Functional Heterogeneity of an Isolated Mitochondrial Population Revealed by Cytofluorometric Analysis at the Single Organelle Level

doi:10.1006/excr.1996.0011

Experimental Cell Research

Volume 222, Issue 1, 10 January 1996, Pages 84-94

https://doi.org/10.1006/excr.1996.0011 Get rights and content

Abstract

Isolated rat liver mitochondria were incubated under various metabolic conditions to determine their membrane potential (MMP) as measured continuously by a tetraphenylphosphonium (TPP⁺)-selective electrode. By flow cytometry, a parallel analysis of fluorescence emissions observing single mitochondria stained with the lipophilic cation 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1) revealed linear correlation between the median orange fluorescence (FL2) due to J-aggregate formations and MMP values measured by TPP⁺. No correlation was detected with the green fluorescence (FL1) emission. A significantly higher correlation appeared between the FL2/FL1 ratio and MMP values. Within the same mitochondrial population, cytofluorimetric analysis revealed the presence of various classes of organelles with different MMP, whose distribution was dependent on metabolic condition. The highest functional heterogeneity was found in deenergized mitochondria, while the highest homogeneity was observed during the first phase of the phosphorylative process. Thus, these data suggest that the cytofluorimetric use of JC-1 provides direct experimental evidence for the hypothesis of functional mitochondrial heterogeneity, at least with respect to their membrane potential.

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Citation Excerpt :
It is therefore possible that through the movement of Ca2 + and possibly other ions, the membrane potential across the acrosome vesicle rose sufficiently as to allow for the uptake of JC-1 and the formation of J-aggregates. Traditionally, a ratiometric approach (FL2/FL1 fluorescence) is used to quantify changes in MMP when measured with JC-1 (Cossarizza et al., 1996), however, due to the unusual stain pattern observed for CCCP-treated C. rodgersii sperm, a quadrant approach was required to quantify MMP loss in sea urchin sperm. The proportion of sperm with high MMP (% high MMP) was identified as that with high FL2 and high FL1 fluorescence, i.e. fluorescence signals falling within the top right quadrant of a cytogram of FL1 v FL2.
There is a need within marine research areas for more rapid techniques to assess the health of sperm from marine invertebrates. Originating in medical research, flow cytometry has been applied to rapidly measure a range of cellular processes within a plethora of different cell types. To date, the transfer of that knowledge to marine research has been limited. A method has been developed to assess mitochondrial membrane potential (MMP) in sea urchin (Centrostephanus rodgersii) sperm using the stain 5,5′,6,6′-tetrachloro-1,1′3,3′-tetrathylbenzimidazolyl-carbocyanine iodide (JC-1) and flow cytometry. MMP is a useful indicator of sperm health as the mitochondrion is the single source of ATP production, and the driver of apoptosis. The method was carefully optimised and validated with the use of positive controls. There were strong correlations between MMP measurements and sperm swimming speed and motility (R values of 0.8–0.9, p < 0.001). JC-1 successfully differentiated between sperm with low and high MMP. However, in sperm that were treated with the mitochondrial inhibitor, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), JC-1 fluorescence in stained sperm did not conform to that usually seen for other cell types. Using fluorescence microscopy, it was confirmed that this was due to the formation of J-aggregates in the acrosome vesicle following MMP collapse. To our best knowledge, this is the first report of J-aggregates forming in an organelle other than the mitochondria. This unexpected fluorescence response necessitated the use of a quadrant approach (% high MMP) instead of the usual ratiometric approach (FL2/FL1) to quantify MMP changes. Difficulties overcome during method development are described, many of which were likely related to the required use of seawater as a test medium. The developed method will enable rapid measurement of mitochondrial membrane potential of sea urchin sperm for application in reproductive biology, aquaculture research, and the impact of environmental stressors such as ocean acidification and pollution on sperm development and function.

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Regular ArticleFunctional Heterogeneity of an Isolated Mitochondrial Population Revealed by Cytofluorometric Analysis at the Single Organelle Level

Abstract

Regular Article
Functional Heterogeneity of an Isolated Mitochondrial Population Revealed by Cytofluorometric Analysis at the Single Organelle Level