Studies of molecular mechanisms for activation of amphibian eggs.

在这项研究中，我们从金龟子的精子中提纯了一种精子激动剂来激活非洲爪蛙的卵子。通过多次层析纯化，我们发现该激动剂是一种精子蛋白水解酶，具有丝氨酸蛋白酶的性质，其相对分子质量为170 kDa。精子蛋白水解酶具有SBA-凝集素识别的糖基部分，定位于精子头部顶体区域。蛋白水解酶活性被特定的抑制剂或底物抑制后，卵子的激活受到抑制，这表明它在非洲爪蛙精子激活过程中起着重要作用。由于我们已经在SDS-PAGE下将其纯化为单一条带，因此我们将制备抗它的单抗或获得编码它的c-DNA，以阐明其他脊椎动物中类似的分子。另一方面，精子激动剂在卵子激活时开放氯离子通道和钠离子通道。我们用电生理学方法详细分析了精子激动剂开放的离子通道，证明了卵质膜上钙通道的钙内流是精子激动剂激活精子所必需的。此外，钙离子的增加刺激依赖于内质网IP_3受体的增殖性钙释放。

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.