Project Summary Fertility depends on successful fertilization and early development, processes that occur in the oviduct. Common therapies for human infertility, such as in vitro fertilization and intracytoplasmic sperm injection, are expensive and increase the risks of a variety of problems. More knowledge of how the oviduct interacts with sperm, the cumulus-oocyte complex (COC), and the developing embryo may improve fertility and reduce the need for therapies or lead to the development of improved therapies (i.e. improvements in IVF). The oviduct serves as a reservoir for sperm, after semen deposition and before fertilization. Binding to the oviduct maintains sperm viability and suppresses motility. Sperm are released to move to the upper oviduct (ampulla) to fertilize oocytes. There are many gaps in this model of sperm-oviduct interaction but our studies have begun to fill some of these gaps. We have used a glycomic approach to screen hundreds of glycans and found that glycans with affinity for porcine sperm have either of two motifs, sulfated Lewis X trisaccharide or branched 6- sialylated complex glycans. We also identified two candidate receptors for both glycans on the sperm membrane, PKDREJ and ADAM5, that were not known to bind glycans. Notably, mouse sperm deficient in PKDREJ and other ADAMs do not accumulate beyond the utero-tubal junction, but it is not known if this is due to a problem in binding and retention in the oviduct. Remarkably, if these glycans are immobilized on beads or microscope slides, they can extend sperm lifespan, much like binding to oviduct cells prolongs the lifespan of sperm. Finally, we found that COCs secrete progesterone that signals sperm release from the lower oviduct by inducing hyperactivation so sperm can move toward the site of fertilization. The Specific Aims of this renewal will provide a mechanistic understanding of how sperm bind the oviduct, how binding prolongs sperm lifespan, and how these results may be translated to improve IVF. Aim 1: To determine the function of PKDREJ and ADAM5 in sperm by blocking each protein and mutating each gene in swine. Sperm from pigs that have mutations in these genes have been produced. Sperm that are deficient in each of these proteins will be examined to determine if their ability to bind oviduct cells and their fertility are affected. Aim 2. To determine if sperm binding to glycans diminishes oxidative phosphorylation and the citric acid cycle to lengthen sperm lifespan. Sperm bound to immobilized glycans will be examined to ascertain the specific metabolic changes that are induced and the intracellular signaling that leads to these changes. Aim 3. To determine if oviduct glycans select superior sperm for storage and in vitro fertilization. We will examine whether sperm selected by glycan adhesion have improved characteristics themselves and also produce embryos that more closely resemble in vivo-produced embryos by comprehensive analysis of embryo transcriptome...