To analyze fertilization-induced calcium dynamics in a protostome worm, unfertilized oocytes of the nemertean Cerebratulus lacteus were co-injected with calcium green (CG) and rhodamine (Rh) dextrans for dual-channel confocal imaging of early development. Based on CG/Rh ratioed images collected every 800 msec, fertilization elicits a "cortical flash" of elevated free calcium that spreads rapidly around the oocyte without propagating as a point-source wave. A similar calcium transient occurs in unfertilized oocytes treated with KCl to depolarize the oolemma, and the fertilization-induced cortical flash is eliminated if cobalt is used to block calcium channels, collectively indicating that fertilization initially triggers an influx of calcium ions through voltage-gated calcium channels in the oolemma. However, within minutes after producing a cortical flash, C. lacteus oocytes begin to display a series of point-source, oscillating waves of elevated free calcium that are propagated at about 15 micron/sec. The first two calcium waves arise at the site of sperm fusion and typically fail to reach the antipode, but after sperm incorporation, the waves spread globally throughout the ooplasm and typically shift their origin to a pacemaker region in the vegetal cortex. About 10 oscillations with an average duration of 3.3 +/- 1.2 min are generated for approximately 60-100 min postfertilization as meiotic maturation is completed, and such waves continue to occur in cobalt-containing seawater or calcium-free seawater. Thus, wavelike calcium oscillations induced by fertilization are apparently dependent upon internal calcium stores, which in turn may contain IP3-insensitive and/or IP3-sensitive receptors based on experiments using ryanodine, caged IP3, and heparin. Unfertilized oocytes also display repetitive calcium waves following intracytoplasmic injections of whole sperm, and such oscillations are eliminated if the sperm suspensions are boiled prior to injection, suggesting the possible presence of a heat-labile sperm component that can elicit wavelike oscillations during fertilization.
