O'Brien D A, Gerton G L, Eddy E M
Gamete Biology Section, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
Biol Reprod. 1988 May;38(4):955-67. doi: 10.1095/biolreprod38.4.955.
Monoclonal antibody 1D4, a mouse immunoglobulin M raised against CD-1 mouse spermatogenic cell membranes, recognizes acrosomal constituents in the mouse, rabbit, and guinea pig. In the mouse, acrosomes of round and condensing spermatids were labeled with 1D4 by indirect immunofluorescence on isolated cells and by immunohistochemistry on paraffin sections. During the terminal steps of spermiogenesis, however, acrosomal labeling in mouse germ cells was lost. Little or no 1D4 immunoreactivity was detected by enzyme-linked immunosorbent assays in prepubertal testes, Sertoli cells, or several somatic tissues. To identify antigens recognized by 1D4, mouse spermatogenic cell proteins were separated by one- (1D) and two-dimensional (2D) polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunostained. Multiple antigens larger than 200,000 relative molecular weight (Mr) were resolved on 1D immunoblots from round and condensing spermatids isolated by sedimentation velocity at unit gravity. A smaller antigen (Mr 85,000 isoelectric point approximately 5.7) was also detected on 1D and 2D immunoblots of round spermatid proteins. These antigens can be labeled biosynthetically with [3H] glucosamine and immunoprecipitated, suggesting that they are a set of glycoconjugates that share a common epitope recognized by 1D4. This determinant is no longer detectable in late spermatids, indicating that biochemical modifications of acrosomal constituents occur during the terminal steps of germ cell differentiation.
单克隆抗体1D4是一种针对CD - 1小鼠生精细胞膜产生的小鼠免疫球蛋白M，可识别小鼠、兔子和豚鼠的顶体成分。在小鼠中，通过对分离细胞进行间接免疫荧光以及对石蜡切片进行免疫组织化学检测，发现圆形和浓缩型精子细胞的顶体被1D4标记。然而，在精子发生的末期阶段，小鼠生殖细胞中的顶体标记消失。在青春期前的睾丸、支持细胞或几种体细胞组织中，通过酶联免疫吸附测定几乎检测不到或未检测到1D4免疫反应性。为了鉴定被1D4识别的抗原，通过一维（1D）和二维（2D）聚丙烯酰胺凝胶电泳分离小鼠生精细胞蛋白，转移至硝酸纤维素膜上并进行免疫染色。在通过单位重力沉降速度分离的圆形和浓缩型精子细胞的1D免疫印迹上，分辨出了多种相对分子质量（Mr）大于200,000的抗原。在圆形精子细胞蛋白的1D和2D免疫印迹上还检测到一种较小的抗原（Mr 85,000，等电点约为5.7）。这些抗原可用[3H]葡糖胺进行生物合成标记并进行免疫沉淀，表明它们是一组共享被1D4识别的共同表位的糖缀合物。在晚期精子细胞中不再能检测到这种决定簇，这表明顶体成分的生化修饰发生在生殖细胞分化的末期阶段。
