Approximately one-fifth of the world’s couples encounter fertility problems posing physical and emotional health issues. While half of the infertility problems are attributed to the male, insufficient knowledge of sperm physiology prohibits proper diagnosis in roughly 50% of these cases. A key aspect of fertilization involves capacitation, a sperm maturation step within the female reproductive tract. In this step, the sperm acquires the capacity to fertilize an egg, but many aspects of capacitation are still largely unknown. Our long-term goals are to identify and characterize the mechanisms that regulate sperm capacitation. A key player in the capacitation process is the soluble adenylyl cyclase (sAC), an enzyme that efficiently synthesizes cAMP when exposed to bicarbonate and intracellular calcium. When sAC is activated, cAMP levels increase causing the sperm-specific CatSper channel to open, allowing calcium to flow from the extracellular medium into the flagellum. So, how does calcium initially reach sAC for its activation when CatSper is not yet active? Previous reports show that in CatSper-null mice, cAMP levels can increase similarly to wild-type mice, suggesting that the initial calcium influx does not occur as result of CatSper activation. Our preliminary data show that the initial calcium flow indeed takes place in a CatSper-independent manner. Once sAC is activated, it remains active during the whole capacitation process through an unknown regulation. Our preliminary data place Cdc42, a protein known to control multiple cellular functions at the heart of this sAC regulation. While Cdc42 is expressed in mammalian sperm, its role in this cell is unclear. Our central hypothesis is that sAC is differentially regulated to allow both the initial activation that triggers capacitation and maintenance of sufficient cAMP levels throughout the process. Aim 1 will identify a novel mechanism for the initial activation of sAC. Our hypothesis is that the calcium required for the activation of sAC is delivered by ion channels present in the sperm head. Aim2 will determine the role of Cdc42 on the activation of sAC during capacitation. Our hypothesis is that Cdc42 is essential for cAMP production by sAC in the vicinity of the CatSper complex. These two aims will be achieved by using super-resolution imaging to probe the localization of molecules within the flagellum, flow cytometry to assess the activity of signaling molecules at the single-cell level, and patch clamp electrophysiology to measure calcium currents. In addition, transgenic lines will enable us to abrogate CatSper channels and Cdc42 activity. The outcomes from this work will shed light on the initial activation of sAC, will identify the calcium channel responsible for the first calcium influx during capacitation, and will elucidate the mechanism by which sAC remains active in the same region of the CatSper signaling domain. We expect these fundamental capacitation studies will open new av...