Roles Of Autophagy During Postovulatory Aging Of Mouse Oocyte And Roles Of XRCC1during DNA Demethylation In Mouse Preimplantation Embryo

In most mammals, mature oocytes after ovulatio undergo a process of deterioration in quality, which is called oocyte aging, if fertilization is delayed. The aging oocyte is not degenerate and is still fertilizability, which cause abnormity of embryo development and offspring. Oocyte aging involves a range of cellular and molecular changes, but the mechanism of oocyte aging is not well understood.In this experiment, we used immunofluorescence and immunoblotting to examine the autophagy levels during in vivo and in vitro oocyte aging. The process of oocyte aging that were treated with inhibitor and activator of autophagy was also investigated. The results showed that the autophagy levels in mouse oocytes were markedly increased during in vivo and in vitro postovulatory, and the levels of in vitro aging were gradually increased or decreased when the autophagy was artificially activated or inhibited, respectively. These results indicated that autophagy may be essential for the postovulatory aging.As a significant epigenetic modification, DNA methylation plays a very important role in maintaining genomic stability and regulating gene expression in mammals and flowering plants. It has been reported that active DNA demethylation both in plant and animal cells, and the mechanism of plant behind it is becoming clear. The base excision repair pathway has been shown to participate in the active DNA demethylation process in plants and zebrafish. So far, in mammals we have not found a real enzyme on the activation of DNA demethylation, and the active demethylation pathway is also controversial.In this experiment, we used immunofluorescence to examine the XRCC1levels during from oocytes to preimplantation embryos. Using microinjection techniques put the specific antibody of anti-XRCC1into the cells to block the XRCC1protein function, then detect the level of5-mC after cultured cells. The results showed that, XRCC1protein is expressed in mitotic and meiotic division interphase not in division stage, and colocalization with the nuclear DNA. After the microinjection of anti-XRCC1antibody into the GV oocytes, its GVBD rate decreased significantly. After the microinjection of anti-XRCC1antibody into the MⅡ oocytes, Its5-mC expression levels did not change significantly after cultured in vitro. The5-mC expression levels of2-cell embryos significantly increased by injection the zygotes, which led to its2-cell rate significantly reduced after the zygotes cultured in vitro. These results indicated that XRCC1may play an important role between the DNA demethylation and the growth and development of preimplantation embryos.