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<p>    </p> <p>When mammalian eggs are fertilized, they undergo an egg-to-embryo transition during which different egg activation events take place. Egg activation events include the establishment of blocks to polyspermy, which prevent multiple sperm from fertilizing an egg. One of these blocks to polyspermy occurs at the level of the egg plasma membrane (the membrane block to polyspermy). Previous work in our lab provides evidence that the mammalian membrane block to polyspermy is mediated by sperm-induced calcium signaling and the egg’s actomyosin cytoskeleton (McAvey et al., 2002). This thesis research builds upon this foundation, testing hypotheses about two specific effector molecules, one involved in calcium signaling and one with the actin cytoskeleton, and also developing the use of an actin probe for live-cell imaging, with the goal of imaging actin dynamics in eggs undergoing fertilization. Specifically, we examined the calcium effector molecule Ca2+/Calmodulin-dependent-protein kinase IIg (<strong>CaMKII</strong>g), based on previous studies showing that CaMKII plays a role in the membrane block (Gardner et al., 2007) and that the g isoform of CaMKII is necessary and sufficient for eggs to complete meiosis (Backs et al., 2010). We tested the hypothesis that CaMKIIg would mediate the membrane block to polyspermy but found that egg activation driven by expression of a constitutively active form of CaMKIIg was not sufficient to establish the membrane block. Our studies of the actin cytoskeleton focused on the Arp2/3 complex as a candidate. We tested the hypothesis that Arp2/3, which mediates actin filament branching, was involved in membrane block establishment, building on the finding that disruption of actin with the drug cytochalasin D impairs the membrane block (McAvey et al., 2022). These studies used the Arp2/3 inhibitor CK666, predicting that we would see increased sperm incorporation in CK666-treated eggs. However, an assay of sperm incorporation over time indicated that Arp2/3 may not play a significant role in the membrane block to polyspermy, although follow-up studies will be beneficial. Lastly, the actin probe SiR- Actin was assessed for use on oocytes undergoing live-cell imaging during meiosis I and II. Oocytes were treated with differing concentrations of SiR-Actin and live cell imaged while maturing through meiosis I or completing meiosis II. Higher doses and longer exposure to SiR- Actin caused abnormalities in oocytes during meiosis I but not in eggs completing meiosis II. Together, this work sets the stage of a range of future studies into the mammalian membrane block to polyspermy. </p>