Lin Y, Kan F W
Department of Anatomy and Cell Biology, Faculty of Medicine, Queen's University, Kingston, Ontario, Canada.
Biol Reprod. 1996 Nov;55(5):1133-46. doi: 10.1095/biolreprod55.5.1133.
Fracture-label, surface-replica, and routine freeze-fracture techniques were used in combination with phospholipase A2-colloidal gold (PLA2-CG) and filipin as probes to study changes in the distribution of phospholipids and cholesterol, respectively, in morphologically defined plasma membrane domains of mouse spermatozoa during in vitro capacitation. In noncapacitated spermatozoa, quantitative analysis revealed that the fractured plasma membrane overlying the equatorial segment carried the highest PLA2-CG labeling density. The next highest labeling densities were found in the anterior acrosome region and the post-acrosomal region. On the external surface of the plasma membrane revealed by surface replicas, a uniform distribution of PLA2-CG was confined mainly to the acrosomal region of the head. The plasma membrane of the sperm tail had a relatively low labeling density for PLA2-CG. In freeze-fracture replicas of filipin-treated spermatozoa, the labeling density of filipin/sterol complexes (FSCs) was high in the plasma membrane over the acrosomal region where the FSCs were uniformly distributed. The postacrosomal region was weakly labeled. After in vitro capacitation, the densities of PLA2-CG and FSCs were significantly reduced in the fractured plasma membrane of the sperm head and the middle piece of the tail. However, surface replicas revealed an increased PLA2-CG labeling on the external surface of the plasma membrane covering the postacrosomal region, the middle piece, and the principal piece. Another major change detected in capacitated spermatozoa was the presence of small aggregates and patches of elevated, membrane-associated particles on the surface-replicated plasma membrane in the upper portion of the postacrosomal domain. Here the PLA2-CG labeling density was found to be higher than in noncapacitated spermatozoa. These results provide new information with respect to the reorganization and redistribution of phospholipids in specific regions of the plasma membrane during capacitation and provide further support for the concept that removal or loss of antifusigenic sterol from the sperm plasma membrane constitutes an important step of the capacitation process.
采用骨折标记、表面复型和常规冷冻断裂技术，并结合磷脂酶A2-胶体金（PLA2-CG）和制霉菌素作为探针，分别研究小鼠精子体外获能过程中形态学界定的质膜区域内磷脂和胆固醇分布的变化。在未获能精子中，定量分析显示，覆盖赤道段的断裂质膜具有最高的PLA2-CG标记密度。次高的标记密度出现在顶体前部区域和顶体后区域。在表面复型显示的质膜外表面，PLA2-CG的均匀分布主要局限于头部的顶体区域。精子尾部的质膜对PLA2-CG的标记密度相对较低。在制霉菌素处理的精子的冷冻断裂复型中，制霉菌素/固醇复合物（FSCs）的标记密度在顶体区域的质膜中较高，FSCs在该区域均匀分布。顶体后区域标记较弱。体外获能后，精子头部和尾部中段断裂质膜中的PLA2-CG和FSCs密度显著降低。然而，表面复型显示，覆盖顶体后区域、中段和主段的质膜外表面的PLA2-CG标记增加。在获能精子中检测到的另一个主要变化是，在顶体后区域上部的表面复型质膜上存在小聚集体和膜相关颗粒升高的斑块。此处发现的PLA2-CG标记密度高于未获能精子。这些结果提供了关于获能过程中质膜特定区域磷脂重组和重新分布的新信息，并为精子质膜中抗融合固醇的去除或丧失是获能过程的重要步骤这一概念提供了进一步支持。
