Oviduct Binding and Elevated Environmental pH Induce Protein Tyrosine Phosphorylation in Stallion Spermatozoa Running title: PROTEIN TYROSINE PHOSPHORYLATION IN STALLION SPERM

Sperm-oviduct binding is an essential step in the capacitation process preparing the sperm for fertilization in several mammalian species. In many species, capacitation can be induced in vitro by exposing spermatozoa to bicarbonate, Ca2+ and albumin; however, these conditions are insufficient in the horse. We hypothesized that binding to the oviduct epithelium is an essential requirement for the induction of capacitation in stallion spermatozoa. Sperm-oviduct binding was established by co-incubating equine oviduct explants for 2 h with stallion spermatozoa (2 × 10 spermatozoa/ml), during which it transpired that the highest density (per mm) of oviduct-bound spermatozoa was achieved under non-capacitating conditions. In subsequent experiments, sperm-oviduct incubations were performed for 6 h under non-capacitating versus capacitating conditions. The oviduct-bound spermatozoa showed a time-dependent protein tyrosine phosphorylation response, that was not observed in unbound spermatozoa or spermatozoa incubated in oviduct explant conditioned medium. Both oviduct-bound and unbound sperm remained motile with intact plasma membrane and acrosome. Since protein tyrosine phosphorylation can be induced in equine spermatozoa by media with high pH, the intracellular pH of oviduct explant cells and bound spermatozoa was monitored fluorometrically after staining with BCECF-AM dye. The epithelial