Studies of molecular mechanisms for activation of amphibian eggs.

两栖动物卵激活的分子机制研究。

• 批准号: 05680644
• 负责人: IWAO Yasuhiro
• 金额: $ 1.34万
• 依托单位: YAMAGUCHI UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05680644/
• 关键词: Fertilization; Sperm agonist; Amphibians; Ca channels; Protease; 精子; IP3

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.

在这项研究中，我们从蝾螈的精子中纯化了一种精子激动剂，用于激活青蛙的卵子。通过几种色谱的纯化，我们发现该激动剂是一种具有丝氨酸蛋白酶性质的精子蛋白酶，分子量为170 kDa。精子蛋白酶具有被SBA-凝集素识别的糖部分，并且定位于精子头部的顶体区域。特异性抑制剂或底物对蛋白酶活性的抑制导致卵的激活受到抑制，表明其在Cynops精子激活爪蟾卵中的重要作用。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，精子激动剂在卵子激活时打开Cl离子通道以及Na离子通道。我们已经证明，通过钙离子通道的卵质膜上的钙流出是必要的激活精子激动剂通过详细的分析开放的离子通道的电生理方法。此外，增加的Ca离子刺激增殖性Ca释放依赖于内质网上的IP_3受体。

项目成果

Y.Iwao,A.Miki,M.Kobayshi and K.Onitake: "Activation of Xenopus,eggs by an extract of Cynops sperm." Develp.Growth & Differ.36. 469-479 (1994)

Y.Iwao、A.Miki、M.Kobayshi 和 K.Onitake：“通过蝾螈精子提取物激活爪蟾卵。”

Y.Iwao, A.Miki, M.Kobayashi and K.Onitake: "Activation of Xenopus eggs by an extract of Cynops sperm." Develop.Growth&Differ. 36. 469-479 (1994)

Y.Iwao、A.Miki、M.Kobayashi 和 K.Onitake：“通过蝾螈精子提取物激活爪蟾卵。”

SADAKANE,Y.et al.,: "A Factor that interrupts the cellcycle at G_2 or Prophase is present in Full-grown Frogoocytes" Develop.Growth & Differ.35. 129-138 (1993)

SADAKANE,Y.et al.，：“成熟的青蛙细胞中存在一种在 G_2 或前期中断细胞周期的因子” Develop.Growth

Y.Iwao,A.Miki,M.Kobayashi,and K.Onitake: "Activation of Xenopus eggs by an extract of Cynops sperm." Develp.Growth & Differ.36. 469-479 (1994)

Y.Iwao、A.Miki、M.Kobayashi 和 K.Onitake：“通过蝾螈精子提取物激活非洲爪蟾卵。”

IWAO,Y.et al.,: "The egg nucleus regurates the Behavior of Sperm nuclei as well as cycling of MPF in Physiologically polyspermic newt eggs" Developnental Biology. 160. 15-27 (1993)

IWAO，Y.et al.，：“卵核调节精子核的行为以及生理多精蝾螈卵中 MPF 的循环”发育生物学。