245 EFFECT OF INCUBATION IN OVIDUCTAL EPITHELIAL CELLS AND CO-CULTURE WITH OOCYTES IN THE PROTEIN TYROSINE PHOSPHORYLATION PATTERN OF BOAR SPERM

Abstract

The oviductal epithelial cells (OEC) play an important role in the process of sperm capacitation and the acquisition of fertilizing capacity. One of the most important changes that can be observed in spermatozoa is the increase in protein tyrosine phosphorylation. In previous studies, we have demonstrated changes in the pattern of protein tyrosine phosphorylation in boar sperm after the co-culture with OEC. The aim of this study was to characterise the pattern of protein tyrosine phosphorylation in boar sperm after incubation in oviductal cells in the presence of oocytes. Epithelial cells were stripped from the oviducts of cycling sows and cultivated for 7 days on a petri dish. Sperm cells treated by a discontinuous gradient of isotonic Percoll were added to the OEC and incubated for 30 min. After 30 min, the spermatozoa bound and unbound were separated and co-cultured with oocytes for 18 h. Five 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, sperm unbound OEC after 30 min, sperm unbound after 30 min with oocytes, sperm bound after 30 min with oocytes, and sperm bound after 30 min and detached after culture with oocytes. Five patterns were determined according to their surface distribution: nonphosphorylated spermatozoa (pattern 1), head, tail, or both phosphorylated (pattern 2), equatorial segment phosphorylated (pattern 3), equatorial segment and head, tail, or both phosphorylated (pattern 4), and phosphorylation of the tail alone and in combination with other areas of the sperm (pattern 5). Sperm without any treatment showed lower tyrosine phosphorylation levels (87.04%; P < 0.05). Unbound sperm showed a high level of protein tyrosine phosphorylation in the subequatorial segment and head, tail, or both phosphorylated (47.71%; P < 0.05), and this pattern was associated with a high level of capacitation. The same pattern was reduced when unbound sperm were co-cultured with oocytes for 18 h (25.67%; P < 0.05), and was even lower in sperm bound with oocytes. The study of phosphorylation of the tail (pattern 5) showed no significant differences between different samples. We concluded that sperm-bound OEC have lower levels of phosphorylation, indicating a selective function in the sperm-OEC interaction. In addition, phosphorylation of the tails appears to be related more to the hyperactivation of sperm than to the different culture conditions.