Environmental toxicants cause sperm DNA fragmentation as detected by the Sperm Chromatin Structure Assay (SCSA).
Environmental toxicants cause sperm DNA fragmentation as detected by the Sperm Chromatin Structure Assay (SCSA).
复制标题
DOI：
10.1016/j.taap.2005.03.021
复制
发表时间：
2005-09-01
影响因子：
3.8
通讯作者：
Wixon, Regina
中科院分区：
文献类型：
--
作者：
Evenson, Donald P;Wixon, Regina
过去二十年的研究清楚地表明，生殖毒物会导致精子DNA断裂。这种DNA片段通常可以在观察胚胎中期染色体的改变之前检测到。因此，精子染色质结构分析（SCSA）检测到的DNA损伤被视为在光学显微镜下观察到的后期总体染色体损伤的分子前体。动物或人类精子的SCSA测量包括首先在LN2或干冰中获得新鲜或速冻的纯精液样本。然后将样品送到SCSA诊断实验室，在那里将样品解冻，稀释至约1-2 x 106个精子/ml，用pH为1.2的洗涤剂缓冲液处理30秒，然后用吖啶橙（AO）染色。低pH使DNA链断裂处的DNA部分变性，AO-ssDNA发出红色荧光，AO-dsDNA发出绿色荧光。5000个精子/样本的流式细胞术测量提供了关于红、绿精子比例、DNA断裂程度和测量标准偏差的统计可靠数据。用生殖毒物处理啮齿动物的大量实验清楚地表明，SCSA措施具有高度剂量反应性和非常低的CV。作用于不同发育阶段的生殖细胞的不同药剂通常在生殖细胞片段到达附睾或射精时显示精子DNA断裂。其中一些处理过的样本能够成功地进行体外受精，但经常出现胚胎失败。一项对居住在山谷小镇的男性进行的为期2年的纵向研究表明，据报道，这些男性患有异常水平的不育症和自然流产，同时也受到季节性大气雾霾污染。该研究首次表明，SCSA测量的人类精子DNA片段是可检测到的，并且与空气污染剂量相关，而传统的精液测量则不相关。此外，不带防护装备喷洒农药的年轻男性精子DNA断裂的风险更高。大量的DNA碎片可能无法由卵子修复，如果一个男人有超过30%的精子显示DNA碎片，自然流产率大约会高出2倍。DNA片段是暴露于潜在生殖毒物的极好标记，也是潜在男性不育症的诊断/预后工具。
Studies over the past two decades have clearly shown that reproductive toxicants cause sperm DNA fragmentation. This DNA fragmentation can usually be detected prior to observing alterations of metaphase chromosomes in embryos. Thus, Sperm Chromatin Structure Assay (SCSA)-detected DNA damage is viewed as the molecular precursor to later gross chromosome damage observed under the light microscope. SCSA measurements of animal or human sperm consist of first obtaining a fresh or flash frozen neat semen sample in LN2 or dry ice. Samples are then sent to a SCSA diagnostic laboratory where the samples are thawed, diluted to approximately 1-2 x 106 sperm/ml, treated for 30 s with a pH 1.2 detergent buffer and then stained with acridine orange (AO). The low pH partially denatures DNA at the sites of DNA strand breaks and the AO-ssDNA fluoresces red while the AO-dsDNA fluoresces green. Flow cytometry measurements of 5000 sperm/sample provide statistically robust data on the ratio of red to green sperm, the extent of the DNA fragmentation and the standard deviations of measures. Numerous experiments on rodents treated with reproductive toxicants clearly showed that SCSA measures are highly dose responsive and have a very low CV. Different agents that act on germ cells at various stages of development usually showed sperm DNA fragmentation when that germ cell fraction arrived in the epididymis or ejaculate. Some of these treated samples were capable of successful in vitro fertilization but with frequent embryo failure. A 2-year longitudinal study of men living a valley town with a reported abnormal level of infertility and spontaneous miscarriages and also a seasonal atmospheric smog pollution, showed, for the first time, that SCSA measurements of human sperm DNA fragmentation were detectable and correlated with dosage of air pollution while the classical semen measures were not correlated. Also, young men spraying pesticides without protective gear are at an increased risk for elevated sperm DNA fragmentation. Extensive DNA fragmentation probably cannot be repaired by the egg and the spontaneous abortion rate is approximately 2x higher if a man has more than 30% of sperm showing DNA fragmentation. DNA fragmentation is an excellent marker for exposure to potential reproductive toxicants and a diagnostic/prognostic tool for potential male infertility.