244 PROTEIN TYROSINE PHOSPHORYLATION PATTERN OF SPERM BOUND TO THE PORCINE OVIDUCT DURING THE PERIOVULATION STAGE

Abstract

The interaction of spermatozoa and oviductal epithelial cells (OEC) is a controlled process that regulates sperm capacitation and the acquisition of fertilizing ability until the time of ovulation. A crucial signalling event involved in capacitation is 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 bound or unbound to the oviduct of the sow during the periovulation stage. Eight crossbred multiparous sows were inseminated with 3 × 10⁹ sperm. The animals were anesthetized and laparotomies were performed at 36 h after insemination. Ovaries and oviducts were exposed through a midventral incision for collection. Each oviduct was divided into four parts: the ampulla, ampullary-isthmic junction, isthmus, and utero-tubal junction. All segments of the oviduct were flushed to recover spermatozoa, which were subsequently fixed. Tissue obtained from each of the oviduct segments were fixed and embedded in a paraffin block. Sections were mounted on poly-l-lysine-coated slides and deparaffinized. Flushed sperm and oviductal sections were analysed by indirect immunofluorescence using monoclonal antiphosphotyrosine antibodies. Three different sperm subpopulations were determined according to the distribution of protein tyrosine phosphorylation observed: nonphosphorylated spermatozoa (pattern 1), subequatorial segment or subequatorial segment and flagellum phosphorylation (pattern 2), and subequatorial segment and head or flagellum phosphorylation, or both (pattern 3). Data were analysed with SPSS (IBM, Armonk, NY, USA) using one-way ANOVA. After flushing, most sperm were recovered from the utero-tubal junction segment of the oviduct, and sperm exhibited a higher proportion of pattern 2 (81.62%). Unbound sperm showed a high level of protein tyrosine phosphorylation in the subequatorial segment, head and flagellum in the isthmus (32.34%), ampullary-isthmic junction (37.70%), or ampulla region (35.11%; P < 0.05). Very few sperm were attached to OEC, and sperm oviduct binding was mainly found in the isthmus region. The most common tyrosine phosphorylation distribution observed in sperm attached to OEC was pattern 1 (84.21%), although labelling to the subequatorial segment was also observed. Our results showed that only sperm that did not display tyrosine phosphorylation on the sperm acrosome region (head) were found bound to OEC. In conclusion, distinct protein tyrosine phosphorylation patterns were found on sperm bound to OEC. This interaction could be used as a tool for selecting a population of sperm containing low levels of tyrosine phosphorylation.