Analysis of the function of the glycoprotein, basigin, in the process of male reproduction

1.抗体B5针对外显子5中编码的多肽产生，该多肽被认为在精子成熟过程中是重要的。另外，分别识别basigin的C-末端和N-末端的商业抗体G19和M190购自圣克鲁斯生物技术公司。用这三种抗体对basigin在小鼠睾丸和附睾中的定位进行了研究。用A23187诱发附睾尾精子顶体反应。在这三种抗体中，与M190和B5相比，G19显示出最强的反应。此外，免疫反应性显示抗体之间存在相当大的差异。例如，M190和B5可识别精子头部顶体的主段，而G19则可识别顶体的赤道段。basigin分子的高级结构的修饰可能会影响抗体的识别，以及它们的表位结构域的差异.在体外受精条件下，顶体反应精子头部的抗体B5可检测到basigin，它与透明质酸结合。表明basigin在受精过程中具有重要作用.从小鼠睾丸和附睾头、体、尾精子中提取蛋白质，并通过Western印迹进行分析。抗体M190强烈识别睾丸型basigin（38- 40 kDa）而不是精子型basigin（26- 29 kDa）。经PNGase F处理和顶体反应后，黄芩苷的分子量变小.为了观察basigin蛋白在睾丸中的行为，我们制备了包含睾丸生殖细胞特异性钙调蛋白启动子和随后的Bsg-GFP融合基因（cmg-Bsg-GFP）的表达构建体。本研究利用cmg-Bsg-GFP构建了转基因小鼠。

1. Antibody B5 was produced against the polypetide coded in exon 5, which is considered to be important during sperm maturation. In addition, commercial antibodies G19 and M190, which recognize C-terminal and N-terminal of basigin, respectively, were purchased from Santa Cruz Biotechnology.2. Using these three antibodies, localization of basigin in the mouse testis and epididymis were studied. Sperm from cauda epididimidis, induced acrosome reaction by A23187, were also examined. Among these three antibodies, G19 showed most intensive reaction compared to M190 and B5. Further, the immunoreactivities revealed considerable differences among antibodies. For example, M190 and B5 recognized the principal segment of acrosome of the sperm head, in contrast, G19 immunostained the equatorial segment of the acrosome. It is possible that modification of the higher order structure of basigin molecule can affect the recognition of the antibodies, as well as the difference of their epitope domain.3. Under IVF condition, basigin was detected by antibody B5 in the head of the acrosome-reacted sperm, which binds zona pellucida. It is suggested that basigin has an important role in fertilization.4. Proteins were extracted from mouse testis and sperm from caput, corpus and cauda epididymidis and analyzed by Western blotting. Antibody M190 recognized testis-type basigin (38-40kDa) strongly rather than sperm-type basigin (26-29kDa). After PNGase F treatment and after acrosome reaction, the molecular mass of the baigin became smaller.5. To observe the behavior of basigin protein in the testis, we made the expression construct that contains a testicular germ cell specific calmegin promoter followed by the Bsg-GFP fusion gene (cmg-Bsg-GFP). Now we generate the transgenic mice using cmg-Bsg-GFP.