Assessment of equine sperm mitochondrial function using JC-1

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

Research output: Contribution to journalArticlepeer-review

125 Scopus citations

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 106 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 μM 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. (C) 2000 by Elsevier Science Inc.

Original languageEnglish
Pages (from-to)1691-1703
Number of pages13
JournalTheriogenology
Volume53
Issue number9
DOIs
StatePublished - Jun 2000

Bibliographical note

Funding Information:
The blank corrected orange fluorescence, assessedb y microplate assay, was significantly (P<O.OOOlc) orrelated with the expected (?=0.49) and with the flow cytometric (?=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. 0 2000 by Elsevier Science Inc. Key words: Equine, spermatozoa, mitochondria, fluorescence, flow cytometry Acknowledgements The authors thank Carol Oxford of the UC Davis Optical Biology Laboratory for assistance with flow cytometry of equine spermatozoa. This research was supported by the John P. Hughes Endowment and by the Center for Equine Health with funds provided by the Oak Tree Racing Association, State of California pari-mutuel fund, and contributions by private donors.

Keywords

  • Equine
  • Flow cytometry
  • Fluorescence
  • Mitochondria
  • Spermatozoa

ASJC Scopus subject areas

  • Small Animals
  • Food Animals
  • Animal Science and Zoology
  • Equine

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