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

• Published in: Theriogenology 2018-11, Vol.121, p.160-167
• Main Authors: Wang, Yan-Kui, Yu, Xiao-Xia, Liu, Yun-Hua, Li, Xuan, Liu, Xiao-Man, Wang, Pei-Chao, Liu, Shuai, Miao, Jia-Kun, Du, Zhi-Qiang, Yang, Cai-Xia
• Format: Article
• Language: English
• Subjects: Animals; Betaine - pharmacology; CDC2 Protein Kinase - genetics; CDC2 Protein Kinase - metabolism; Cycloleucine - pharmacology; DNA Methylation; m6A; Meiosis; Meiosis - genetics; Oocyte; Oocytes - cytology; Oocytes - drug effects; Oocytes - growth & development; Pig; Spindle; Swine - embryology
• ISSN: 0093-691X; 1879-3231
• DOI: 10.1016/j.theriogenology.2018.08.009

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. •Endogenous m6A writers Mettl3 and Wtap, eraser Fto elevated their transcript levels in maturing pig oocytes.•Global m6A modification level significantly increased in maturing pig oocytes.•Betaine treatment significantly boosted m6A level, but unaffected oocyte meiosis and subsequent development ability.•Cycloleucine treatment significantly reduced m6A level, that impaired meiotic maturation and subsequent development potency.•Cycloleucine could down-regulate Lin28 and disturb MPF-regulated chromosome/spindle organization to affect porcine oocytes.