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

胞质Ca^<2+>是调控重要细胞功能的关键因子。细胞内Ca^<2+>浓度（[Ca^<2+>]_i）的反复升高称为“Ca^<2+>振荡”，发生在各种细胞的刺激下。在卵子中，在受精时，一个戏剧性的[Ca^<2+>]_i升高，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ca^<2+>的振荡。本研究旨在阐明一种Ca^<2+>振荡诱导蛋白（COIP）的作用机制，并鉴定被认为来自精子的COIP。我们得到了以下结果。1)我们对小鼠卵子在受精时（发表在Dev. Biol.），在将精子（Cell Calcium）或精子提取物（Dev. Biol.）注入卵子后[Ca^<2+>]_i升高进行了时空分析，发现COIP存在于精子细胞质中，并产生与受精时类似的Ca^<2+>振荡。卵皮质对COIP敏感，通过IP_3受体从内质网（ER）释放Ca^<2…More +>。钙<2+>从卵子外部进入是补充ER（细胞钙）所必需的。我们发现COIP也在海鞘（发育）中起作用。2)我们发现黄体酮和环GMP/AMP在小鼠精子中引起a [Ca^<2+>]_i升高，导致顶体反应，而顶体反应是精子与卵子融合的先决条件。天线转换开关)。在缺乏表面蛋白CD9的小鼠中，精卵融合缺陷，缺乏[Ca^<2+>]_i升高，提示COIP是通过精卵融合引入的（Nature Genetics）。3)圆形精子（未成熟精子）缺乏COIP。我们将精子与精子提取物一起注射到小鼠卵子中，成功地受精、胚胎发育和后代（Mol. Human reproduction .）。因此，COIP可用于实际应用。4)采用硫酸铵处理和各种色谱法纯化了仓鼠精子提取物中的COIP。在确定末端氨基酸序列后，我们克隆了COIP候选基因cDNA，并在大肠杆菌中进行了合成。然而，该蛋白单独不表现出COIP活性。影响COIP功能的因素可能多种多样。我们一直在继续从猪睾丸中纯化COIP，从而提供更多的样本。少

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