WGBS of embryonic gonads revealed that long non-coding RNAs in the MHM region might be involved in cell autonomous sex identity and female gonadal development in chickens.

DNA methylation plays a key role in sex determination and differentiation in vertebrates. However, there are few studies on DNA methylation involved in chicken gonad development, and most focused on male hypermethylated regions (MHM). It is unclear whether there are specific differentially methylated regions (DMRs) in chicken embryonic gonads regulating sex determination and differentiation. Here, the DNA methylation maps showed that the difference of DNA methylation level between sexes was much higher at embryonic day 10 (E10) than that at embryonic day 6 (E6), and the significant differentially methylated regions at both stages were mainly distributed on the Z chromosome, including MHM1 and MHM2. The results of bisulphite sequencing PCR (BSP) and qRT-PCR showed hypomethylation of female MHM and upregulation of long non-coding RNAs (lncRNAs) whose promoter in the MHM region was consistent with the sequencing results, and similar results were in brain and muscle. In female sex-reversed gonads, the methylation pattern of MHM remained unchanged, and the expression levels of the three candidate lncRNAs were significantly decreased compared with those in females, but were significantly increased compared to males. The fluorescence in situ hybridization (FISH) results also showed that these lncRNAs were highly expressed in female embryonic gonads. The results of methyltransferase inhibitor and dual-luciferase reporter assay suggest that lncRNA expression may be regulated by DNA methylation within their promoters. Therefore, we speculated that MHM may be involved in cell-autonomous sex identity in chickens, and that lncRNAs regulated by MHM may be involved in female sexual differentiation.