PROPAGATION OF TRANSIENT CA2+ INCREASE IN SEA-URCHIN EGGS UPON FERTILIZATION AND ITS REGULATION BY MICROINJECTING EGTA SOLUTION

Upon fertilization, the concentration of intracellular Ca2+ (Ca(i)) in sea urchin eggs increased up to 3-mu-M when measured with fura-2, a fluorescent Ca indicator and the increase in Ca(i) traversed from the sperm entry point as a wave over the entire egg at the mean propagation velocities of 5.0-mu-m/sec in C. japonicus egg and 5.3-mu-m/sec in H. pulcherrimus egg. However, the velocity was not uniform; i.e., it was rapid in the vicinity of the sperm entry point and the opposite point, but slow in the central region of the egg. Microinjecting a Ca-EGTA buffer and an IP3 solution into the C. japonicus egg induced the transient Ca(i) increase more rapidly than that upon fertilization, due perhaps to the diffusion of the injectates. In order to investigate Ca2+ release during Ca(i) increase upon fertilization, EGTA solutions were microinjected into unfertilized or fertilizing eggs. Microinjecting 100 mM EGTA (final concentration of 1 mM) not only suppressed the transient Ca(i) increase, but also reduced the increased Ca(i) rapidly, and never induced egg activation after insemination, whereas 10 mM EGTA (final concentration of 0.1 mM) did not significantly affect the Ca(i) increase or the activation. Ca2+ released upon fertilization was estimated to be 150-170-mu-M in the egg cytoplasm from the amount of microinjected EGTA and fura-2. It was concluded that although more than 150-mu-M of Ca2+ was released intracellularly upon fertilization, Ca(i) increased to only a few mu-M because most of the released Ca2+ was sequestered by intracellular Ca2+ binding substances.