ABSTRACT
One difficulty in analyzing the damage response is that the effect of damage itself and that of cellular response are hard to distinguish in irradiated cells. In mouse zygotes, damage can be introduced by irradiated sperm, while damage response can be studied in the unirradiated maternal pronucleus. We have analyzed the p53-dependent damage responses in irradiated-sperm mouse zygotes and found that a p53-responsive reporter was efficiently activated in the female pronucleus. [3H]thymidine labeling experiments indicated that irradiated-sperm zygotes were devoid of G1/S arrest, but pronuclear DNA synthesis was suppressed equally in male and female pronuclei. p53−/− zygotes lacked this suppression, which was corrected by microinjection of glutathione S-transferase-p53 fusion protein. In contrast, p21−/− zygotes exhibited the same level of suppression upon fertilization by irradiated sperm. About a half of the 6-Gy-irradiated-sperm zygotes managed to synthesize a full DNA content by prolonging S phase, while the other half failed to do so. Regardless of the DNA content, all the zygotes cleaved to become two-cell-stage embryos. These results revealed the presence of p53-dependent pronuclear cross talk and a novel function of p53 in the S-phase DNA damage checkpoint of mouse zygotes.
Pronuclear Microinjection during S-Phase Increases the Efficiency of CRISPR-Cas9-Assisted Knockin of Large DNA Donors in Mouse Zygotes