The development of new contraceptive technologies is essential for meeting global reproductive health and family planning needs. The Copper-Zinc-Lactide Intra-vaginal Ring (CZL-IVR) will prevent pregnancy, sexually transmitted disease transmission, and maintain vaginal health through the release of Zn2+, Cu2+, and lactic acid. These substances are known to inhibit sperm motility, but their effects on other aspects of sperm physiology are unknown. To fertilize an egg, sperm must undergo functional maturation, termed “capacitation,” within the female reproductive tract, a process that involves many physiological changes including increasing cytosolic pH, membrane hyperpolarization, and increasing cytosolic Ca2+. Many of these processes are mediated by plasma membrane proteins. However, our current list of sperm plasma membrane proteins, and thus our knowledge of the molecular mechanisms involved in capacitation, is incomplete. Because capacitation is essential for fertility, it is an intriguing target for contraceptive development. Yet, the effects of contraceptives on sperm capacitation are rarely examined, even though sperm are propelled into the upper female tract minutes after copulation, where they may be able to recover from the effects of motility inhibiting contraceptives. In this proposal we will examine how the active pharmaceutical ingredients (APIs) of the CZLIVR affect capacitation (Aim1). We will briefly treat ejaculated sperm with the APIs, and then perform five independent assays measuring various aspects of sperm capacitation. We will perform the CapScore Assay (Androvia Lifesciences LLC), and an acrosome exocytosis assay to ascertain the overall ability of API-treated sperm to capacitate. We will also measure cytosolic alkalinization, hyperpolarization, and intracellular Ca2+ to examine the effects of these APIs on specific signaling events underlying capacitation. The combination of assays described here will also provide a framework for future studies examining the effects of other contraceptive APIs on capacitation. Finding API-induced defects in one or more of these assays will provide evidence that these APIs have contraceptive function beyond motility inhibition. Characterizing such effects provides a more comprehensive view of these API's contraceptive functionality, and will aid in further CZL-IVR development, and potential FDA approval. Additionally, to gain insight into potential mechanisms of API activity, and obtain new targets for capacitation inhibiting contraceptives, we will generate a comprehensive sperm membrane proteome (Aim 2). We will isolate membrane proteins from separated sperm heads and tails, and subject them to mass spectrometry. This subcellular approach will allow us to detect less abundant membrane proteins that may have been missed in previous studies. Beyond contraceptives, the new membrane proteins identified here will also be of interest to those studying infertility and sperm physiology. Together, these studies will aid in the development of the CZL-IVR as well as lay the groundwork for future contraceptives targeting sperm capacitation.