Contributions of suboolemmal acidic vesicles and microvilli to the intracellular Ca(2+) increase in the sea urchin eggs at fertilization

The onset of fertilization in echinoderms is characterized by instantaneous increase of Ca(2+) in the egg cortex, which is called 'cortical flash', and the subsequent Ca(2+) wave. While the cortical flash is due to the ion influx through L-type Ca(2+) channels in starfish eggs, its amplitude was shown to be affected by the integrity of the egg cortex. Here, we investigated the contribution of cortical granules (CG) and yolk granules (YG) to the sperm-induced Ca(2+) signals in sea urchin eggs. To this end, prior to fertilization, Paracentrotus lividus eggs were treated with agents that disrupt or relocate CG beneath the plasma membrane: namely, glycyl-L-phenylalanine 2-naphthylamide (GPN), procaine, urethane, and NH(4)Cl. All these pretreatments consistently suppressed the cortical flash in the fertilized eggs, and accelerated the decay kinetics of the subsiding Ca(2+) wave in most cases. By contrast, centrifugation of the eggs, which stratifies organelles but not the CG, did not exhibit such changes except that the CF was much enhanced in the centrifugal pole where YG are localized. Surprisingly, we noted that pretreatment of the eggs with these CG-disrupting agents or with the inhibitors of L-type Ca(2+) channels all drastically reduced the density of the microvilli and their individual shapes on the egg surface. Taken together, our results suggest that the integrity of the egg cortex ensures successful generation of the Ca(2+) responses at fertilization, and that modulation of microvilli shape and density may serve as a mechanism of controlling ion flux across the plasma membrane.