Caffeine alleviates the deterioration of Ca2+ release mechanisms and fragmentation of in vitro-aged mouse eggs

The developmental competence of mammalian eggs is compromised by postovulatory aging. We and others have found that in these eggs, the intracellular calcium ([Ca2+]i) responses required for egg activation and initiation of development are altered. Nevertheless, the mechanism(s) underlying this defective Ca2+ release is not well known. Here, we investigated if the function of IP3R1, the major Ca2+ release channel at fertilization, was undermined in in vitro‐aged mouse eggs. We found that in aged eggs, IP3R1 displayed reduced function as many of the changes acquired during maturation that enhance IP3R1 Ca2+ conductivity, such as phosphorylation, receptor reorganization and increased Ca2+ store content ([Ca2+]ER), were lost with increasing postovulatory time. IP3R1 fragmentation, possibly associated with the activation of caspase‐3, was also observed in these eggs. Many of these changes were prevented when the postovulatory aging of eggs was carried out in the presence of caffeine, which minimized the decline in IP3R1 function and maintained [Ca2+]ER content. Caffeine also maintained mitochondrial membrane potential, as measured by JC‐1 fluorescence. We therefore conclude that [Ca2+]i responses in aged eggs are undermined by reduced IP3R1 sensitivity, decreased [Ca2+]ER, and compromised mitochondrial function, and that addition of caffeine ameliorates most of these aging‐associated changes. Understanding the molecular basis of the protective effects of caffeine will be useful in elucidating, and possibly reversing, the signaling pathway(s) compromised by in vitro culture of eggs. Mol. Reprod. Dev. 78:684–701, 2011. © 2011 Wiley‐Liss, Inc.