The Effect Of TP,ALC And NAC Combination On The Oxidation-reduction System Of Mouse Early Embryo In Vitro Culture

In the process of IVC operation,the embryo will be exposed to OS for a long time due to the growth environment of the embryo in vivo cannot be completely simulated.Therefore,OS is one of the key factors affecting the quality and quantity of IVC embryo.To sum up,adding antioxidants to the culture medium is undoubtedly the most direct and effective method to reduce OS,but OS problem still seriously restricts the IVC technology of embryos.Studies have shown that TP,ALC and NAC have antioxidant effects on embryos under oxidative stress,but they act on different stages of oxidation-reduction mechanism respectively.For example,TP can effectively promote the expression of antioxidant enzymes such as SOD and CAT,NAC can improve the activity of GPx enzyme,while ALC can accelerate the production of ATP that provides energy for the in vivo reaction.In conclusion,these three antioxidants play roles in different links of the oxidation-reduction system.Whether the combination of TP,ALC and NAC antioxidant can improve the quality and quantity of early embryo in mice during IVC process more than the addition of one antioxidant alone or without any antioxidant,and what effect does it have on the endogenous oxidation-reduction reaction system of embryos?The experiment was divided into five groups:negative control group,positive control group(including TP treatment group,ALC treatment group and NAC treatment group),and combined treatment group.To evaluate the quality and quantity of IVC embryos,three kinds of antioxidants were added alone or in combination in this experiment.Through the observation of relevant indexes(Such as 2-cell development rate,4-cell development rate and blastocyst development rate)of the embryos in the above five groups,it was verified that the embryos added with TP,ALC and NAC antioxidants were in better development state than those with one or no antioxidants;Inside the embryo research on the effects of oxidation-reduction reaction system,the test by detecting the blastocyst groups of five embryos that endogenous oxidase gene,antioxidant enzymes and antioxidant enzymes key regulatory factor Nrf2 gene expression,to explore TP and ALC and NAC,three kinds of combination of antioxidants to add to the influence of oxidation-reduction reaction system;in addition,by comparing the above five groups of intracystic ROS and GSH levels,it was further proved that the three antioxidant combinations of TP,ALC and NAC had better antioxidant effects.We detected the expression of endogenous genes in embryos by qRT-PCR,and we determined the levels of reactive oxygen species and glutathione in embryos by 2 ’,7’-dichlorofluorescein diacetate(DCHFDA)and 4-chloromethyl-6,8-difluoro-7-hydroxy coumarin assays.Main experimental results:Embryos were treated with different concentrations of TP,ALC and NAC,we found that the 4-cell development rate(P<0.05)and blastocyst development rate(P<0.01)of the 5 TP group were higher than that of the control group,the 4-cell development rate(P>0.05)and blastocyst development rate(P<0.01)of the 10 μM ALC group were higher than that of the control group,the 4-cell development rate(P<0.01)and blastocyst development rate(P<0.01)of the 10 μM NAC group were higher than that of the control group;the 4-cell development rate and blastocyst development rate of TP,ALC and NAC group were significantly higher than those of TP,ALC and NAC group(P>0.05)and significantly higher than that of control group(P<0.01);the addition group of TP,ALC and NAC could efficiently up-regulate the expression of oxidase(NOX1,NOX2,NOX4,Duoxl and Duox2)genes in the embryo,and effectively promote the expression of antioxidant enzymes(SOD1,SoD2,CAT,GPx1,GPx2 and GPx4)genes in the embryo;in addition,the addition groups of TP,ALC and NAC can also up-regulate the expression of Nrf2 gene in embryos(P<0.01);the addition of TP,ALC and NAC significantly decreased the RoS level(P<0.01)and increased the GSH level(P<0.01).The above studies indicated that supplementation with three synergistic antioxidants in vitro culture medium can further facilitate the maintenance of IVC embryo survival and development in vitro,which is easy to be associated that these phenomena may be achieved by weakening oxidative stress.