流式细胞术评估精子参数与生育潜力的关系。
Flow cytometric evaluation of sperm parameters in relation to fertility potential.
作者信息
Gillan Lindsay, Evans Gareth, Maxwell W M C
机构信息
RMC Gunn Building (B19), The Faculty of Veterinary Science, University of Sydney, Camperdown, NSW 2006, Australia.
出版信息
Theriogenology. 2005 Jan 15;63(2):445-57. doi: 10.1016/j.theriogenology.2004.09.024.
Most laboratory methods used to evaluate semen quality have not correlated highly with fertilizing capacity. The discovery of a variety of fluorochromes and compounds conjugated to fluorescent probes has enabled a more widespread analysis of sperm attributes, and in conjunction with the flow cytometer, permit the evaluation of a large number of spermatozoa. A number of characteristics of sperm integrity, viability and function can be assessed by flow cytometry. The DNA status of spermatozoa has been determined using the metachromatic properties of acridine orange (AO). AO staining, when used in the sperm chromatin structure assay (SCSA), correlates with fertility in a number of species. DNA fragmentation can also be assessed using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, which identifies DNA strand breaks by labeling free 3'-OH termini with modified nucleotides. The status of the sperm acrosome can be determined using fluorescently labeled lectins and LysoTracker Green DND-26, a fluorescent acidotropic probe. Capacitation status has been observed through calcium-mediated changes using chlortetracycline (CTC) or by changes in membrane fluidity monitored by the binding of the fluorescent amphiphilic probe, Merocyanine 540. Fluorescently labeled annexin-V, C6NBD and Ro-09-0198 can also be used to detect changes in membrane phospholipid distribution. Cell viability can be determined using the propensity of propidium iodide (PI), ethidium homodimer-1 (EthD-1) or Yo-Pro-1 to permeate damaged membranes. These are generally more adaptable to clinical flow cytometry than the bisbenzimide membrane impermeable stain, Hoechst 33258, which excites in the ultraviolet range and requires UV laser equipment. Mitochondrial function can be determined using rhodamine 123 (R123) and MitoTracker Green FM (MITO) and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1). Flow cytometry is a tool that may be used in the future to monitor many new potential markers of sperm function.
大多数用于评估精液质量的实验室方法与受精能力的相关性都不高。多种荧光染料和与荧光探针结合的化合物的发现，使得对精子属性的分析更为广泛，并且结合流式细胞仪，可以对大量精子进行评估。精子完整性、活力和功能的一些特征可以通过流式细胞术进行评估。精子的DNA状态已使用吖啶橙（AO）的异染特性来确定。AO染色用于精子染色质结构分析（SCSA）时，与许多物种的生育力相关。DNA片段化也可以使用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）试验进行评估，该试验通过用修饰的核苷酸标记游离的3'-OH末端来识别DNA链断裂。精子顶体的状态可以使用荧光标记的凝集素和溶酶体追踪染料绿色DND-26（一种荧光嗜酸探针）来确定。通过使用金霉素（CTC）的钙介导变化或通过荧光两亲性探针部花青540的结合监测膜流动性变化来观察获能状态。荧光标记的膜联蛋白-V、C6NBD和Ro-09-0198也可用于检测膜磷脂分布的变化。细胞活力可以使用碘化丙啶（PI）、乙锭同二聚体-1（EthD-1）或Yo-Pro-1渗透受损膜的倾向来确定。与双苯甲酰亚胺这种不透膜的染料（在紫外范围内激发且需要紫外激光设备的Hoechst 33258）相比，这些通常更适用于临床流式细胞术。线粒体功能可以使用罗丹明123（R123）、线粒体追踪染料绿色FM（MITO）和5,5',6,6'-四氯-1,1',3,3'-四乙基苯并咪唑基羰花青碘化物（JC-1）来确定。流式细胞术是一种未来可能用于监测许多精子功能新潜在标志物的工具。
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