Sase I, Okinaga T, Hoshi M, Feigenson G W, Kinosita K
Department of Physics, Faculty of Science and Technology, Keio University, Yokohama, Japan.
J Cell Biol. 1995 Nov;131(4):963-73. doi: 10.1083/jcb.131.4.963.
The acrosome reaction in many animals is a coupled reaction involving an exocytotic step and a dramatic change in cell shape. It has been proposed 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 (pHi), and morphological changes in a single starfish sperm (Asterina pectinifera). [Ca2+]i and pHi were monitored with the fluorescent probes indo-1 and SNARF-1, respectively. The acrosome reaction was induced with ionomycin. After the introduction of ionomycin in the medium, [Ca2+]i increased gradually and reached a plateau in approximately 30 s. The fusion of the acrosomal vacuole took place abruptly before the plateau, during the rising phase. Although the speed of the [Ca2+]i increase varied among the many sperm tested, exocytosis in all cases occurred at the same [Ca2+]i of approximately 2 microM (estimated using the dissociation constant of indo-1 for Ca2+ of 1.1 microM). This result suggests that the exocytotic mechanism in starfish sperm responds to [Ca2+]i rapidly, with a reaction time of the order of one second or less. Unlike the change in [Ca2+]i, an abrupt increase in pHi was observed immediately after exocytosis, suggesting the presence of a proton mobilizing system that is triggered by exocytosis. The rapid increase in pHi coincided with the formation of the acrosomal rod and the beginning of vigorous movement of the flagellum, both of which have been proposed to be pHi dependent. The exocytotic event itself was visualized with the fluorescent membrane probe RH292. The membrane of the acrosomal vacuole, concealed from the external medium in an unreacted sperm, was seen to fuse with the plasma membrane.
在许多动物中，顶体反应是一种偶联反应，涉及胞吐步骤和细胞形状的显著变化。有人提出，这些形态变化受细胞内离子如Ca2+和H+的调节。我们在此报告，在多视角显微镜下同时观察单个海星精子（pectinifera海星）的细胞内游离Ca2+浓度（[Ca2+]i）、细胞内pH值（pHi）和形态变化。[Ca2+]i和pHi分别用荧光探针indo-1和SNARF-1监测。用离子霉素诱导顶体反应。在培养基中加入离子霉素后，[Ca2+]i逐渐增加，并在约30秒内达到平台期。顶体小泡的融合在平台期之前、上升阶段突然发生。尽管在所测试的许多精子中[Ca2+]i增加的速度各不相同，但在所有情况下，胞吐作用都发生在相同的[Ca2+]i约2 microM时（使用indo-1对Ca2+的解离常数1.1 microM估算）。这一结果表明，海星精子中的胞吐机制对[Ca2+]i反应迅速，反应时间约为一秒或更短。与[Ca2+]i的变化不同，胞吐作用后立即观察到pHi突然升高，这表明存在一个由胞吐作用触发的质子动员系统。pHi的快速升高与顶体杆的形成以及鞭毛剧烈运动的开始同时发生，这两者都被认为依赖于pHi。胞吐事件本身用荧光膜探针RH292进行了观察。未反应精子中与外部介质隔离的顶体小泡膜被观察到与质膜融合。
