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


CONCLUSIONS
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.
Department of Toxicology, Anhui Medical University, Hefei, Anhui, P. R. China. xudex@mail.hf.ah.cn
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Xu
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Shen
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Wang
D004249:DNA Damage; D004586:Electrophoresis; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008297:Male; D013094:Spermatozoa / Q000737:chemistry*
D006861:Hydrogen Peroxide