METTL3-mediated m6A methylation regulates granulosa cells autophagy during follicular atresia in pig ovaries

Follicular atresia is a normal physiological event in mammals, yet its mechanism remains to be studied. Granulosa cell (GC) autophagy is closely associated with follicular atresia. The N6-methyladenosine (m6A) modification is the most common post-transcriptional modification in eukaryotes, but its role in follicular atresia is still unknown. In this study, the possible relationship amongst follicular atresia, GC autophagy and m6A modification was studied. Our results showed that the level of autophagy in GCs increased with the degree of follicle atresia, whereas the overall m6A level decreased. Rapamycin treatment induced atresia in vitro cultured follicles, whereas 3-Methyladenine inhibited follicular atresia. Progressed atretic follicle (PAF) GCs had significantly lower METTL3 levels and significantly higher FTO levels than healthy follicle (HF) GCs. Differential gene expression analysis of GCs in PAF and HF by RNA sequencing was showed that the autophagy-related genes ULK1, ULK2, ATG2A, and ATG2B were significantly elevated in the PAF. In cultured GCs, overexpression of METTL3 significantly decreased the mRNA level of ULK1, as well as the autophagy level, whereas knockdown of METTL3 by RNAi significantly increased the mRNA level of ULK1, as well as the autophagy level. Our results indicate that m6A modification can regulate autophagy in GCs and play a role in the process of porcine follicular atresia.