Cortical granule exocytosis in sea urchin eggs is initiated at the point of sperm entry, and sweeps over the egg surface 15 to 45 seconds after insemination. This biologically important event releases enzumes and structural proteins that lead to assembly of the fertilization envelope, an extracellular matrix which prevents polyspermy and protects the early embryo. It is an excellent model system for studying the mechanisms of exocytosis. We have taken our previous observations that hyperosmotic sea water inhibits cortical granule exocytosis at different levels depending upon the molecular weight of the osmoticant to the ultrastructural level. We have determined by electron microscopy the exact point at which exocytosis is arrested. We find that granule fusion in hyperosmotic sea water is prevented by formation of a granule-free zone at the cortex that separates the cortical granules from the plasma membrane. The few granules that do fuse are arrested at early stages of pore formation which have not been previously visualized. In contrast, polymer solutions do not block fusion. Instead, exocytic pockets are wide open but contain undischarged granule matrix cores. This suggests that polymers retard water entry into the matrix thus preventing proper dispersal of granule components. These observations allow us to separate cortical granule exocytosis, normally a single continuous process, into four consecutive but distinct steps.
