Comparision Of DNA Methylation In The Oct4 Promoter Region Between Mouse IVF And In Vivo Derived Preimplantation Embryos

DNA methylation,one of the major epigenetic modifications,is essential for mammalian normal development.The DNA methylation pattern is stable in somatic cells,but de novo methylation and demethylation,namely reprogramming,occur in early embryos.Oct4 is one of the critical pluripotency regulators and plays key roles in the establishment and maintenance of pluripotency during normal embryogenesis. So the promoter region of Oct4 was chosen as targets to determine methylation patterns in somatic cells,germ lines and early mouse embryos by using bisulfite sequencing,and to analyze the difference of methylation between in vivo and IVF embryos.All materials for analysis were amplified by nested PCR after being mixed with low melting point agarose and modified with sodium metabisulfite.The entire PCR products were purified,and then ligated into the PGEM-T Easy plasmid for sequencing.The results were as the following:(1)The promoter of Oct4 was hypermetylated in somatic cells(73.8±5.9%,mean±S.D.),significantly higher than that of germ cells(P＜0.01).There was a distinct difference(P＜0.05)between the methylation level of the two kinds of germ cells,with sperm being mildly methylated(18.75±4.1%)and oocytes essentially unmethylated(2.5±1.4%).(2)The Oct4 promoter region displayed hypomethylation pattern in in vivo derived embryos, and the pattern was gradually lost as the embryos development from 2-cell (5.63±2.5%)to 4-cell(4.38±2.0%),8-to16-cell(3.75±1.4%),morulae(1.88±0.2%), but without significant changes(P＞0.05).After this stage,de novo methylation took place and the methylation status was recovered to 6.88±2.4%in blastocysts.(3) There were similar methylation patterns in IVF embryos compared to those in in vivo ones.But the methylation level was different,respectively 2-cell(11.25±4.5%), 4-cell(6.25±2.1%),8-tol6-cell(5.63±3.7%),morulae(1.88±0.2%),blastocysts (7.5±2.4%).(4)The methylation value was higher at each stage in IVF embryos compared to those in in vivo embryos,but without significant difference except for 2-cell stage embryos(P＜0.05).These results suggest that an efficient reprogramming of DNA methylation occurs during early development in IVF embryos too,and the IVF embryos can develop to blastocysts.However,the DNA methylation level is higher in IVF embryos than that in in vivo derived embryos,especially at 2-cell stage.Possibly, this is the reason of the rate of IVF embryos developing to term lower than that in vivo derived embryos.