Fertilization and Inositol 1,4,5-Trisphosphate (IP3)-Induced Calcium Release in Type-1 Inositol 1,4,5-Trisphosphate Receptor Down-Regulated Bovine Eggs

It is widely believed that stimulation of the phosphoinositide pathway and production of 1,4,5-inositol trisphosphate (IP3) underlies the oscillatory changes in the concentration of intracellular free calcium ions ([Ca2+]i) seen during mammalian fertilization. IP3 promotes Ca2+ release in eggs by binding to its receptor, the type-1 IP3 receptor (IP3R-1, also known as ITPR1), a ligand-gated Ca2+ channel located in the membrane of the endoplasmic reticulum, the main Ca2+ store of the cell. While IP3R-1 has been shown to mediate all Ca2+ release during mouse fertilization, whether or not it plays such an essential role in fertilization-induced Ca2+ release in large domestic species such as bovine and porcine is presently not known. Accordingly, we have generated metaphase II bovine eggs with a ~70%–80% reduction in the number of intact IP3R-1 by inducing receptor down-regulation during oocyte maturation. We did so by injecting the nonhydrolyzable IP3 analogue, adenophostin A. Functional Ca2+ release analysis revealed that IP3R-1 is the predominant Ca2+ release channel in bovine eggs, requiring as little as 20% of total intact receptor to mount persistent [Ca2+]i oscillations in response to fertilization, expression of PLC(zeta) (also known as PLCZ1), and adenophostin A. However, lower concentrations of IP3 and near-physiological concentrations of porcine sperm extract were unable to trigger [Ca2+]i oscillations in this reduced IP3R-1 model. Furthermore, we present evidence that the sensitivity of bovine IP3R-1 is impaired at the first embryonic interphase. Together, these results demonstrate the essential role of IP3R-1-mediated Ca2+ release during fertilization in bovine eggs, and identify cell cycle regulatory mechanisms of [Ca2+]i oscillations at the level of IP3R-1.