Biological nature and cDNA cloning of sperm ligand binding to pZP1

透明带是哺乳动物卵母细胞周围的细胞外基质，具有多种重要的受精功能。小鼠透明带（mZP2）的一个组分已被证明具有次级精子受体的功能。最近，我们成功克隆了一个编码猪透明带糖蛋白（pZP1）的cDNA。cDNA序列与mZP2同源性为54%。本研究的目的是利用重组pZP1 （r-pZP1）来检测pZP1的功能。本研究使用的重组蛋白为pZP1的nh_2末端区（氨基酸位置1-198）。用含牛血清白蛋白（3mg/ml）的BWW培养基洗涤离心制备人精子。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）的培养基中孵育15min，诱导顶体反应（AR）。精子悬液中加入r-pZp1(1-198)，终浓度为10 μ g/ml。用单克隆抗体（MAb-5H4）处理甲醇固定精子，该单克隆抗体已被证明可以识别pZP1上的表位（50-59），然后用FITC偶联的抗小鼠IgG处理。PSA凝集素染色评价AR。将r-pZP1（1-198）加入到AR诱导的人精子悬液中，在孵育过程中结合该蛋白的精子数量逐渐增加。将r-pZP1（1-198）添加到体外受精培养基中，既不影响精子与人类透明带的结合，也不影响精子进入无透明带的仓鼠卵。r-pZP1（1-198）与顶体反应的精子结合，但不与顶体完整的精子结合。这表明pZP1的nh2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。由此得出结论，pZP1（1-198）结构域与mZP2一样作为精子的次级受体。

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.