Detection Of Reactive Oxygen Species(ROS) In Different Developmental Stage Mouse Embryos In Vivo And In Vitro

The excessive Reactive Oxygen Species (ROS) produced in vivo and in vitro conditions have a toxic effect on embryo. The Oxidative damage induced by ROS may result in development block of embryo. We investigate into the effect of ROS on the development block of embryo in vitro. We detect ROS levels within embryos using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) or hydroethidine (HE), and search after the relation between ROS and development block of Kunming mouse embryos in vivo and which cultured in mCZB in vitro in the experiments. We compare H₂O₂ levels within embryos on the different stages with on the same stages using DCFH-DA in vivo and in vitro. The DCF fluorescence density of early 2-cell stage, late 2-cell stage, 4-cell stage, morula and blastula are 31.33, 32.60, 39.55, 28.73 and 25.79 in vivo, respectively, and are 38.56, 45.49, 41.13, 30.2 and 29.21 on the same stages in vitro, respectively. The result suggests that H₂O₂ levels rise gradually when cultured in vitro from early 2-cell stage to late 2-cell stage. However, H₂O₂ levels decrease gradually after late 2-cell stage. There are significantly differences on H₂O₂ levels between embryos in vivo and cultured in vitro on the early 2-cell stage and late 2-cell stage, but there are no significantly differences among embryos those on the 4-cell stage, morula and blastula using SPSS statistics software. We also detect superoxide anion (O^2-) levels within embryos on the different stages in vivo and in vitro using HE. The EB fluorescence density of early 2-cell stage, late 2-cell stage, 4-cell stage, morula and blastula are52.92, 50.77, 48.84, 39.96 and 30.36 in vivo, respectively, and are 74.85, 82.62, 82.98, 81.28 and 54.09 on the same stages in vitro, respectively. The result suggests that O^2- levels within embryos decrease gradually from early 2-cell stage to blastula in vivo. There are significantly differences on O^2- levels between embryos on the early 2-cell stage, late 2-cell stage, 4-cell stage and morula, blastula, furthermore there are significantly differences between morula and blastula in vivo, the EB fluorescence density are higher among early 2-cell stage, late 2-cell stage, 4-cell stage and morula in vitro, but there are no significantly differences among those embryos, nevertheless there are significantly differences when compare with blastula, in addition, there are significantly differences among embryos on the same stage in vivo and in vitro using SPSS statistics software. So we conclude that 1) ROS levels within embryos are lower in vivo than those cultured in vitro, 2)...