MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] 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 basckground, 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 versus extra-mitochondrial redox activity that, in turn, defines the functional qualities of these cells.

中文翻译：

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哺乳动物精子中 MTT 减少的模式

MTT [3-(4,5-二甲基噻唑-2-基)-2,5-二苯基溴化四唑]因其在强氧化还原酶位点产生不溶性甲臜沉积物的能力而在生物学中广泛用作细胞活力的探针活动。这种反应通常被认为反映了线粒体氧化还原活性，但在某些细胞类型中也记录了线粒体外 MTT 减少。鉴于此背景，我们着手确定哺乳动物精子中甲臜沉积的主要位点。在小鼠中，大部分 MTT 减少发生在广泛的线粒体回旋中，精子头部有一个较小的甲臜沉积位点。相比之下，人类精子通常表现出小的杂乱无章的中段，表现出适度的 MTT 减少活性，伴随着从颈部到前顶体的精子头部不同位置的主要线粒体外甲臜沉积物。马精子呈现出这两种模式的组合，线粒体中的主要甲臜沉积伴随着约 20% 细胞的线粒体外甲臜沉积。人类精子的功能与线粒体外甲臜颗粒的存在呈正相关。随后的研究表明，二亚苯基碘鎓、锌、2-脱氧葡萄糖、辅酶 Q、SOD 模拟物和 NADPH 氧化酶抑制剂的存在抑制了这种线粒体外活性。