There is a growing concern over the ability to detect early cellular changes that may be indicative of damage within embryos and gametes incurred from very low levels of environmental reproductive hazards such as low-level ionizing radiation. This proposal uses a novel, very sensitive assay in which effects of radiation are revealed as a decrease in cellular proliferation in preimplantation embryos. The assay utilizes mouse embryo aggregation chimeras and has the proven ability to detect .01-.05 Gy (1 to 5 rad) given to embryos in vitro or with embryos produced from sperm irradiated in vivo. Our primary objective is to determine whether the assay is reflecting a nuclear or extra nuclear radiosensitive target in gametes and preimplantation embryos. The radiation source will be tritium, attached to a carrier (thymidine) that will concentrate irradiation to the nucleus, or, (water) that will deliver a uniform irradiation. A third carrier (polyethylene glycol, which does not enter cells) will be used in the in vitro studies to limit irradiation to the plasma membrane/cytocortex to examine the hypothesis that this might be the radiosensitive target responsible for the decrease in cellular proliferation in the chimera assay. Three different studies will be undertaken to attain our primary objective. The first one is an in vitro study that asks whether the target is nuclear or extra-nuclear (the plasma membrane/cytocortex?) for the cell proliferation decrease exhibited by cleaving embryos in the chimera assay. The second one is an in vivo study that asks 3 questions of the oocyte: 1) will a radiation exposure to the oocyte result in a proliferative disadvantage of the embryo in the chimera assay?; 2) if so, is the radiosensitive target nuclear?; 3) will the sensitivity of the oocyte--and the nature of the radiosensitive target-differ during different periods of oocyte development? The last study is also in vivo and asks whether the radiosensitive target observed with X-irradiation in spermatogenesis is nuclear. The results of these studies are crucial for determining whether this radiation-associated decrease in cellular proliferation is indicative of heritable genetic change.
