Use of JC-1 to assess mitochondrial membrane potential in sea urchin sperm

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.