Activation of sea urchin eggs by microinjection of calcium buffers

Abstract

The effects of changing the intracellular Ca2+ concentration on unfertilized sea urchin eggs were investigated by microinjecting calcium buffers, i.e., aqueous solutions for stabilizing concentrations of free calcium ions (Ca2+) containing calcium salt and calcium-chelating substances (ethyleneglycol bis(β-aminoethylether)-N,N′-tetraacetic acid (EGTA) or N-hydroxyethyl-ethylenediaminetriacetic acid (HEDTA)) at various ratios. When the intracellular Ca2+ concentration was raised to more than 0.2 μM, the elevation of fertilization membrane was observed in more than 50% of eggs in normal sea water. The threshold Ca2+ concentration inducing membrane-elevation was 0.5 μM in eggs in Ca2+-free sea water. Activation accompanying the formation of a monaster was induced by raising the intracellular Ca2+ concentration to 3–4 times the above threshold levels in both eggs in normal sea water and eggs in Ca2+-free sea water. Eggs were not fertilized when the intracellular Ca2+ concentration had been clamped below 0.1 μM by microinjecting calcium buffers before insemination. When the egg was put into sea water containing 5 mM procaine, the threshold Ca2+ concentration for inducing the membrane-elevation increased. It was concluded that the rise of Ca2+ concentration in the cytoplasm to a definite level is the primary cause of the activation of the sea urchin egg at fertilization.