Donnelly E T, O'Connell M, McClure N, Lewis S E
Department of Obstetrics & Gynaecology, The Queen's University of Belfast, Northern Ireland, UK.
Hum Reprod. 2000 Jul;15(7):1552-61. doi: 10.1093/humrep/15.7.1552.
Sperm DNA integrity is essential for accurate transmission of genetic material to offspring. Fragmentation of genomic DNA is an initial hallmark of apoptosis (programmed cell death). The aim of this study was to determine sperm nuclear DNA integrity and mitochondrial function, to quantify possible apoptosis and to investigate any relationship between these parameters. Semen samples (n = 25) were prepared by discontinuous Percoll density centrifugation (95.0:47.5). DNA integrity was determined using a modified alkaline single cell gel electrophoresis (Comet) assay. DNA fragmentation, possibly indicative of apoptosis, was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL). Mitochondrial transmembrane potential was determined using the mitochondrial probe 5,5',6,6'-tetrachloro-1,1', 3,3'-tetraethylbenzimidazolyl carbocyanine iodide (JC-1). The DNA integrity of prepared spermatozoa was significantly greater than that of semen (P < 0.005). Further, the percentage of spermatozoa with fragmented DNA and the degree of fragmentation within these cells in prepared spermatozoa is significantly less than in semen (P < 0.005). There is a significant correlation between DNA damage quantified using the Comet assay and DNA fragmentation determined using TUNEL (R = 0.562, P < 0.01). The percentage of spermatozoa with dysfunctional, possibly apoptotic, mitochondria was significantly lower in prepared spermatozoa than in neat semen samples (P < 0.001). There was a negative correlation between the percentage of spermatozoa with dysfunctional mitochondria and the percentage of progressively motile spermatozoa (R = -0.67, P < 0.01).
精子DNA完整性对于将遗传物质准确传递给后代至关重要。基因组DNA片段化是细胞凋亡（程序性细胞死亡）的一个初始标志。本研究的目的是确定精子核DNA完整性和线粒体功能，量化可能的细胞凋亡，并研究这些参数之间的任何关系。精液样本（n = 25）通过不连续Percoll密度离心法（95.0:47.5）制备。使用改良的碱性单细胞凝胶电泳（彗星）试验测定DNA完整性。通过末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）检测可能指示细胞凋亡的DNA片段化。使用线粒体探针5,5',6,6'-四氯-1,1',3,3'-四乙基苯并咪唑基碳菁碘化物（JC-1）测定线粒体跨膜电位。制备的精子的DNA完整性显著高于精液（P < 0.005）。此外，制备的精子中DNA片段化的精子百分比以及这些细胞内的片段化程度显著低于精液（P < 0.005）。使用彗星试验量化的DNA损伤与使用TUNEL测定的DNA片段化之间存在显著相关性（R = 0.562，P < 0.01）。制备的精子中功能异常、可能凋亡的线粒体的精子百分比显著低于纯精液样本（P < 0.001）。线粒体功能异常的精子百分比与进行性运动精子百分比之间存在负相关（R = -0.67，P < 0.01）。
