Our study aimed to develop embryo models to evaluate the impact of oxidative stress on embryo development. Mouse zygotes, which stayed at G1 phase, were treated with prepared culture medium (containing 0.00, 0.01, 0.02, 0.03, 0.04, 0.05, or 0.1 mM hydrogen peroxide (H2O2)) for 30 min in experiment 1. The dose-effects of H2O2 on embryo development were investigated via comparisons of the formation rate at each stage (2- and 4-cell embryos and blastocysts). Experiment 2 was carried out to compare behaviors of embryos in a mild oxidative-stressed status (0.03 mM H2O2) with those in a control (0 mM H2O2). Reactive oxygen species (ROS) levels, variation of mitochondrial membrane potential (MMP), expression of γH2AX, and cell apoptosis rate of blastocyst were detected. We observed a dose-dependent decrease on cleavage and blastocyst rates. Besides, higher level of ROS, rapid reduction of MMP, and the appearance of γH2AX revealed that embryos are injured early in mild oxidative stress. Additionally, γH2AX may involve during DNA damage response in early embryos. And the apoptotic rate of blastocyst may significantly increase when DNA damage repair is inadequate. Most importantly, our research provides embryo models to study cell cycle regulation and DNA damage response under condition of different levels of oxidative stress.

No abstract

In zygotes, DNA damage delays the first cleavage to enable repair. Our previous study found that 0.03 mM hydrogen peroxide (H2O2) was the minimum concentration required for induction of oxidative DNA damage in mouse zygotes and that this represented the most similar situation to the clinical phenomenon. In this study, we quantified the cleavage rates of cells in blastocysts at different developmental stages, followed by immunofluorescence to detect activation of γ-H2A histone family member X (a marker of DNA damage) in zygotes to confirm that oxidative DNA damage was induced in H2O2-treated zygotes. Monitoring H3S10P (phosphorylation of Ser10 on histone H3; a prometaphase/metaphase marker) levels at different hour postinsemination revealed that treatment of zygotes with 0.03 mM H2O2 resulted in a prometaphase/metaphase delay. Furthermore, immunofluorescence staining for mitotic arrest deficient 2-like 1 and the protein kinase TTK, components of the spindle assembly checkpoint (SAC), suggested that this delay possibly involved SAC activation. These studies of the relationships between oxidative stress and SAC can promote the success rate of in vitro fertilization.