Reduced nucleic acid methylation impairs meiotic maturation and developmental potency of pig oocytes

Oocyte meiosis is a complex process coordinated by multiple endocrinal and molecular circuits. Recently, N⁶-methyladenosine (m⁶A) epigenetic modification on RNA is revealed to be important for meiotic maturation. However, the molecular mechanism of how m⁶A modification exerts its effect on oocyte maturation is largely unknown. Here, we showed that endogenous m⁶A writers (Mettl3 and Wtap) and eraser (Fto) elevated their transcript levels during meiotic maturation of pig oocytes. From germinal vesicle (GV) to metaphase II (MII) stages, global m⁶A level significantly increased, and existed mostly in ooplasm. Methyl donor (betaine, 16 mM) treatment of porcine cumulus-oocyte complexes (COCs) during in vitro maturation (IVM) significantly boosted nucleic acid m⁶A level within oocytes, but unchanged meiotic process and oocyte subsequent development. By contrast, methylation inhibitor (cycloleucine, 20 mM) reduced nucleic acid m⁶A level, and significantly decreased the germinal vesicle breakdown (GVBD) rate, the extrusion rate of the first polar body, and the cleavage and blastocyst rates of parthenotes. In addition, in cycloleucine-treated oocytes Wtap increased but Lin28 decreased their abundances significantly, along with the higher incidence of spindle defects and chromosome misalignment. Furthermore, pT161-CDK1 protein level in pig oocytes was confirmed to be decreased after cycloleucine treatment for 24 h. Taken together, chemical induced reduction of nucleic acid m⁶A methylation during pig oocyte meiosis could impair meiotic maturation and subsequent development potency, possibly through down-regulating pluripotency marker Lin28 mRNA abundance and disturbing MPF-regulated chromosome/spindle organization.