HAMSTER OVIDUCTIN ENHANCES TYROSINE PHOSPHORYLATION OF SPERM PROTEINS DURING CAPACITATION

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.

IN SITU AND IN VITRO IMMUNOLOCALIZATION OF OVIDUCTIN BINDING SITES ON HAMSTER UTERINE EPITHELIAL CELLS AND DETECTION OF A HAMSTER OVIDUCTIN HOMOLOGUE IN THE FEMALE RAT REPRODUCTIVE TRACT

Oviductin is an oviduct-specific and high-molecular-weight glycoprotein that has been suggested to play important roles in the early events of reproduction. The present study was undertaken to localize the oviductin binding sites in the uterine epithelial cells of the golden hamster (Mesocricetus auratus) both in situ and in vitro, and to detect a hamster oviductin homologue in the female rat reproductive tract. Immunohistochemical localization of oviductin in the hamster uterus revealed certain uterine epithelial cells reactive to the monoclonal anti-hamster oviductin antibody. In order to study the interaction between hamster oviductin and the endometrium in vitro, a method for culturing primary hamster uterine epithelial cells has been established and optimized. Study with confocal microscopy of the cell culture system showed a labeling pattern similar to what was observed using immunohistochemistry. Pre-embedding immunolabeling of cultured uterine epithelial cells also showed gold particles associated with the plasma membrane and microvilli. These results demonstrated that hamster oviductin can bind to the plasma membrane of certain hamster uterine epithelial cells, suggesting the presence of a putative oviductin receptor on the uterine epithelial cell surface. In the second part of the present study, using the monoclonal anti-hamster oviductin antibody that cross-reacts with the rat tissue, we have been able to detect an oviduct-specific glycoprotein, with a molecular weight of 180~300kDa, in the female rat reproductive tract. Immunohistochemical labeling of the female rat reproductive tract revealed a strong immunolabeling in the non-ciliated oviductal epithelial cells and a faint immunoreaction on the cell surface of some uterine epithelial cells. Ultrastructurally, immunogold labeling was restricted to the secretory granules, Golgi apparatus, and microvilli of the non-ciliated secretory cells of the oviduct. In the uterus, immunogold labeling was observed on the cell surface of some uterine epithelial cells. Furthermore, electron micrographs of ovulated oocytes showed an intense immunolabeling for rat oviductin within the perivitelline space surrounding the ovulated oocytes. The findings of the present study demonstrated that oviductin is present in the rat oviduct and uterus, and it appears that, in the rat, oviductin is secreted by the non-ciliated secretory cells of the oviduct.