PROJECT SUMMARY/ABSTRACT Cortical ER structure is a key aspect of oocyte quality as exemplified in mammals where unique `ER clusters' become positioned in close proximity to the plasma membrane (PM). Our recent in vivo studies demonstrate the presence of hereto unknown actin fenestrae within cortical actin layer, in addition the ER clusters were centrally located within these fenestrae and sperm binding occurred preferentially over these fenestrae. Another novel observation was that upon sperm fusion the oocyte ER clusters undergo a maturational event involving an increase in volume, docking to fenestrae in the actin layer. This led us to a hypothesis that the PM overlying ER clusters is enriched in sperm binding proteins that promote close interaction between the sperm, oocyte PM, and the ER cluster. Temporal and mechanistic studies are necessary to elucidate the role of the oocyte cortical actin fenestrae in the establishment of sperm binding sites as well as the role of the sperm PLCζ released from the sperm head in mediating the ER cluster maturational changes. The presence of unique actin fenestrae led to a second hypothesis that actin remodeling proteins, RhoA and cdc42, could modulate sperm binding sites, cortical ER cluster formation via the actin fenestrae. Lastly, we observed that oocytes from aged female mice exhibited abnormal ER structure and varied considerably in their ability to form ER clusters in response to fertilization. This finding led us to propose a third hypothesis where maternal aging disrupts actin fenestrae and Ca2+ oscillations, causing defects in ER cluster maturation and reduced fertility. Our findings suggest a new paradigm wherein the oocyte responds to sperm binding/fusion by ER cluster maturation, then establishes close interaction with the sperm during its incorporation into the egg. Testing of the hypotheses presented in this proposal will lead to an enhanced understanding of the fertilization process, and potential mechanisms for improving Assisted Reproduction Technologies such as ICSI and protocols for reproductively aged oocytes as well as potential new contraceptive targets that block fertilization.