Function And Regulatory Mechanism Of Neuronatin(NNAT) During Porcine Oocyte Postovulatory Aging

Oocyte aging occurs if oocytes are not fertilized in time after ovulation.Oocyte aging significantly affects fertilized embryo development.DNA methylation plays an important role in regulating many physiological behaviors;however,few studies have focused on the changes of DNA methylation during oocyte aging.The types of oocyte aging are divided into pre-ovulatory or post-ovulatory aging,and in vivo or in vitro aging.In this study,pig oocytes at M? stage after maturation are selected to study the changes of DNA methylation and the function of related genes during oocyte aging.Our main results are shown as follows:1.Detection the expression of imprinted genes(GRB10,IGF2,PEG1,PEG10,H19 and NNAT),the maternal effect genes(BRG1,ZAR1,BMP15,TET3)and stem cell pluripotency genes(POU5F1,SOX2,CDX2)by quantitative Real-time Polymerase Chain Reaction(qRT-PCR)during porcine oocyte aging in vitro.We found that different aging time(24 h or 48 h)and cumulus cells had no effects on gene expression in porcine oocyte.So our studies focused on the oocyte aging for 24 h in vitro without cumulus cells in the following experiments.2.After comparing the expression of imprinted genes during oocyte aging,we found that the expression of PEG1 and NNAT in oocytes aged 24 h and 48 h in vitro were down-regulated,and there was no significant difference between oocytes aged for 24 h and 48 h.It has been proved that oocyte quality determined the embryo's developmental potential and oocyte aging was a key factor to affect oocyte quality.We found that 45.0%fresh oocytes could develop to blastocyst after parthenogenetic activation,however,there were only 14.0% oocytes aged for 24 h in vitro that developed to blastocysts.Next,we detected the expression of these imprinted genes in blastocysts from fresh oocytes and oocytes aged for 24 h.The results showed that the expression of NNAT was decreased significantly in blastocysts from aged oocytes.3.To detect DNA methylation pattern of NNAT and H19 in aged oocytes and blastocysts by parthenogenetic activation,we employed both bisulfite DNA sequencing(BSP)and combined bisulfite restriction analysis(COBRA)methods.The data showed the same results that NNAT was hypermethylated in aged oocytes.However,H19 has no significant change.BSP and COBRA data showed that oocyte aging had no effect on DNA methylation pattern of NNAT and H19 in parthenogenetic blastocysts.4.After detecting the expression of NNAT protein in aged oocytes,we found that the exprssion of NNAT protein was decreased significantly in aged oocytes by immunofuorescence staining.5.To determine whether oocyte aging affect glucose transport,2-NBDG was used to track glucose transport in oocytes.The results showed that 2-NBDG uptake was reduced with the time of aging,which indicated that oocyte aging blocked glucose transport in oocytes.6.To determine whether NNAT affect glucose transport in oocytes,we injected NNAT antibody into the cytoplasm of fresh M? oocytes to block the function of NNAT.The fluorescence intensity of 2-NBDG in oocytes injected with NNAT antibody was significantly decreased compared to oocytes injected with IgG,suggesting that the loss of NNAT affected glucose transport in aged oocytes.These results suggested that the expression of NNAT was down regulated by elevating DNA methylation,which affected oocyte quality by decreasing the ability of glucose transport in aged oocytes.