PROJECT SUMMARY Rather than the traditional approach of studying the function of one gene in a proposal, our team’s discovery- based approaches have allowed us to define the functions of many novel genes simultaneously. Our bioinformatic discovery of novel reproductive tract-enriched genes and definition of their functions in mouse models provide powerful means to place these gene products into unique biological pathways and gain insight into the molecular processes of mammalian spermatogenesis. With the support of this R01 grant, we created knockouts of over 150 novel genes using CRISPR/Cas9-mediated genome engineering. We prioritized 27 genes that encode secreted, glycosylphosphatidylinositol (GPI)-anchored, and transmembrane proteins that are potential contraceptive targets and/or act in spermiogenesis or for sperm maturation or function; knockouts of 17 of these 27 genes resulted in fertility defects. These classes of genes were chosen because of their potential relevance to infertility in men and their likelihood as targets for male contraception; ~70% of FDA- approved drugs target either membrane-bound or secreted proteins, and thus these proteins could be targets for antibody-based or small molecule contraceptives. To date, these R01 studies have resulted in 38 papers, including 9 papers in Science, PNAS, and PLoS Genetics. In this R01 grant renewal proposal, we will continue to utilize our combined expertise in informatics, biochemistry, molecular biology, chemical biology, and manipulation of the mouse genome. The overall goals of this grant renewal are to mechanistically define the in vivo functions of six novel male reproductive tract-essential proteins that are required for sperm transit through the uterotubal junction (UTJ) and to use DNA-Encoded Chemistry Technology (DEC-Tec) to find chemical entities that can serve as non-hormonal contraceptives. Our hypothesis is that these novel reproductive tract- specific proteins act in evolutionarily conserved pathways required for sperm function, processes that are vulnerable to targeting strategies for contraception. Using DEC-Tec, we have already screened multi-billion compound libraries with contraceptive target proteins in this proposal and identified hits with excellent structure-enrichment relationships (both predicted specific and pan-inhibitor molecules). Our hypothesis will be tested in the following Specific Aims: 1) Define the molecular mechanisms of action of five testis-specific proteins in sperm transit through the UTJ; 2) Determine the functions and functional domains of an epididymis- specific gene in sperm epididymal maturation and transit through the UTJ; and 3) Use small molecules identified through DEC-Tec screens to block sperm transit through the UTJ. Our proof-of-principle studies have important translational implications for human reproductive genetics and contraceptive development: these essential sperm UTJ transit proteins and interacting pathway proteins are potent...