The Capacity to Repair Sperm DNA Damage in Zygotes is Enhanced by Inhibiting WIP1 Activity

Abstract

Maintaining genome integrity in germ cells is essential not only for successful fertilization and embryo development, but also to ensure proper transmission of genetic information across generations. However, unlike oocytes, sperm are incapable of repairing DNA damage. Therefore, sperm DNA damage is repaired after fertilization in zygotes using maternal DNA repair factors. In this study, we found that zygotic repair of paternal DNA damage is enhanced by inhibiting WIP1 activity. Oxidative stress induced DNA damage in sperm and severely impaired motility. Although DNA damage in sperm did not compromise fertilization, it increased DNA damage in the paternal pronucleus of zygotes. However, WIP1 inhibition during fertilization reduced DNA damage in the paternal pronucleus, improving the rate of two-cell development, and subsequent zygotic genome activation. Therefore, our results suggest that WIP1 inhibition could enhance maternal DNA repair capacity and thereby decrease paternal DNA damage in zygotes.

Keywords: DNA repair; WIP1; sperm dna damage; zygote; zygotic genome activation.

FIGURE 1

Oxidative stress induces DNA damage in sperm and impairs sperm motility. Sperm were treated with 0, 25, 50, and 100 μM hydrogen peroxide (H₂O₂) for 1 h. Sperm DNA fragmentation was assessed by TUNEL assay and 8-OHdG staining. (A–C) TUNEL-positive sperm and TUNEL intensity were quantified and shown with representative images. (D–F) 8-OHdG-positive sperm and 8-OHdG fluorescence intensity were quantified and shown in representative images. Scale bar, 10 μm. Data are expressed as mean +SEM. *p < 0.05, **p < 0.001, and ***p < 0.0001, compared to control (0).

FIGURE 2

WIP1 inhibition enhances DNA damage repair in zygotes. (A) Schematic diagram of the experiment. After insemination with sperm treated with 100 μM hydrogen peroxide (H₂O₂) for 1 h, oocytes were cultured with or without GSK for 9 h. Fresh sperm were used as a control. (B,C) The rate of pronuclear (PN) formation was shown with representative images at 9 h after ICSI. Scale bar, 100 μm. Arrowheads indicate pronucleus. (D,E) The γ-H2AX intensity in paternal and maternal pronuclei was normalized to the mean DAPI intensity of two independent experiments and shown with representative images. ♂ and ♀ indicate the paternal PN and maternal PN, respectively. Scale bar, 20 μm. (F) The γ-H2AX intensity ratio of the paternal/maternal pronuclei was quantified. Data are expressed as mean +SEM. *p < 0.05, **p < 0.001, and ***p < 0.0001, ns; not significant.

FIGURE 3

WIP1 inhibition improves two-cell development and zygotic genome activation. (A,B) The rate of two-cell embryos was shown with representative images at 24 h after ICSI. Scale bar, 100 μm. (C–E) The γ-H2AX intensity and nuclear size were quantified and shown with representative images. Scale bar, 20 μm. (F,G) The Zscan4 intensity was quantified and shown with representative images. Scale bar, 20 μm.

FIGURE 4

A working model for the effect of WIP1 inhibition on the repair of sperm-derived DNA damage in zygotes. Oxidative stress in sperm increases paternal DNA damage in zygotes and causes ZGA failure. However, WIP1 inhibition during fertilization reduces paternal DNA damage in zygotes and restores ZGA. PN, pronucleus.