p53-Dependent S-Phase Damage Checkpoint and Pronuclear Cross Talk in Mouse Zygotes with X-Irradiated Sperm

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. [ 3 H]thymidine labeling experiments indicated that irradiated-sperm zygotes were devoid of G 1 /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.

Suppression of replication fork progression in low-dose-specific p53-dependent S-phase DNA damage checkpoint

“…Therefore, slowing down of replication fork movement and the resulting suppression of DNA synthesis are the consequence of cellular response to DNA damage, rather than the result of the mechanical block of replication fork movement at the sites of DNA double strand breaks. A very similar conclusion was reached in our previous study of mouse zygotes fertilized by irradiated sperm in which the suppression of DNA synthesis was observed even in the unirradiated female pronuclei (Shimura et al, 2002a).…”

“…However, one cannot draw such conclusion on the role of p53, as ATM protein, a key factor in S-phase DNA damage checkpoint, was undetectable in HL60 (Gately et al, 1998). Low dose specificity of the p53-dependent S-phase DNA damage checkpoint was found previously in p53 þ / þ and p53À/À mouse zygotes fertilized with irradiated sperm (Shimura et al, 2002a). Now the same checkpoint is demonstrated in mouse fibroblasts.…”

“…In contrast, the role of p53 in the S-phase DNA damage checkpoint was reported only in a few studies in which the checkpoint was induced in tissue culture cells by depletion of the nucleotide substrates or in mouse zygotes by fertilization with X-ray-irradiated sperm (Agarwal et al, 1998;Shimura et al, 2002a). p53 was shown to respond to the replication block induced by aphidicolin (Gottifredi et al, 2001;Nayak and Das, 2002) and colocalized with BLM, Rad51 and 53BP1 at the sites of stalled DNA replication fork (Sengupta et al, 2003(Sengupta et al, , 2004.…”

“…Indeed, we have described a novel p53-dependent S-phase DNA damage checkpoint in mouse zygotes fertilized with X-irradiated sperm. The S-phase DNA damage checkpoint in mouse zygotes operated in a biphasic manner such that 3 H-TdR uptake was sharply suppressed at sperm doses below 2 Gy but less sharply at higher doses and the first portion was found to be dependent on p53 (Shimura et al, 2002a).…”

Apoptosis and DNA damage in human spermatozoa

“…Thus, immediately after fertilization, the oocyte surveys the integrity of the DNA introduced into the zygote by the spermatozoon and, if damaged, puts DNA replication on hold in both male and female pronuclei, until DNA repair has been completed. 19 If the oocyte makes a mistake at this point, there is the potential to create a mutation which, because it precedes S phase of the first mitotic division, will be in every cell in the body. Through such a mechanism, DNA damage induced in spermatozoa by such factors as smoking, 20 exposure to radiofrequency electromagnetic radiation 21 or age 22 could increase the mutational load carried by the embryo, resulting in miscarriage or serious disease in the offspring, including cancer.…”