Research has shown that MTNR1A plays an essential role in the estrus cycle and seasonal reproduction changes in sheep. However, few people have focused on the DNA methylation of MTNR1A by season or photoperiod. In this study, using qPCR and Western blotting, we measured the MTNR1A expression in the hypothalamus of ovariectomized and estradiol-treated (OVX + E2) sheep under different photoperiod treatment conditions. Subsequently, the core promoter of the MTNR1A gene was identified, and its methylation level in sheep exposed to different photoperiod treatments was measured by pyrosequencing. The results showed that MTNR1A gene expression significantly differed between the short 42-day photoperiod (SP42) and the LP42 or combined SP-LP42 treatment groups (p < 0.05). In addition, we determined that the core MTNR1A promoter region was within 540 bp upstream of the transcriptional start site (TSS) and that the DNA methylation levels at CpG sites in the SP42 vs. LP42 and SP42 vs. SP-LP42 groups significantly differed. Finally, a significant negative correlation (p < 0.001) between gene expression and DNA methylation levels was revealed, suggesting that photoperiod may induce DNA methylation of the MTNR1A gene and thus change its expression. The findings provide valuable bases for the further study of seasonal reproduction in sheep.
Keywords: DNA methylation; hypothalamus; melatonin receptor 1A; photoperiod; promoter.