Investigating Effects Caused By Cryopreservation On Bovine MⅡ Oocytes’ Epigenomes And Their Subsequent Development

The oocyte is an important component of mammalian germplasm resources. In vitroculture and operation of animal eggs have been extensively used in somatic cell nucleartransplantation and clinical treatment of infertility. These eggs can also be applied toresearches about mechanisms of the oocyte maturation and early embryonic development.Oocyte cryopreservation makes it possible to get enough eggs we need without the limit oftime and conditions. Whenever and wherever, oocytes cryopreserved can be used forproduction of transgenic cloned animals, human in vitro fertilization and intracytoplasmicsperm injection, so it is of great significance to support human assisted reproductive, keep thegood animal germplasm resources and protect endangered species etc. Epigeneticmodifications occurred during eggs and early embryonic development are importantreprogramming events, which is necessary to erase epigenetic memory and reach to anembryonic pluripotent state. Epigenetic events are easily affected by environmental factors.Oocyte cryopreservation as a strong physical stimulation may affect the stability of epigeneticreprogramming, which will interfere with normal embryonic development. Epigeneticmodifications include DNA methylation, histone modification, genomic imprinting and so on.In this study, with the cryotop vitrification method, bovine MⅡ oocytes werecryopreserved. Then effects of oocyte cryopreservation on epigenetic modification and earlyembryo development were examined. After thawing, the oocytes’ recovery time and activationcondition were explored to improve the efficiency. Anti-oxidative resveratrol (Res) was alsointroduced during oocyte cryopreservation and warming to improve its development potential.On the one hand, through analyzing DNA methylation, histone modification, imprinted geneexpression in the cryopreserved eggs and embryos, the effects of cryopreservation onepigenetic modifications were evaluated. On the other hand, by examing the survival rate,cleavage rate, blastocyst rate, blastocyst cell count and apoptosis, as well as the change ofcytoskeleton and other indicators, effects of oocyte cryopreseration on embryo developmentwere evaluated. At the same time, effects of measures adopted to improve thecryopreservation efficiency were identified. The following results were achived: 1. The cryopreservation made widespread effects on DNA methylation, H3K9modification and expression of imprinted genes. Specifically, the level of H3K9me3decreased significantly (P <0.05) in eggs cryopreserved and before the8-cell embryo it wasall significantly lower than control group (C-group)(P <0.05). Prior to8-cell stage, embryosin cryopreservation group (T-group) had higher acH3K9level than C-group (P <0.05). As forDNA methylation level, it was significantly lower in the T-group than that of C-group (P<0.05). In addition, the expression level of imprinted genes XIST and SNRPN in blastocystsof T-group was significantly higher than the C-group, while H19was significantly lower thanthat of the C-group (P <0.05). Results from DNA methylation and H3K9changes showed thatthe embryonic genome in T-group approched to an excessive “open” state during the earlyembryo development. Genomic imprinting loss and imprinted gene expression disorders maybe associated with decreased DNA methylation level in the blastocyst stage.2. The cryopreservation reduced the cleavage rate and blastocyst rate, decreasedblastocyst cell number and increased apoptotic rate (P <0.05). The cytoskeleton stability couldbe disrupted by cryopreservation. In detail, the cross-linked network organized bymicrofilaments and microtubulins got reduced or even disappeared, while the distribution ofvimentin also decreased. After thawing of oocytes, immediate activation with ionomycin for1min without extra recovery achieved the most ideal effect, with which cleavage rate andblastocyst rate were74.0%and15.7%, respectively. However, the cleavage rate andblastocyst rate were only49.7%and7.0%, respectively, when these oocytes were recoveredfor2h and ionomycin for normal4min. In addition, the supplement of2μM antioxidantresveratrol during oocyte cryopreservation and thawing process could increase both thecleavage rate and blastocyst rate (87.6%vs73.7%;21.1%vs15.3%, P <0.05). Besides, theblastocyst cell number was increased and the apoptotic rate became lower (P <0.05).