| Project/Area Number |
16390055
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General physiology
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| Research Institution | Tokyo Women's Medical University |
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Principal Investigator |
MIYAZAKI Shunichi Tokyo Women's Medical University, School of Medicine, Research Assistant, 医学部, 教授 (80010081)
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| Co-Investigator(Kenkyū-buntansha) |
ITO Masahiko Tokyo Women's Medical University, School of Medicine, Research Assistant, 医学部, 助手 (50385423)
AWAJI Takeo Tokyo Women's Medical University, School of Medicine, Assistant Professor, 医学部, 講師 (60297546)
SHIRAKAWA Hideki University of Electro-Communications, Department of Applied Physics and Chemistry Associate Professor, Associate Professor, 電気通信学部, 助教授 (40241070)
ODA Shoji University of Tokyo, Graduate School of Frontier Science, Assistant Professor, 大学院・新領域創成科学研究科, 講師 (50266714)
河内 全 東京女子医科大学, 医学部, 助手 (70322485)
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| Project Period (FY) |
2004 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥14,700,000 (Direct Cost: ¥14,700,000)
Fiscal Year 2006: ¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 2005: ¥4,600,000 (Direct Cost: ¥4,600,000)
Fiscal Year 2004: ¥6,300,000 (Direct Cost: ¥6,300,000)
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| Keywords | Fertilization / Intracellular Calcium Ion / Mammalian Eggs / Calcium Oscillation / Cytosolic Sperm Factor / Egg-activating Protein / Phospholipase C-zeta / Nuclear transpotation abity / 精子因子 / ホスフォリパーゼCゼータ / 核移行 |
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| Research Abstract |
Repetitive transient increase in intracellular Ca^<2+> concentration ([Ca^<2+>]_i) called Ca^<2+> oscillations occur at fertilization of mammalian eggs, and trigger egg activation. Each [Ca^<2+>]_i rise is due to Ca^<2+> release from the endoplasmic reticulum mainly through inositol 1,4,5-trisphosphate (IP_3) receptors. Evidence indicates that Ca^<2+> oscillations are caused by cytosolic sperm factor driven into the ooplasm upon sperm-egg fusion. The Ca^<2+> oscillation-inducing factor is the egg-activating protein (EAP). A current strong candidate of EAP is a novel isozyme of IP_3-producing enzyme, phospholipase C-zeta (PLCζ). This study aims to characterize PLCζ and confirm whether PLCζ operates as EAP at fertilization. The following results were obtained. 1) Injection of recombinant PLCζ that was synthesized by baculovirus/Sf9 cell expression system induced Ca^<2+> oscillations in mouse eggs at low concentrations. 2) PLCζ has such a high Ca^<2+>-sensitivity of PLC activity that the
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enzyme can be active in resting cells at 〜100 nM [Ca^<2+>]_i, suitable for a putative EAP to be introduced into the egg at fertilization. 3) The N-terminal EF-hand domain 1 (EF1) and EF2 are important for the PLCζ activity, and EF3 is responsible for its high Ca^<2+> sensitivity. 4) C2 domain has substantial affinity to PI(3)P and, to the lesser extent, to PI(5)P. 5) Ca^<2+> oscillations were induced by injection of RNA encoding PLCζ fused with a fluorescent protein "Venus". The expressed PLCζ corresponded to the estimated amount contained in a single spermatozoon. 6) Expressed PLCζtranslocated into the formed pronucleus. 7) The nuclear localization signal was located at the X-Y linker region of PLCζ molecule. A highly coordinated three-dimensional structure involving a folding in the middle is thought to be required for not only Ca^<2+> oscillation-inducing activity but also nuclear translocation ability. These results support PLCζ as a strong candidate of EAP. To confirm whether PLCζ operates as EAP at fertilization, PLCζ-knockout mice were produced, and PLCζ-transgenic mice were prepared for reserve PLCζ in the knockout mice sperm. Less
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