Oxidative Stress Delays Prometaphase/Metaphase of the First Cleavage in Mouse Zygotes via the MAD2L1‐Mediated Spindle Assembly Checkpoint

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.

AMPK Activity Contributes to G2 Arrest and DNA Damage Decrease via p53/p21 Pathways in Oxidatively Damaged Mouse Zygotes

“…Regarding the blastocyst formation rate, inhibition of AMPK activity with Compound C led to a 36.12% reduction compared to the control group (p < 0.001) and a 17.92% reduction compared to the H 2 O 2 -treated group (p < 0.05), inhibition of AMPK activity with SBI-0206965 led to a 50.72% reduction compared to the control group (p < 0.001) and a 32.66% reduction compared to the H 2 O 2 -treated group (p < 0.001). In addition, consistently with our previous findings (Qian et al, 2016;Wu et al, 2017), the blastocyst formation rate in the H 2 O 2 -treated group was 18.06% lower than that of the control group (p < 0.05), while we observed no statistical differences in the 2-and 4-cell embryo formation rate between the control and H 2 O 2 -treated groups (Figures 7A,B).…”

“…Our previous results also showed that 0.03 mM H 2 O 2 induced DNA damage and caused a G2/M delay in mouse zygotes. Moreover, the delay occurred during the M phase due to the activation of the spindle assembly checkpoint (Zhang et al, 2016;Wu et al, 2017). However, these results did not confirm the existence of G2 arrest in zygotes subjected to oxidative stress, which is the typical mark of activation of the G2/M checkpoint.…”

“…In the clinic, IVF-derived embryos subjected to excessive ROS exposure can appear normal by day 3 but have a low blastocysts formation rate (Ventura-Junca et al, 2015). Our previous studies used different doses of hydrogen peroxide (H 2 O 2 ) to treat mouse zygotes at 7 h post-insemination (hpi), and found that 0.03 mM H 2 O 2 was the minimum concentration required to generate elevated ROS levels and cause oxidative damage, which reduced the rate of blastocyst formation but did not affect the formation rate of 2-, 4-, and 8-cell embryos (Qian et al, 2016;Wu et al, 2017). We concluded that this was the condition most similarly to the physiological oxidative damage observed in the clinical practice.…”

High Dosages of Equine Chorionic Gonadotropin Exert Adverse Effects on the Developmental Competence of IVF-Derived Mouse Embryos and Cause Oxidative Stress-Induced Aneuploidy

“…MAD2L1, a crucial component of the SAC complex, ensures chromosomal stability by regulating Aurora B (Shandilya et al, 2016 ). In a previous study, we first confirmed that ROS can arrest IVF-obtained mouse embryos in prophase/metaphase at the first mitotic cleavage through MAD2L1-mediated SAC activation (Wu et al, 2017 ). We then illustrated that SAC and DDR assist in repairing sex chromosome aneuploidy through the MAD2-mediated pathway (Huang et al, 2019 ).…”

“…Only Aurora B overexpression (not Aurora C overexpression) can rescue chromosome alignment defects (Shuda et al, 2009 ). Aurora B has been reported to potentiate Mps1 (also known as TTK) activation to quickly establish the mitotic checkpoint (Saurin et al, 2011 ); moreover, our previous findings have shown that mitotic arrest deficient 2 like 1 (MAD2L1; also referred to as MAD2) recruitment to kinetochores by TTK occurs at the onset of oxidative damage in embryos during SAC activation (Wu et al, 2017 ). In addition, MAD2 prevents chromosome segregation defects and maintains chromosome stability through modulation of the mitotic functions of Aurora B (Shandilya et al, 2016 ).…”