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Measurement of DNA fragmentation in human sperm and their exclusion by means of in vitro processing

测量人类精子中的 DNA 碎片并通过体外处理排除它们

基本信息

批准号：
15591781
负责人：
KANEKO Satoru
金额：
$ 2.18万
依托单位：
TOKYO DENTAL COLLEGE
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15591781/
关键词：
human sperm DNA DNA damage electrophoresis thin layer gel

项目摘要

The assurance of the integrity in DNA structure is minimum premise for the clinical application of ICSI. The establishment of the quality certification system is certainly the most urgent task. We newly developed single cell pulse field gel electrophoresis(SCPFGE) to observe double strand breaks in DNA. The specimen (2 X10^5 sperm/ml) was suspended into 2.0% melted agarose, and thin film of agarose was made on a glass slide with hyddrophylic surface. Our preliminary experiments by SCPFGE have revealed that the mode of DNA fragmentation in human sperm nuclei have been extremely variable. They were classified as follows ; 1.intact (continuous DNA fibers were elongated form the origin), 2.light fragmentation (a few long fragments were found beyond the continuous fibers), 3.moderate fragmentation (two or three mass of short fragments were extended from the origin) and 4.severe fragmentation (origin was almost disappeared and the mass of short fragment was found).Human sperm with DNA fragmentation was excluded by means of differential velocity sedimentation in Percoll density gradient, equilibrium sedimentation in Optidenz and swim up procedure. The sediment of Percoll density gradient was farther separated by equilibrium sedimentation, the sperm were distributed into two peaks. The profiles of SCPFGE showed that almost the sperm in lower peak showed DNA fragmentation, whereas those in the upper peak gave elongated continuous DNA fibers. The motile sperm was further separated by means of the swim up method.In conclusion, DNA denature in human sperm accompanied with the alteration of apparent density and the sperm with double strand breaks in DNA structure could be excluded by the in vitro processings.

DNA结构完整性的保证是ICSI临床应用的最低前提。建立质量认证制度当然是当务之急。我们新发展了单细胞脉冲场凝胶电泳（SCPFGE）来观察DNA双链断裂。将样本（2 × 10^5精子/ml）悬浮于2.0%融化的琼脂糖中，并在具有亲水表面的载玻片上制备琼脂糖薄膜。我们用SCPFGE进行的初步实验表明，人精子核中DNA的断裂方式是极其多变的。它们被分类如下; 1.intact（连续的DNA纤维从起始处开始伸长），2.轻片段化（在连续纤维之外发现了一些长碎片），3.适度破碎（2 ~ 3团短碎片）和（4）严重碎裂用Percoll密度梯度差速沉降法排除DNA断裂的人精子，Optidenz中的平衡沉降和上游程序。Percoll密度梯度的沉淀物经平衡沉降进一步分离，精子呈双峰分布。SCPFGE图谱显示，下峰的精子DNA几乎全部断裂，而上峰的精子DNA纤维延长。用上游法进一步分离活动精子，结果表明，体外处理可排除人精子中DNA变性、表观密度改变和DNA结构双链断裂的精子。