The Effect Of Tet Proteins On DNA Methylation And Hydroxymethilation Of Vitrified Oocyte

Oocyte vitrification has extensive application in the field of embryo engineering and can be used it in the genetic resources of fine livestock preservation,the field of human reproductive medicine,thus providing these techniques with more sufficient resources of oocyte.However,oocyte have to experience the violent surrounding stress in vitro in the process of vitrified-thawed,which can lead to oocyte's DNA hypomethylation,DNA and cellular ultrastructure damage,these changes make oocyte expose to huge challenge when it is thawed,fertilized and development.This study due to the previous research result that the level of oocyte DNA methylation after being vitrified was significantly decreased,through immunofluorescent staining,qRT-PCR and RNAi techniques,exploring the dynamic change of active demethylation proteins TET1/2/3(ten-eleven-translocation 1/2/3),5-mC(5-methylcytosine)and 5-hmC(5-hydroxymethycytosine)after vitrified and thawed.Meanwhile,in order to verified the influence of TET3 proteins on 5-mC and 5-hmC in the process of vitrified-thawed,we change the activity and level of TET3 protein that mediate active demethylation by used two small molecular active regulator(Vitamin C and dimethyloxaloyl glycine)and siRNA(short small RNA).Acquiring research results here:1,After being vitrified and thawed for 0h,1h,2h,we detected the level of 5-mC and 5-hmC by immunofluorescent staining,both have got significant increase.Moreover,TET1/2 did not change during this process(P>0.05).The level of TET3 protein was significantly accumulated after being thawed for 2h.The qRT-PCR results showed that the relative gene expression of TET1 yielded no significantly change(P>0.05)during 2h after being vitrified-thawed;The TET2/3 was also no significantly change in the last hour,but their gene relative expression yielded significantly increase at 2h(P<0.05).2,VC was added to the process of vitrification and recovery,in two hours,Relative to blank group,VC was not able to significantly improve the level of TET3 relative gene expression,and reduce the levels of 5-mC and 5-hmC(P>0.05).Moreover,DMOG was also added the solutions of vitrification,thawing and recovery.t was similar with the groups of VC-treatment,the levels of 5-mC and 5-hmC and the relative gene expression and protein expression of TET3 were affected relative to blank group,(P>0.05).Then we transferred TET3 siRNA into GV oocytes by microinjection and after in vitro maturation to M?and implement vitrification.The results showed that TET3 gene was suppressed significantly(P<0.01),the level of 5-mC was no change,but the 5-hmC yielded significant accumulated(P<0.05)between the group of interference and noninterference.Conclusion: The DNA methylation and hydroxymethylation in vitrified M?oocyte were significantly decreased.Among TET proteins,the relative gene expression and proteins of TET3 yielded significantly increased after being vitrified.Therefore,the reason why M?oocytes DNA methylation and hydroxymethylation was decreased is TET3 as active methylation protein increased after being vitrified and thawed.By using the activator(VC)and depressor(DMOG)of TET3,we found that these molecular have no effect on the status of DNA methylation and hydroxymethylation of M? oocytes after being vitrified.The microinjection of TET3 siRNA has influence on the level of DNA hydroxymethylation after being vitrified which makes the level of M ? oocytes' hydroxymethylation changed dramatically.