Carrageta David F, Freire-Brito Laís, Oliveira Pedro F, Alves Marco G
Clinical and Experimental Endocrinology, UMIB-Unit for Multidisciplinary Research in Biomedicine, ICBAS-School of Medicine and Biomedical Sciences, University of Porto, Porto, Portugal.
Laboratory for Integrative and Translational Research in Population Health (ITR), University of Porto, Portugal.
Curr Protoc. 2022 Sep;2(9):e531. doi: 10.1002/cpz1.531.
Mitochondria are fundamental for human spermatozoa motility and fertilizing ability. Mitochondria participate not only in ATP production, but also in reactive oxygen species production, redox equilibrium, and calcium regulation, all of which are central for human spermatozoa motility, capacitation, acrosome reaction, and ultimately, oocyte fertilization. Mitochondrial membrane potential is a key indicator of mitochondrial health and activity. Most commonly used methods for the study of mitochondrial membrane potential, however, cannot be applied to human spermatozoa due to their unique characteristics, including high motility and time-dependent decay of quality, limiting the study of this important parameter in these cells. Here, we describe an easy, fast, and cheap protocol for the quantitative evaluation of human spermatozoa mitochondrial membrane potential, using the fluorescent cationic dye 5,5,6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimi-dazoylcarbocyanine iodide (JC-1). JC-1 is a sensitive marker for mitochondrial membrane potential, exhibiting a potential-dependent accumulation in the mitochondria. At high mitochondrial membrane potential, JC-1 forms J-aggregates, which emit red fluorescence, whereas at low mitochondrial membrane potential, JC-1 remains at its monomer state, which emits green fluorescence. We first describe how to evaluate human spermatozoa mitochondrial membrane potential using JC-1 and a fluorescence plate reader, for high-throughput studies. The calculation of the JC-1 ratio (indicative of the J-aggregates/monomers ratio) is then used to quantitatively evaluate mitochondrial health and activity. In addition, we describe an imaging protocol for the qualitative evaluation of human spermatozoa mitochondrial membrane potential using a fluorescence microscope. This allows for a visual analysis of the results that can complement the quantitative data. These protocols can be used to study the effects of spermatozoa exposure to compounds of interest, and alterations due to diseases or different conditions. While these protocols are illustrated with human spermatozoa, they can be adapted and used on spermatozoa of different species. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Quantitative evaluation of human spermatozoa mitochondrial membrane potential using the JC-1 dye and a fluorescence plate reader Basic Protocol 2: Qualitative evaluation of human spermatozoa mitochondrial membrane potential using the JC-1 dye and fluorescence microscopy Support Protocol: Preparation of the JC-1 working solution.
线粒体对于人类精子的运动能力和受精能力至关重要。线粒体不仅参与三磷酸腺苷（ATP）的产生，还参与活性氧的产生、氧化还原平衡和钙调节，所有这些对于人类精子的运动、获能、顶体反应以及最终的卵母细胞受精都至关重要。线粒体膜电位是线粒体健康和活性的关键指标。然而，由于人类精子具有独特的特性，包括高运动性和质量随时间衰减，大多数常用的研究线粒体膜电位的方法无法应用于人类精子，这限制了对这些细胞中这一重要参数的研究。在此，我们描述了一种使用荧光阳离子染料5,5,6,6'-四氯-1,1',3,3'-四乙基苯并咪唑羰花青碘化物（JC-1）对人类精子线粒体膜电位进行定量评估的简便、快速且廉价的方案。JC-1是线粒体膜电位的敏感标志物，在线粒体中表现出电位依赖性积累。在线粒体膜电位高时，JC-1形成J聚集体，发出红色荧光，而在线粒体膜电位低时，JC-1保持单体状态，发出绿色荧光。我们首先描述如何使用JC-1和荧光酶标仪评估人类精子线粒体膜电位，用于高通量研究。然后，通过计算JC-1比率（指示J聚集体/单体比率）来定量评估线粒体的健康和活性。此外，我们描述了一种使用荧光显微镜对人类精子线粒体膜电位进行定性评估的成像方案。这允许对结果进行视觉分析，以补充定量数据。这些方案可用于研究精子暴露于感兴趣的化合物的影响，以及由于疾病或不同条件引起的改变。虽然这些方案以人类精子为例进行说明，但它们可以进行调整并用于不同物种的精子。© 2022威利期刊有限责任公司。基本方案1：使用JC-1染料和荧光酶标仪对人类精子线粒体膜电位进行定量评估 基本方案2：使用JC-1染料和荧光显微镜对人类精子线粒体膜电位进行定性评估 支持方案：JC-1工作溶液的制备
