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 on various locations on the sperm head in approximately 20% of cells. The quality, and capacitation status of stallion spermatozoa was 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.

Predicting the Outcome of Equine Artificial Inseminations Using Chilled Semen

“…Unlike other cell types, WST-1 in the presence of equine spermatozoa does not appear to need an intermediate electron acceptor to facilitate its reduction and instead is directly reduced at the cell surface [ 16 , 17 , 23 ]. Therefore, it was hypothesized that WST-1 reduction would not only determine sperm concentration but might also reveal the fundamental biochemical status of these cells, as other tetrazolium salts such as MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) has performed before [ 24 ]. As predicted, WST-1 reduction within this isolated sperm population generated highly significant correlations ( p < 0.001; R 2 = 0.69) between WST-1 reduction and motile sperm count ( Figure 3 A).…”

Synthesis and Discovery of Ligustrazine–Heterocycle Derivatives as Antitumor Agents

“…The in vitro antiproliferative activity of 33 TMPH compounds was evaluated by MTT assay ( Medica et al, 2022 ). Five types of TNBC-related cell lines (human breast cancer MCF-7 and MDA-MB-231, mouse breast cancer 4T1, mouse fibroblast L929, and human umbilical vein endothelial HUVEC cell lines) were used.…”