PROJECT SUMMARY/ABSTRACT Docosahexaenoic acid (DHA) is an omega-3 (ω-3) dietary-derived essential polyunsaturated fatty acid (PUFA) present in particularly high levels in testes. DHA is a critical component of mitochondrial and cellular membranes that regulates membrane fluidity, susceptibility to oxidative stress, and membrane-protein interactions. Multiple studies using mouse models or analyzing human populations have clearly shown that DHA deficiency negatively impacts spermatogenesis to cause male infertility, and that supplementation with DHA improves semen quality and fertility. Unfortunately, Western diets are remarkably deficient in w-3 fatty acids, and therefore a majority of the world’s population is at risk for DHA-deficiency. Despite these clearly established links between DHA and male fertility, DHA’s role and mechanism of action in spermatogenic cells remain unknown. To address this, the Geyer lab (experts in spermatogenesis) has partnered with the Ellis lab (experts in lipid metabolism), and this unique partnership has provided the applicant (Dr. Ben Hale) with formal training in both disciplines. This training will enable Dr. Hale to pursue the long-term goals to define the roles of PUFAs in germ cell metabolism and sperm formation and function. The proposed project herein is an outgrowth of work from our group characterizing a new diet-independent genetic mouse model of DHA deficiency by whole-body deletion of ‘acyl-CoA synthetase 6’ (Acsl6), which encodes an enzyme essential for cellular DHA metabolism. We recently reported whole-body Acsl6 knockout (KO) mice have significantly reduced testicular DHA levels, multiple defects in spermatogenesis, and are severely subfertile. These Acsl6 KO mice are the ideal tool to define the specific mechanisms underlying DHA function in spermatogenesis. We will directly correlate defects in sperm function and morphogenesis to changes in germ cell lipid distribution. We will test the central hypothesis that ACSL6 is required to incorporate DHA into germ cell lipid membranes to facilitate sperm morphogenesis. This hypothesis will be tested in the following Aims: 1) Determine how Acsl6 KO sperm are defective, and 2) determine the roles of DHA during spermatogenesis. The completion of these Aims will provide an unprecedented understanding of how incorporation of DHA into the germ cell facilitates sperm morphogenesis and function.