Identification of sperm-derived egg-activating protein at fertilization of mammalian eggs

哺乳动物卵子受精时精子来源的卵子激活蛋白的鉴定

• 批准号: 13470010
• 负责人: MIYAZAKI Shunichi
• 金额: $ 9.09万
• 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: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13470010/
• 关键词: fertilization; mammalian egg; egg activation; intracellular calcium ion; calcium oscillation; sperm factor; phospholipase C zeta; nuclear translocation; 卵活性化蛋白質; 前核; 精子一卵融合; CD9; 卵細胞内精子注入法(ICSI)

Mature mammalian eggs are arrested at the metaphase of the second meiosis, and resume meiosis by fertilization, leading to formation of the second polar body and male and female pronuclei. Egg activation is induced by repetitive increase in intracellular Ca^<2+> concentration (Ca^<2+> oscillation). The present study aimed to identify the sperm-derived egg activating protein (EAP) that induces Ca^<2+> oscillations in mouse eggs. We obtained the following results.1)Extract from the hamster sperm or boar testis was separated using various chromatography and Ca^<2+> oscillation-inducing activity of each fraction was assayed by injecting it into mouse eggs. The activity was lost during purification, suggesting that there may be two compensatory components for the function of EAP.2)In CD9-(an egg surface protein) deficient mouse eggs, sperm-egg fusion is defective, and no Ca^<2+> response occurs. The ability of sperm-egg fusion was found to be restored by expressing CD9 in mouse eggs by inje … More ction of CD9 RNA.3)Egg activation is induced by injection of a mouse spermatozoon but not a round spermatid. EAP is thought to be expressed at the stage between the round spermatid and the sperm in the mouse. After fertilization the HAP activity is concentrated to the male and female pronucleus from the egg cytoplasm.4)Actin filaments are involved in sperm-egg fusion and sperm incorporation into the egg. The involvement of Rho family small G proteins in sperm incorporation was demonstrated by inhibition with Clostridium dfficile toxin B.5)A report showed that novel type of phospholipase C (PLCζ) is a strong candidate of EAP. Expression of PLCζ fused with a fluorescent protein induced Ca^<2+> oscillations and is accumulated into the pronucleus.6)PLCζ was synthesized using baculovirus/Sf9 cell system. Recombinant PLCζ induced Ca^<2+> oscillations in eggs. In vitro PLC activity of PLCζ was active at the Ca^<2+> concentration of resting cells. 5 and 6 supported PLCζ as a strong candidate of EAP. Less

成熟的哺乳动物卵子在第二次减数分裂中期被阻滞，通过受精恢复减数分裂，形成第二极体和雌雄原核。卵的活化是由细胞内Ca^<2+>浓度的反复增加引起的（Ca^<2+>振荡）。本研究旨在鉴定在小鼠卵子中诱导Ca^<2+>振荡的精子源性卵子激活蛋白（EAP）。我们得到了以下结果。1)采用各种色谱法分离仓鼠精子或公猪睾丸提取物，并将其注射到小鼠卵细胞中，检测其Ca^<2+>振荡诱导活性。纯化过程中失去了活性，这表明eap的功能可能有两个补偿成分。2)在CD9-（一种卵子表面蛋白）缺陷的小鼠卵子中，精卵融合缺陷，没有Ca^<2+>反应发生。结果表明，小鼠卵细胞内注射CD9可恢复精子与卵细胞的融合能力。EAP被认为在小鼠的圆形精细胞和精子之间的阶段表达。受精后，HAP活性从卵细胞浆集中到雄性和雌性原核。肌动蛋白丝参与精子与卵子的融合和精子与卵子的结合。Rho家族的小G蛋白参与了精子合并，通过抑制艰难梭菌毒素B.5)一份报告显示，新型磷脂酶C （PLCζ）是EAP的一个强有力的候选者。plcs ζ与荧光蛋白融合的表达诱导Ca^<2+>振荡并积累到原核中。6)采用杆状病毒/Sf9细胞体系合成PLCζ。重组PLCζ在卵中诱导Ca^<2+>振荡。在静息细胞Ca^<2+>浓度下，PLCζ在体外具有活性。5和6支持PLCζ作为EAP的有力候选。少

项目成果

Yoda A, Oda S, Shikano T.Kouchi Z.Awaii T, Shirakawa H, Kinoshita K, Miyazaki S.: "Ca^<2+> oscillation-inducing phospholipase C zeta expressed in mouse eggs is accumulated to the pronucleus during egg activation"Developmental Biology. 印刷中.

Yoda A、Oda S、Shikano T.Kouchi Z.Awaii T、Shirakawa H、Kinoshita K、Miyazaki S.：“小鼠卵子中表达的 Ca^<2+> 振荡诱导磷脂酶 C zeta 在卵子激活过程中积累到原核“发育生物学。正在出版。

Kaji, K., Oda, S., Miyazaki, S., Kudo, A.: "Infertility of CD9-deficient mouse eggs is reversed by mouse CD9, human CD9, or mouse CD81 ; polyadenylated mRNA injection developed for molecular analysis of sperm-egg fusion."Developmental Biology. 247. 327-33

Kaji, K.、Oda, S.、Miyazaki, S.、Kudo, A.：“小鼠 CD9、人类 CD9 或小鼠 CD81 可以逆转缺乏 CD9 的小鼠卵子的不育；为精子分子分析而开发的聚腺苷酸化 mRNA 注射

Kouchi Z, Fukami K, Shikano T, Oda S, Nakamura Y, Takenawa T, Miyazaki S.: "Recombinant phospholipase C-zeta has high Ca^<2+> sensitivity and induces Ca^<2+> oscillations in mouse eggs"Journal of Biological Chemistry. 印刷中.

Kouchi Z、Fukami K、Shikano T、Oda S、Nakamura Y、Takenawa T、Miyazaki S.：“重组磷脂酶 C-zeta 具有高 Ca^<2+> 敏感性，可诱导小鼠卵子中的 Ca^<2+> 振荡”生物化学杂志正在出版。

Yoda, A., Oda, S., Shikano, T., Kouchi, Z., Awaji, T., Shirakawa, H., Kinoshita, K., Miyazaki, S.: "Ca^<2+> oscillation-inducing phospholipase C zeta expressed in mouse eggs is accumulated to the pronucleus during egg activation."Developmental Biology. (i

Yoda, A.、Oda, S.、Shikano, T.、Kouchi, Z.、Awaji, T.、Shirakawa, H.、Kinoshita, K.、Miyazaki, S.：“Ca^<2> 振荡诱导磷脂酶

Yoda A, Oda S, Shikano T, Kouchi Z, Awaji T, Shirakawa H, Kinoshita K, Miyazaki S.: "Ca^<2+> oscillation-inducing phospholipase C zeta expressed in mouse eggs is accumulated to the pronucleus during egg activation"Developmental Biology. (印刷中).

Yoda A、Oda S、Shikano T、Kouchi Z、Awaji T、Shirakawa H、Kinoshita K、Miyazaki S.：“小鼠卵子中表达的 Ca^<2+> 振荡诱导磷脂酶 C zeta 在卵子激活过程中积累到原核“发育生物学。（正在出版）。