Herr J C, Spell D R, Conklin D J, Flickinger C J
Department of Anatomy and Cell Biology, University of Virginia Medical Center, Charlottesville 22908.
Biol Reprod. 1989 Feb;40(2):333-42. doi: 10.1095/biolreprod40.2.333.
Seminal vesicle-specific antigen (SVSA) has been shown to be a polymorphic antigen represented by multiple immunoreactive peptides when fresh human semen is probed with monoclonal antibody (MHS-5) on Western blots. Semen samples collected directly into sodium dodecyl sulfate (SDS) demonstrate major immunoreactive peptide bands at 69-71 kDa and 58 kDa as well as a series of peptides of lower molecular mass. As semen liquefies, the higher molecular mass forms of SVSA are transformed into lower molecular mass bands, with 10-13 kDa immunoreactive peptides predominating after 8 h of liquefaction (McGee and Herr, Biol. Reprod. 37:431-439, 1987). In the present study, the 10-13 kDa form of SVSA was purified by preparative electrophoresis from SDS gels and a polyclonal antibody was generated in guinea pigs. Human seminal vesicle was fixed by immersion in combinations of glutaraldehyde and paraformaldehyde and embedded in Araldite or LR Gold. Both the guinea pig polyclonal antibody and the murine monoclonal antibody MHS-5 were employed to localize SVSA in human seminal vesicle by immunoelectron microscopy using Protein-A gold complexes. Gold particles were quantified in various subcellular compartments by a Videoplan computer. With either antibody probe, SVSA was found predominantly in the central electron-dense cores of secretory granules, with no staining evident over the electron lucent halo surrounding the granule core. With preimmune serum, the mean number of gold particles overlying secretory granules was 3/microns2; with polyclonal anti-SVSA, the mean number of particles observed over secretory granules was 182/microns2. This study represents, to our knowledge, the first fine-structural localization of a specific secretory protein to the electron-dense cores of secretory granules in principal cells of the human seminal vesicle.
精囊特异性抗原（SVSA）已被证明是一种多态性抗原，当用单克隆抗体（MHS-5）在Western印迹上检测新鲜人精液时，它由多种免疫反应性肽段组成。直接收集到十二烷基硫酸钠（SDS）中的精液样本在69 - 71 kDa和58 kDa处显示出主要的免疫反应性肽带以及一系列较低分子量的肽段。随着精液液化，SVSA的高分子量形式转变为低分子量条带，液化8小时后，10 - 13 kDa的免疫反应性肽段占主导（McGee和Herr，《生物繁殖》37:431 - 439，1987）。在本研究中，通过制备性电泳从SDS凝胶中纯化出10 - 13 kDa形式的SVSA，并在豚鼠中产生了多克隆抗体。将人精囊用戊二醛和多聚甲醛的组合进行浸泡固定，然后包埋在环氧树脂或LR Gold中。豚鼠多克隆抗体和鼠单克隆抗体MHS-5都被用于通过使用蛋白A金复合物的免疫电子显微镜在人精囊中定位SVSA。通过Videoplan计算机对各种亚细胞区室中的金颗粒进行定量。使用任何一种抗体探针，都发现SVSA主要存在于分泌颗粒的中央电子致密核心中，在围绕颗粒核心的电子透明晕圈上没有明显的染色。使用免疫前血清时，覆盖分泌颗粒的金颗粒平均数为3/μm²；使用多克隆抗SVSA时，在分泌颗粒上观察到的颗粒平均数为182/μm²。据我们所知，本研究首次对人精囊主细胞中一种特定分泌蛋白在分泌颗粒的电子致密核心进行了精细结构定位。
