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Studies on the extracellular ubiquitin-proteasomes system involved in fertilization

Research Project

Project/Area Number 15390023
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Biological pharmacy
Research InstitutionNagoya University

Principal Investigator

SAWADA Hitoshi  Nagoya University, Graduate School of Science, Professor, 大学院・理学研究科, 教授 (60158946)

Project Period (FY) 2003 – 2005
Project Status Completed (Fiscal Year 2005)
Budget Amount *help
¥15,400,000 (Direct Cost: ¥15,400,000)
Fiscal Year 2005: ¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 2004: ¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 2003: ¥10,500,000 (Direct Cost: ¥10,500,000)
KeywordsFertilization / Sperm / Proteasome / Ubiquitin / Ascidian / マボヤ / 卵黄膜 / カタユウレイボヤ / ライシン / プロテアーゼ
Research Abstract

We have previously reported that the sperm ubiquitin-proteasome system plays a key role in fertilization, particularly in the process of sperm penetration of the vitelline coat, of the ascidian, Halocynthia roretzi. In this studies, we attempted to identify sperm enzymes involved in ubiquitination of the vitelline coat during ascidian fertilization. First, we tried to isolate the ubiquitin-conjugating enzyme complex from sperm exudate, a supernatant fraction upon sperm reaction or sperm activation. However, the amount of the purified enzyme was not sufficient for the analysis of the N-terminal sequence of the isolated enzymes.
Then, we were interested in the cognate enzymes or proteins expressed in the ascidian Ciona intestinalis, since the draft genomic DNA sequence in this species has already been published. In order to identify ubiquitin lygases involved in ascidian fertilization, we explored a RING-domain-containing proteins among gene models exressed in C.intestinalis testis. We se … More lected 2 gene models, which were highly expressed in testis as revealed by RT-PCR. One (ci100130063) of the gene models with a molecular mass of 86 kDa contains a TRP domain and a LON doman in addition to a RING domain. The other gene model (ci100137400) with a molecular mass of 57 kDa contains a FERM domain in addtition to a RING domain. In order to investigate the biological functions of these proteins during fertilization, polyclonal antibodies were raised in rabbits against the respective fusion proteins, which were expressed in E.coli. By using these antibodies, we found that the extent of the expression level of these proteins in C.intestinalis sperm appears to be low and that there are several cross-reacted bands in sperm of C.intestinalis. On the other hand, we examined the cross-reactivity toward the proteins expressed in sperm of Halocynthia roretzi, and we found that there are cross-reactable bands in H.roretzi sperm, implying that RING-domain-containing E3-like protein may be expressed in H.roretzi sperm and may be involved in ascidian fertilization.
In addition to investigate candidate E3-like proteins, we explored E2-like ubiquitin-conjugating enzymes in C.intestinalis gene models. We revealed that several E2-like proteins are highly expressed in testis of this species. Further studies are necessary to clarify whether these proteins are involved in fertilization of the ascidian C.intestinalis and also whether the congate enzymes involved in fertilization are expressed in H.roretzi sperm. Less

Report

(4 results)
  • 2005 Annual Research Report   Final Research Report Summary
  • 2004 Annual Research Report
  • 2003 Annual Research Report
  • Research Products

    (19 results)

All 2005 2004 2003 Other

All Journal Article (14 results) Book (1 results) Publications (4 results)

  • [Journal Article] Highly polymorphic vitelline-coat protein HaVC80 from the ascidian, Halocynthia aurantium : Structural analysis and involvement in self/nonself recognition during fertilization.2005

    • Author(s)
      Ban, S., Harada, Y., Yokosawa, H., Sawada, H.
    • Journal Title

      Developmental Biology 286-2

      Pages: 440-451

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Annual Research Report 2005 Final Research Report Summary
  • [Journal Article] Sever cell fragmentation in the endotherial cell apoptosis induced by snake apoptosis toxin VAP1 is an apoptotic characteristic controlled by caspases.2005

    • Author(s)
      Murayama, J., Hayashi, H., Miao, J., Sawada, H., Araki, S.
    • Journal Title

      Toxicon 46-1

      Pages: 1-6

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Highly polymorphic vitelline-coat protein HaVC80 from the ascidian, Halocynthia aurantium.2005

    • Author(s)
      Ban, S., Harada, Y., Yokosawa, H., Sawada, H.
    • Journal Title

      Developmental Biology 286-2

      Pages: 440-451

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Modification of ascidian fertilization related gamete proteins by ubiquitination, proteolysis, and glycosylation.2005

    • Author(s)
      Sawada, H., Akasaka, M., Yokota, N., Sakai, N.
    • Journal Title

      New Impact on Protein Modifications in the Regulation of Reproductive System (Tokumoto ed.)

      Pages: 61-81

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Sever cell fragmenttion in the endothelial cell apoptosis induced by snake apoptosis toxin VAP1 is an apoptotic characteristic controlled by caspases.2005

    • Author(s)
      Maruyama, J., Hayashi, H., Miao, J., Sawada, H., Araki, S.
    • Journal Title

      Toxicon 46-1

      Pages: 1-6

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Modifications of Ascidian Fertilization Related Gamete Proteins by Ubiquitination, Proteolysis, and Glycosylation2005

    • Author(s)
      Sawada, H., Akasaka, M., Yokota, N., Sakai, N.
    • Journal Title

      New Impact on Protein Modifications in the Regulation of Reproductive System(T.Tokumoto ed.)(Research Signpost) (印刷中)

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Non-traditional roles of ubiquitin-proteasome system in fertilization and gametogenesis.2004

    • Author(s)
      Sakai, N., Takagi Sawada, M., Sawada, H.
    • Journal Title

      Int. J. Biochem. Cell. Biol. 36-5

      Pages: 776-784

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Self/nonself recognition in ascidian fertilization : vitelline coat protein HrVC70 is a candidate allorecognition molecule.2004

    • Author(s)
      Sawada H, Tanaka E, Ban S, Yamasaki C, Fujino J, Ooura K, Abe Y, Matsumoto K, Yokosawa H.
    • Journal Title

      Proc. Natl. Acad. Sci. USA 101-44

      Pages: 15615-15620

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Non-traditional roles of ubiquitin-proteasome system in fertilization and gametogenesis.2004

    • Author(s)
      Sakai, N., Takagi Sawada, M., Sawada, H.
    • Journal Title

      Int.J.Biochem.Cell.Biol. 36-5

      Pages: 776-784

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Self/nonself recognition in ascidian fertilization : vitelline coat protein HrVC70 is a candidate allorecognition molecule.2004

    • Author(s)
      Sawada H, Tanaka E, Ban S, Yamasaki C, Fujino J, Ooura K, Abe Y, Matsumoto K, Yokosawa H.
    • Journal Title

      Proc.Natl.Acad.Sci.USA, 101-44

      Pages: 15615-15620

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Self/nonself Recognition in ascidian fertilization : Vitelline coat protein HrVC70 is a candidate allorecognition molecule.2004

    • Author(s)
      Sawada, H., Tanaka, E., Ban, S., Yamazaki, C., Fujino, J., Ooura, K., Abe, Y., Yokosawa, H.
    • Journal Title

      Proc.Natl.Acad.Sci.USA, 101

      Pages: 15615-15620

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Non-traditional roles of ubiquitin-proteasome system in fertilization and gametogenesis.2004

    • Author(s)
      Sakai, N., Takagi Sawada, M., Sawada, H.
    • Journal Title

      Int.J.Biochem.Cell Biol. 36

      Pages: 776-784

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Spermosin2004

    • Author(s)
      Sawada, H.
    • Journal Title

      Handbook of Proteolytic Enzymes(A.J.Barrett, N.D.Rawlings, J.F.Woessner eds.)(Elsevier-Academic Press) Vol.2

      Pages: 1567-1569

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Extracellular ubiquitin system implicated in fertilization of the ascidian, Halocynthia roretzi : isolation and characterization.2003

    • Author(s)
      Sakai, N., Sawada, H., Yokosawa, H.
    • Journal Title

      Developmental Biology 264-1

      Pages: 299-307

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Book] New Impact on Protein Modifications in the Regulation of Reproductive System2005

    • Author(s)
      Sawada, H., Akasaka, M., Yokota, N., Sakai, N.
    • Total Pages
      167
    • Publisher
      Research Signpost, Kerala, India
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Annual Research Report 2005 Final Research Report Summary
  • [Publications] N.Sakai, H.Sawada, H.Yokosawa: "Extracellular ubiquitin system implicated in fertilization of the ascidian, Halocynthia roretzi : isolation and characterization"Developmental Biology. 264. 299-307 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] H.Sawada, K.Nagahiro, Y.Kikukawa, S.Ban, R.Kakefuda, T.Shiomi, H.Yokosawa: "Therapeutic effects of camostat mesilate on Duchenne muscular dystrophy in mdx mice"Biological & Pharmaceutical Bulletin. 26. 1025-1027 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] H.Sawada, Y.Kikukawa, S.Ban, T.Kakudo, H.Yokosawa: "Expression of trypsin-like proteases and protease nexin-1 mdx mouse muscles"Biochemical and Biophysical Research Communications. 314. 654-658 (2004)

    • Related Report
      2003 Annual Research Report
  • [Publications] N.Sakai, M.T.Sawada, H.Sawada: "Non-traditional roles of ubiquitin-proteasome system in fertilization and gametogenesis"International Journal of Biochemistry and Cell Biology. 36. 776-784 (2004)

    • Related Report
      2003 Annual Research Report

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Published: 2003-04-01   Modified: 2016-04-21  

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