significantly decreased mature spermatozoa. This finding is 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, nanos2 and piwil1, markers of spermatogonia, were upregulated while sycp3 and odf3b, markers of spermatids, were downregulated in the testes of cyp11a2 homozygous mutants. The expression analysis is consistent with the histological results suggesting spermatogonia are the dominant cell type in the testes of cyp11a2 homozygous mutants. Our work demonstrates the crucial role of Cyp11a2 in interrenal and gonadal steroid hormone biosynthesis in zebrafish larvae and adults. It establishes an in vivo model allowing studies of systemic consequences of altered steroid hormone deficiency.
19 - 21 Sep 2019
European Society for Paediatric Endocrinology