Effect Of Vitrification On In Vitro Development And Imprinted Gene Grb10 In Mouse Embryo

Objectives:1.Establish appropriate vitrification and thawing conditions;2.Investigate the effect of vitrification on the in vitro development of embryos;3.Investigate the effect of vitrification on the embryo DNA methylation level;4.Investigate the effect of vitrification on the transcription level of embryo imprinted gene Grb10 and DNA methylation level.Methods: The experiment was divided into three groups,natural mating 3.5 dpc(days post coitus)blastocyst in vivo was the control group;8-cell embryo processed by vitrification growing to blastocyst and 8-cell embryo directly cultured in vitro to the blastocyst were set as experimental groups.among them,natural mating 3.5 dpc blastocyst in vivo was the in vivo group,in vivo 8-cell embryo cultured in vitro to blastocyst was the in vitro group,8-cell embryo processed by vitrification treatment growing to blastocyst was the frozen group.After successful vitrification,calculated the blastocyst rate and hatching rate of the experimental group embryos;calculated the fluorescence intensity of each group's 5-Me C methylation by immunofluorescence technique;through selecting the appropriate "relative standard preparation",established the relative standard curve after series of 5-fold dilutions,detected the transcription level changes of each group's imprinted gene Grb10 with real-time PCR;through the modification results of bisulfite on unmethylated cytosine,conducted PCR sequencing and sequence blast for the modified products,detected the methylation modification changes of imprinted gene Grb10 CGI1(Cp G island)in a single blastocyst.Results: Vitrification results showed that there is no significant difference between frozen group and in vitro group on blastocyst rate,the hatching rate of frozen group was significantly lower than the in vitro blastocyst group;embryo immunofluorescence results showed that the 5-Me C fluorescence intensity of the frozen group was lower than in vitro and in vivo group.Real-time PCR analysis showed that the transcription level of imprinted gene Grb10 in frozen group decreased compared with the in vivo and in vitro group,there was no significant difference between in vitro and in vivo group of tanscription level of imprinted gene Grb10;after a trace amount of bisulfite modification,sequencing showed that the methylation level of imprinted gene Grb10 CGI1 in the frozen group was obviously lower,and the methylation level of the in vitro group was even lower.Conclusions: Vitrification may reduce the in vitro development of 8-cell embryos and the DNA methylation of frozen blastocyst,immunofluorescence showed that genome DNA methylation level of the frozen blastocyst was significantly lower than in vivo blastocyst.Vitrification affect the transcription of imprinted gene Grb10 expressed in frozen embryo,both vitrification group and the in vitro group showed low methylation status in CGI1 area of imprinted gene Grb10,indicating that in addition to the methylation control,the expression of imprinted gene Grb10 may have other regulatory factors.