In vivo post-ovulatory aging of oocytes significantly affects the development of oocytes and embryos. Also, oocyte aging alters the regulation of the intracellular calcium concentration, thus affecting Ca2+ oscillations in fertilized oocytes. Because reactive oxygen species (ROS) are known to significantly perturb Ca2+ homeostasis mainly through direct effects on the machinery involved in intracellular Ca2+ storage, we hypothesized that the poor development of aged oocytes that may have been exposed to oxidative stress for a prolonged time might arise from impaired Ca2+-oscillation-dependent signaling. The fertilization rates of aged oocytes and of fresh oocytes treated with 100 muM hydrogen peroxide (H2O2) for 10 min were significantly lower than that of fresh oocytes. Comparing within the fertilized oocytes, blastocyst formation was decreased while embryo fragmentation was increased similarly in the aged and H2O2-treated fresh oocytes. The frequency of Ca2+ oscillations was significantly increased whereas the amplitude of individual Ca2+ transients was lowered in the aged and H2O2-treated fresh oocytes. The rates of rise And decline in individual Ca2+ transients were decreased in these oocytes, indicating impaired Ca2+ handling. When lipid peroxidation was assessed using 4,4-difluoro-5-(4-phenyl-1,3-buttadienyl)-4-bora-3a, 48-diaza-s-indacene-3-undecanoic acid (C11-BODIPY) in unfertilized oocytes placed in a 5% CO2 in air atmosphere, the green fluorescence (indicating lipid peroxidation) increased faster in the aged oocytes than in the fresh oocytes. Furthermore, the green fluorescence in the aged oocytes was already approximately 20 times higher than that in the fresh oocytes at the beginning of the measurements. These findings support the idea that Ca2+ oscillations play a key role in the development of fertilized aged oocytes. (C) 2003 Wiley-Liss, Inc.