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

卵冻成分通过诱导顶体反应（AR）为精子受精做准备。由于海胆精子AR过程中的形态变化轻微，且活精子游动速度快，因此一直难以检测活精子的AR。用荧光猝灭法检测了精子AR过程中胞吐膜融合的情况。该方法基于5-N-（十八烷酰基）-氨基荧光素（AF 18）荧光的浓度依赖性自猝灭（去猝灭）缓解。结果表明：1）荧光染料染色的海胆精子质膜上的AF 18荧光发生自猝灭，2）卵冻（EJ）的加入使AF 18荧光去猝灭;（3）扫描电镜观察到顶体突起的出现与去淬灭的时间相一致; 4）Ca^<2+>通道阻断剂可抑制AF 18荧光的去猝灭（硝苯地平）、K^+通道阻断剂（TEA）和高K^+培养基; 5）在肌动蛋白聚合抑制剂latrunculin A的存在下，AF 18荧光的增加没有受到抑制，顶体颗粒的成分被胞吐，用Fura-2测定细胞内Ca^2+浓度和AR过程中细胞内K^+浓度，结果表明，[Ca^2+] i和[K^+] i的增加先于AF 18荧光的去猝灭。结果表明，[Ca^<2+>] i和[K^+] i升高是AR的原因。

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.