Fluorometric assessments of mitochondrial function and viability in cryopreserved bovine spermatozoa
Fluorometric assessments of mitochondrial function and viability in cryopreserved bovine spermatozoa
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DOI：
10.1095/biolreprod57.6.1401
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发表时间：
1997-12-01
影响因子：
3.6
通讯作者：
Marshall, CE
中科院分区：
文献类型：
--
作者：
Garner, DL;Thomas, CA;Marshall, CE
用荧光分光光度技术对12头公牛冷冻保存的精子进行了线粒体功能和精子活力的测定。用罗丹明123(R123)、5，5‘，6，6’-tetrachloro-1，1‘，3，3’-tetraethylbenzimidazolyl-carbocyanine碘化物(JC-1)和MitoTracker Green FM(MITO)三种不同的荧光团对精子线粒体进行荧光染色。用流式细胞仪对染色的精子和SYBR-14(活细胞核酸染色)和碘化丙啶染色的等价物进行定量。线粒体功能的荧光测量结果与解冻后立即进行的精子活力的显微镜评估、37℃孵育3小时后的活力以及精子活力的荧光评估进行了比较。用R123或Mito染色得到单一的绿色种群。相比之下，线粒体的JC-1染色产生了绿色和红橙色的精子群体，有时还会在两个群体之间产生渐进的梯度。与其他两种荧光染色相比，IC-L区分高膜电位和中膜电位线粒体的能力提供了更严格的代谢功能估计。总体而言，线粒体功能的三种荧光测量彼此之间高度相关，与SYBR-14对生存能力的评估，以及对运动性的微观估计。
Mitochondrial function and sperm viability were quantified in samples of cryopreserved bovine spermatozoa from 12 bulls using fluorometric techniques. The active mitochondria of the spermatozoa were fluorescently stained using three different fluorophores: rhodamine 123 (R123), 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) or MitoTracker Green FM (MITO). The stained spermatozoa, and companion aliquots that had been stained with SYBR-14 (a living-cell nucleic acid stain) and propidium iodide to assess viability, were quantified using flow cytometry. The resulting fluorescent measurements of mitochondrial function were compared with microscopic assessments of progressive sperm motility immediately after thawing, with motility after 3-h incubation at 37 degrees C, and with the fluorescent assessment of sperm viability. Staining with either R123 or MITO resulted in a single green population. In contrast, the JC-1 staining of mitochondria produced both green and red-orange populations of spermatozoa and sometimes a progressive gradient between the two populations. The ability of IC-l to discriminate between mitochondria exhibiting high membrane potential from those having low to medium membrane potential provided a more rigorous estimate of metabolic function than the other two fluorescent stains. Overall, the three fluorometric measurements of mitochondrial function were highly correlated with each other, with the SYBR-14 assessment of viability, and with the microscopic estimates of motility.