A Ca2+ wave at fertilization triggers cortical granule exocytosis in sea urchin eggs. New methods for visualizing exocytosis of individual cortical granules were developed using fluorescent probes and confocal microscopy. Electron microscopy previously provided evidence that cortical granule exocytosis results in the formation of long lived depressions in the cell surface. Fluorescent dextran or ovalbumin in the sea water seemed to label these depressions and appeared by confocal microscopy as disks. FM 1-43, a water soluble fluorescent dye which labels membranes in contact with the sea water, seemed to label the membrane of these depressions and appeared as rings. In double labeling experiments, the disk and ring labeling by the two types of fluorescent dyes were coincident to within 0.5 sec. The fluorescent labeling is coincident with the disappearance of cortical granules by transmitted light microscopy, demonstrating that the labeling corresponds to cortical granule exocytosis. Fluorescent labeling was simultaneous with an expansion of the space occupied by the cortical granule, and labeling by the fluorescent dextran was found to take 0.1-0.2 sec. These results are consistent with, and reinforce the previous electron microscopic evidence for long lived depressions formed by exocytosis; in addition, the new methods provide new ways to investigate cortical granule exocytosis in living eggs.
The fluorescence labeling methods were used with the Ca2+ indicator Ca Green dextran to test if Ca2+ and cortical granule exocytosis are closely related spatially and temporally. In any given region of the cortex, Ca2+ increased relatively slowly. Furthermore, Ca2+ began to rise about 8 seconds before the first fluorescent labeling of exocytosis, suggesting that it takes a relatively long time for the Ca2+ concentration to reach a threshold value that triggers fusion. No evidence was found for highly localized Ca2+ increases that could account for the pattern of individual exocytic events. These results indicate that there is a long lag period between Ca2+ rise and cortical granule exocytosis, and that Ca2+ and cortical granule exocytosis are not tightly coupled spatially and temporally.