Saxena N K, Saxena N, Hunt W, Peterson R N, Henry L, Russell L D
J Cell Sci. 1986 Jun;82:295-308. doi: 10.1242/jcs.82.1.295.
High-resolution two-dimensional polyacrylamide gel electrophoresis analyses have indicated that several major boar sperm plasma membrane polypeptides (PMPs) increased in concentration during maturation in the epididymis. To investigate this further, monoclonal antibodies (MAbs) to two of these PMPs/glycoproteins referenced as 4.85 and 5.0 and polyclonal antisera (PCA) raised against PMP 5.0 were used to quantify these changes. ELISA assays for both PMPs, using solubilized plasma membrane (PM) and intact PM as antigens, indicated that both PMPs were present in greater concentration in cauda PM than in caput PM. ELISA assay using PCA against 5.0 and whole sperm from the cauda and caput epididymis also showed an increase in the concentration of this protein during transit through the epididymis. Indirect FITC fluorescence microscopy showed that MAbs reacted specifically with the luminal aspect of secretory cells of the corpus epididymis and, with greater intensity, with secretory cells of the cauda segment. When MAb tags were used the fluorescence technique indicated that PMP 5.0 was restricted to a narrow area of PM overlying the principal segment of the head of caput sperm but fluorescence extended further into the principal segment and into the flagellum of cauda PM. Since MAb binding sites appear to be partially masked, PCA were also used to localize antigenic sites. PCA to PMP 5.0 showed antigen localization in both head and flagellar PM of cauda and caput sperm. The fluorescence of cauda sperm PM, however, was significantly more intense than that of caput sperm PM. MAb to PMP 4.85 also bound to antigen more extensively on cauda sperm. The sum of these analyses suggests that major sperm PMPs are secreted by the epididymis and absorbed by sperm. The magnitude of surface protein changes occurring during epididymal transit in boar sperm appears to be greater than heretofore recognized.
高分辨率二维聚丙烯酰胺凝胶电泳分析表明，几种主要的公猪精子质膜多肽（PMPs）在附睾成熟过程中浓度增加。为了进一步研究这一现象，针对其中两种分别称为4.85和5.0的PMPs/糖蛋白的单克隆抗体（MAbs）以及针对PMP 5.0产生的多克隆抗血清（PCA）被用于量化这些变化。使用溶解的质膜（PM）和完整的PM作为抗原对两种PMPs进行的ELISA分析表明，尾部PM中这两种PMPs的浓度均高于头部PM。使用针对5.0的PCA和来自附睾尾部和头部的完整精子进行的ELISA分析也显示，该蛋白在通过附睾的过程中浓度增加。间接荧光素异硫氰酸酯（FITC）荧光显微镜检查显示，MAbs与附睾体分泌细胞的管腔面特异性反应，并且与尾部段的分泌细胞反应强度更大。当使用MAb标签时，荧光技术表明PMP 5.0局限于覆盖头部精子主段的PM的狭窄区域，但荧光进一步延伸到主段并进入尾部PM的鞭毛。由于MAb结合位点似乎被部分掩盖，PCA也被用于定位抗原位点。针对PMP 5.0的PCA显示抗原定位于尾部和头部精子的头部和鞭毛PM中。然而，尾部精子PM的荧光明显比头部精子PM的荧光更强。针对PMP 4.85的MAb也在尾部精子上更广泛地结合抗原。这些分析的结果表明，主要的精子PMPs由附睾分泌并被精子吸收。公猪精子在附睾转运过程中发生的表面蛋白变化程度似乎比以往认识的更大。
