使用JC-1评估马精子的线粒体功能。

Assessment of equine sperm mitochondrial function using JC-1.

作者信息

Gravance C G, Garner D L, Baumber J, Ball B A

机构信息

Department of Population, Health and Reproduction, University of California, Davis, USA.

出版信息

Theriogenology. 2000 Jun;53(9):1691-703. doi: 10.1016/s0093-691x(00)00308-3.

DOI:10.1016/s0093-691x(00)00308-3

PMID:10968415

Abstract

The fluorescent carbocyanine dye, JC-1, labels mitochondria with high membrane potential orange and mitochondria with low membrane potential green. Evaluation of mitochondrial membrane potential with JC-1 has been used in a variety of cell types, including bull spermatozoa; however, JC-1 staining has not yet been reported for equine spermatozoa. The aim of this study was to apply JC-1 staining and assessment by flow cytometry or a fluorescence microplate reader for evaluation of mitochondrial function of equine spermatozoa. Six ejaculates from four stallions were collected and centrifuged through a Percoll gradient (PERC). Spermatozoa were resuspended to 25 x 10(6) cells/mL, samples were split, and one sample was repeatedly flash frozen (FF) in LN2 and thawed. The following gradients of PERC:FF were prepared: 100:0 (100), 75:25(75), 50:50 (50), 25:75 (25) and 0:100 (0). Samples were stained with 2.0 microM JC-1 and assessed for staining by flow cytometry and by a fluorescence microplate reader. A total of 10,000 gated events was analyzed per sample with flow cytometry. The mean percentage of cells staining orange for the 100, 75, 50, 25 and 0 treatments was 92.5, 72.8, 53.4, 27.3 and 7.3, respectively. The expected percentage of spermatozoa forming JC-1 aggregates was correlated with the actual percentage of orange labeled sperm cells determined by flow cytometry (r2=0.98). Conversely, JC-1 monomer formation was negatively correlated with expected mitochondrial membrane potential (r2=-0.98). The blank corrected orange fluorescence, assessed by microplate assay, was significantly (P<0.0001) correlated with the expected (r2=0.49) and with the flow cytometric (r2=0.50) determination of percentage of spermatozoa with mitochondria of high membrane potential. Total orange and orange:green fluorescence was also correlated with mitochondrial function. These results indicate that JC-1 staining can accurately detect changes in mitochondrial membrane potential of equine spermatozoa. The relative fluorescence of JC-1 labeling patterns of equine spermatozoa can be accurately and objectively determined by flow cytometry and by a fluorescence microplate reader assay.

摘要

荧光碳菁染料JC-1可将具有高膜电位的线粒体染成橙色，将具有低膜电位的线粒体染成绿色。用JC-1评估线粒体膜电位已应用于多种细胞类型，包括公牛精子；然而，马精子的JC-1染色尚未见报道。本研究的目的是应用JC-1染色并通过流式细胞术或荧光酶标仪进行评估，以评价马精子的线粒体功能。采集了4匹种公马的6份精液，通过Percoll梯度（PERC）离心。将精子重悬至25×10⁶个细胞/mL，将样本分开，一份样本在液氮中反复速冻（FF）并解冻。制备了以下PERC:FF梯度：100:0（100）、75:25（75）、50:50（50）、25:75（25）和0:100（0）。样本用2.0微摩尔的JC-1染色，并通过流式细胞术和荧光酶标仪评估染色情况。每个样本用流式细胞术分析总共10000个门控事件。100、75、50、25和0处理组中染成橙色的细胞平均百分比分别为92.5%、72.8%、53.4%、27.3%和7.3%。形成JC-1聚集体的精子预期百分比与通过流式细胞术测定的橙色标记精子细胞的实际百分比相关（r²=0.98）。相反，JC-1单体的形成与预期的线粒体膜电位呈负相关（r²=-0.98）。通过酶标仪测定的空白校正橙色荧光与预期的（r²=0.49）以及通过流式细胞术测定的（r²=0.50）具有高膜电位线粒体的精子百分比显著相关（P<0.0001）。总橙色荧光和橙色:绿色荧光也与线粒体功能相关。这些结果表明，JC-1染色能够准确检测马精子线粒体膜电位的变化。马精子JC-1标记模式的相对荧光可以通过流式细胞术和荧光酶标仪测定准确、客观地确定。