Delayed execution of apoptosis and impaired developmental competence of the embryos in response to fertilization with x-irradiated sperm

Ionizing radiation induces DNA double-strand breaks and inflicts a variety of damage responses, which include induction of cell cycle checkpoint and apoptosis in damaged cells. However, damage response in preimplantation stage mouse embryo is not yet elucidated. We have previously shown p53 dependent S-checkpoint, which reduced the pronuclear DNA synthesis in zygotes fertilized with irradiated sperm. Interestingly, the zygotes with sub-optimal DNA have progressed beyond implantation but a majority of the fetus showed post implantation demise. These observations made us to undertake the present study to find out whether apoptotic machinery functions in the embryos, and if so, is it p53 dependent, and whether developmental potential is compromised in the embryos fertilized with irradiated sperm. In this study we used sexually mature male ICR mice, X-irradiated at 6 Gy and immediately mated with female ICR mice.Embryos were recovered from the pregnant mice on day 3.5, 4.0 and 4.5 post conception. Embryonic development and total cell number were assessed in addition to the incidence of apoptosis and micronuclei frequency in both wild type and p53 knock out mice. The early cleavage in the embryos is not affected by the X-irradiation of sperm until 6-8 cell stage, however, we observed delayed development and reduced total cell number in the embryos at peri-implantation period. This is in agreement with our previous study where we found delayed cleavage and reduced fetus-placental ratio in embryos fertilized by X-irradiated sperm. We observed high incidence of apoptosis in wild type embryos fertilized with irradiated sperm and the apoptosis was higher in blastocysts than in arrested or slow cleaving embryos. However, apoptosis was not evident in p53-/- embryos suggesting that activation of functional p53 mediated apoptosis pathway is possible only after blastocyst formation. Incidence of apoptosis increased significantly after day 4.0 post conception and it was associated with the expression of active caspases. We have been doing DNA content analysis of the embryos at peri- implantation stages to find out the cause of post implantation fetal death observed in our previous study and the results will be discussed