| N~6-methyladenosine(m6A)is the most abundant methylation modification in eukaryotic mRNA,and it is widely involved in a variety of physiological and pathological regulation processes in humans and animals.However,little have been known about the relationship between m6A and mammalian embryonic development until now.In recent years,as an ideal model animal,pigs have been paid more and more attention in biomedical research.However,reports about the function and mechanism of m6A in porcine early embryonic development are very rare.Therefore,based on the overexpression strategy,the mRNA of m6A methyltransferase METTL3 was injected into porcine pronuclear embryos by microinjection technique to induce an increase in m6A levels.Then,we explore the effects of increased m6A levels on the developmental efficiency,quality,lineage differentiation,apoptosis and autophagy of porcine preimplantation embryos.First of all,METTL3 mRNA obtained by in vitro transcription successfully induced an increase in m6A levels in porcine early embryos.METTL3 overexpression(OE)significantly decreased the blastocyst rate of porcine parthenogenetic activated(PA)embryos.Secondly,METTL3 OE significantly increased the expression level of NANOG,and reduced the levels of CDX2,ELF5 and TFAP2C mRNA.In addition,the expression levels of CDX2 protein were also significantly decreased.Cell number analysis revealed that there was no significant difference in the ratio of ICM cells to TE cells between the OE group and the control group,but the total cell number,TE cell number and ICM cell number in the OE group were significantly lower than those in the control group.Finally,there were no significant differences in the expression levels of apoptosis-related factors between the OE group and the control group.However,the mRNA and protein level of an autophagy core gene ATG5 significantly decreased.In addition,METTL3 OE had no influence on the expression of autophagy marker LC3,but significantly decreased the levels of LC3-II protein.In conclusion,METTL3 OE successfully increased m6A levels in porcine early embryos,reduced blastocyst rate and quality,disrupted embryonic lineage differentiation,and inhibited autophagy,indicating that m6A plays an important role in the development of porcine early embryos.The specific mechanism underlying METTL3 overexpression inhibiting early embryonic development needs to be further studied in future. |
