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

基本信息

批准号：
06454481
负责人：
KOYAMA Koji
金额：
$ 2.88万
依托单位：
Hyogo College of Medicine
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
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

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.

Zona pellucida是围绕哺乳动物卵母细胞的细胞外基质，具有施肥的各种重要功能。小鼠Zona pellucida（MZP2）的组成部分已显示出二级精子受体的功能。最近，我们成功地克隆了编码猪Zona pellucida糖蛋白（PZP1）的cDNA。 cDNA序列与MZP2同源54％。这项研究的目的是使用重组PZP1（R-PZP1）检查PZP1的功能。本研究中使用的重组蛋白是PZP1的NH_2-末端区域（氨基酸位置1-198）。人类精子是通过在包含BSA（3mg/mL）的BWW培养基中洗涤和离心制备的。在5％CO_2在空气中预孵育3小时后，将精子在含有离子载体A23187（10 mu M）的培养基中孵育15分钟，以诱导Acrosome反应（AR）。将R-PZP1（1-198）添加到精子悬浮液中，最终浓度为10 mu g/ml。用单克隆抗体（MAB-5H4）处理了甲醇固定的精子，该抗体已被证明可以识别PZP1（50-59）上的表位，然后用FITC共轭抗小鼠IgG.AR进行psa lectin swectains psa psa rectin swith进行了theTreatement。当将R-PZP1（1-198）添加到AR诱导的人类精子悬浮液中时，孵育过程中蛋白质的精子数逐渐增加。而用R-PZP1（1-198）对精子的处理并没有增加AR。当将R-PZP1（1-198）添加到体外受精培养基中时，精子与人Zona pelludica的结合也不是与人Zona pelludica结合，也没有将精子渗透到无Zona的无鼠仓鼠卵中。 R-PZP1（1-198）被证明与Acrosome反应的精子结合，但与Acrosme完整精子没有结合。这表明PZP1的NH2末端部分（1-198）可能对无碳水化合物残基的受精作用。因此得出的结论是，PZP1（1-198）的结构域以与MZP2相同的方式充当次级精子受体。