Dual cyp17a1/a2 knockout unveils paralog-specific steroid pathways in fish reproduction

Cyp17a is a key enzyme in steroidogenesis and reproduction in vertebrates. In teleosts, two paralogous genes, cyp17a1 and cyp17a2, have been identified, and while the potential impact of mutations in these genes have been studied, a comprehensive comparative analysis of their regulatory mechanisms in teleost reproduction remains lacking. In this study, we established a double mutant (dko) of cyp17a1 and cyp17a2 and conducted a transcriptome analysis of their gonads to systematically investigate the differences in steroidogenesis and gene expression between the two genes. Phenotypic analysis revealed that both cyp17a1^−/− and dko mutants exhibited a reduced gonadosomatic index, abnormal Leydig cell proliferation, and male infertility. RNA sequencing of the testes identified 2688 differentially expressed genes (DEGs), with significant variations between the mutants and wild-type males. KEGG enrichment analysis indicated that DEGs were enriched in pathways related to steroid biosynthesis, HIF-1, PPAR, and Hippo signaling. Furtherly, steroid profiling confirmed cyp17a1^−/−-specific androgen and estrogen deficiency (T, 11-KT, E1, and E2), while revealing dko-specific deficiencies in DHP, cortisol, and cortisone, alongside an overaccumulation of corticosterone, highlighting paralog-specific substrate preferences. These findings underscore the Cyp17a paralogs as critical regulators of testicular metabolic-transport networks, thereby offering insights into male infertility linked to steroid biosynthesis defects.