Voltage-gated calcium channels (VGCC) regulate exocytosis in cells ranging from sperm to pancreatic beta cells and neurons. There is growing recognition that sterols and the ganglioside GM1 modulate the activity of VGCCs with significant physiological and pathological consequences. However, determining the mechanisms by which lipids regulate these channels, and thereby exocytosis, remains a major challenge in the field. These questions are critical to our understanding of medical issues ranging from diabetes and pain management to infertility. In the field of reproduction, several recent findings have attacked long-held beliefs. Patch clamping data have been interpreted to suggest that CatSper is the only calcium (Ca2+) channel in sperm, as opposed to a model in which multiple channels contribute to Ca2+ transients and waves that culminate in release from internal stores and activation of store-operated channels. The model of the acrosome reaction, in which physical contact between receptors on the sperm membrane are triggered by ligands on the zona pellucida to induce exocytosis, is challenged by new data showing that regulated exocytosis is a process beginning much earlier, as sperm penetrate the cumulus cells. Until recently, it remained unclear how Ca2+ flux and exocytosis are triggered in the absence of contact with the zona. Relevant to both of these controversies, we showed that removal of sperm sterols spurs a functional interaction between GM1 and CaV2.3, an R-type VGCC. These interactions result in transient influx of Ca2+ that promotes acrosome exocytosis in response to a subsequent Ca2+ wave. Mechanistically, we showed that this interaction relies on both the pore forming α1E and auxiliary α2δ subunits of CaV2.3, and both the lipid and sugar components of GM1. Based on these published data, we now propose to utilize state-of-the-art genetically encoded Ca2+ indicators (GECI) in generating novel research resources to resolve these controversies and further our understanding of how lipids regulate Ca2+ signaling in sperm. Specifically, we will generate 2 lines of mice that express new generation GECIs (i.e. GCaMPs) in fusion with mCherry (a Ca2+ insensitive, red-shifted fluorescent protein), to be targeted to the sperm acrosome (Aim 1) or the cytosol (Aim 2). The advantages of the new-generation GECIs over synthetic dyes and the use of GCaMP-mCherry fusion sensors that enable ratiometric Ca2+ measurements will greatly advance our efforts to investigate lipid modulation of sperm signaling and function. Specifically, these new tools will allow us to explore major questions through future R01s, including the relative roles of Ca2+ influx and intra-acrosomal Ca2+ stores during the signaling processes that lead to exocytosis, and the properties and signaling pathways associated with the CaV2.3 mediated Ca2+ transients. Importantly, these novel transgenic mouse models will enable investigation of these questions in live sperm under physiological conditions utilizing state-of-the-art imaging. Looking forward, we can cross these lines with other genetic models to investigate the activities and mechanisms of function of various channels, resolving major questions about sperm function and fertilization.