Identification of Ca^<2+>-oscillation-inducing Protein and Analysis of Its Action

Ca^2-振荡诱导蛋白的鉴定及其作用分析

• 批准号: 10470011
• 负责人: MIYAZAKI Shunichi
• 金额: $ 7.42万
• 依托单位: Tokyo Women's Medical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-10470011/
• 关键词: Fertilization; Mammalian Egg; Intracellular Ca^<2+>; Ca^<2+> Oscillation; Egg Activation; Sperm Factor; 細胞内カルシウムイオン; カルシウムオシレーション; 精子蛋白質

Cytoplasmic Ca^<2+> is a key factor that regulates important cellular functions. Repetitive rises in intracellular Ca^<2+> concentration ([Ca^<2+>]_i) called "Ca^<2+> oscillations" occur upon stimulation of various cells. In eggs, a dramatic [Ca^<2+>]_i rise occurs at fertilization and triggers egg activation, commom to all species. We have found that fertilized mammalian eggs show ca^<2+> oscillations. This research aimed to elucidate the mechanism of the action of a Ca^<2+> oscillations-inducing protein (COIP) and identify COIP which is thought be derived from the sperm. We obtained the following results.1) We performrd spatiotemporal analysis of [Ca^<2+>]_i rises in mouse eggs at fertilization (published in Dev. Biol.), upon injection of a spermatozoon (Cell Calcium) or sperm extract (Dev. Biol.) into eggs, and found that COIP exists in the sperm cytoplasm and produces Ca^<2+> oscillations similar to those seen at fertilization. The egg cortex is sensitive to COIP which causes Ca^<2 … More +> release from the endoplasmic reticulum (ER) via IP_3 receptors. Ca^<2+> entry from outdide the egg is necessary to refill the ER (Cell Calcium). We found that COIP also functions in the ascidia (Development).2) We found that progesterone and cyclic GMP/AMP cause a [Ca^<2+>]_i, rise in the mouse sperm, leading to the acrosome reaction which is prerequisite for sperm-egg fusion (Biol. Reprod.). In the mice which lack surface protein CD9, sperm-egg fusion was defective, and [Ca^<2+>]_i rise was lacking, suggesting that COIP is introduced through sperm-egg fusion (Nature Genetics).3) Round spermatids (immature sperm) lack COIP.We injected a spermatid together with sperm extract into a mouse egg and succeeded in fertilization, enbryonic development, and offspring (Mol. Human Reprod.). Thus, COIP is available for practical use.4) We purified COIP from hamster sperm extract by treating with ammonium sulfated and passing various chromatography. After determinating amino-acid sequence in the terminus, we cloned cDNA of a COIP candidate, and synthesized it in E.Coli. However, this protein alone did not exhibit the COIP activity. Multiple factors may be necessary for function of COIP.We have been continuing to purifiy COIP from pig testis which provides much more samples. Less

胞质钙离子是调节细胞重要功能的关键因子。当刺激不同的细胞时，细胞内钙离子浓度([Ca^&lt；2+&gt；]_i)反复升高，称为“钙振荡”。在卵子中，[Ca~(2+)]_i在受精时显著升高，并触发卵子激活，这在所有物种中都是常见的。我们发现，哺乳动物受精卵表现出明显的振荡。本研究旨在阐明一种钙振荡诱导蛋白(COIP)的作用机制，并鉴定一种被认为来自精子的COIP。我们得到了以下结果：1)我们对受精时小鼠卵子中[Ca^&lt；2+&gt；]_i的升高进行了时空分析(发表在Dev.在注射精子(细胞钙)或精子提取液(Dev.Biol.)进入卵子，发现COIP存在于精子细胞质中，并产生与受精时类似的钙振荡。卵皮质对引起钙离子通道阻滞剂…的COIP敏感更多的+&gt；通过IP_3受体从内质网释放。从鸡蛋中提取钙是补充内质网(细胞钙)所必需的。我们发现COIP也在腹水中发挥作用(发育)2)我们发现孕酮和环状GMP/AMP引起小鼠精子中[Ca^&lt；2+&gt；]_i升高，导致顶体反应，这是精子-卵子融合的先决条件(Biol.报告。)。在缺乏表面蛋白CD9的小鼠中，精子-卵子融合有缺陷，[Ca^&lt；2+&gt；]_i升高，这表明COIP是通过精卵融合(自然遗传学)导入的。3)圆形精子细胞(未成熟精子)缺乏COIP。人类报告。)。4)采用硫酸铵盐析和层析的方法从金黄地鼠精子提取液中分离纯化得到CoIP。在确定末端氨基酸序列后，我们克隆了一个COIP候选基因的cDNA，并在E.Coli中合成了它。然而，该蛋白本身并不表现出COIP活性。COIP的功能可能需要多种因素，我们一直在继续从猪睾丸中纯化COIP，这提供了更多的样本。较少

项目成果

Deguchi,R.,Shirakawa,H.,Oda,S.,Mohri,T.and Miyazaki,S.: "Spatiotemporal analysis of Ca^<2+> waves in relation to the sperm entry site and animal-vegetal axis during Ca^<2+> oscillations in fertilized mouse eggs."Developmental Biology. 218. 299-313 (2000)

Deguchi,R.、Shirakawa,H.、Oda,S.、Mohri,T. 和 Miyazaki,S.：“Ca^<2> 波在 Ca^ 过程中与精子进入部位和动植物轴相关的时空分析

Kaiji, K., Oda, S., Shikano, T., Ohnuki, T., Uematsu, Y., Sakagami, J., Tada, N., Miyazaki, S.and Kudo, A: "The gamete fusion process is defective in eggs of Cd9-deficient mice."Nature Genetics. 24. 279-282 (2000)

Kaiji, K.、Oda, S.、Shikano, T.、Ohnuki, T.、Uematsu, Y.、Sakagami, J.、Tada, N.、Miyazaki, S. 和 Kudo, A：“配子融合过程是

Oda,S.,Deguchi,R.,Mohri,T.,Shikano,T.,Nakanishi,S. and Miyazaki,S.: "Spatiotemporal dynamics of the [Ca^<2+>]_i rise induced by microinjection of sperm extract into the mouse egg: preferential induction of a Ca^<2+> wave from the cortex"Developmental Biol

小田 S.、出口 R.、毛利 T.、鹿野 T.、中西 S.

Kaji,K.,Oda,S.,Shikano,T.,Ohnuki,T.,Uematsu,Y.,Sakagami,J.,Tada,N.,Miyazaki,S.and Kudo,A.: "The gamete fusion process is defective in eggs of Cd9-deficient mice."Nature Genetics. 24. 279-282 (2000)

Kaji,K.、Oda,S.、Shikano,T.、Ohnuki,T.、Uematsu,Y.、Sakagami,J.、Tada,N.、Miyazaki,S. 和 Kudo,A.：“配子融合过程

Kyozuka,K.,Deguchi,R.,Mohri,T.and Miyazaki,S.: "Injection of sperm extract mimics spatiotemporal dynamics of Ca^<2+> responses and progression of meiosis at fertilization of ascidian oocytes."Development. 125. 4099-4105 (1998)

Kyozuka,K.、Deguchi,R.、Mohri,T. 和 Miyazaki,S.：“注射精子提取物可模拟海鞘卵母细胞受精时 Ca^2 反应和减数分裂进展的时空动态。”开发。