Molecular characterization of the starfish inositol 1,4,5-trisphosphate receptor and its role during oocyte maturation and fertilization
Molecular characterization of the starfish inositol 1,4,5-trisphosphate receptor and its role during oocyte maturation and fertilization
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DOI：
10.1074/jbc.m108839200
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发表时间：
2002-01-25
影响因子：
4.8
通讯作者：
Mikoshiba, K
中科院分区：
文献类型：
--
作者：
Iwasaki, H;Chiba, K;Mikoshiba, K
钙离子（Ca 2+）从其细胞内储存的释放对于各种物种的卵母细胞的受精是必不可少的。钙池可以通过两种主要类型的钙通道受体诱导释放Ca 2+：肌醇1，4，5-三磷酸受体（IP 3R）和兰尼碱受体。海星卵母细胞经常被用于研究卵母细胞成熟和受精过程中的细胞内钙动员，但细胞内钙通道如何促进细胞内钙动员从未被完全理解，因为这些分子尚未被鉴定，也没有发现这些通道的特异性抑制剂。在这项研究中，我们利用一种新的IP 3受体拮抗剂，“IP 3海绵”，研究IP 3在海星卵母细胞受精过程中的作用。IP 3海绵强烈地和特异性地与内源性IP 3R竞争结合IP 3。将IP 3海绵注射到海星卵母细胞中，虽然卵母细胞的成熟没有被阻止，但胞内钙的增加和受精被膜的形成都被显著地阻止。为了更精确地研究IP 3R在海星卵母细胞中的作用，我们从海星卵巢中克隆了IP 3R，预测的氨基酸序列表明海星IP 3R与哺乳动物IP 3R 1、2和3型具有58-68%的同源性。然后，我们提出了识别海星IP 3R的抗体，并使用该抗体进行免疫印迹分析显示，IP 3R的表达水平在整个卵母细胞成熟过程中保持不变。然而，一项免疫细胞化学研究显示，海星IP 3R的分布在卵母细胞成熟过程中发生变化。
The release of calcium ions (Ca2+) from their intracellular stores is essential for the fertilization of oocytes of various species. The calcium pools can be induced to release Ca2+ via two main types of calcium channel receptor: the inositol 1,4,5-trisphosphate receptor (IP3R) and the ryanodine receptor. Starfish oocytes have often been used to study intracellular calcium mobilization during oocyte maturation and fertilization, but how the intracellular calcium channels contribute to intracellular calcium mobilization has never been understood fully, because these molecules have not been identified and no specific inhibitors of these channels have ever been found. In this study, we utilized a novel IP3R antagonist, the "IP3 sponge," to investigate the role of IP3 during fertilization of the starfish oocyte. The IP3 sponge strongly and specifically competed with endogenous IP3R for binding to IP3. By injecting IP3 sponge into starfish oocyte, the increase in intracellular calcium and formation of the fertilization envelope were both dramatically blocked, although oocyte maturation was not blocked. To investigate the role of IP3R in the starfish oocyte more precisely, we cloned IP3R from the ovary of starfish, and the predicted amino acid sequence indicated that the starfish IP3R has 58-68% identity to mammalian IP3R types 1, 2, and 3. We then raised antibodies that recognize starfish IP3R, and use of the antibodies to perform immunoblot analysis revealed that the level of expression of IP3R remained unchanged throughout oocyte maturation. An immunocytochemical study, however, revealed that the distribution of starfish IP3R changes during oocyte maturation.