236 PROTEIN TYROSINE PHOSPHORYLATION IN BOAR SPERM DURING CO-CULTURE WITH OVIDUCTAL EPITHELIAL CELLS

Abstract

Essentials steps of the capacitation are coordinated in the caudal isthmus of the oviduct, where sperm are stored in intimate contact with the epithelium. A crucial event involved in capacitation is the protein tyrosine phosphorylation. The aim of this study was to characterise the different protein tyrosine phosphorylation patterns in boar sperm after the co-culture with oviducal epithelial cells (POEC). Epithelial cells were stripped from the oviducts of cycling sows and cultivated for 7 days on Petri dishes. Sperm (with and without treatment by discontinuous gradient of isotonic Percoll) were added and co-incubated for 1 h. Six different samples were analysed by indirect immunofluorescence to determine the localization of proteins phosphorylated in tyrosine residues (Tardif et al. 2001 Biol. Reprod. 65, 784–792): ejaculated control, ejaculated bound and unbound POEC, sperm washed through Percoll, and sperm Percoll-washed bound and unbound POEC. Four patterns were determined according to their surface distribution: nonphosphorylated spermatozoa, head and/or tail phosphorylated, equatorial segment phosphorylated, equatorial segment, and head and/or tail phosphorylated. Sperm without any treatment showed lower tyrosine phosphorylation levels than sperm washed through Percoll (66.90% v. 1.90%; P < 0.01). The most common tyrosine phosphorylation distribution in sperm adhesion to POEC was located in equatorial segment (57.78%; P < 0.01). A significant difference regarding head phosphorylation (P < 0.01) was found between sperm bound to oviductal epithelium and unbound sperm (0.00% v. 4.75%). Binding to POEC occurred mainly in sperm with nonphosphorylated heads and tails, whereas higher proportions of equatorial segment and head and/or flagellum phosphorylated cells were found in unbound population (50.25% v. 83.75%; P < 0.01). The data of this study showed differences in the sperm-POEC binding in relation to protein tyrosine phosphorylation patterns, indicating a selective function in the sperm-oviductal epithelial cells interaction.