Methylation Imprinting Of Postovulatory Aging Mouse Oocyte And ICSI Embryo

Genomic imprinting plays important roles in the regulation of fetal growth, development,placental function,and postnatal behaviour.Once the methylation imprints are established,they will be maintained,even during the epigenetic reprogramming after fertilization,while genomic DNA undergoes global demethylation.Hence,parent-specific methylation imprints may play an important role during genomic reprogramming,which is an essential factor that determines developmental potentials.However,the disrupted methylation imprinting results in abnormal development and imprinting defects disease. Postovulatory oocyte aging and intracytoplasmic sperm injection(ICSI) manipulations may interfere in the methylaiton imprinting.We carried out the following experiments to assess these problems.Experiment 1:Prolonged residence of postovulatory oocyte in the oviduct or prolonged culture in vitro can lead to oocyte aging,which significantly affects pre- and post-implantation embryo development.In this study,we employed bisulfite sequencing and COBRA methods to investigate the DNA methylation status of differentially methylated regions(DMRs)of Snrpn and Peg1/Mest,two maternally imprinted genes,in postovulatory oocytes aged in vivo and in vitro. The results showed that Snrpn DMR was clearly demethylated in oocytes aged in vivo at 29 h post-hCG and in denuded oocytes aged in vitro for the same time period.However,Peg1/Mest did not show any demethylation in all aged groups at 29 h post-hCG.These data indicate that oocytes undergo time-dependent demethylation of Snrpn DMR during the process of postovulatory aging.Experiment 2:Since the first baby derived from intracytoplasmic sperm injection was successfully given birth in 1990s,this technology has been the most effective method to overcome male infertility problems.Now,people concern more and more about the safety of assisted reproductive technologies (ARTs)including in vitro fertilization and intracytoplasmic sperm injection. Especially many documents recently been reported indicate that ARTs associating with imprinting defects.It is largely unknown that imprinted gene DNA methylation profiles of embryo derived from intracytoplasmic sperm injection.In order to assess the effects of intracytoplasmic sperm injection procedures on imprinted methylation status of individual embryo,we employed genomic bisulfite sequencing to investigate the methylaiton status of H19 differentially methylation domain,which is a paternally imprinted and maternally expressed gene.In this study,different experimental groups were established to obtain embryos from in vivo,in vivo fertilization zygote and in vitro culture,in vivo MII oocytes and ICSI and IVM combining ICSI, respectively.The results showed that H19 methylation pattem of in vivo embryo inherits faithfully from that of parental gametes.In vitro culture embryo with KSOM didn't affect the normal methylaiton of H19.In contrast to in vivo and in vivo fertilization zygote embryo group,ICSI gives rise to loss methylation of H19.Similarly,IVM combining with ICSI also resulted in demthylaiton and abnormal methylaiton patterns of H19.Compared to previous reports utilizing pools of embryos,our study enables us to emphasize a high individual variability of blastocysts in the H19 differentially methylation domain.These results provide practical cues for clinic use of ICSI.We concluded that in vitro environments or ARTs manipulation result in abnormal methylation imprint of gamete and embryo.These problems should be taken into account when clinic manipulation of gamete or embryo.