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

细胞质Ca^<2+>是调节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”，在刺激不同细胞时发生。在卵子中，受精时[Ca^<2+>] i显著升高，并触发卵子激活，这是所有物种的共同现象。我们发现哺乳动物受精卵显示出钙离子的振荡。本研究旨在阐明钙振荡诱导蛋白（COIP）的作用机制，并鉴定被认为来源于精子的COIP。我们获得了以下结果：1）我们对小鼠受精卵中[Ca^<2+>]_i的上升进行了时空分析（发表在Dev. Biol.），在注射精子（Cell Calcium）或精子提取物（Dev. Biol.）结果发现COIP存在于精子细胞质中，并产生与受精时相似的Ca^2+振荡。卵皮质对COIP敏感，导致Ca^<2 ...更多信息 +>通过IP_3受体从内质网（ER）释放。Ca^<2+>从卵细胞进入是补充ER（细胞钙）所必需的.我们发现COIP在海鞘中也起作用（发育）。2）我们发现孕酮和环GMP/AMP引起小鼠精子中[Ca^<2+>]_i升高，导致顶体反应，这是精卵融合的先决条件（Biol. Reprod.）。在缺乏表面蛋白CD 9的小鼠中，精卵融合是缺陷的，并且[Ca^<2+>]_i缺乏升高，这表明COIP是通过精卵融合引入的（Nature Genetics）。人类生殖）。4）从仓鼠精子中提取COIP，经硫酸铵处理后，通过各种层析方法纯化得到COIP。在测定了其末端氨基酸序列后，我们克隆了一个COIP候选基因的cDNA，并在大肠杆菌中进行了人工合成。然而，这种蛋白单独不显示COIP活性。COIP的功能可能需要多种因素的影响，我们一直在继续从猪睾丸中纯化COIP，这为COIP的功能提供了更多的样品。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