Abstract

Understanding the gonadal differentiation and development of fish is critical for both basic biology and aquaculture. Sox9 has been associated with the determination and subsequent development of fish testes. In teleosts, genome duplication events produced two co-orthologs of the sox9 gene, known as sox9a and sox9b . However, direct evidence of their exact roles at the protein level remains limited. In this study, the bioactive recombinant Sox9a protein from the olive flounder Paralichthys olivaceus , an important mariculture fish in Asia, was obtained. Expression analysis showed that the flounder Sox9a protein exhibits sexually dimorphic expression, with high levels observed in testicular Sertoli cells and low levels in ovarian oocytes. In vitro assays demonstrated that Sox9a suppressed cyp19a and upregulated dmrt1 , which was accompanied by alterations in sex steroid levels, including 17β-estradiol (17β-E2), testosterone (T), and 11-ketotestosterone (11-KT). Furthermore, cell co-transfection showed that the flounder Sox9a can inhibit the Nr5a2-mediated cyp19a transcription and cooperate with Dmrt1 and Amh to enhance this repression. Subsequent intraperitoneal injection of the recombinant Sox9a protein in females downregulated the female-biased genes cyp19a , foxl2 , and nr5a2 , and upregulated the male-biased gene dmrt1 , as well as the HPG axis regulator gene arβ . Histological analysis revealed ovarian developmental alterations in 12.5% (1/8) of the fish that were treated with the protein. These results provide novel evidence that Sox9a may act as a transcriptional repressor of cyp19a in fish. This regulation is likely to be associated with changes in the production of sex hormones and may contribute to alterations in ovarian development.