San Agustin J T, Witman G B
Male Fertility Program, Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.
Cell Motil Cytoskeleton. 1993;24(4):264-73. doi: 10.1002/cm.970240407.
A procedure for preparing cytosol-free ram sperm models was developed. Sperm are introduced to a Triton X-100-containing demembranation medium layered on top of a discontinuous Percoll gradient. After brief exposure to the demembranating solution, the sperm are separated from the detergent-soluble components by centrifugation through a 55% Percoll layer, finally collecting on top of a 90% Percoll cushion from where they are recovered. Optimum conditions consisted of Triton X-100 at 0.20% and a demembranation time of 35 sec. Cross-sections of midpieces and principal pieces of the demembranated sperm were examined by electron microscopy. With 0.20% Triton X-100 in the demembranation medium, 86% of the cross-sections showed no plasma membranes and the rest had broken plasma membranes. The remaining tail structures appeared to be morphologically intact. Assay of phosphoglucose isomerase as a marker enzyme confirmed that at least 98% of the cytosolic protein was removed. Ram sperm models obtained by this procedure could be reactivated, and the percent motility and beat parameters were similar to those of the intact sperm. Reconstitution with the detergent-soluble components was neither required for, nor enhanced, reactivation. Therefore, demembranated ram sperm do not require a detergent-soluble protein factor for reactivation.
开发了一种制备无细胞质的公羊精子模型的方法。将精子引入置于不连续Percoll梯度之上的含Triton X-100的去膜介质中。在短暂暴露于去膜溶液后，通过55%Percoll层离心将精子与去污剂可溶成分分离，最终收集在90%Percoll垫层之上并从那里回收。最佳条件包括0.20%的Triton X-100和35秒的去膜时间。通过电子显微镜检查去膜精子的中段和主段的横截面。在去膜介质中含有0.20%Triton X-100时，86%的横截面显示没有质膜，其余的质膜已破裂。其余的尾部结构在形态上似乎完好无损。以磷酸葡萄糖异构酶作为标记酶的测定证实至少98%的细胞质蛋白被去除。通过该方法获得的公羊精子模型可以重新激活，其活力百分比和摆动参数与完整精子相似。用去污剂可溶成分进行重组既不是重新激活所必需的，也不会增强重新激活。因此，去膜的公羊精子重新激活不需要去污剂可溶蛋白因子。
