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Biological nature and cDNA cloning of sperm ligand binding to pZP1

Research Project

Project/Area Number 06454481
Research Category

Grant-in-Aid for General Scientific Research (B)

Allocation TypeSingle-year Grants
Research Field Obstetrics and gynecology
Research InstitutionHyogo College of Medicine

Principal Investigator

KOYAMA Koji  HYOGO COLLEGE OF MEDICINE,DEPT.OF OB/GYN,PROFESSOR, 医学部, 教授 (00068496)

Co-Investigator(Kenkyū-buntansha) KOMORI Shinji  HYOGO COLLEGE OF MEDICINE,DEPT.OF OB/GYN,ASSISTANT, 医学部, 助手 (60195865)
YAMASAKI Noriyuki  HYOGO COLLEGE OF MEDICINE,DEPT.OF OB/GYN,ASSISTANT, 医学部, 助手 (50174644)
HASEGAWA Akiko  HYOGO COLLEGE OF MEDICINE,DEPT.OF OB/GYN,ASSISTANT, 医学部, 助手 (50212402)
Project Period (FY) 1994 – 1995
Project Status Completed (Fiscal Year 1995)
Budget Amount *help
¥4,500,000 (Direct Cost: ¥4,500,000)
Fiscal Year 1995: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1994: ¥3,900,000 (Direct Cost: ¥3,900,000)
KeywordsFertilization / Acrosome Reaction / Zona Pellucida / Sperm Ligand / Secondary Sperm Receptor / Proacrosin / pZP1 / 卵透明帯 / ブタ透明帯エピトープ / ブタZP1 / 避妊ワクチン
Research Abstract

The zona pellucida is an extracellular matrix surrounding mammalian oocytes and possesses various important functions for fertilization. A component of mouse zona pellucida (mZP2) has been shown to function a secondary sperm receptor. Recently, we succeeded in cloning a cDNA coding for a porcine zona pellucida glycoprotein (pZP1). The cDNA sequence was 54% homologous to mZP2. The aim of this study is to examine the function of pZP1 using recombinant pZP1 (r-pZP1). The recombinant protein used in this study was the NH_2-terminal region (amino acid positions 1-198) of pZP1. Human sperm was prepared by washing and centrifugation in BWW medium containing BSA (3mg/ml). After preincubation in 5% CO_2 in air for 3 hours, the sperm was incubated for 15min in the medium containing ionophore A23187 (10 mu M) to induce the acrosome reaction (AR). r-pZp1 (1-198) was added to the sperm suspension at a final concentration of 10 mu g/ml. The methanol-fixed sperm was treated with a monoclonal antibody (MAb-5H4), which has been proven to recognize an epitope on pZP1 (50-59), and followed by thetreatment with FITC conjugated anti-mouse IgG.AR was assessed by PSA lectin staining. When r-pZP1 (1-198) was added to the AR induced human sperm suspension, the number of sperm bound the protein gradually increased during the incubation. While, the treatment of sperm with r-pZP1 (1-198) did not increase AR.When the r-pZP1 (1-198) was added to in vitro fertilization medium, neither sperm binding to human zona pelludica nor sperm penetration into zona-free hamster eggs was interfered. r-pZP1 (1-198) was shown to bind to the acrosome reacted sperm but not to acrosme intact sperm. This suggests that the NH2-terminal portion (1-198) of pZP1 might be functional for fertilization without carbohydrate residues. It is thus concluded that the domain of pZP1 (1-198) serves as a secondary sperm receptor in the same manner to mZP2.

Report

(3 results)
  • 1995 Annual Research Report   Final Research Report Summary
  • 1994 Annual Research Report
  • Research Products

    (18 results)

All Other

All Publications (18 results)

  • [Publications] HASEGAWA,A.,INOUE,M.,KOYAMA,K. and ISOJIMA,S.: "Binding of a recombinant pZP1(I-198)zona protein to acrosomereacted human sperm." Proceedings of the 8th Annual Meeting of Japan Society for Immunology of Reproduction.8. 63-66 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] HASEGAWA,A.,YAMASAKI,N.,INOUE,M.,KOYAMA,K. and ISOJIMA,S.: "Analysis of an epitope sequence recognized by a fertilization blocking monoclonal antibody (MAb-5H4) against a porcine zona pellucida glycoprotein (pZP1)." Journal of Reproduction and Fertility.105. 295-302 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] TAYA,T.,YAMASAKI,N.,TSUBAMOTO,H.,HASEGAWA,A.and KOYAMA,K.: "Cloning of a cDNA coding for porcine zona pellucida glycoprotein ZP1 and its genomic organization." Biochemical and Biophysical Research Communications. 207. 790-799 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] TSUBAMOTO,H.,HASEGAWA,A. INOUE,M.,YAMASAKI,N. and KOYAMA,K.: "Bindings of a recombinant porcine zona protein (pZP1) to acrosomereacted spermatozoa." Proceedings of International Symposium on Prospects of Zona Pellucida Glycoproteins for Immunocontraception and 7th Annual Conference of Indian Society for the Study of Reproduction and Fertility.(in press). (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] 長谷川昭子、井上みゆき、山崎則行、香田 浩二: "卵透明帯の第二精子レセプターと反応する精子側リガンドについて" 日本受精着床学会雑誌. 13(発表予定). (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] HASAGAWA,A., INOUE,M., KOYAMA,K.and ISOJIMA,S.: "Binding of a recombinant pZP1 (1-198) zona protein to acrosome-reacted human sperm." Proceedings of the 8th Annual Meeting of Japan Society for Immunology of Reproduction.63-66 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] HASEGAWA,A., YAMASAKI,N., INOUE,M., KOYAMA,K.and ISOJIMA,S.: "Analysis of an epitope sequence recognized by a fertilization blocking monoclonal antibody (MAb-5H4) against a porcine zona pellucida glycoprotein (pZP1)." Journal of Reproduction and Fertility.105. 295-302 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] TAYA,T., YAMASAKI,N., TSUBAMOTO,H., HASEGAWA,A.and KOYAMA,K.: "Cloning of a cDNA coding for porcine zona pellucida glycoprotein ZP1 and its genomic organization." Biochemical and Biophysical Research Communications. 207. 790-799 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] TSUBAMOTO,H., HASEGAWA,A., INOUE,M., YAMASAKI,N.and KOYAMA,K.: "Binding of a recombinant porcine zona protein (pZP1) to acrosome-reacted spermatozoa." Proceedings of International Symposium on Prospects of Zona Pellucida Glycoproteins for Immunocontraception and 7th Annual Conference of Indian Society fo the Study of Reproduction and Fertility.(in press). (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] HASEGAWA,A., INOUE,M., YAMASAKI,N.and KOYAMA,K.: "The sperm ligand to the secondary sperm receptor in the zona pellucida." Journal of Fertilization and Implantation.13 (1), (in press). (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] HASEGAWA, A., INOUE, M., KOYAMA, K. and ISOJIMA, S.: "Binding of a recombinant pZP1 (1-198) zona protein to acrosome-reacted human sperm." Proceedings of the 8th Annual Meeting of Japan Society for Immunology of Reproduction.8. 63-66 (1994)

    • Related Report
      1995 Annual Research Report
  • [Publications] HASEGAWA, A., YAMASAKI, N., INOUE, M., KOYAMA, K. and ISOJIMA, S.: "Analysis of an epitope sequence recognized by a fertilization blocking monoclonal antibody (MAb-5H4) against a porcine zona pellucida glycoprotein (pZP1)." Journal of Reproduction and Fertility.105. 295-302 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] TAYA, T., YAMASAKI, N., TSUBAMOTO, H., HASEGAWA, A. and KOYAMA, K.: "Cloning of a cDNA coding for porcine zona pellucida glycoprotein ZP1 and its genomic organization." Biochemical and Biophysical Research Communications. 207. 790-799 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] TSUBAMOTO, H., HASEGAWA, A., INOUE, M., YAMASAKI, N. and KOYAMA, K.: "Bindings of a recombinant porcine zona protein (pZP1) to acrosome-reacted spermatozoa." Proceedings of International Symposium on Prospects of Zona Pellucida Glycoproteins for Immunocontraception and 7th Annual Conference of Indian Society for the Study of Reproduction and Fertility.(in press.). (1996)

    • Related Report
      1995 Annual Research Report
  • [Publications] 長谷川昭子、井上みゆき、山崎則行、香山浩二: "卵透明帯の第二精子レセプターと反応する精子側リガンドについて" 日本受精着床学会雑誌. 13(発表予定). (1996)

    • Related Report
      1995 Annual Research Report
  • [Publications] Koji Koyama: "Peptide mapping and cDNA cloning for a glycoprotein(pZP4)of porcine zona pellucida" Advances in Contraceptive Delivery Systems. 10. 235-253 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Akiko Hasegawa: "Amino acid sequbnce of a porcine zora pellucida glycoprotein ZP4 determined by peptide mappimg and CDNA cloning" Journal of Reproduction and Fertility. 100. 245-255 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] 長谷川 昭子: "卵透明帯の生合成部位と発現時期に関する免疫組織学的研究" 哺乳動物卵子学会誌. 11. 239-246 (1994)

    • Related Report
      1994 Annual Research Report

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Published: 1994-04-01   Modified: 2016-04-21  

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