Patterns of MTT reduction in mammalian spermatozoa.
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.
Publication Date: 2020-06-23 PubMed ID: 32567557DOI: 10.1530/REP-20-0205Google Scholar: Lookup
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- Journal Article
Summary
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This research investigates the patterns of MTT reduction in mammalian sperm, focusing on the sites of formazan deposition and how various factors influence this process. The findings suggest that these patterns are species-specific and give insights into the functionality of sperm cells.
Understanding MTT and its Role
- MTT or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide is an important compound used in biology as an indicator of cell viability. This is because it transforms into a compound called formazan at locations of intense oxidoreductase activity.
- This transformation generally signifies mitochondrial redox activity.
- Despite this, certain cell types also show MTT reduction outside the mitochondria (referred to as extra-mitochondrial MTT reduction).
Probing Formazan Deposition in Mammalian Spermatozoa
- The study seeks to identify where the primary sites of formazan deposition are within mammalian sperm cells.
- In mice, most MTT reduction happens in the mitochondrial gyres (spiral formations), with a minor deposit site on the sperm head.
- Contrastingly, human sperm generally shows disorganized midpieces with moderate MTT reduction, and a primary extra-mitochondrial formazan deposit can occur anywhere from the neck to the anterior acrosome on the sperm head.
- Equine sperm cells show both patterns, with significant formazan deposition in the mitochondria and about 20% of cells showing an extra-mitochondrial deposit.
Effect of Various Factors on Extra-mitochondrial Activity
- The study observes a positive association between the functionality of human sperm cells and the presence of an extra-mitochondrial formazan granule.
- Further investigation shows that various factors can suppress this extra-mitochondrial activity. These include diphenylene iodonium, zinc, 2-deoxyglucose, co-enzyme Q, an SOD mimetic, and NADPH oxidase inhibitors.
Conclusions of the Research
- The pattern of MTT reduction to formazan in sperm cells is species-specific, i.e., it varies significantly among different species.
- These patterns provide vital information about the relative significance of mitochondrial and extra-mitochondrial redox activity.
- These activities, in turn, help define the functional attributes of sperm cells, thus making this a significant area of research in biology and reproduction studies.
Cite This Article
APA
Aitken RJ, Gregoratos D, Kutzera L, Towney E, Lin M, Wilkins A, Gibb Z.
(2020).
Patterns of MTT reduction in mammalian spermatozoa.
Reproduction, 160(3), 431-445.
https://doi.org/10.1530/REP-20-0205 Publication
Researcher Affiliations
- Priority Research Centre for Reproductive Science, Faculty of Science and Faculty of Health and Medicine, University of Newcastle, University Drive, Callaghan, New South Wales, Australia.
- Priority Research Centre for Reproductive Science, Faculty of Science and Faculty of Health and Medicine, University of Newcastle, University Drive, Callaghan, New South Wales, Australia.
- Priority Research Centre for Reproductive Science, Faculty of Science and Faculty of Health and Medicine, University of Newcastle, University Drive, Callaghan, New South Wales, Australia.
- Priority Research Centre for Reproductive Science, Faculty of Science and Faculty of Health and Medicine, University of Newcastle, University Drive, Callaghan, New South Wales, Australia.
- Priority Research Centre for Reproductive Science, Faculty of Science and Faculty of Health and Medicine, University of Newcastle, University Drive, Callaghan, New South Wales, Australia.
- Priority Research Centre for Reproductive Science, Faculty of Science and Faculty of Health and Medicine, University of Newcastle, University Drive, Callaghan, New South Wales, Australia.
- Priority Research Centre for Reproductive Science, Faculty of Science and Faculty of Health and Medicine, University of Newcastle, University Drive, Callaghan, New South Wales, Australia.
MeSH Terms
- Animals
- Cell Proliferation
- Female
- Formazans / chemistry
- Horses
- Humans
- Male
- Mice
- Mitochondria / physiology
- Oxidation-Reduction
- Sperm-Ovum Interactions
- Spermatozoa / cytology
- Spermatozoa / physiology
- Tetrazolium Salts / chemistry
Citations
This article has been cited 3 times.- Buranaamnuay K. The MTT assay application to measure the viability of spermatozoa: A variety of the assay protocols. Open Vet J 2021 Apr-Jun;11(2):251-269.
- Mohammadi T, Soltani L. Effects of hydroethanolic extracts of Terminalia chebula and Thymbra spicata on ram fresh semen under normal and oxidative stress conditions. Vet Med Sci 2021 Sep;7(5):1778-1785.
- Oeschger TM, Erickson DC. Visible colorimetric growth indicators of Neisseria gonorrhoeae for low-cost diagnostic applications. PLoS One 2021;16(6):e0252961.
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