Patterns of MTT reduction in mammalian spermatozoa
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Published on: February 6, 2018
Robert John Aitken1, Diatsendoula Gregoratos1, Leslie Kutzera1
1Priority Research Centre for Reproductive Science, Faculty of Science and Faculty of Health and Medicine, University of Newcastle, University Drive, Callaghan, New South Wales, Australia.
Abstract:
MTT is widely used in biology as a probe for cell viability by virtue of its ability to generate deposits of insoluble formazan at sites of intense oxidoreductase activity. This response is generally held to reflect mitochondrial redox activity; however, extra-mitochondrial MTT reduction has also been recorded in certain cell types. Given this background, we set out to determine the major sites of formazan deposition in mammalian spermatozoa. In the mouse, most MTT reduction took place within the extensive mitochondrial gyres, with a single minor site of formazan deposition on the sperm head. By contrast, human spermatozoa generally displayed small disorganized midpieces exhibiting moderate MTT reduction activity accompanied by a major extra-mitochondrial formazan deposit on various locations in the sperm head from the neck to the anterior acrosome. Equine spermatozoa presented a combination of these two patterns, with major formazan deposition in the mitochondria accompanied by an extra-mitochondrial formazan deposit in around 20% of cells. The functionality of human spermatozoa was positively associated with the presence of an extra-mitochondrial formazan granule. Subsequent studies indicated that this extra-mitochondrial activity was suppressed by the presence of diphenylene iodonium, zinc, 2-deoxyglucose, co-enzyme Q, an SOD mimetic and NADPH oxidase inhibitors. We conclude that the pattern of MTT reduction to formazan by spermatozoa is species specific and conveys significant information about the relative importance of mitochondrial vs extra-mitochondrial redox activity that, in turn, defines the functional qualities of these cells.
Insights
The MTT assay reveals species-specific formazan deposition patterns in sperm. Extra-mitochondrial activity in human sperm correlates with functionality, offering insights into cell redox status.
Area of Science:
- Cell Biology
- Reproductive Biology
- Biochemistry
Background:
- The MTT assay is a common probe for cell viability, typically reflecting mitochondrial redox activity.
- However, extra-mitochondrial MTT reduction has been observed, necessitating further investigation into its sites and significance.
- Spermatozoa possess unique metabolic and functional characteristics relevant to MTT assay interpretation.
Purpose of the Study:
- To identify the primary locations of formazan deposition in mammalian spermatozoa using the MTT assay.
- To investigate the relationship between MTT reduction sites and sperm functionality across different species.
- To explore the biochemical pathways influencing extra-mitochondrial MTT reduction in spermatozoa.
Main Methods:
- Spermatozoa from mice, humans, and horses were subjected to MTT assay.
- Formazan deposition sites were visualized and quantified using microscopy.
- The impact of various inhibitors (e.g., diphenylene iodonium, NADPH oxidase inhibitors) on MTT reduction was assessed.
- Sperm functionality was correlated with formazan deposition patterns.
Main Results:
- Mouse spermatozoa showed predominant formazan deposition in mitochondria.
- Human spermatozoa exhibited significant extra-mitochondrial formazan deposits on the sperm head, associated with higher functionality.
- Equine spermatozoa displayed a mixed pattern of mitochondrial and extra-mitochondrial deposition.
- Extra-mitochondrial activity was sensitive to specific inhibitors, suggesting involvement of NADPH oxidase and other pathways.
Conclusions:
- MTT reduction patterns in spermatozoa are species-specific, highlighting varied redox activity.
- Extra-mitochondrial formazan deposition in human sperm is a key indicator of cellular functionality.
- The MTT assay provides valuable insights into the interplay between mitochondrial and extra-mitochondrial redox processes in sperm viability and function.
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