2003 Fiscal Year Final Research Report Summary
Identification of sperm-derived egg-activating protein at fertilization of mammalian eggs
| Project/Area Number |
13470010
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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 Univ, Dept of Physiology, Professor, 医学部, 教授 (80010081)
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| Co-Investigator(Kenkyū-buntansha) |
AWAJI Takeo Tokyo Women's Medical Univ, Dept of Physiology, Assistant Professor, 医学部, 講師 (60297546)
KOHCHI Zen Tokyo Women's Medical Univ, Dept of Physiology, Research Assistant, 医学部, 助手 (70322485)
ODA Shoji Tokyo Women's Medical Univ, Dept of Physiology, Research Assistant, 医学部, 助手 (50266714)
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| Project Period (FY) |
2001 – 2003
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| Keywords | fertilization / mammalian egg / egg activation / intracellular calcium ion / calcium oscillation / sperm factor / phospholipase C zeta / nuclear translocation |
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| Research Abstract |
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
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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
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[Publications] Ogonuki, N., Sankai T., Yagami, K., Shikano, T., Oda, S., Miyazaki, S., Ogura, A.: "Activity of a sperm-borne oocyte-activating factor in spermatozoa and spermatogenic cells from cynomolgus monkeys and its localization after oocyte activation."Biology of Reproduction. 65. 351-357 (2001)
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[Publications] 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. (in press).
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「研究成果報告書概要(欧文)」より
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[Publications] 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. (in press).
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「研究成果報告書概要(欧文)」より
