Project Summary The identification of interacting male-female reproductive proteins is imperative for a molecular understanding of fertilization. The goal of this R21 proposal is to identify a set of human gamete recognition proteins using genomic and proteomic approaches, and then perform evolutionary guided computational bioinformatic searches for a homolog of abalone sperm lysin. It is known from three-dimensional protein crystallographic studies of the major proteins from the mammalian Zona Pellucida (ZP) and abalone Vitelline Envelope (VE) that they contain homologous ZP-N domains. Given the molecular homology of ZP-N domains and overall biochemical similarity of the egg coats, an obvious question to address is whether sperm use similar mechanisms (or homologous proteins) to generate a path through the egg coat. The dissolution of the abalone egg VE by the sperm protein lysin has been extensively characterized. However, it remains unknown how mammalian sperm penetrate egg coats, and no lysin homolog is currently identifiable in any mammalian genome. Our proposal address this question by (1) using long-read sequencing to generate deep coverage of full-length non-chimeric cDNAs for use in sperm proteomics, (2) using these resources as a database to perform detailed bioinformatic analyses aimed at generating a database of putative human gamete recognition proteins and identification of a human homolog of abalone sperm lysin and (3) functional tests of predicted protein's ability to disrupt the mouse ZP. If we identify a human sperm lysin, it will immediately open up new avenues of research into mammalian gamete recognition and how the human sperm passes the ZP. If we are unsuccessful at identifying a human lysin, we will have generated a valuable genomic and proteomic resource database for future studies of human sperm biology.