m6A RNA methylation dynamics during in vitro maturation of cumulus-oocyte complexes derived from adult or prepubertal sheep

Purpose: N6-methyladenosine (m6A) is the most prevalent base epigenetic modification within eukaryotic mRNAs. It participates in post-transcriptional regulation, including maternal RNA maintenance and decay in mouse oocytes and during maternal-to-zygotic transition. The landscape in other mammalian species remains largely unexplored. The present work analyzed m6A dynamics in sheep cumulus oocyte complexes (COCs), during in vitro maturation. To explore potential relationships with oocyte developmental competence, a previously established model consisting of oocytes derived from adult and prepubertal sheep was adopted. Methods: m6a dynamics were analyzed in terms of m6A RNA methylation abundance in cumulus cells (CCs) by colorimetric assay and expression of key m6A methylation-related proteins (METTL3, METTL14, METTL16, VIRMA, YTHDC1, YTHDC2, YTHDF2, YTHDF3, ALKBH5, and FTO) in both cumulus cells and oocytes by real-time PCR. Results: We report the dynamics of m6A in sheep COCs, and reveal alterations in both oocytes and cumulus cells derived from prepubertal donors. These changes were observed in terms of m6A RNA methylation levels and transcript dynamics of several m6A methylation-related proteins. Notably, our study shows that dysregulations occur after IVM. Conclusion: Overall, this work describes for the first time the dynamics of m6A in sheep COCs and uncovers the involvement of m6A RNA methylation in oocyte developmental potential.