HUMAN SPERM SURFACE GLYCOPROTEIN INVOLVED IN SPERM ZONA-PELLUCIDA INTERACTION

To identify peptide-specific antibodies which define sperm surface ant igens, hybridomas were derived from the splenocytes of mice immunized with swollen human spermatozoa which had been subjected to limited pro teolytic cleavage under reducing conditions prior to immunization. A t otal of 13.7% of the hybrid clones secreted antibodies which reacted w ith deglycosylated human seminal plasma glycoproteins when screened by an ELISA. A monoclonal antibody, designated mAb 4A8 sp., specifying a peptide epitope of human epididymal and a sperm surface glycoprotein was selected which inhibited human sperm-egg binding in a dose-depende nt manner, and totally blocked sperm penetration in vitro. This inhibi tion did not result from an effect of the antibody on the motility of spermatozoa, nor was it due to premature induction of the acrosome rea ction. Exclusion of oligosaccharide chains by chemical hydrolysis with trifluoromethane sulphonic acid (TFMS), enzymatic degradation and bin ding of lectins, did not abrogate the reactivity of mAb 4A8 to the cog nate epitope whereas antibody binding was precluded upon digestion wit h proteolytic enzymes. In Western immunoblots of human seminal plasma glycoproteins, the antigen presented as a set of immunoreactive polype ptides, a major glycoprotein of M-r 78 kDa and less prominent bands of M-r 56 and 44 kDa. Immunocytochemical staining of a number of human r eproductive and somatic tissues revealed strong immunostaining of the luminal epithelium of the epididymis as well as of spermatozoa in the lumen. Immunolocalization to the plasma membrane of ejaculated human s permatozoa was demonstrated by immunofluorescence, although on undiges ted spermatozoa the antigen epitope was less accessible. Upon capacita tion the antigen persisted on the sperm surface and was present on the head of capacitated acrosome-intact spermatozoa. The pronounced perip heral immunostaining of the sperm head was accentuated after DTT/tryps in treatment, implicating the dynamic accessibility of the epitope on the plasma membrane of capacitated spermatozoa. It is suggested that t he protein in question appears on the sperm membrane as a consequence of its modification in the epididymis (insertion and processing), and may be involved in the processes leading to sperm attachment and inter action with the human zona pellucida.