Jassim A, Gillott D J, al-Zuhdi Y, Gray A, Foxon R, Bottazzo G F
Department of Immunology, London Hospital Medical College, UK.
Hum Reprod. 1992 Jan;7(1):86-94. doi: 10.1093/oxfordjournals.humrep.a137566.
The isolation and biochemical characterization of the human sperm tail fibrous sheath (FS) is described for the first time. Initially, the solubilization properties of the FS were assessed immunocytochemically using GDA-J/F3 and RT97 monoclonal antibodies (MoAbs) and morphologically by electron microscopy. Following extensive investigations to optimize the conditions for the FS isolation, a simple method was developed which involved sequential extraction of the flageller components with Triton-dithiothreitol (DTT) and urea-DTT. The procedure was monitored by phase contrast microscopy and the purity of the FS preparations was confirmed by electron microscopy. SDS-PAGE of the isolated FS revealed seven major protein bands with mol. wt of 97, 76, 62, 55, 33, 28 and 25 kDa. In Western blotting, the reaction of RT97 MoAb with supernatants from the various extraction steps and the isolated FS indicated that its target antigen (AJ-p97) was an integral FS product and that disulphide bonding was probably involved in its stabilization. The reactivity of normal and aprotruded sperm tails with GDA-J/F3 and RT97 MoAbs was not affected by Triton while the GDA-J/F3 staining of the cytoplasmic matrix of other abnormal spermatids was abolished, thus suggesting variation in the biochemical properties of GDA-J/F3 in normal and abnormal germ cells. These and other data indicate that the FS could be a modified form of intermediate filament.
首次描述了人精子尾部纤维鞘（FS）的分离及其生化特性。最初，使用GDA-J/F3和RT97单克隆抗体（MoAbs）通过免疫细胞化学方法评估FS的溶解特性，并通过电子显微镜进行形态学评估。在广泛研究以优化FS分离条件后，开发了一种简单的方法，该方法包括用Triton-二硫苏糖醇（DTT）和尿素-DTT依次提取鞭毛成分。通过相差显微镜监测该过程，并通过电子显微镜确认FS制剂的纯度。分离的FS的SDS-PAGE显示出七条主要蛋白带，分子量分别为97、76、62、55、33、28和25 kDa。在蛋白质印迹中，RT97 MoAb与各个提取步骤的上清液和分离的FS的反应表明，其靶抗原（AJ-p97）是FS的一种整合产物，二硫键可能参与其稳定化。正常和突出精子尾部与GDA-J/F3和RT97 MoAbs的反应性不受Triton影响，而其他异常精子细胞胞质基质的GDA-J/F3染色被消除，这表明正常和异常生殖细胞中GDA-J/F3的生化特性存在差异。这些以及其他数据表明，FS可能是中间丝的一种修饰形式。
