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Biological nature and cDNA cloning of sperm ligand binding to pZP1

Biological nature and cDNA cloning of sperm ligand binding to pZP1
精子配体与 pZP1 结合的生物学性质和 cDNA 克隆
批准号:
06454481
负责人:
KOYAMA Koji
金额:
$2.88万
依托单位:
依托单位国家:
日本
项目类别:
Grant-in-Aid for General Scientific Research (B)
财政年份:
1994
资助国家:
日本
项目状态:
已结题
起止时间:
1994 至 1995

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中文摘要
翻译
透明质酸是哺乳动物卵母细胞周围的细胞外基质,具有受精的多种重要功能。小鼠透明质酸(mZP 2)的一个组成部分已被证明是一个次要的精子受体。最近,我们成功地克隆了一个编码猪透明质酸糖蛋白(pZP 1)的cDNA。cDNA序列与mZP 2的同源性为54%。本研究的目的是利用重组pZP 1(r-pZP 1)检测pZP 1的功能。本研究所用的重组蛋白是pZP 1的NH_2端(氨基酸位置1-198)。人精子经洗涤、离心后在含BSA(3 mg/ml)的BWW培养基中制备。精子在5%CO_2空气中预孵育3小时后,在含离子载体A23187(10 μ M)的培养液中孵育15分钟,诱导顶体反应(AR)。将r-pZp 1(1-198)以10 μ g/ml的终浓度加入精子悬浮液中。用单克隆抗体(MAb-5 H4)处理甲醇固定的精子,然后用FITC标记的抗鼠IgG处理,PSA凝集素染色检测AR。将r-pZP 1(1-198)加入AR诱导的人精子悬液中,在孵育过程中结合蛋白的精子数量逐渐增加。而r-pZP 1(1-198)处理精子不增加AR。将r-pZP 1(1-198)加入体外受精培养液中,既不干扰精子与人精子的结合,也不干扰精子对去透明带仓鼠卵的穿透。r-pZP 1(1-198)与顶体反应的精子结合,而与顶体完整的精子不结合。这表明pZP 1的NH 2-末端部分(1-198)可能在没有碳水化合物残基的情况下具有受精功能。因此得出结论,pZP 1(1-198)的结构域以与mZP 2相同的方式作为二级精子受体。
英文摘要
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.
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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 Fer
HASEGAWA, A.、YAMASAKI, N.、INUE, M.、KOYAMA, K. 和 ISOJIMA, S.:“针对猪透明带糖蛋白的受精阻断单克隆抗体 (MAb-5H4) 识别的表位序列的分析(
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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
HASEGAWA,A.、YAMASAKI,N.、INUE,M.、KOYAMA,K. 和 ISOJIMA,S.:“针对猪透明带糖蛋白(MAb-5H4)的受精阻断单克隆抗体(MAb-5H4)识别的表位序列的分析(
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16
    Production of transgenic mice expressing human ZPA and its application to sperm function diagnosis
    • 批准号:
      10470350
    • 项目类别:
      Grant-in-Aid for Scientific Research (B).
    • 资助金额:
      $7.81万
    • 财政年份:
      1998
    • 负责人:
      KOYAMA Koji
    • 依托单位:
    ESTABLISHMENT AND CHARACTERIZATION OF HUMAN SPERM SPECIFIC MONOCLONAL ANTIBODY 1G1 WITH INHIBITORY EFFECT OF SPERM-ZONA PELLUCIDA INTERACTION
    • 批准号:
      04671026
    • 项目类别:
      Grant-in-Aid for General Scientific Research (C)
    • 资助金额:
      $1.34万
    • 财政年份:
      1992
    • 负责人:
      KOYAMA Koji
    • 依托单位:
    Gene Cloning of a Porcine Zona Antigen Which Cross-Reacts With Human Zona Pellucida
    • 批准号:
      01570944
    • 项目类别:
      Grant-in-Aid for General Scientific Research (C)
    • 资助金额:
      $1.34万
    • 财政年份:
      1989
    • 负责人:
      KOYAMA Koji
    • 依托单位:
    Production of human monoclona. antibody to spermatozoa and its application for purification of the corresponding sperm antigen.
    • 批准号:
      61570815
    • 项目类别:
      Grant-in-Aid for General Scientific Research (C)
    • 资助金额:
      $1.34万
    • 财政年份:
      1986
    • 负责人:
      KOYAMA Koji
    • 依托单位:
    海外基金