Oxidative stress-induced DNA damage of mouse zygotes triggers G2/M checkpoint and phosphorylates Cdc25 and Cdc2.

Abstract

In vitro fertilized (IVF) embryos show both cell cycle and developmental arrest. We previously showed oxidative damage activates the ATM → Chk1 → Cdc25B/Cdc25C cascade to mediate G2/M cell cycle arrest for repair of hydrogen peroxide (H2O2)-induced oxidative damage in sperm. However, the mechanisms underlying the developmental delay of zygotes are unknown. To develop a model of oxidative-damaged zygotes, we treated mouse zygotes with different concentrations of H2O2 (0, 0.01, 0.02, 0.03, 0.04, 0.05mM), and evaluated in vitro zygote development, BrdU incorporation to detect the duration of S phase. We also examined reactive oxygen species level and used immunofluorescence to detect activation of γH2AX, Cdc2, and Cdc25. Oxidatively damaged zygotes showed a delay in G2/M phase and produced a higher level of ROS. At the same time, γH2AX was detected in oxidatively damaged zygotes as well as phospho-Cdc25B (Ser323), phospho-Cdc25C (Ser216), and phospho-Cdc2 (Tyr15). Our study indicates that oxidative stress-induced DNA damage of mouse zygotes triggers the cell cycle checkpoint, which results in G2/M cell cycle arrest, and that phospho-Cdc25B (Ser323), phospho-Cdc25C (Ser216), and phospho-Cdc2 (Tyr15) participate in activating the G2/M checkpoint.