DNA methylation plays a critical role in testicular maintenance, but not in sex determination of male tilapia Oreochromis niloticus

The significance of DNA methylation in fish sex determination and differentiation is increasingly being recognized. Exploring the role of DNA methylation in sex determination and differentiation is advantageous for the advancement of sex control techniques in aquaculture. In this study, the global DNA methylation levels of XX and XY gonads at different developmental stages of tilapia (Oreochromis niloticus) was assessed using an anti-5-methylcytosin (5-mC) antibody. The results indicated that the difference of global DNA methylation levels between XX and XY gonads was established after morphological sex differentiation had occurred at 30 dah (days after hatching). The bisulfite sequencing PCR (BSP) analysis revealed a significant disparity in the DNA methylation level of cyp19a1a between the XX and XY gonads after 30 dah, rather than before. The Dnmts (DNA methyltransferases) inhibitor 5-aza-2′-deoxycytidine (5-aza-dC, 20 μg/g diet), the aromatase inhibitor letrozole (AI, 150 μg/g diet), and 17β-estradiol (E2, 150 μg/g diet) were employed for treating XX and XY tilapia in order to investigate the involvement of DNA methylation in sex determination and differentiation. Treatment of XX and XY fish with 5-aza-dC from 5 to 50 dah did not result in any sex reversal, either female-to-male or male-to-female. For XX tilapia, both the use of AI alone and its combination with 5-aza-dC resulted in sex reversal from female-to-male when treated from 30 to 60 dah. This indicates that the addition of 5-aza-dC did not prevent the sex reversal from female-to-male induced by AI treatment, which is different from the results observed in zebrafish (Danio rerio). Intriguingly, treatment of XY fish from 30 to 90 dah with either E2 or 5-aza-dC alone resulted in 6% and 0% sex reversal, respectively, while simultaneous treatment with 5-aza-dC and E2 resulted in significant decrease of cyp19a1a methylation level and increase of cyp19a1a expression and 65% sex reversal. Taken together, these results suggest that DNA methylation plays a critical role in gonadal maintenance, but not in sex determination in tilapia. This study deepens our understanding of the role of epigenetic modifications in fish sex differentiation and maintenance, providing an approach to achieve sex reversal in testis-differentiated male fish and improving the available methods for sex control in aquaculture.