| With the continuous improvement of technology, in recent years, in vitro fertilization (IVF) technology is widely used in large-scale production of fine properties of livestock embryo, human infertility treatment, and so on. Despite researchers have made a great effort to optimize the medium and culture conditions, the culture condition can’t completely simulate the fertilization conditions in vivo, so the embryo development rate in culture systerm is difficult to achieve that in vivo. It is well showed that the effect of ROS on embryo development, however, the molecular mechanism is unclear. This study was focused on the molecular mechanisms of oxidative stress induced by oxidizer, AAPH and the recovery role of the antioxidant activity of glutathione GSH in mouse early embryos.1. The study about the effect of AAPH on early embryo development of mouse and its molecular mechanism.The zygotes and 2-cell were treated by 0.1、1.0、1.5、2.0mM AAPH. Results showed that zygotes treated by 1.5mM AAPH for 24h were blocked in 2-cell stage and most of embryos died after 48h. Meanwhile,2-cell treated by 1.5mM AAPH for 24h also blocked in 2-cell stage. Therefore, zygotes treated by 1.5mM AAPH for 24h were selected for the following study. A significant increase in the level of ROS was observed by DCFH-DA staining (P<0.05) and the expression of Gpx4、Gpx6、Prdx2 and genes which related to ROS were detected, compared to the control group, the expression of Gpx4、Prdx2 were increased, while Gpx6 were decreased (P<0.05), which shows that the oxidative stress model was successfully constructed.The mitochondrial membrane potential of embryo decreased significantly by JC-1 staining, the expression levels of genes associated with apoptosis (Bax、 Caspase8、Caspase3、Caspase9) was significantly increased (P<0.05) and early apoptotic signal was detected. The expression levels of genes associated with ZGA (Zygotic genome activation)Zscan4d、eIF-1A、Hspalb、H2afz was significantly decreased (P<0.01)2. The recovery test of GSH on early embryos treated by AAPHThe zygotes and 2-cell were treated by 1mM,2mM,3mM GSH respectively with 1.5mM AAPH. For treated zygote, all concentrations of GSH failed to recovery the arrested 2-cell embryos after 24h treatment, but significantly increased the first cleavage rate. The first cleavage rate is the highest after treating with 2mM GSH+1.5mM AAPH; When 2-cells were treated after 24h, some of embryos can released from the arrested, but did not develop to the blastocyst stage. Therefore, zygotes treated by 2mM GSH+1.5mM AAPH for 24h were selected for the following study, The results about the levels of ROS and the expression levels of genes associated with ROS show that compared with the control, the level of reactive oxygen species was still significantly increased(P<0.05).The mitochondrial membrane potential was significantly increased (P<0.05). In addition, earlier period apoptosis signal was not detected and supplementation of GSH did not reverse the expression tendency of ZGA related genes.When zygotes were treated by 0.5mM AAPH, only 8% of zygotes can develop to 4-cell, after added GSH,23.7% of zygotes can develop to 4-cell, but these embryos can not develop to the blastocyst stage.In conclusion, higher concentration of AAPH can cause an increase in the level of ROS, resulting in oxidative stress, mitochondrial membrane potential decreased and apoptosis. Finally, embryo development of mouse blocked in the 2- cell stage, which may cause by the failed of ZGA. At high level of ROS, GSH can not release embryos from arrested, but at low level of ROS the arrested rate of embryos can be reduced and the development rate of embryos could be improved. This study primarily explored the molecular mechanism of ROS impacting on early embryo development of mouse. These results laid a basis for further research and will be benefit to optimization of the culture system of mammalian in vitro fertilization. |
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