Organelle-specific probe JC-1 identifies membrane potential differences in the mitochondrial function of bovine sperm.
The mitochondrial probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyan ine iodide (JC-1) not only identifies mitochondria exhibiting low membrane potentials by the emission of green fluorescence (range, 510-520 nm) but also differentiates these from mitochondria exhibiting relatively high membrane potentials. This discrimination occurs because JC-1 forms aggregates at high membrane potentials. These J-aggregates emit a bright red-orange fluorescence at 590 nm. In this study, JC-1 was combined with the classical dead cell stain, propidium iodide (PI), to identify a spectrum of functional sperm along with degenerate sperm. Flow cytometric analysis of bull sperm showed that the aggregate:monomer ratio differed among bulls before cryopreservation (P < 0.001) but not afterwards (P > 0.05). The effects of stain equilibration time, sperm concentration, and live:dead ratios were examined. The addition of SYBR-14 to the JC-1 and PI combination enhanced the distinction between the red PI-stained and red-orange JC-1-stained populations. This discrimination between J-aggregates and the PI-stained sperm was affected by sperm concentration. These studies show that JC-1 can be useful in monitoring mitochondrial function in bovine sperm.
Garner
D L
DL
School of Veterinary Medicine, University of Nevada, Reno, USA.
Thomas
C A
CA
D000818:Animals; D001562:Benzimidazoles; D002232:Carbocyanines; D002417:Cattle; D002470:Cell Survival; D015925:Cryopreservation; D005456:Fluorescent Dyes; D007425:Intracellular Membranes / Q000502:physiology; D008297:Male; D008564:Membrane Potentials; D008928:Mitochondria / Q000502:physiology*; D015388:Organelles; D009930:Organic Chemicals; D013094:Spermatozoa / Q000502:physiology*; D013194:Staining and Labeling / Q000379:methods
D001562:Benzimidazoles; D002232:Carbocyanines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C093207:SYBR-14; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine