title: Assessment of equine sperm mitochondrial function using JC-1.
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.
authors.affiliation: Department of Population, Health and Reproduction, University of California, Davis, USA.
authors.forename: C G
authors.initials: CG
authors.lastname: Gravance
authors.forename: D L
authors.initials: DL
authors.lastname: Garner
authors.forename: J
authors.initials: J
authors.lastname: Baumber
authors.forename: B A
authors.initials: BA
authors.lastname: Ball
mesh_terms: D000818:Animals; D001562:Benzimidazoles / Q000737:chemistry*; D002232:Carbocyanines / Q000737:chemistry*; D005434:Flow Cytometry / Q000662:veterinary; D005456:Fluorescent Dyes / Q000737:chemistry*; D006736:Horses / Q000502:physiology*; D008297:Male; D008856:Microscopy, Fluorescence / Q000662:veterinary; D008928:Mitochondria / Q000737:chemistry / Q000502:physiology*; D011419:Propidium / Q000737:chemistry; D012044:Regression Analysis; D012661:Semen / Q000737:chemistry; D013094:Spermatozoa / Q000737:chemistry / Q000502:physiology*
chemical_list: D001562:Benzimidazoles; D002232:Carbocyanines; D005456:Fluorescent Dyes; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine; D011419:Propidium