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Identification of sperm-derived egg-activating factor and analysis of molecular mechanisms of intracellular Ca^<2+> increase at fertilization of mammalian eggs

Research Project

Project/Area Number 08457016
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field General physiology
Research InstitutionTokyo Women's Medical College

Principal Investigator

MIYAZAKI Shunichi  Tokyo Women's Medical College, School of Medicine, Professor, 医学部, 教授 (80010081)

Co-Investigator(Kenkyū-buntansha) AWAJI Takeo  Tokyo Women's Medical College, School of Medicine, Researchi assistant, 医学部, 助手 (60297546)
SHIRAISHI Koichi  Tokyo Women's Medical College, School of Medicine, Researchi assistant, 医学部, 助手 (50256476)
SHIRAKAWA Hideki  Tokyo Women's Medical College, School of Medicine, Researchi assistant, 医学部, 助手 (40241070)
ODA Shoji  Tokyo Women's Medical College, School of Medicine, Research assistant, 医学部, 助手 (50266714)
Project Period (FY) 1996 – 1997
Project Status Completed (Fiscal Year 1997)
Budget Amount *help
¥6,900,000 (Direct Cost: ¥6,900,000)
Fiscal Year 1997: ¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 1996: ¥3,600,000 (Direct Cost: ¥3,600,000)
KeywordsFERTILIZATION / EGG / SPERM / EGG ACTIVATION / INTRACELLUAR Ca^<2+> / Ca^<2+> WAVE / Ca^<2+> OSCILLATION / 哺乳動物卵 / 哺乳動物精子 / 卵活性化因子 / 細胞内カルシウム / カルシウムオシレーション / 精子細胞質因子
Research Abstract

This study was performed as a step to elucidate the molecular mechanism by which the sperm induces an increase in intracellular Ca^<2+>, the pivotal signal for egg activation at fertilization. The following results were obtainde by microinjection techniques, Ca^<2+> image analysis, and confocal laser scanning microscopy.
1)As a phenomenological observation, we established the experimental condition in which sperm-egg fusion is formed only transiently without sperm entry into the sea urchin egg in the presence of a gamete fusion inhibitor, jaspisin. A localized Ca^<2+> rise restricted at the sperm binding site was recorded, separated fron the Ca^<2+> wave which spreads throughout the egg at normal fertilization. Spatiotemporal aspects of the sprem-induced localized Ca^<2+> rise were characterized in detail.
2) Injection of a mouse spermatozoon into an egg (intracytoplsmic sperm injection, ICSI) caused repetitive transient Ca^<2+> rises (Ca^<2+> oscillalions) as seen at fertilization, star … More ting 15 to 30 minutes after sperm injection and persisting for several hours. Each Ca^<2+> rise occurred almost synchronously in the whole egg. The results suggested that a sperm cytosolic factor leaks out from the injected spermatozoon into the egg cytoplasm and can induce Ca^<2+> oscillations, even if sperm-egg binding is bypassed by ICSI.
3) We found that injection of a precursor sperm, round spermatids without flagellum (round spermatid injection, ROSI), can not induce any Ca^<2+> response in the egg but that combined injection of a potent agonist of the inositol 1,4,5-trisphosphate (IP_3) receptor resulted in egg activation (fertilization) associated with male and female pronucleus formation. About 25% of two-cell embryos transplanted into foster mothers developed to normal offspring. The newborn infants grew up to normal adults, which reproduced normal second generations.
4)We partially purified a cytosolic protein from hamster and ascidian spermatozoa that possesses the activity of inducing Ca^<2+> oscillations when injected into hamster, mouse, and ascidian eggs. Microinjection of the sperm factor into a localized region of the egg showed that the peripheral cytoplasm is more sensitive to the sperm factor to generate Ca^<2+> increase (possibly due to Ca^<2+> release from the endoplasmic reticulum through IP_3 receptors), compared with the central region of the egg.
These results suggested that, at fertilization, a sperm cytosolic factor could be introduced into the cortical region of the egg cytoplasm through cytoplasmic continuity formed between the sperm and egg, and induce Ca^<2+> release in the egg. This study provided useful information for advancing further studies in future. Less

Report

(3 results)
  • 1997 Annual Research Report   Final Research Report Summary
  • 1996 Annual Research Report
  • Research Products

    (22 results)

All Other

All Publications (22 results)

  • [Publications] Nakano, T., Shirakawa, H. Mitsuhashi, N. Kuwabara, Y.and Miyazaki, S.: "Spatiotemporal dynamics of intracellular calcium in the mouse egg injected with a spermatozoon." Molecular Human Reproduction. 3. 1087-1093 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Mohri, T., Miyazaki, S., Shirakawa, H.and Ikegami, S.: "Sperm-induced local 〔Ca^<2+>〕i rise speparated from the Ca^<2+> wave in sear urchin eggs in the presence of a gamete fusion inhibitor,jaspisin." Development. 125. 293-300 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Sato, Y., Miyazaki, S.Shikano, T., Mitsuhashi, N., Takeuchi, Y. Mikoshiba, K.Kuabara, Y.: "Adenophostin,a potent agonist of the inositol 1,4,5-trisphosphate receptor,is usful for bertilization of mouse eggs injected with roand spcematids leading to normal offspring" Biology of Reproduction. (印刷中). (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Shirakawa, H.and Miyazaki, S.: "Spatiotemporal analysis of intracellular calcium increases in hamster sperm induced by solxcbilized" Japanese Journal of Phyiology zona pellucida.47. 562- (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Sato, Y., Shirakawa, H.and Miyazaki, S.: "The use of in agonist of inosital 1,4,5-trisphosphate receptor,adenophestin,for activation of mouse eggs injected with round spermatid." Japanese Journal of Phyiology. 47. S61- (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Mohri, T.Ikegami, S.and Miyazaki, S.: "Analysis of sperm-induced Ca^<2+> response and membrane current in sear urchin eggs in the presence of jaspisin." Cell structure and Function. 22. 782- (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] 白川 英樹, 宮崎 俊一(分担執筆): "細胞内カルシウム実験プロトコール" 工藤佳久編 羊土社, 201 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Nakano, Y., Shirakawa, H., Mitsuhashi, N., Kuwabara, Y.and Miyazaki, s: "Spatiotemporal kynamics of intracellular calcium in the mouse egg injected with a spermatozoon." Mol.Human Reprod. 3. 1087-1093 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Mohri, T., Miyazaki, S., Shirakawa, H.and Ikegami S.: "Sperm-induced local [Ca^<2+>] _j rise separated from the Ca^<2+> wave in sea urchin eggs in the presence of a gamete fusion inhibitor, jaspisin" Development. 125. 293-300 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Sato, Y., Myazaki, S., Shikano, T., Mitsuhashi, N., Takeuchi, Y., Mikoshiba, Y.and Kuwabara, Y.: "Adenophostin, a potent agonist of the inositol 1,4,5-trisphosphate receptor, is useful for fertilization of mouse eggs injected with round spermatids leading to normal offspring" Biol.Reprod.in press. (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Shirakawa, H.and Miyazaki, S.: "Spatiotemporal analysis of intracellular calcium increases in hamster sperm induced by solubilized zona pellucida." Jpn.J.Physiol.47, Suppl.2. S62 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Sato, Y., Shirakawa, H.and Miyazaki, S.: "The use of an agonist of inositol 1,4,5-trisphosphate receptor, adeno-phostin, for activation of mouse egg injected with round spermatids." Jpn.J.Physiol.47, Suppl.2. S61 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Mohri, T., Ikegami, S.and Miyazaki, S.: "Anaysis of sperm-induced Ca^<2+> response and membrane current in sea urchin eggs in the presence of jaspisin" Cell Struct.Func.22. 782 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Kyozuka, K., Degichi, R., Mohri, T.and Miyazaki, S.: "Intracellular calcium oscillations and meiosis resumption from metaphase I induced by injection of the sperm extract in the ascidian Ciona savignyi" Zool.Sci.in press. (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Nakano, T., Shirakawa, H., Mitsuhashi, N., Kuwabara, Y.and Miyazaki, S.: "Spatiotemporal dynamics of intracellular calcium in the mouse egg injected with a spermatojoon." Molecular Human Reproduction. 3. 1087-1093 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Mori, T., Miyazaki, S., Shirakawa, H.and, Ikegami, S.: "Sperm-induced local[Ca^<2+>]rise speparated from the Ca^<2+> wave in sear urcbin eggs in the presence of a garnete fusion inhibitor,jaspisin." Development. 125. 293-300 (1998)

    • Related Report
      1997 Annual Research Report
  • [Publications] Sato, Y., Miyazaki, S., Shikano, T., Mitsuhashi, N., Takeuchi, Y., Mikoshiba, K.: "A denopohostin,a potent aganist of the inosital 1,4,5-trisphosphate receptor,is Kuwabara,Y.usful for fertilization of mouse eggs injected with to and spermatids reading to non mal offsring" Biology of Reproduction. 58. 867-873 (1998)

    • Related Report
      1997 Annual Research Report
  • [Publications] Shirakawa, H.and Miyazaki, S.: "spatiotemporal analysis of intracellular calcium increases in hamster sperm induced by soblbilized jona pellucida." Japanese Journal of Physiology. 47. S62 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Sato, Y., Shirakawa, H.and Miyazaki, S.: "The use of an agonist of inositol 1,4,5-trisphosphate receptor,adenophastin,for activation of mouse eggs injected with round spermatid." Japanese Journal of Physiology. 47. S61 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Mohri, T., Ikegami, S and Miyazaki, S.: "Analysis of sperm-induced Ca^<2+> response and membrance current in sear urchim eggs in the presence of jaspisin." Cell structure and Function. 22. 782 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] 宮崎俊一: "受精と卵細胞内情報伝達機構" 日本受精着床学会雑誌. (印刷中). (1997)

    • Related Report
      1996 Annual Research Report
  • [Publications] 尾田正二,宮崎俊一: "受精とカルシウム" 細胞工学. 16. 87-93 (1997)

    • Related Report
      1996 Annual Research Report

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Published: 1996-04-01   Modified: 2016-04-21  

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