Determination of high mitochondrial membrane potential in spermatozoa loaded with the mitochondrial probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) by using fluorescence-activated flow cytometry.
A flow cytometric method was developed to identify viable, energized sperm cells with high mitochondrial inner transmembrane potential (Deltapsi(m)), >80-100 mV using the mitochondrial probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) and the impermeant nuclear stain propidium iodine (PI). This flow cytometric method is described in detail here. When in contact with membranes possessing a high Deltapsi(m), JC-1 forms aggregates (J(agg)) that are fluorescent at 590 nm in response to 488 nm excitation. We found that the reactive oxygen species generator, menadione reduced sperm motility and reduced Deltapsi(m) in a dose responsive fashion that was closely correlated with the loss of motility.
Guthrie
H David
HD
US Department of Agriculture, Animal Bioscience and Biotechnology Laboratory, Agricultural Research Service, Beltsville, Maryland, USA.
Welch
Glenn R
GR
D000818:Animals; D001562:Benzimidazoles / Q000378:metabolism*; D002232:Carbocyanines / Q000378:metabolism*; D002465:Cell Movement / Q000187:drug effects; D002498:Centrifugation; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism*; D007091:Image Processing, Computer-Assisted; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D011419:Propidium / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D012661:Semen / Q000187:drug effects / Q000378:metabolism; D013094:Spermatozoa / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D013194:Staining and Labeling; D013552:Swine; D013997:Time Factors; D024483:Vitamin K 3 / Q000494:pharmacology
D001562:Benzimidazoles; D002232:Carbocyanines; D005456:Fluorescent Dyes; D017382:Reactive Oxygen Species; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine; D011419:Propidium; D024483:Vitamin K 3