An analysis of the cell signaling during sperm acrosome reaction using a fluorescence dequenching method

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Components of egg jelly prime spermatozoa for fertilization by inducing acrosome reactions (AR). Because the morphological changes during AR of sea urchin sperm are slight and the living sperm swim quickly, it has been difficult to detect AR of living sperm. Using a fluorescence dequenching method, we detected the exocytotic membrane fusion during AR of living sperm. The method is based on the relief from concentration-dependent self-quenching (dequenching) of fluorescence of 5-N-(octadecanoyl)-aminofluorescein (AF18). The validity and usefulness of this method were shown by the following results: l) self-quenching of AF18 fluorescence occurred in the plasma membrane of sea urchin sperm, which were heavily stained with the fluorescent dye; 2) dequenching of AF18 fluorescence occurred by the addition of egg-jelly (EJ); 3) the time course of these dequenching coincided with the appearance of acrosomal processes which were observed by a scanning electron microscope; 4) the dequenching of AF18 fluorescence were inhibited by the addition of a Ca^<2+>-channel blocker (nifedipine), a K^+-channel blocker (TEA) and high K^+- media; 5) In the presence of an inhibitor of actin polymerization, latrunculin A, an increase of AF18 fluorescence was not inhibited, and the ingredients of acrosomal granule were exocytosed, but no process was observed.Measurement of the intracellular Ca^<2+> concentration with fura-2 and the intracellular K^+ concentration during AR revealed that the increases of [Ca^<2+>]_i and [K^+]_i preceded the dequenching of AF18 fluorescence. These results showed that the [Ca^<2+>]_i and [K^+]_i elevation caused the AR.