Sperm adhesion and release from oviduct in vitro.

Sperm adhesion and release from oviduct in vitro Gualtieri R. and Talevi R. Dpt. Structural and Functional Biology. University of Naples Federico II, Italy The mammalian oviduct is the natural site where crucial reproductive events, i.e., final gamete maturation, fertilization, and early embryo development, naturally take place. In several mammals the interval from the onset of estrus to ovulation may cover several hours or even days. The oviduct provides a suitable environment where spermatozoa transiently adhere to the epithelial cells of isthmus and the motility and capacitation of adhering spermatozoa remain suppressed until ovulation-associated signals induce detachment of selected and stored spermatozoa allowing their migration toward the ampulla for fertilization. Early in vivo oviduct transillumination studies in naturally mated small rodents, such as mice and hamsters, that represent favourable animal models, provided fundamental information about the sperm ascension through the oviduct. However, the development of in vitro sperm oviduct interaction systems has been indispensable to understand the cellular and molecular mechanisms involved. To this end, two oviductal epithelial culture stages have generally been used: explants an early in vitro culture stage and cellular monolayers that form after several days of culture. Although culture of explants until they attach to the substrate and progressively form a monolayer is accompanied by some signs of de-differentiation such as the regression of cilia on the cell apical surfaces and decrease of the cell height, we generally adopted monolayers for ease of handling, fluorescence analysis and bound sperm counting. The choice to use this culture stage to study bovine sperm-oviduct binding and release in vitro has been substantiated by numerous experiments demonstrating the maintainance of several important features expressed by explants, as well as by the oviduct in vivo. Monolayers retain the ability to bound spermatozoa, to maintain their motility far longer than in unbound spermatozoa, to depress their capacitation, and spermatozoa adhering to monolayers or to explants respond to the treatment with releasing signals in a similar manner. Our studies have been focused on the mechanisms involved in sperm adhesion, selection and release. Main result demonstrate that two different class of molecules, reportedly represented in the oviductal fluid, are capable to induce the sperm release from oviductal monolayers in vitro: sulfated glycoconjugates and disulfide reductants. The possibility to release the sperm sub population selected by in vitro adhesion allowed to demonstrate its superior ability in zona pellucida binding and fertilization. Moreover, we recently showed that disulfide-reductants and sulfated glycosaminoglycans release spermatozoa bound to the fallopian tube epithelium in vitro through the reduction of sperm surface proteins. More interestingly, released spermatozoa promptly recover the ability to adhere after removal of the disulfide-reductant and such a recovery is associated to reoxidation of sperm surface proteins sulfhydryls and to reversal of capacitation. These results suggest that the ascension of spermatozoa through the oviduct might be fine tuned by different reversible and irreversible releasing signals.