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

使用荧光去猝灭方法分析精子顶体反应期间的细胞信号传导

• 批准号: 11680718
• 负责人: KURODA Hideyo
• 金额: $ 2.3万
• 依托单位: University of Toyama
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11680718/
• 关键词: sperm; acrosome reaction; fertilization; sea urchin; fluorescence dequenching; membrane fusion; exocytosis; 蛍光; 脱消光; 細胞内情報伝達; 情報伝達

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.

卵冻的成分通过诱导顶体反应(AR)为受精的精子提供准备。由于海胆精子在AR过程中形态变化轻微，活体精子游动速度快，因此活体精子AR的检测一直比较困难。我们用荧光去猝灭的方法检测了活体精子AR过程中胞膜融合的情况。该方法基于5-N-(十八酰)氨基荧光素(AF18)的荧光不依赖于浓度的自猝灭(去猝灭)。结果表明：(1)被荧光染料染色的海胆精子质膜上的AF18荧光发生了自猝灭；(2)蛋冻(EJ)对AF18荧光的猝灭；(3)这种猝灭的时间进程与扫描电子显微镜观察到的顶体突起的出现相吻合；(4)钙通道阻滞剂(硝苯地平)、K~(2+)通道阻滞剂(TEA)和高K~(+)介质的加入抑制了AF18荧光的猝灭。5)在肌动蛋白聚合抑制剂Latrunculin A存在下，AF18荧光增强不受抑制，顶体颗粒成分被排出，但没有观察到这一过程。用Fura-2测定细胞内Ca~(2+)浓度和AR过程中细胞内K~(++)浓度发现，[Ca~(++)]_i和[K~(++)]_i的升高先于AF18荧光的熄灭。这些结果表明，[Ca~(2+)]_i和[K~(++)]_i升高引起AR。

项目成果

H.Tanaka,R.Kuroda,K.Onitake,K.Takemoto and H.Kuroda: "Increases In pH, and [Ca^<2+>]_i of Sea Urchin Sperm Precede The Exocytosis of Acrosomal Reaction"Jpn.J.Physiol.. 49(Suppl). S36 (1999)

H.Tanaka、R.Kuroda、K.Onitake、K.Takemoto 和 H.Kuroda：“海胆精子的 pH 值增加，[Ca^<2>]_i 先于顶体反应的胞吐作用”Jpn.J.Physiol

Takemoto, K. et al.: "A fluorescence dequenching method for monitoring exocytotic membrane fusion in fertilization of single sea urchin eggs"Biology of the Cell. 91. 5-15 (1999)

Takemoto, K. 等人：“用于监测单个海胆卵受精过程中胞吐膜融合的荧光去猝灭方法”细胞生物学。

Takemoto, K: "A fluorescence dequenching method for monitoring exocytoric membrane fusioninfertilization in single sedurchin egg"Biology of the Cell. 91. 5-15 (1999)

Takemoto, K：“一种用于监测单个 sedurchin 卵受精过程中胞外膜融合的荧光去猝灭方法”《细胞生物学》。