Maintenance of motility in mouse sperm permeabilized with streptolysin O.
Maintenance of motility in mouse sperm permeabilized with streptolysin O.
复制标题
用链球菌溶血素 O 透化小鼠精子活力的维持。
DOI：
10.1095/biolreprod60.3.683
复制
发表时间：
1999
影响因子：
3.6
通讯作者：
Gerton,GL
中科院分区：
文献类型：
--
作者：
Johnson,LR;Moss,SB;Gerton,GL
研究精子运动机制的一种方法是使精子透化，并通过操纵细胞的细胞内环境来检查运动的调节。最常见的精子透化方法，洗涤剂处理，具有从细胞中提取膜和许多蛋白质的缺点。为了避免这个问题，我们开发了一种方法，使用链球菌溶血素O在质膜内产生稳定的孔，同时保持内膜完整。将精子透化，预孵育，然后用0.6 U/ml的链球菌溶血素O处理。通过荧光染料技术和使用外源性添加的[γ-32 P]ATP的内源性蛋白磷酸化来评估透化。链球菌溶血素O诱导的透化使精子不动，其作用是钙依赖性的。当细胞同时用含有ATP的培养基处理时，链球菌溶血素O处理的精子保持鞭毛运动。这些结果表明，链球菌溶血素O透化模型系统是一个有用的实验方法，用于研究调节精子活力的机制，因为它允许鞭毛装置暴露于各种外源添加的分子。
One approach to studying the mechanisms governing sperm motility is to permeabilize sperm and examine the regulation of motility by manipulating the intracellular milieu of the cell. The most common method of sperm permeabilization, detergent treatment, has the disadvantage that the membranes and many proteins are extracted from the cell. To avoid this problem, we have developed a method that uses streptolysin O to create stable pores within the plasma membrane while leaving internal membranes intact. Sperm were permeabilized, preincubated, and then treated with 0.6 U/ml of streptolysin O. Permeabilization was assessed by fluorescent dye technologies and endogenous protein phosphorylation using exogenously added [γ-32P]ATP. Streptolysin O-induced permeabilization rendered the sperm immotile, and the effect was Ca2+-dependent. When the cells were treated simultaneously with a medium containing ATP, streptolysin O-treated sperm maintained flagellar movement. These results demonstrate that the streptolysin O permeabilization model system is a useful experimental method for studying the mechanisms that regulate sperm motility since it allows the flagellar apparatus to be exposed to various exogenously added molecules.