Capacitation is essential for fertilization of ovulated oocytes. Capacitation is correlated with activation of a signal transduction pathway leading to protein tyrosine phosphorylation, an essential prerequisite for fertilization. Oviductin has been shown to bind to the acrosomal cap and the equatorial segment region of the sperm head. In light of findings reported in previous studies, we hypothesized that estrus stage-specific oviductin (EOV) enhances tyrosine phosphorylation. Immunofluorescent detection by light and confocal microscopy and immunogold labeling by electron microscopy and surface replica techniques were used to localize tyrosine phosphorylated proteins to the equatorial segment region and midpiece after incubation in medium in the presence or absence of EOV. In the presence of EOV, an increase in tyrosine phosphorylation in the equatorial segment region was observed as early as 5 minutes after incubation. On prolonging incubation in medium containing EOV immunostaining further increased, indicative of increased levels of tyrosine phosphorylation of sperm proteins as capacitation proceeds. Regardless of the presence or absence of EOV, phosphotyrosine expression was observed along the tail, specifically at the midpiece. However, this reactivity was enhanced in the presence of EOV. Western blot analysis of NP-40 extractable and non-extractable sperm proteins confirmed these observations. NP-40 extractable sperm proteins (25, 37, 44kDa) and non-extractable sperm proteins (70, 83, 90kDa) showed increased intensity when sperm were capacitated in the presence of EOV after 5-, 60-, 120- and 180-minutes of capacitation. Mass spectrophotometric analysis identified enolase, ATP-specific succinyl CoA, succinate CoA ligase, zona pellucida binding protein, heat shock protein 90, aconitase and hexokinase as proteins that undergo enhancement in tyrosine phosphorylation in the presence of EOV. The proteins identified are known to be involved in specific functions including cellular metabolism, molecular chaperoning and normal sperm development. In summary, the present investigation has provided new evidence showing that sperm capacitated in vitro in the presence of EOV display an enhanced expression of tyrosine phosphorylation compared to sperm incubated in capacitating medium alone. These results indicate that inclusion of oviductin in media used for in vitro fertilization (IVF) may improve success rates of IVF by enhancing the signaling pathways involved in sperm capacitation.
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