Characterization and isolation of SOB2, a human sperm protein with a potential role in oocyte membrane binding

G12 monoclonal antibody (mAb), one of a library of constructed mAb directed against human sperm proteins, was found by immunoperoxidase staining to label the post-acrosomal and neck regions of fixed human cauda epididymal and ejaculated spermatozoa. Epithelium and fluid of caput epididymis were strongly labelled while there was no staining on testis and efferent ducts. Western lot analysis revealed that G12 antibody reacted with proteins of 17.5, 18 and 19 kDa in human spermatozoa. This pattern seems to be specific for mature human spermatozoa, as it has not been observed either in other human tissues tested, or in spermatozoa from different animals. SOB2, the corresponding protein, was isolated from NP40-extracted human spermatozoa by using preparative electrophoresis, followed by isoelectrofocusing according to its isoelectric point of 6.4 G12 Fab fragments strongly inhibited binding of human spermatozoa to zona-free hamster oocytes (up to 86% inhibition at 200 micrograms/ml). Impairment of binding was dependent on the concentration of purified G12 immunoglobulin (Ig)G1, and significant even at 10 micrograms/ml. There was no inhibitory effect of G12 antibody on sperm motility parameters or triggering of the acrosome reaction and it did not inhibit binding to human zona pellucida. These results indicate that SOB2 is likely to participate in membrane oocyte binding, and my be potential candidate for the development of a contraceptive vaccine.