High temperature induced masculinization of zebrafish by down-regulation of sox9b and esr1 via DNA methylation

Elevated temperature could influence the sex differentiation by altering the expression of sex-related genes in fish.However, the underlying mechanisms by which the gene expression is altered remain poorly understood.Here, we aimed to explore the role of DNA methylation in sex differentiation of zebrafish(Danio rerio) in response to elevated temperature.The results showed that high temperature(33℃) exposure of fish from 20 to 30 days post fertilization(dpf), compared to normal temperature(28℃), resulted in male-biased sex ratio and decreased expression of female-related genes including cyp19a1a, sox9b and esr1.Meanwhile, the expressions of DNA methyltransferases dnmt3a1 and dnmt3a2, and the DNA methylation levels in sox9b and esr1 promoter were significantly increased by high temperature, strongly implying that DNA methylation is involved in high temperature-induced masculinization of zebrafish.Co-treatment with 5-aza-2-deoxycytidine(a DNA methylation inhibitor) attenuated the high temperature-induced masculinizing effect, recovered the expression of esr1 and sox9b, suppressed the transcription of dnmt3a1 and dnmt3a2, and decreased the methylation of esr1 and sox9b promoter, further confirming that DNA methylation plays an important role in high temperature-induced masculinization of zebrafish.Furthermore, the methylation of sox9b promoter decreased the enrichment of transcription factor CREB(cAMP-responsive element binding proteins).Overall, these findings suggest that high temperature induce masculinization of zebrafish by down-regulation of femalerelated genes via DNA methylation, providing a new insight in understanding the epigenetic mechanism of thermal-mediated sex differentiation in fish.