The intracellular calcium increase at fertilization in Urechis caupo oocytes: Activation without waves

Abstract

The intracellular Ca2+ (Ca(i)) increase at fertilization of the marine worm Urechis caupo (Echiura) was studied with conventional and confocal epifluorescence microscopy in oocytes microinjected with calcium green dextran or dually labeled with the calcium-insensitive dye tetramethylrhodamine dextran. Calcium green fluorescence was also measured with a photomultiplier system while the oocyte membrane potential was recorded and manipulated. The results show that Ca(i) rises simultaneously around the oocyte cortex and peaks slightly later in the nucleoplasm. The Ca(i) rise coincides with the initiation of the fertilization potential and we conclude that it is due primarily to external Ca2+ entering through the voltage-gated Ca2+ action potential channels that open during the fertilization potential because: (1) current clamping the oocyte membrane potential to positive values in the absence of sperm produces a similar Ca(i) increase, (2) external Ca2+ is required, (3) and the confocal images are consistent with this mechanism. External application of sperm acrosomal peptide (P23) also caused a Ca(i) increase that was inhibited in the presence of CoCl2. Ca(i) and pH(i) (measured with BCECF dextran) were manipulated in experiments employing microinjection of BAPTA (to chelate Ca(i)), external application of NH4Cl (to increase pH(i)) and COCl2 (to block Ca2+ channels), and fertilization of eggs in pH 7 seawater (Ca(i) increase without pH(i) increase). The results showed that increases in both Ca(i) and pH(i) are required for GVBD; neither alone is sufficient. However, although nuclear and cytoplasmic Ca2+ levels tended to parallel each other in oocytes fertilized at pH 7, and during the initial Ca(i) response in oocytes fertilized at pH 8, there was a disproportionate fluorescence increase in the nucleoplasm of the latter prior to GVBD which could not be explained by any artifact we tested, suggesting there may be a selective increase in nuclear Ca2+ associated with GVBD. Finally, electrophysiological experiments with BAPTA-injected oocytes showed that the opening of the fertilization potential Na+ channels was Ca2+-independent, (although they did not close at the normal time). These and earlier results suggest that Urechis sperm may activate oocytes by interacting directly with the Na+ channels or associated receptors.