There is a persistent gap in our knowledge of the molecules and mechanisms that mediate Ca2+ influx into oocytes and eggs. Ca2+ influx is required for egg activation and embryo development in all mammals, including human beings. This gap in our knowledge therefore represents a serious impediment, as until it is filled we cannot physiologically modulate Ca2+ influx or objectively diagnose and treat infertility associated with disturbances in Ca2+ homeostasis. The long-term goal is to understand how Ca2+ homeostasis is regulated in oocytes and eggs and identify its molecular effectors. The objective here is to identify the Ca2+ channel(s) that mediate Ca2+ influx during maturation and fertilization and characterize their regulatory mechanisms. Mouse oocytes/eggs are a great model because they display distinctive Ca2+ entry during maturation and fertilization, and Ca2+ release is required for egg activation. The central hypothesis is that expression and/or distinct regulation of underdetermined Ca2+ channels on the plasma membrane underlies Ca2+ influx in oocytes, its inactivation during maturation and its recovery after fertilization. This hypothesis was conceived based on extensive preliminary data. The rationale for this research is that once the channels are identified, a better understanding of the molecular determinants of oocyte maturation and fertilization will be gained. The findings here also have the potential to translate into therapeutic methods to assist infertile couples in this country. We plan to test our central hypothesis by pursuing the following specific aims: 1) Identify the Ca2+ channel(s) that mediate Ca2+ influx in oocytes prior to and during maturation; and 2) Identify the Ca2+ influx channel(s) that support oscillations after fertilization. Under Aim 1, Ca2+ imaging, pharmacology, conditional knockout mice, and electrophysiology will be used to identify the active channel(s) and to assess the impact of Ca2+ influx on maturation; the role of a TRPM7-like current recently discovered by the applicant and collaborators will be closely examined. Under Aim 2, the signaling mechanism(s) whereby fertilization stimulates Ca2+ influx and the contributing channel(s) will be determined. A novel approach that overcomes the inactivation of Ca2+ influx in eggs will facilitate these studies. Genetic models and electrophysiology will confirm the function of these channel(s) during fertilization. The research in this application is innovative because it combines electrophysiology, pharmacology and new KO lines, approach that has served to identify two new channels in oocytes, including TRPM7, whose global deletion is embryonic lethal at E7.5. The contribution of the proposed project is significant because it is expected to allow physiological modulation of Ca2+ entry in oocytes and eggs that will produce new conditions and activation protocols for use in the clinic. It will also expand our understanding of the impact of Ca2+ homeostasis on ...