| Abstract: | Monoclonal antibodies (mAbs) have been generated to determine the sperm components responsible for interaction with an egg that results in fertilization. Here, we report upon a group of six different mAbs, all of which localize to a restricted region of the sperm head, the equatorial segment. Several of these mAbs demonstrated cross-reactivity with sperm from the other species tested (human, hamster, rabbit); when cross-reaction occurred, the mAb distribution was restricted to the equatorial segment despite the various configurations that this homologous region assumes in different species. When tested for an effect upon the fertilization process in vitro, ascites fluids containing two of the six mAbs, M29 and M37, displayed significant inhibition. The concentration dependency of this inhibition was observed using purified M29 immunoglobulin M, over a range of 0 to 0.2 mg/ml. The mAb inhibition of fertilization was independent of the presence of either the cellular (the cumulus) or acellular (the zona pellucida) layers surrounding the egg, indicating that the specific locus of inhibition for both of these antisperm mAbs was the egg plasma membrane. Immunologic detection of sperm components separated by electrophoresis on 12% sodium dodecyl sulfate-polyacrylamide gels followed by transfer to nitrocellulose sheets was used to identify the sperm components recognized by two of the mAbs in this group: M29, which inhibited fertilization, and M2, which did not inhibit fertilization. Using M29 mAb, a single sperm component with an apparent subunit molecular weight of approximately 40,000 was detected, whereas in the nitrocellulose strips incubated with M2 mAb two components displayed reactivity, a very prominent band at approximately 44,000 and a tight cluster of bands at approximately 36,000. Parallel nitrocellulose strips of mouse liver did not display these reactivities, consistent with indirect immunofluorescence data in which only testis and sperm, and not liver, kidney, ovary, and epididymal epithelium, demonstrated positive reactivity. These results indicate that the use of mAbs permits identification of sperm components that participate, putatively, in individual events of the fertilization process. Furthermore, using this strategy, we have identified a specific sperm component that appears to be a candidate for a role in sperm fusion with the egg plasma membrane. |
