Analysis Of The Correlation Between Embryo Arrest In High Glucose,Reactive Oxygen Species And Adaptor Protein P66^shc In Mouse

Glucose as the energy source is important for the cell growth, but research indicated that early embryos exposed to high level glucose could experience significant developmental arrest. Diabetes can cause pregnency failure, which may be also associated with the adverse effect of high concentration of glucose on the embryonic development.The recent studies have shown that the detrimental effects on embryos occurred as early as preimplantation stage.It is reported that high glucose might induce overproduction of reactive oxygen species (ROS), caused the oxidative stress and blocked the development of embryos. ROS can induce the lipid peroxidation,nuclear DNA strand broken which may lead to growth retardation.The formation of ROS is associated with the oxidative stress adaptor protein p66shc, which is encoded by gene SHCA. p66shc controls life span in mammals, and regulates the cellular response to oxidative stress. Now it is comfirmed that p66shc was involved the diabetes mellitus,the main mechanism was that p66shc could induce overprodction of ROS and oxidative stress. Additionally, research has indicated that p66shc played a centre role in the regulation of the oxidative stress and embryo development. But whether the embryo arrest in high glucose caused by oxidative injury is also linked to the protein p66shc, it is unknown yet.ObjectivesIn this experiment, we observed the effect of different concentrations of glucose on the mouse embryo development, and detected the ROS and p66shc in different development stage embryos to discuss the relationship between embryo arrest in high glucose, ROS and p66shc. By this experiment, we tried to explore the mechanism by which abnormal embryonic development caused by high glucose, and to provide supported evidence for clinical treatment.MethodsZygotes from SPF Kunming female mouse were cultured in vitro with different concentration of glucose (0,5,15,25mM) medium. Zygote,2-cell,4-cell and blastocyst stage embryos were collected from different groups to do eximinations. ROS inside of embryos were examined by flurescent dye DCHF-DA, the expression of protein p66shc was detected by immunofluorescence and the amount of p66shc mRNA was quantified by Real Time PCR.Results1.Outcomes of embryos cultured in different levels of glucose.Exposure of zygotes to the increasing concentrations of glucose resulted in significantly fewer blastocysts formation, and more arrested embryos. The blastocyst rates in15and25mM groups were significantly decreased compared with control and5mM group(P<0.05); the arrested embryo rates in15,25mM were significantly increased compared with other groups(P<0.05). 2.Outcomes of ROS measurementIn each groups, embryos from zygotes to blastocyst stage, the level of ROS in2-cell stage rised to the peak, and were significantly higher to other developmental stages(P<0.05). During the different groups, the expression of ROS in2-cell and4-cell stage in15,25mM groups were higher than0,5mM groups(P<0.05). The ROS in blastocyst stage was increasing with a rising concentration of glucose, and there were statistically significant among the four groups (P<0.05).3.Detection of p66shc protein and mRNA expressionDuring p66shc detection by Immunofluorescence, we found at protein level p66shc expressed in embryos at all developmental stages. Quantification of p66Shc at RNA level indicated that p66shc expression was similar to ROS, p66shc RNA in2-cell embryos was increasing to peak, when development process continued, p66shc RNA was decreasing gradually. In the different concentrations of glucose, the expression of p66shc RNA in2-cell and4-cell stage between15,25mM groups are higher than0,5mM groups (P＜0.05). To the blastocyst stage, there were not statistically significant between four groups.Conclusions1.High level glucose had an adverse effect on embryo development, which might be associated with embryo arrest.2.High glucose might induce oxidative stress by over production of ROS, to cause embryo arrest.3.In the process of oxidative injury induced by high concentration of glucose, p66shc as an adaptor protein might be involved in regulating embryo arrest.