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Theriogenology2000; 53(9); 1691-1703; doi: 10.1016/s0093-691x(00)00308-3

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.
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Publication Date: 2000-09-01 PubMed ID: 10968415DOI: 10.1016/s0093-691x(00)00308-3Google Scholar: Lookup
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  • Journal Article
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  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The study examines and validates the use of a specific dye called JC-1 in identifying the presence of high or low mitochondrial function in equine sperm cells. The application of this fluorescent dye has been used in other cell types but not yet on horse sperm cells.

Objective of the Study

  • The central aim of this research was to apply JC-1 staining, a method not previously used with equine spermatozoa, and assess it by flow cytometry or a fluorescence microplate reader. The goal was to evaluate the mitochondrial function of equine spermatozoa.

Methodology

  • Six ejaculates from four stallions were collected and centrifuged through a Percoll gradient (PERC).
  • The sperm cells were then resuspended to 25 x 10(6) cells/mL and split into samples. Among them, one sample was repeatedly flash frozen in LN2 and thawed.
  • These prepared samples were stained with 2.0 microM JC-1 and then evaluated for staining by flow cytometry and by a fluorescence microplate reader.
  • For each sample, 10,000 events were analysed.

Findings

  • The results showed that the percentage of cells staining orange, an indication of high mitochondrial function, decreased as the ratio of freeze-thawed cells increased in the sample mix.
  • 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 sperm cells forming JC-1 aggregates correlated strongly with the actual percentage of orange labeled sperm cells, as determined by flow cytometry (r2=0.98).
  • A negative correlation (r2=-0.98) was identified between JC-1 monomer formation and the expected mitochondrial membrane potential.
  • The microplate reader assessment of the blank-corrected orange fluorescence, or high mitochondrial membrane potential, strongly correlated with both the expected (r2=0.49) and the flow cytometric (r2=0.50) determinations.

Conclusion

  • Overall, the researchers concluded that JC-1 staining can accurately detect changes in mitochondrial membrane potential of equine spermatozoa and the relative fluorescence of JC-1 labelling patterns of equine spermatozoa.
  • This can be accurately and objectively determined by flow cytometry and by a fluorescence microplate reader assay, suggesting possibilities for its practical application in equine reproduction and genetics.

Cite This Article

APA
Gravance CG, Garner DL, Baumber J, Ball BA. (2000). Assessment of equine sperm mitochondrial function using JC-1. Theriogenology, 53(9), 1691-1703. https://doi.org/10.1016/s0093-691x(00)00308-3

Publication

Theriogenology
ISSN: 0093-691X
NlmUniqueID: 0421510
Country: United States
Language: English
Volume: 53
Issue: 9
Pages: 1691-1703

Researcher Affiliations

Gravance, C G
  • Department of Population, Health and Reproduction, University of California, Davis, USA.
Garner, D L
    Baumber, J
      Ball, B A

        MeSH Terms

        • Animals
        • Benzimidazoles / chemistry
        • Carbocyanines / chemistry
        • Flow Cytometry / veterinary
        • Fluorescent Dyes / chemistry
        • Horses / physiology
        • Male
        • Microscopy, Fluorescence / veterinary
        • Mitochondria / chemistry
        • Mitochondria / physiology
        • Propidium / chemistry
        • Regression Analysis
        • Semen / chemistry
        • Spermatozoa / chemistry
        • Spermatozoa / physiology

        Citations

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