Paper-Based Resazurin Assay of Inhibitor-Treated Porcine Sperm
Paper-Based Resazurin Assay of Inhibitor-Treated Porcine Sperm
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DOI：
10.3390/mi10080495
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发表时间：
2019-08-01
期刊：
影响因子：
3.4
通讯作者：
Cheng, Chao-Min
中科院分区：
文献类型：
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作者：
Matsuura, Koji;Wang, Wen-Hsin;Cheng, Chao-Min
使用我们自己设计的新型纸基测定法，通过精子细胞中刃天青还原颜色变化来评估猪精子活力。我们将刃天青溶液和猪精液的混合物应用于我们的纸基设备上的亲水性测试环上，并研究了所产生的还原反应，表示为红色和蓝色强度（RBCI）。我们使用来自我们的8 x 3 = 24位RGB彩色图像的蓝色/粉红色比率来量化该反应。为了检测精子细胞中的酶反应性，我们使用了两种抑制剂：3-硝基丙酸（3-NPA）和3-溴代丙酸（3-BP）。3-NPA抑制线粒体中的柠檬酸循环和电子转移反应，但在我们的试验中并没有强烈地降低精子活力。3-BP降低细胞质中线粒体电子传递酶和糖酵解酶的反应性，从而显著降低猪精子活力。3-NPA-和3-BP-处理的样品的RBCI与我们未处理的对照相比显著更低（p < 0.025）。基于这些结果，我们认为刃天青可用于估计有和无抑制剂处理的还原剂的量。为了继续研究评估猪精子中刃天青还原的分子机制，使用两种或更多种氧化还原指示剂（例如，刃天青和噻唑蓝溴化四唑（MTT））嵌入我们的纸基装置中可以进一步我们对精子细胞生物能量学的理解。
Porcine sperm motility was assessed via resazurin reduction color change in sperm cells using a novel paper-based assay of our own design. We applied mixtures of resazurin solution and porcine semen onto hydrophilic test circles on our paper-based device and investigated the resulting reduction reaction expressed as red and blue color intensity (RBCI). We quantified this reaction using a blue/pink color ratio from our 8 x 3 = 24 bit RGB color image. To examine enzymatic reactivity in sperm cells, we used two inhibitors: 3-Nitropropanoic acid (3-NPA) and 3-Bromopyruvic acid (3-BP). 3-NPA inhibits the citric acid cycle and electron transfer reaction in mitochondria, but did not strongly reduce sperm motility in our tests. 3-BP decreases reactivity of both mitochondrial electron transfer and glycolytic enzymes in cytosol, which significantly lowers porcine sperm motility. RBCIs of 3-NPA- and 3-BP-treated samples were significantly lower compared to our untreated control (p < 0.025). Based on these results, we feel that resazurin can be used to estimate the amount of reductants with and without inhibitor treatment. For continued research assessing the molecular mechanisms of resazurin reduction in porcine sperm, a combination assay using two or more redox indicators (e.g., resazurin and Thiazolyl Blue Tetrazolium Bromide (MTT)) embedded into our paper-based device could further our understanding of sperm cell bioenergetics.