[利用单细胞凝胶电泳检测人类精子中的DNA链断裂]

[Detection of DNA strand breakage in human spermatozoa by use of single-cell gel electropheresis].

作者信息

Xu D, Shen H, Wang J

机构信息

Department of Toxicology, Anhui Medical University, Hefei, Anhui, P. R. China.

出版信息

Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2000 Aug;17(4):281-4.

PMID:10932016

Abstract

OBJECTIVE

To establish the single-cell gel electropherosis (SCGE) protocol for detection of DNA strand breakage in human spermatozoa.

METHODS

The slides on which sperm cells and agarose were layered were immersed in a cold lysing solution of pH10 to lyse the sperm cells. Sperm nuclei were then pretreated with 10 mmol/L of DTT for 1 h, 10 microg/ml of RNase A for 4 h and 200 microg/ml of proteinase K for 15 h. Lastly, electrophoresis was performed in electrophoresis running buffer(pH10) at 12V (0.46V/cm) and 100 mA for 1 h. Sperm nuclei were stained with 15 microg/ml of EtBr for 5 min. The percentage of comet cells was counted. The in vitro hydrogen peroxide-induced DNA damage in human spermatozoa was measured with SCGE established by this laboratory.

RESULTS

The comet sperm cells in human spermatozoa ranged from 2% to 38%. There was a significant variance on the percentage of comet cells between different subjects. Hydrogen peroxide increased the percentage of comet sperm in a dose- and time-dependent manner.

CONCLUSION

SCGE may be used to detect DNA strand breakage in human spermatozoa. Hydrogen peroxide-induced DNA damage in human sperm cells was detected successfully using SCGE protocol established by this laboratory.

摘要

目的

建立用于检测人类精子DNA链断裂的单细胞凝胶电泳（SCGE）方法。

方法

将铺有精子细胞和琼脂糖的载玻片浸入pH10的冷裂解液中裂解精子细胞。然后精子核先用10 mmol/L二硫苏糖醇（DTT）处理1小时，再用10 μg/ml核糖核酸酶A（RNase A）处理4小时，接着用200 μg/ml蛋白酶K处理15小时。最后，在pH10的电泳缓冲液中于12V（0.46V/cm）、100 mA条件下进行1小时电泳。用15 μg/ml溴化乙锭（EtBr）对精子核染色5分钟。计数彗星样细胞的百分比。用本实验室建立的SCGE方法检测体外过氧化氢诱导的人类精子DNA损伤。