Assessment of mitochondrial potential: implications for the correct monitoring of human sperm function

Mitochondrial membrane potential (MMP) is an indicator of sperm functionality that can be assessed using specific fluorescent markers. However, the ability of distinct probes to dynamically evaluate sperm MMP has not been determined. In the present study, human sperm samples were independently labelled with MitoTracker Green, MitoTracker Red and JC-1. The ability of each probe to correctly monitor MMP was determined by incubating sperm with MMP disruptors (KCN, FCCP and valinomycin). Similarly, the effect of distinct fixatives (formaldehyde and methanol) was also tested. The three mitochondrial probes provided similar results, and were able to monitor changes in MMP when sperm had been previously incubated with MMP-disrupting agents. However, only JC-1 could, to a small extent, mirror MMP alterations after sperm labelling. Unexpectedly, the three probes were able to stain some pre-fixed sperm, even though this behaviour was very variable, especially for MitoTrackers. On the other hand, none of the probes was shown to be reliably fixable. Of the three probes tested JC-1 seems to be the most adequate, nevertheless, the choice of an MMP-specific probe may depend on the aim of each experimental setting and appropriate controls must always be performed.