Causative mechanisms and functional correlates of MTT reduction in stallion spermatozoa.
Abstract: MTT is a commonly used cell vitality probe, due to its ability to form insoluble formazan deposits at cellular locations of intense oxidoreductase activity. Although this response is considered a reflection of mitochondrial redox activity, extra-mitochondrial sites of MTT reduction have been recognized within the spermatozoa of several mammalian species. Therefore, the aim of this study was to determine the major sites and causative mechanisms of MTT reduction in stallion spermatozoa. Our results show that stallion spermatozoa displayed substantial mitochondrial formazan deposition, as well as a single extra-mitochondrial formazan deposit in various locations on the sperm head in approximately 20% of cells. The quality and capacitation status of stallion spermatozoa were positively correlated with the presence of an extra-mitochondrial formazan granule. Additionally, extra-mitochondrial formazan deposition was suppressed by the presence of an NADPH oxidase (NOX) inhibitor (VAS2870; active against NOX2, NOX4 and NOX5), MnTMPyP (SOD mimetic) and zinc (NOX5 inhibitor) suggesting that extra-mitochondrial MTT reduction may be facilitated by NOX-mediated ROS generating activity, conceivably NOX5 or NOX2. When comparing MTT to resazurin, another well-known probe used to detect metabolically active cells, MTT reduction had a higher correlation with sperm concentration and motility parameters (R2= 0.91), than resazurin reduction (R2 = 0.76). We conclude that MTT reduction in stallion spermatozoa follows a species-specific pattern due to a high dependence on oxidative phosphorylation and a degree of NOX activity. As such, MTT reduction is a useful diagnostic tool to assess extra-mitochondrial redox activity, and therefore, the functional qualities of stallion spermatozoa.
Publication Date: 2022-04-22 PubMed ID: 35333772DOI: 10.1530/REP-21-0464Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research investigates the primary sites and causes of MTT (a cell vitality probe) reduction in stallion sperm, revealing that it holds a correlation with sperm quality and status. The results could make MTT reduction a useful diagnostic tool for assessing the functional qualities of stallion sperm.
Objective and Background
- MTT, or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, is a common probe used to assess cell vitality. It creates formazan deposits at locations of high oxidoreductase activity within cells.
- While MTT response is typically linked to mitochondrial redox activity, several mammalian species, including stallions, have displayed extra-mitochondrial sites of MTT reduction in the spermatozoa (male reproductive cells).
- This research aims to pinpoint the primary sites and causes of MTT reduction in stallion spermatozoa.
Findings
- Stallion sperm showed a substantial amount of mitochondrial formazan deposition, along with a single extra-mitochondrial formazan deposit in different locations on the sperm head in roughly 20% of cells.
- The study found a positive correlation of the presence of an extra-mitochondrial formazan granule with the quality and capacitation status (the process that allows sperm to fertilize an egg) of the sperm.
- The deposition of extra-mitochondrial formazan was found to be suppressed by the presence of NADPH oxidase (NOX) inhibitor and an SOD mimetic (MnTMPyP) and zinc, both of which inhibit NOX5. This suggested that extra-mitochondrial MTT reduction could be facilitated by ROS generation activity mediated by NOX, conceivably NOX5 or NOX2.
Comparing MTT to Resazurin
- The researchers compared MTT to resazurin, another known probe used to measure metabolically active cells.
- The reduction of MTT was found to have a higher association with sperm concentration and motility parameters than resazurin reduction, pointing to its potentially superior diagnostic capabilities.
Conclusion
- The pattern of MTT reduction in stallion sperm was found to be species-specific, relying heavily on oxidative phosphorylation and a degree of NOX activity.
- The research concludes that MTT reduction can serve as a valuable diagnostic tool to assess extra-mitochondrial redox activity, and as a result, the functional qualities of stallion spermatozoa.
Cite This Article
APA
Medica AJ, Gibb Z, Sheridan A, Harrison N, Aitken RJ.
(2022).
Causative mechanisms and functional correlates of MTT reduction in stallion spermatozoa.
Reproduction, 163(6), 341-350.
https://doi.org/10.1530/REP-21-0464 Publication
Researcher Affiliations
- Priority Research Centre for Reproductive Science, College of Engineering, Science and Environmental, and Faculty of Health and Medicine, University of Newcastle, Callaghan, New South Wales, Australia.
- Priority Research Centre for Reproductive Science, College of Engineering, Science and Environmental, and Faculty of Health and Medicine, University of Newcastle, Callaghan, New South Wales, Australia.
- Priority Research Centre for Reproductive Science, College of Engineering, Science and Environmental, and Faculty of Health and Medicine, University of Newcastle, Callaghan, New South Wales, Australia.
- Priority Research Centre for Reproductive Science, College of Engineering, Science and Environmental, and Faculty of Health and Medicine, University of Newcastle, Callaghan, New South Wales, Australia.
- Priority Research Centre for Reproductive Science, College of Engineering, Science and Environmental, and Faculty of Health and Medicine, University of Newcastle, Callaghan, New South Wales, Australia.
MeSH Terms
- Animals
- Formazans
- Horses
- Male
- Mammals
- Mitochondria / metabolism
- Oxidative Phosphorylation
- Sperm Motility
- Spermatozoa / metabolism
Citations
This article has been cited 2 times.- Medica AJ, Lambourne S, Aitken RJ. Predicting the Outcome of Equine Artificial Inseminations Using Chilled Semen. Animals (Basel) 2023 Mar 30;13(7).
- Ma S, Zhang N, Hou J, Liu S, Wang J, Lu B, Zhu F, Wei P, Hong G, Liu T. Synthesis and Discovery of Ligustrazine-Heterocycle Derivatives as Antitumor Agents. Front Chem 2022;10:941367.
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