Identification and isolation of boar sperm specific antigens with potential role in sperm-egg interaction

Investigations on specific and functionally active sperm antigens would bring about the elucidation of the mechanisms of gamete interaction and help the search to new approaches for prognosis and regulation of fertility. Previously, we have produced a polyclonal rabbit anti-boar spermatozoa antibody (RABSA) that might affect the fertilizing capacity of boar spermatozoa. The sperm specificity of RABSA was demonstrated by double immunodiffusion, immunoelectrophoresis and ELISA against boar spermatozoa, as well as against saline extracts of boar reproductive and somatic organs. Using indirect immunofluorescence (IIF) test, here we provide evidence that RABSA stained the acrosomes of ejaculated and capacitated boar and human spermatozoa, the fluorescence being intensified on the equatorial region after the acrosome reaction. The RABSA cognate antigen/s is a subject of interest because of their specific localization in sperm structures, which is shown to be a binding and/or fusion competence region. Using ion-exchange (Heparin-Sepharose) chromatography, we eluted an antigen with molecular mass 60 kDa (Ag60) in SDS-PAGE from NP40 extracts of capacitated boar spermatozoa. In Western blot, RABSA recognized specifically this antigen. The Ag60 did not affect the sperm-ligand activity of zona pellucida in a porcine sperm-zona binding assay. IIF experiments showed that zona-free porcine oocytes preincubated with Ag60 and RABSA presented fluorescent labeling over the entire egg surface. The biological and IIF experiments provide evidence supporting the involvement of Ag60 in functional steps required for sperm-egg binding and/or fusion, but not sperm-zona pellucida binding.