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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.
Smyth, Jeremy T, "Using the mouse egg as a model system for the study of intracellular calcium signaling mechanisms" (2004). Doctoral Dissertations Available from Proquest. AAI3136780.