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Molecular basis of spermatogenesis and fertilization in mammals

Research Project

Project/Area Number 15208033
Research Category

Grant-in-Aid for Scientific Research (A)

Allocation TypeSingle-year Grants
Section一般
Research Field Applied molecular and cellular biology
Research InstitutionUniversity of Tsukuba

Principal Investigator

BABA Tadashi  University of Tsukuba, Graduate School of Life and Environmental Sciences, Professor, 大学院・生命環境科学研究科, 教授 (40165056)

Co-Investigator(Kenkyū-buntansha) YANAGISAWA Junn  University of Tsukuba, Graduate School of Life and Environmental Sciences, Professor, 大学院・生命環境科学研究科, 教授 (50301114)
KASHIWABARA Shin-ichi  University of Tsukuba, Graduate School of Life and Environmental Sciences, Associate Professor, 大学院・生命環境科学研究科, 助教授 (00254318)
YAMAGATA Kazuo  University of Tsukuba, Graduate School of Life and Environmental Sciences, Assistant Professor, 大学院・生命環境科学研究科, 講師 (10361312)
NAKANISHI Tomoko  University of Tsukuba, Graduate School of Life and Environmental Sciences, Assistant Professor, 大学院・生命環境科学研究科, 助手 (10344863)
TAKAHASHI Satoru  University of Tsukuba, Graduate School of Comprehensive Human Sciences, Professor, 大学院・人間総合科学研究科, 教授 (50271896)
Project Period (FY) 2003 – 2005
Project Status Completed (Fiscal Year 2005)
Budget Amount *help
¥43,680,000 (Direct Cost: ¥33,600,000、Indirect Cost: ¥10,080,000)
Fiscal Year 2005: ¥10,010,000 (Direct Cost: ¥7,700,000、Indirect Cost: ¥2,310,000)
Fiscal Year 2004: ¥15,470,000 (Direct Cost: ¥11,900,000、Indirect Cost: ¥3,570,000)
Fiscal Year 2003: ¥18,200,000 (Direct Cost: ¥14,000,000、Indirect Cost: ¥4,200,000)
KeywordsSperm / Egg / Fertilization / Spermatogenesis / Egg activation / Cell fusion / Oviduct / Knockout mouse / 精子成熟 / 輪卵管
Research Abstract

To elucidate the molecular basis of mammalian spermatogenesis and fertilization, we have examined the synthesis, processing, and functions of sperm proteins involved in fertilization. The following experimental results have been obtained :
1. In mouse, two different isoforms of ADAM1, ADAM1a and ADAM1b, are produced in the testis. ADAM1a is localized within the endoplasmic reticulum of testicular germ cells (TGC), whereas epididymal sperm contain only ADAM1b on the plasma membrane. We show that the loss of ADAM1a results in the male infertility because of the severely impaired ability of sperm to migrate from the uterus into the oviduct through the uterotubal junction. Among testis (sperm)-specific proteins examined, only the level of ADAM3 was strongly reduced in ADAM1a-deficient mouse sperm. Moreover, the appearance of ADAM3 on the sperm surface was dependent on the formation of a fertilin protein complex between ADAM1a and ADAM2 in TGC. These results suggest that ADAM1a/ADAM2 fertili … More n may be implicated in the selective transport of specific sperm proteins including ADAM3 from the endoplasmic reticulum of testicular germ cells onto the cell surface.
We also show that mutant male mice lacking ADAM1b are fertile, and the loss of ADAM1b results in no significant defect in the sperm functions. ADAM1b-deficient epididymal sperm showed a severe reduction of ADAM2 on the cell surface, despite the normal presence of ADAM2 in TGC. The appearance of ADAM1b and ADAM2 on the sperm surface depended on formation and abundance of ADAM1b/ADAM2 fertilin in TGC. Thus, mouse ADAM1b/ADAM2 fertilin may play the crucial role not in the sperm/egg fusion, but in the appearance of these two ADAMs on the sperm surface.
2. A glycosylphosphatidylinositol (GPI)-anchored hyaluronidase, PH-20, on the sperm surface has long been believed to assist sperm penetration through the cumulus mass surrounding the eggs. However, mouse sperm lacking PH-20 were still capable of penetrating the cumulus mass despite a delayed dispersal of cumulus cells. Intriguingly, a 55-kDa hyaluronan-hydrolyzing protein was abundantly present in wild-type and PH-20-deficient mouse sperm. In this study, we have purified the 55-kDa mouse protein from soluble protein extracts released from epididymal sperm by acrosome reaction, and have identified as a novel hyaluronidase, Hyal5. Hyal5 was exclusively expressed in the testis, and formed a 160-kbp gene cluster together with Hyalp1, Hyal4, and Ph-20 on mouse chromosome 6. Hyal5 was a single-chain hyaluronidase present on the plasma and acrosomal membranes of sperm presumably as a GPI-anchored protein. Moreover, hyaluronan zymography revealed that Hyal5 is enzymatically active in the pH range 5-7, and inactive at pH 3 and 4. Both Hyal5-enriched, PH-20-free soluble protein extracts, and PH-20-deficient mouse sperm were capable of dispersing cumulus cells from the cumulus mass. The cumulus cell dispersal was strongly inhibited by the presence of a hyaluronidase inhibitor, apigenin. These results suggest that in the mouse, Hyal5 may function principally as a "cumulus matrix depolymerase" in the sperm penetration through the cumulus mass, and in the local hyaluronan hydrolysis near or on the surface of the egg zona pellucida to enable the proximal region of sperm tail to move freely. PH-20 may compensate in part for the functional roles of Hyal5.
3. We have previously identified a 42-kDa serine protease, TESP5, identical to testisin, esp-1, or tryptase 4 as a candidate enzyme involved in the sperm penetration of egg ZP. Mouse TESP5 is glycosylphosphatidylinositol (GPI)-anchored on the cell surface of cauda epididymal sperm. To elucidate the role(s) of TESP5 in fertilization, we have produced mutant mice carrying a targeted mutation in the TESP5 gene by homologous recombination in embryonic stem cells. The TESP5-deficient male and female mice showed a normal fertility. However, in vitro fertilization assay revealed that epididymal sperm lacking TESP5 barely fertilize metaphase II-arrested oocytes. The loss of TESP5 on sperm resulted in the reduced ability to bind the ZP in vitro. Even when TESP5-deficient sperm bound to the ZP, the rate of ZP-induced acrosome reaction was very low. Moreover, the ability of sperm to fuse with egg in vitro was severely impaired by the TESP5 loss. These results suggest that TESP5 may act in the acrosome reaction, and imply that the impaired function of TESP5-deficient sperm may be compensated for during the transit through the uterus and/or oviduct. Less

Report

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

    (31 results)

All 2006 2005 2004 2003 2001 Other

All Journal Article (24 results) Publications (7 results)

  • [Journal Article] Possible role of mouse poly(A) polymerase mGLD-2 during oocyte maturation.2006

    • Author(s)
      Tomoko Nakanishi, Tadashi Baba
    • Journal Title

      Developmental Biology 289

      Pages: 115-126

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Mouse sperm lacking ADAM1b/ADAM2 fertilin can fuse with the egg plasma membrane and effect fertilization.2006

    • Author(s)
      Ekyune Kim, Tadashi Baba
    • Journal Title

      Journal of Biological Chemistry 281

      Pages: 5634-5639

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Possible role of mouse poly(A) polymerase mGLD-2 during oocyte maturation.2006

    • Author(s)
      Tomoko Nakanishi, Tadashi Baba
    • Journal Title

      Dev.Biol. 289

      Pages: 115-126

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Mouse sperm lacking ADAM1b/ADAM2 fertilin can fuse with the egg plasma membrane and effect fertilization.2006

    • Author(s)
      Ekyune Kim, Tadashi Baba
    • Journal Title

      J.Biol.Chem. 281

      Pages: 5634-5639

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Possible role of mouse poly(A) polymerase mGLD-2 during oocyte maturation.2006

    • Author(s)
      Tomoko Nakanishi, Tadasi Baba
    • Journal Title

      Developmental Biology 289

      Pages: 115-126

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Mouse sperm lacking ADAM1b/ADAM2 fertilin can fuse with the egg plasma membrane and effect fertilization.2006

    • Author(s)
      Ekyune Kim, Tadasi Baba
    • Journal Title

      Journal of Biological Chemistry 281

      Pages: 5634-5639

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Identification of a hyaluronidase, Hyal5, involved in penetration of mouse sperm through cumulus mass.2005

    • Author(s)
      Ekyune Kim, Tadashi Baba
    • Journal Title

      Proceedings of the National Academy of Sciences of the United States of America 102

      Pages: 18028-18033

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Gene expression in spermiogenesis.2005

    • Author(s)
      Hiromitsu Tanaka, Tadashi Baba
    • Journal Title

      Cellular and Molecular Life Science 62

      Pages: 344-354

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Identification of a hyaluronidase, Hyal5, involved in penetration of mouse sperm through cumulus mass.2005

    • Author(s)
      Ekyune Kim, Tadashi Baba
    • Journal Title

      Proc.Natl.Acad.Sci.USA 102

      Pages: 18028-18033

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Gene expression in spermiogenesis.2005

    • Author(s)
      Hiromitsu Tanaka, Tadashi Baba
    • Journal Title

      Cell.Mol.Life Sci. 62

      Pages: 344-354

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Identification of a hyaluronidase, Hyal5, involved in penetration of mouse sperm through cumulus mass.2005

    • Author(s)
      Ekyune Kim, Tadasi Baba
    • Journal Title

      Proceedings of the National Academy of Sciences of the United States of America 102

      Pages: 18028-18033

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Gene expression in spermiogenesis.2005

    • Author(s)
      Hiromitsu Tanaka, Tadasi Baba
    • Journal Title

      Cellular and Molecular Life Sciences 62

      Pages: 344-354

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Birth of mice produced by germ cell nuclear transfer.2005

    • Author(s)
      Hiromi Miki, Tadasi Baba
    • Journal Title

      Genesis 41

      Pages: 81-86

    • Related Report
      2005 Annual Research Report
  • [Journal Article] The proacrosin-binding protein, sp32, is tyrosine phosphorylated during capacitation of pig sperm.2005

    • Author(s)
      Charlotte Dube, Tadasi Baba
    • Journal Title

      Journal of Andrology 26

      Pages: 519-528

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Possible function of the ADAM1a/ADAM2 fertilin complex in the appearance of ADAM3 on the sperm surface.2004

    • Author(s)
      Hitoshi Nishimura, Tadashi Baba
    • Journal Title

      Journal of Biological Chemistry 279

      Pages: 34957-34962

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Possible function of the ADAM1a/ADAM2 fertilin complex in the appearance of ADAM3 on the sperm surface.2004

    • Author(s)
      Hitoshi Nishimura, Tadashi Baba
    • Journal Title

      J.Biol.Chem. 279

      Pages: 34957-34962

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Possible function of the ADAM1a/ADAM2 fertilin complex in the appearance of ADAM3 on the sperm surface2004

    • Author(s)
      Hitoshi Nishimura
    • Journal Title

      Journal of Biological Chemistry 279

      Pages: 34957-34962

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Transgenic expression of testis-specific poly(A) polymerase TPAP in wild-type and TPAP-deficient mice2004

    • Author(s)
      Tiangang Zhuang
    • Journal Title

      Journal of Reproduction and Development 50

      Pages: 207-213

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Selective passage through the uterotubal junction of sperm from a mixed population produced by chimeras of calmegin-knockout and wild-type mice2004

    • Author(s)
      Tomoko Nakanishi
    • Journal Title

      Biology of Reproduction 71

      Pages: 959-965

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Synthesis, processing, and subcellular localization of mouse ADAM3 during spermatogenesis and epididymal sperm transport2004

    • Author(s)
      Ekyune Kim
    • Journal Title

      Journal of Reproduction and Development 50

      Pages: 571-578

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Cytoplasmic asters are required for progression past the first cell cycle in cloned mouse embryos2004

    • Author(s)
      Hiromi Miki
    • Journal Title

      Biology of Reproduction 71

      Pages: 2022-2028

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Differential localization of ADAM1a and ADAM1b in the endoplasmic reticulum of testicular germ cells and on the surface of epididymal sperm.2003

    • Author(s)
      Ekyune Kim, Tadashi Baba
    • Journal Title

      Biochemical and Biophysical Research Communications 304

      Pages: 313-319

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Differential localization of ADAM1a and ADAM1b in the endoplasmic reticulum of testicular germ cells and on the surface of epididymal sperm.2003

    • Author(s)
      Ekyune Kim, Tadashi Baba
    • Journal Title

      Biochem.Biophys.Res.Commun. 304

      Pages: 313-319

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Birth of mice produced by germ cell nuclear transfer2001

    • Author(s)
      Hiromi Miki
    • Journal Title

      Genesis 41

      Pages: 81-86

    • Related Report
      2004 Annual Research Report
  • [Publications] Sugiura Shin: "Expression of a testis-specific form of TBP-related factor 2 (TRF2) mRNA during mouse spermatogenesis"Journal of Reproduction and Development. 49. 107-111 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Doi Hirotoshi: "Functional activation of arylhydrocarbon receptor (AhR) in primary T cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin"Chemosphere. 52. 655-662 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Fukuzawa Noriho Honda: "Effects of 3,3',4,4',5-pentachlorobiphenyl, a coplanar polychlorinated biphenyl congener, on cultured neonatal mouse testis"Toxicology In Vitro. 17. 259-269 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Kim Ekyune: "Differential localization of ADAM1a and ADAM1b in the endoplasmic reticulum of testicular germ cells and on the surface of epididymal sperm"Biochemical and Biophysical Research Communications. 304. 313-319 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Fujita Toshihide: "Full activation of estrogen receptor α activation function-1 induces proliferation of breast cancer cells"Journal of Biological Chemistry. 278. 26704-26714 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Zhuang Tiangang: "Transgenic expression of testis-specific poly(A) polymerase TPAP in wild-type and TPAP-deficient mice"Journal of Reproduction and Development. (in press). (2004)

    • Related Report
      2003 Annual Research Report
  • [Publications] 柏原 真一, 馬場 忠: "実験医学(羊上社)"精巣特異的細胞質ポリ(A)ポリメラーゼTPAPによる精子形態形成制御. 4 (2003)

    • Related Report
      2003 Annual Research Report

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

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