荧光探针JC-1与PI联合染色法在猪精子线粒体膜电位检测中的应用

目前,对于精子线粒体膜电位(MMP)的检测分析,通常采用JC-1单独染色,这种染色方法可以准确地区分高、低MMP精子,但不能明确区分精子的存活状态。本研究采用JC-1与碘化丙啶(propidium iodide,PI)2种荧光染料联合染色的方法,对6种不同保存条件下的猪精子进行染色,分别使用流式细胞仪与荧光显微镜2种检测手段对猪精子MMP进行检测。结果表明:死亡精子头部呈红色,高MMP精子尾部呈橙红色,低MMP精子尾部为绿色。2种检测手段的结果比较显示,荧光显微镜能够有效地区分出高、低MMP死精子,但对于活精子MMP的高低检测效果不甚理想。相反,流式细胞仪则能够有效地区分活精子MMP的高低,但对于死精子MMP高低的检测仍较困难。因此,对于猪精子线粒体MMP的检测,采用JC-1与PI双染的方法,用流式细胞仪与荧光显微镜进行联合检测,能比较全面的反映出精子线粒体的功能状态。

Application of Co-staining Method with JC-1 and PI in detection of Boar Sperm Mitochondrial Membrane Potential

At present,JC-1 single staining method is usually used for the detection of sperm mitochondrial membrane potential(MMP).However,this method is able to distinguish between high-and low-MMP sperm,but can not indentify the live sperm and dead one.In this study,co-staining method using two fluorescent dyes of JC-1 and propidium iodide(propidium iodide,PI) was developed and applied to the boar semen under six different conditions.Flow cytometry and fluorescence microscopy were utilized to detect the function status of these sperms.The results showed that,the head of dead sperm fluoresce red,and the head of high-MMP sperm fluoresce orange,whereas the tail of low MMP fluoresces green.The compared results of two detection means found that the fluorescence microscopy could separate high-and low-MMP effectively,but the detection level for the MMP of live sperm was less than ideal results.In contrast,flow cytometry is able to effectively distinguish the level of MMP of live sperm,but the detection for dead sperm of high and low MMP was still difficult.Therefore,it is effective to use co-staining method with JC-1 and PI,using flow cytometry and fluorescence microscopy joint detection,to detect the sperm mitochondrial membrane potential(MMP) and comprehensive reflect the functional status of boar sperm mitochondria.