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Updated: Jun 20, 2026
High-Resolution Respirometry to Assess Mitochondrial Function in Human Spermatozoa
Published on: June 23, 2023
Assessment of mitochondrial potential: implications for the correct monitoring of human sperm function
1Department of Zoology, Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal.
Abstract:
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.
Insights
This study evaluated fluorescent probes for assessing sperm mitochondrial membrane potential (MMP). JC-1 showed the most promise for dynamic MMP monitoring, though fixative compatibility remains a challenge.
Area of Science:
- Reproductive Biology
- Cellular Biology
- Biochemistry
Background:
- Mitochondrial membrane potential (MMP) is crucial for sperm functionality.
- Assessing MMP dynamically requires reliable fluorescent probes.
- Distinct probes' performance in dynamic MMP evaluation is not well-established.
Purpose of the Study:
- To determine the dynamic evaluation capabilities of MitoTracker Green, MitoTracker Red, and JC-1 for sperm MMP.
- To assess the impact of MMP disruptors and fixatives on these probes.
- To identify the most suitable probe for sperm MMP assessment.
Main Methods:
- Human sperm samples were labeled with MitoTracker Green, MitoTracker Red, and JC-1.
- Probes' ability to monitor MMP was tested using disruptors (KCN, FCCP, valinomycin).
- The effect of fixatives (formaldehyde, methanol) on probe performance was evaluated.
Main Results:
- All three probes monitored MMP changes induced by disruptors.
- JC-1 partially reflected MMP alterations post-labeling.
- Probes stained pre-fixed sperm variably; none were reliably fixable.
Conclusions:
- JC-1 appears most adequate for sperm MMP assessment, but probe choice depends on experimental goals.
- Careful selection of probes and appropriate controls are essential for accurate MMP evaluation.
- Fixation methods significantly impact the reliability of MMP assessment using these probes.
