Effects Of ROS Induced P53 Mediated Signaling Pathway On Early Embryonic Development In Mice

In recent years, embryo transfer technology, embryonic segmentation, and embryonic stem cell engineering greatly promoted the development of animal husbandry. However, application of these new technologies is restricted by embryonic source.During embryo developmentin vitro, the embryonic blocking phenomenon often occurs, and the underlying mechanism has not been clear so far.Our previous studies showed that during embryo culture in vitro, ROS is often produced and can cause embryonic oxidative stress damage. However, the exact mechanism of action is unclear.a tumor suppressor protein P53, as cell cycle and apoptosis regulators, is associated with oxidative stress response. However,it islittle known that p53 acts role on embryos. So we assume that P53 plays an important role in embryonic oxidative stress.In the study, we will research the regulation role of signaling pathways between the ROS and P53, and further detect mechanisms of suppression of embryo development by ROS. Therefore, this study made the following experiment:Experiment One:induction of oxidative stress response in embryos by hydrogen peroxide (H2O2)We explore the effect of the different concentrations of H2O2 and various concentrations of antioxidant NAC on embryo development, and screen the optimal embryonic oxidative stress model for next study. Our result showed that the 4-cell embryo and blastocyst development rate significantly decrease with increasing concentrations (25μM,50μM,100μM) of hydrogen peroxide, and up to 50μM H2O2 concentration has reached a tipping point for inhibiting embryo development. Adding different concentrations (1mM、5mM、25mM) of NAC to the medium containing 50μM H2O2, we found that 4-cell embryo and blastocyst development rate of the 5mM NAC group was obviously higher than those of other groups, indicating 5mM concentration as optimal antioxidant contrations. Further, we find that 50μM H2O2 can increase ROS level in embryos and supplement with 5mM NAC can reduce the ROS rise of embryos induced by H2O2. Meanwhile, the fluorescenceintensity of embryos in H2O2 treatment group is also higher than those in H2O2+NAC group after embryos of all groups are stained with PI. To further assess the extent of embryonic oxidative damage, we detected lipid peroxidation products MDA in embryos, and found MDA level in embryo exposed to H2O2 signanificantly rise. All together, H2O2 can cause oxidative stress and inhibit embryo development by inducing ROS and MDA production.Experiment two:Effect of ROS induced P53 signaling on embryonic development.In the study, we analyzes the impact of ROS on the P53 gene expression and its regulation of cell cycle-related genes and P53-ser15 expression. Our results indicated that hydrogen peroxide can activate gene P53 and lead cell cycle gene such as, cdc2, CDK4 and CDK6 gene down-regulation. Then,we further detect the P53-ser15 and P53 protein expression in embryos by western blot, and find H2O2 can cause P53-serl5 phosphorylation protein and the corresponding P53 protein expressionincrease. In order to further investigate whether the changes of above genesby ROS ismediated by P53, we added P53 inhibitor PFT-a to the above experiment groups, and found that P53 and P53-ser15 phosphorylation protein expression was significantly inhibited. At the same time, we also found that GADD45a and P21 mRNA expression levels was significantly down-regulated, and cell cycle gene cdc2 is elevated, suggesting these genes are directly regulated by P53. There fore, we presumed to ROS-induced oxidative stress in embryos may be mediated by the P53.Experiment three:Effect of P53 on early embryos redox.In the study, to explore effect of P53 on redoxof embryos, we research oxidase and anti-oxidase expression levels of embryos exposed to PFT-a or H2O2 by qPCR and western blot analyse. Our results suggest that inhibition of p53 expression increase ROS levels, and suppress antioxidant enzymes SOD, GPX expression. Meanwhile, we also find that for embryo without oxidative stress, low level of P53 can increase antioxidant enzymes SOD and GPX expression, and has no effect on oxidase BAX. PUMA expression. In addition, when embryos suffer from oxidative stress, p53 expression is highly induced, leading to suppression of antioxidant enzymes SOD, GPX expressions and activation of pro-oxidant enzymes BAX. PUMA expression.Together, our researchreveals that H2O2 can induce oxidative stress ofearly embryo and suppress the embryos development in vitro by regulating P53 expression, which further effects the expression of its some downstream genes such as, cell cycles genes(P21,cdc2,CDK4),antioxidant enzymes (SOD, GPX, SESN2) and pro-oxidant enzymes (BAX, PUMA).