Assessment of equine sperm mitochondrial function using JC-1

• Published in: Theriogenology Vol. 53; no. 9; pp. 1691 - 1703
• Main Authors: Gravance, CG, Garner, DL, Baumber, J, Ball, BA
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.06.2000
• Subjects: Animals; Benzimidazoles - chemistry; Carbocyanines - chemistry; Equine; flow cytometry; Flow Cytometry - veterinary; fluorescence; Fluorescent Dyes - chemistry; Horses - physiology; Male; Microscopy, Fluorescence - veterinary; mitochondria; Mitochondria - chemistry; Mitochondria - physiology; Propidium - chemistry; Regression Analysis; Semen - chemistry; spermatozoa; Spermatozoa - chemistry; Spermatozoa - physiology; Equine; flow cytometry; spermatozoa; fluorescence; mitochondria
• ISSN: 0093-691X; 1879-3231
• DOI: 10.1016/S0093-691X(00)00308-3

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 Percoli gradient (PERC). Spermatozoa were resuspended to 25×10 6 cells/mL, samples were split, and one sample was repeatedly flash frozen (FF) in LN 2 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 μJC-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 (r 2 = 0.98). Conversely, JC-1 monomer formation was negatively correlated with expected mitochondrial membrane potential (r 2 = −0.98). The blank corrected orange fluorescence, assessed by microplate assay, was significantly (P<0.0001) correlated with the expected (r 2 = 0.49) and with the flow cytometric (r 2 = 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