Studies of molecular mechanisms for activation of amphibian eggs.

在这项研究中，我们从蝾螈（Cynops pyrrhogaster）的精子中纯化了一种精子激动剂，用于激活非洲爪蟾（Xenopus laevis）的卵子。经多次层析纯化，我们发现激动剂是一种具有丝氨酸蛋白酶特性的精子蛋白酶，分子量为170 kDa。精子蛋白酶具有被sba -凝集素识别的糖片段，定位于精子头的顶体区域。特异性抑制剂或底物对蛋白酶活性的抑制导致卵细胞活化受到抑制，说明其在爪蟾精子活化爪蟾卵细胞中起重要作用。由于我们已经在SDS-PAGE下将其纯化为单个条带，我们将制作针对它的单克隆抗体或获得编码它的c-DNA，以澄清其他脊椎动物中的类似分子。另一方面，精子激动剂在激活卵子时打开Cl离子通道和Na离子通道。我们通过电生理方法详细分析了离子通道的打开，证明了钙离子通过卵质膜上的钙通道的流出是精子激动剂激活所必需的。此外，钙离子的增加刺激钙的增殖释放依赖于内质网上的ip_3受体。

In this study, we have purified a sperm agonist from sperm of the newt, Cynops pyrrhogaster to activate eggs of the frog, Xenopus laevis. By the purification with several chromatographies, we have found that the agonist is a sperm protease which shares properties of serine-proteases and have a molecular weight of 170 kDa. The sperm protease has a sugar moiety recognized by SBA-lectin and is localized at the acrosomal region of sperm heads. The inhibition of the protease activity by specific inhibitors or substrates caused the inhibition of egg activation, indicating its important role for activation of Xenopus eggs by Cynops sperm. Since we have purified it as a single band under SDS-PAGE,we are going to make a monoclonal antibody against it or to obtain c-DNA which encodes it to clarify the similar molecules in other vertebrates. On the other hand, the sperm agonist open Cl ion channels as well as Na ion channels at egg activation. We have demonstrated that Ca eflux through Ca channels on the egg plasm membrane is necessary for activation by the sperm agonist by detailed analysis of opening of ion channels by electrophysiological methods. Furthermore, increased Ca ions stimulate a propagative Ca-release dependent upon IP_3-receptors on endoplasmic reticulum.