Use of JC-1 to assess mitochondrial membrane potential in sea urchin sperm
Use of JC-1 to assess mitochondrial membrane potential in sea urchin sperm
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DOI：
10.1016/j.jembe.2013.12.008
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发表时间：
2014-03-01
影响因子：
2
通讯作者：
Schlegel, P.
中科院分区：
文献类型：
--
作者：
Binet, M. T.;Doyle, C. J.;Schlegel, P.
海洋研究领域需要更快速的技术来评估海洋无脊椎动物精子的健康状况。起源于医学研究，流式细胞术已被应用于快速测量大量不同细胞类型内的一系列细胞过程。迄今为止，向海洋研究转让这种知识的情况有限。建立了一种用5，5 '，6，6'-四氯-1，1 '3，3'-四乙基苯并咪唑碘化碳菁（JC-1）染色和流式细胞术测定海胆（Centrostephanus rodgersii）精子线粒体膜电位的方法。MMP是精子健康的一个有用指标，因为它是ATP产生的唯一来源，也是细胞凋亡的驱动因素。使用阳性对照对该方法进行了仔细优化和验证。MMP测量值与精子游泳速度和活力之间存在强相关性（R值为0.8-0.9，p < 0.001）。JC-1成功地区分了具有低MMP和高MMP的精子。然而，在用线粒体抑制剂羰基氰3-氯苯腙（CCCP）处理的精子中，染色精子中的JC-1荧光与通常在其他细胞类型中观察到的荧光不一致。使用荧光显微镜，它被证实，这是由于顶体囊泡中的J-聚集体的形成后，MMP崩溃。据我们所知，这是第一次报告的J-聚集体形成的细胞器以外的线粒体。这种非预期的荧光响应需要使用象限法（高MMP %）而不是通常的比率法（FL 2/FL 1）来定量MMP变化。描述了方法开发过程中克服的困难，其中许多可能与所需使用海水作为试验介质有关。开发的方法将能够快速测量海胆精子的线粒体膜电位，用于生殖生物学，水产养殖研究以及海洋酸化和污染等环境压力因素对精子发育和功能的影响。皇冠版权所有（C）2013由爱思唯尔B. V.出版保留所有权利。
There is a need within marine research areas for more rapid techniques to assess the health of sperm from marine invertebrates. Originating in medical research, flow cytometry has been applied to rapidly measure a range of cellular processes within a plethora of different cell types. To date, the transfer of that knowledge to marine research has been limited. A method has been developed to assess mitochondrial membrane potential (MMP) in sea urchin (Centrostephanus rodgersii) sperm using the stain 5,5',6,6'-tetrachloro-1,1'3,3'-tetrathylbenzimidazolyl-carbocyanine iodide (JC-1) and flow cytometry. MMP is a useful indicator of sperm health as the mitochondrion is the single source of ATP production, and the driver of apoptosis. The method was carefully optimised and validated with the use of positive controls. There were strong correlations between MMP measurements and sperm swimming speed and motility (R values of 0.8-0.9, p < 0.001). JC-1 successfully differentiated between sperm with low and high MMP. However, in sperm that were treated with the mitochondrial inhibitor, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), JC-1 fluorescence in stained sperm did not conform to that usually seen for other cell types. Using fluorescence microscopy, it was confirmed that this was due to the formation of J-aggregates in the acrosome vesicle following MMP collapse. To our best knowledge, this is the first report of J-aggregates forming in an organelle other than the mitochondria. This unexpected fluorescence response necessitated the use of a quadrant approach (% high MMP) instead of the usual ratiometric approach (FL2/FL1) to quantify MMP changes. Difficulties overcome during method development are described, many of which were likely related to the required use of seawater as a test medium. The developed method will enable rapid measurement of mitochondrial membrane potential of sea urchin sperm for application in reproductive biology, aquaculture research, and the impact of environmental stressors such as ocean acidification and pollution on sperm development and function. Crown Copyright (C) 2013 Published by Elsevier B.V. All rights reserved.