Asymmetric inhibition of spicule formation in sea urchin embryos with low concentrations of gadolinium ion.

As gastrulation proceeds during sea urchin embryogenesis, primary mesenchyme cells (PMCs) fuse to form syncytial cables, within which calcium is deposited as CaCO3, and a pair of spicules is formed. Earlier studies suggested that calcium, previously sequestered by primary mesenchyme cells, is secreted and incorporated into growing spicules. We examined the effects of gadolinium ion (Gd(3+)), a Ca(2+) channel blocker, on spicule formation. Gd(3+) did not lead to a retardation of embryogenesis prior to the initiation of gastrulation and did not inhibit the ingression of PMCs from the blastula wall or their migration along the inner blastocoel surface. However, when embryos were raised in seawater containing submicromolar to a few micromolar Gd(3+), of which levels are considered to be insufficient to block Ca(2+) channels, a pair of triradiate spicules was formed asymmetrically. At 1-3 µmol/L Gd(3+), many embryos formed only one spicule on either the left or right side, or embryos formed a very small second spicule. Induction of the spicule abnormality required the presence of Gd(3+) specifically during late blastula stage prior to spicule formation. An accumulation or adsorption of Gd(3+) was not detected anywhere in the embryos by X-ray microanalysis, which suggests that Ca(2+) channels were not inhibited. These results suggest that Gd(3+) exerts an inhibitory effect on spicule formation through a mechanism that does not involve inhibition of Ca(2+) channels.

Development of calcium releasing activity induced by inositol trisphosphate and cyclic ADP-ribose during in vitro maturation of sea urchin oocytes.

During fertilization of sea urchin eggs, the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) transiently increases (Ca(2+) transient). Increased [Ca(2+)](i) results from a rapid release from intracellular stores, mediated by one or both of two signaling pathways; inositol 1,4,5-trisphosphate (IP(3)) and IP(3) receptor (IP(3)R) or cyclic GMP (cGMP), cyclic ADP-ribose (cADPR) and ryanodine receptor (RyR). During fertilization, cGMP and cADPR increase preceding the Ca(2+) transient, suggesting their contribution to this. If the RyR pathway contributed to the Ca(2+) transient, its Ca(2+) releasing activity would develop in parallel with that of the IP(3) system during maturation of oocytes. Sea urchin oocytes were cultivated in vitro and Ca(2+) transients induced by photolysis of caged IP(3) or caged cADPR were measured during maturation. Oocytes spontaneously began to maturate in seawater. More than 50% of oocytes underwent germinal vesicle breakdown within 25 h and the second meiosis within 35 h, but it took more than 24 h until they became functionally identical to in vivo-matured eggs. Both IP(3) and cADPR induced Ca(2+) transients comparable to those of in vivo-matured eggs later than 24 h from the second meiosis. However, cADPR induced a small Ca(2+) transient even before meiosis, whereas IP(3) and sperm almost did not.