INCREASED GENERATION OF REACTIVE OXYGEN SPECIES IN EMBRYOS CULTURED INVITRO

Previous studies have suggested that oxygen toxicity is closely related to the developmental blockage of embryos cultured in vitro. In this study, to obtain an actual proof of the increase in production of reactive oxygen species within embryos, we have measured the level of H2O2 in individual embryos using a fluorimetric method. Mouse (ICR) pronuclear stage embryos from the oviducts were cultured for a specified time under various conditions in a medium to which 2',7'-dichlorodihydrofluorescin diacetate was added. After washing the embryos, the fluorescence emissions of the H2O2-dependent oxidative product in embryos were measured. The fluorescent emissions were lowest in embryos cultured under 5% O2 and highest under 40% O2 (5% < 20% < 40%), just the inverse of the culture efficacy. The fluorescence emissions of embryos cultured in Ham's F-10, which contains hypoxanthine and transition metals such as Cu and Fe, were higher than those cultured in BWW and alphaMEM, which do not contain these components (alphaMEM < BWW < Ham's F-10; again this is the inverse of the culture efficacy). The fluorescence emissions of embryos increased with the time of the exposure to visible light. L-cysteine and thioredoxin, both of which have been shown to promote embryo development, decreased the fluorescence emissions of embryos. All of these results would provide direct evidence for the hypothesis that oxygen radicals are involved in the developmental blockage.