Male infertility is well-recognized as a major health problem in the United States. Clinically, the major cause is poor semen quality, that is, oligozoospermia, asthenozoospermia, and/or teratozoospermia. About half of infertility cases are thought to be genetic in nature, although the molecular bases are mostly unknown. Remarkably, these sperm defects are not limited to infertile men; ~2% of all men exhibit suboptimal sperm parameters, and the sperm counts among men in many developed countries have more than halved in the past 40 years. Again, the underlying causes are unknown. Thus, it is a priority to understand the molecular basis of these disturbing increases in reproductive problems in men. With respect to genetic and epigenetic contributions to infertility, there are many stages and molecular processes that can be affected, including all stages of spermatogenesis, and roles for thousands of genes required for fertility. This project focuses on post- transcriptional gene regulation during spermiogenesis, the elaborate process by which round haploid spermatids undergo dramatic morphological development to become mature, flagellated spermatozoa with a highly compacted, inert genome. Post-transcriptional gene regulation plays a substantial role in spermiogenesis. The 3′UTR (3′ untranslated region) is central in this respect, affecting mRNA stability, translation and localization by influencing polyadenylation, access to regulatory proteins and noncoding RNAs, and loading onto ribosomes or ribonuclear protein particles (RNPs). This project seeks to understand if and how variation/mutation in the 3′UTRs of infertility patients plays a role in haploid gene expression and infertility. Aim I will determine if there are systematic or specific alterations in the post-transcriptional regulatory function of 3′UTRs in fertile versus infertile men, using 3′RNA-seq of round spermatids purified from ejaculates, and public genome sequence data. Based on these data, we will identify and prioritize variants that reside within motifs predicted bioinformatically to impact mRNA stability or other relevant attributes, focusing on genes implicated in spermiogenesis. In Aim II, candidate variants will be screened via a high-throughput in vitro system to identify those that indeed have an impact on mRNA stability and/or translation. Finally, Aim III will create mouse models of these putative pathogenic variants, and assess molecular and phenotypic effects upon reproductive parameters such as sperm number, morphology, motility, and fertility. Overall, if successful, this will be the largest effort to date to identify genetic lesions that cause infertility via disruption of post- transcriptional gene expression.