A monoclonal antibody that recognizes mammalian cortical granules and a 32-kilodalton protein in mouse eggs

The fertilization-induced exocytosis of egg cortical granules (CGs) is resp onsible for a block to polyspermy, crucial to the viability of many species . The contents of mammalian CGs have been an elusive target for analysis be cause of picogram quantities of CG proteins. By using media enriched in sec reted CG contents from calcium ionophore-induced eggs as an immunogen, a mo noclonal antibody was raised that immunolocalized to structures in the mous e egg cortex with all the hallmarks of CGs. These structures were the corre ct size, absent from the region over the metaphase II spindle, and greatly reduced after fertilization. Double-labeling experiments confirmed that the antibody recognized the same population of CGs as those recognized by Lens culinaris agglutinin. On Western blots, the antibody primarily recognized a 32-kDa protein (and secondarily one at similar to 25 kDa) in mouse eggs. Analysis of biotin-labeled secreted proteins from activated eggs confirmed that CGs release only a small number of major proteins (45, 34, 32, 28, and similar to 20 kDa by SDS-PAGE). We therefore propose that the 32-kDa prote in identified by this antibody is likely to correspond to the 32-kDa protei n released from activated eggs and that it may be involved in the block to polyspermy. These methods should make it possible to generate additional an tibodies to study the structure of CG components as well as their roles in the polyspermy block and CG biogenesis.