The role of DNA methylation reprogramming during sex determination and sex reversal in the Pacific oyster Crassostrea gigas

DNA methylation is instrumental in vertebrate sex reversal. However, the mechanism of DNA methylation regulation regarding sex reversal in invertebrates is unclear. In this study, we used whole genome bisulfite sequencing (WGBS) to map single -base resolution methylation profiles of the Pacific oyster, including female -tomale (FMa-to-FMb) and male -to -female (MFa-to-MFb) sex reversal, as well as sex non -reversed males and females (MMa-to-MMb and FFa-to-FFb). The results showed that global DNA methylation levels increase during femaleto -male sex reversals, with a particular increase in the proportion of high methylation levels (mCGs >0.75) and a decrease in the proportion of intermediate methylation levels (0.25 < mCGs <0.75). This increase in DNA methylation was mainly associated with the elevated expression of DNA methylase genes. Genome-wide methylation patterns of females were accurately remodeled to those of males after sex reversal, while the opposite was true for the male -to -female reversal. Those findings directly indicate that alterations in DNA methylation play a significant role in sex reversal in Pacific oysters. Comparative analysis of the DNA methylomes of pre- and post- sex reversal gonadal tissues (FMb-vs-FMa or MFb-vs-MFa) revealed that differentially methylated genes were mainly involved in the biological processes of sex determination or gonadal development. However critical genes such as Dmrt1, Foxl2 and Sox -like, which are involved in the putative sex determination pathway in Pacific oysters, showed almost an absence of methylation modifications, varying greatly from vertebrates. Additionally, comparative analysis of the DNA methylomes of sexual reversal and sex non -reversal (FMa-vs-FFa or MFa-vs-MMa) revealed that heat shock protein genes, such as Hsp68-like and Hsp70B, were important for the occurrence of sex reversal. These findings shed light on the epigenetic mechanisms underlying the maintenance of gonadal plasticity and the reversal of organ architecture in oysters.