Biological nature and cDNA cloning of sperm ligand binding to pZP1

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。

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.