REGULATORY MECHANISMS OF THE ACROSOME REACTION REVEALED BY MULTIVIEW MICROSCOPY OF SINGLE STARFISH SPERM

The acrosome reaction in many animals is a coupled reaction involving an exocytotic step and a dramatic change in cell shape. It has been pr oposed that these morphological changes are regulated by intracellular ions such as Ca2+ and H+. We report here simultaneous visualization, under a multiview microscope, of intracellular free Ca2+ concentration ([Ca2+](i)), intracellular pH (pH(i)), and morphological changes in a single starfish sperm (Asterina pectinifera). [Ca2+](i) and pH(i) wer e monitored with the fluorescent probes indo-1 and SNARF-1, respective ly, The acrosome reaction was induced with ionomycin. After the introd uction of ionomycin in the medium, [Ca2+](i) increased gradually and r eached a plateau in similar to 30 s, The fusion of the acrosomal vacuo le took place abruptly before the plateau, during the rising phase. Al though the speed of the [Ca2+](i) increase varied among the many sperm tested, exocytosis in all cases occurred at the same [Ca2+](i) of sim ilar to 2 mu M (estimated using the dissociation constant of indo-1 fo r Ca2+ of 1.1 mu M). This result suggests that the exocytotic mechanis m in starfish sperm responds to [Ca2+](i) rapidly, with a reaction tim e of the order of one second or less. Unlike the change in [Ca2+](i), an abrupt increase in pH(i) was observed immediately after exocytosis, suggesting the presence of a proton mobilizing system that is trigger ed by exocytosis. The rapid increase in pH(i) coincided with the forma tion of the acrosomal rod and the beginning of vigorous movement of th e flagellum, both of which have been proposed to be pH(i) dependent. T he exocytotic event itself was visualized with the fluorescent membran e probe RH292. The membrane of the acrosomal vacuole, concealed from t he external medium in an unreacted sperm, was seen to fuse with the pl asma membrane.