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一种细胞外基质，具有多种重要的受精功能。小鼠透明带(MZP2)的一种成分已被证明具有次级精子受体的功能。最近，我们成功地克隆了猪透明带糖蛋白(PZP1)的编码基因。该序列与mZP2的同源性为54%。本研究的目的是利用重组pZP1(r-pZP1)检测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)。人精子在含牛血清白蛋白(BSA)(3 mg/ml)的BWW培养液中洗涤和离心法获得。精子在5%CO_2空气中预孵育3小时后，在含有离子载体A23187(10 MU M)的培养液中孵育15分钟，以诱导顶体反应(AR)。将重组pZp1(1-198)以10微克/毫升的终浓度加入精子悬液中，甲醇固定的精子用识别pZP1(50-59)表位的单抗(MAb-5H4)处理，然后用FITC标记的抗鼠IgG处理，用PSA凝集素染色评估AR。在AR诱导的人精子悬液中加入r-pZP1(1-198)后，与蛋白质结合的精子数在孵育过程中逐渐增加。在体外受精液中加入r-pZP1(1-198)，既不影响精子与人透明带的结合，也不影响精子对去透明带金黄地鼠卵子的穿透。R-pZP1(1-198)能与顶体反应的精子结合，但不能与顶体完整的精子结合。这表明pZP1的NH2末端(1-198)可能对无碳水化合物残基的受精起作用。因此，pZP1(1-198)的结构域与mZP2一样具有次级精子受体的功能。

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.