Althouse G C, Bruns K A, Evans L E, Hopkins S M, Hsu W H
Department of Veterinary Clinical Sciences, Iowa State University, Ames 50011, USA.
Prep Biochem. 1995 Feb-May;25(1-2):69-80. doi: 10.1080/10826069508010108.
Spermatozoa were initially separated from fresh boar ejaculates using a 1.0 M sucrose density gradient. Spermatozoa (1 x 10(8) cells/ml) were subjected to gas cavitation (650 psi, 10 minutes), followed by a 4-step centrifugation technique to yield the final plasma membrane preparation. Purity of the plasma membrane isolate was determined using microscopic techniques (i.e. differential interference contrast and transmission electron microscopy) and marker enzymes for biochemical characterization. Plasma membranes were found to be removed primarily from the periacrosomal region of the sperm. Acrosomes appeared to remain intact on the cavitated spermatozoa. Transmission electron microscopy yielded a homogenous population of 100-200 microns unilamellar vesicles. Enzyme markers specific for plasma, acrosome and mitochondrial membranes substantial the purity observed under visual examination.
首先使用1.0M蔗糖密度梯度从新鲜公猪精液中分离精子。将精子（1×10⁸个细胞/毫升）进行气体空化处理（650磅力/平方英寸，10分钟），随后采用四步离心技术以获得最终的质膜制剂。使用显微镜技术（即微分干涉对比和透射电子显微镜）以及用于生化表征的标记酶来测定质膜分离物的纯度。发现质膜主要从精子的顶体周围区域去除。顶体在空化后的精子上似乎保持完整。透射电子显微镜观察到一群均匀的100 - 200微米单层囊泡。针对质膜、顶体膜和线粒体膜的酶标记证实了在视觉检查下观察到的纯度。
