Effects Of Mouse Oviduct Epithelial Cells CO-Culture On The Development Of Early Embryos In Vitro

Objective:The major objective of this study was to investigate the effects of OECs on the development and quality of mouse embryos, as well as to study the impacts of embryos on the co-cultured OECs and evaluate the effects of oxidative stress on the OECs co-culture system.Methods:Differential adherent method was used to purify the OE cell line, purified mouse OECs were used to co-culture the zygotes until blastocyst, developed and arrested embryos were evaluated, H2O2content in embryos was measured by DCHFDA, MTT was used to measure epithelial cell activity, RT-PCR was applied to test the transcriptions of CAT and GPX gene, exogenous H2O2was used as oxidative stress to dispose OECs and embryos, applied Hochest33342and PI staining to detect cellular apoptosis.Results:1. Clear colony of paving stone shape can be seen from the third passage. Purified OE cell line was established from the third passage and be used for co-culture.2. Co-culture of OECs can improve the development of embryo in vitro, as it can improve the early2-cell and4-cell rates (P<0.05), as well as the blastocyst (P>0.05)rate compared with control group.3. There was a deposition of endogenous H2O2in the fragments of arrested embryo. Generally the fluorescence intensity was higher than that in normal embryos.4. The rates of type â…  and â…¡ arrested embryos were lower in co-cultured embryos compared with control embryos (P>0.05). However, the rate of type â…¢ arrested embryos was higher in co-cultured embryos compared with control embryos (P<0.05), therefore, co-culture with OECs can change the distribution of arrested embryos and decrease the damaging extent of arrested embryos. 5. H2O2content was significantly higher of early2-cell embryos in vitro than in vivo ones(P<0.05). Co-culture of OECs can decrease the H2O2content of embryos at MZT phase. H2O2content of co-cultured early2-cell embryos was significantly lower than in vitro embryos (P<0.05).6. The co-cultured embryos also had some effects on OECs, the activity of OECs in co-cultured group was higher than control group (P>0.05) when they developed to the early2-cell phase (36ph hCG).7. The antioxidative ability of OECs was influenced by co-culture, as the transcriptions of CAT and GPX gene in co-cultured OECs were higher than that in control group (P>0.05).8. Embryos have their own ability to protect themselves from the damage of ROS, treated with0.12ppM of exogenous H2O2for30min can induce development arrest of mouse zygotes, therefore, this condition was used as oxidative stress.9. The development-improving ability can be decreased by oxidative stress, the early2-cell and4-cell rates were not significantly increased (P>0.05) when OECs were treated with H2O2.10. OECs are very sensitive to ROS as well, the activity of H2O2treated OECs was lower than control group at36(P>0.05)and60h phCG(P<0.05), and embryo co-culture will decrease the activity of H2O2treated OECs further, when those cells were used for co-culture (P<0.05).11. Oxidative stress could induce apoptosis and death of embryos and OECs by Hochest33342and PI detection. Therefore, this kind of OECs cannot promote the development of zygotes.Conclusions:Those results indicated that OECs co-culture can improve the development of embryos and decrease the damaging extent of arrested embryos, also the co-culture of OECs can decrease the H2O2content of embryos at MZT phase and improve the quality of embryos. There is a cell-cell contact in which the antioxidative ability and activity of OECs were influenced by co-culture. Oxidative stress could induce apoptosis and death of embryos and OECs, so OECs cannot exert this effect when they have experienced oxidative stress.