Using the mouse egg as a model system for the study of intracellular calcium signaling mechanisms

Mouse metaphase II (MII)-stage eggs exhibit oscillatory Ca2+ responses ([Ca2+]i oscillations) following fertilization. The wealth of information regarding Ca2+ signaling pathways in eggs has allowed these cells to become an ideal model system for the study of general Ca2+ signaling pathways. This dissertation provides data that contribute to the elucidation of the mechanism that culminates in Ca2+ release at fertilization, and to our understanding of the functional regulation of the inositol 1,4,5-trisphosphate receptor (IP 3R) using the mouse egg as a model system.; We first present data indicating that injection of mouse eggs with porcine sperm factor (SF) induces [Ca2+]i oscillations through activation of a phospholipase C (PLC). U73122, a PLC inhibitor, prevented SF-induced [Ca2+]i oscillations whether SF or eggs were treated with the inhibitor. We also show that SF injection elicits inositol 1,4,5-trisphosphate (IP3) production and Ca2+ release in single Xenopus oocytes. Thus, SF induces [Ca2+]i release by stimulating the phosphoinositide pathway.; We next show that KN-93, a Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor, antagonizes IP3R function independently of effects on CaMKII in mouse eggs and permeabilized A7r5 cells. This inhibition is not due to a block of IP3 production, Ca2+ store filling, or IP3 binding to the IP3R. KN-93 interferes with Ca2+-induced Ca2+ release by the IP 3R, indicating that KN-93 may prevent the ability of IP3 and/or Ca2+ to induce activatory conformational changes to the IP3R. KN-93 directly interacts with and alters the conformation of the IP3R, based on in vitro and in vivo proteolysis experiments. Finally, KN-93 does not interact with the IP3R via a calmodulin binding site, as hypothesized based on its mechanism of CaMKII inhibition.; Lastly, we present preliminary data toward the development of an IP 3R overexpression system in mouse eggs. We successfully generated enhanced yellow fluorescent protein (eYFP)-tagged murine IP3R-1 mRNA in vitro. Injection of eYFP-IP3R-1 mRNA into mouse eggs resulted in expression of exogenous IP3R-1 protein. This system will provide new opportunities for the use of the mouse egg as a model system for the study of IP3R signaling, and may help facilitate the elucidation of the mechanism by which KN-93 inhibits the IP3R.