Shortening of alkaline DNA unwinding time does not interfere with detecting DNA damage to mouse and human spermatozoa in the comet assay

Abstract

The comet assay was performed on mouse and human spermatozoa to examine the effect of alkaline DNA unwinding time. The spermatozoa were treated in vitro with the DNA-damaging agents, methyl methanesulfonate (MMS) or hydrogen peroxide (H 2 O 2 ), and then embedded in agarose gel on glass slides. The slides were immersed in alkaline solution (.pH 13) for 1, 5, 10 and 20 min, and then subjected to the electrophoresis under neutral conditions. In mouse spermatozoa, comet tails seen in solvent controls became brighter and longer as the alkaline DNA unwinding time increased. However, in the MMS-treated mouse spermatozoa, a smaller difference in the damage from that in the solvent control was seen with time within a dose. DNA damage induced by H 2 O 2 could also be detected accurately after alkali treatment for 1-20 min. In human spermatozoa, DNA damage induced by MMS and H 2 O 2 could be detected in a dose-dependent manner after alkali treatment for 1 min. The ability of the comet assay to detect DNA damage was not adversely affected by the short period (1 min) of the alkaline DNA unwinding time. Keywords: alkaline; comet assay; DNA unwinding; human; mice; spermatozoa INTRODUCTION Single-cell gel electrophoresis assay (the comet assay) is a simple and high-throughput technique to detect the cellular DNA damage induced by various types of genotoxicants in situ. The comet assay is performed in one of two versions, alkaline and neutral. Alkaline comet assays visualize single-and double-strand breaks in cellular DNA, whereas the neutral comet assay reveals mainly double-strand breaks. Nowadays, the alkaline comet assay of reproductive cells has been recommended to screen genotoxic hazards. Furthermore, it is more practical in larger studies to evaluate the DNA integrity of cryopreserved mammalian spermatozoa. 1 The standard alkaline comet assay includes a step of alkali treatment to unwind the DNA before electrophoresis under the alkaline condition. In another version of the assay, cells embedded in agarose gel are treated with an alkali (.pH 13) followed by electrophoresis under neutral conditions. This protocol is named the 'A/N protocol'. 2 Lower background levels of DNA damage and better dose responses are obtained by the A/N protocol. [2][3][4] Recently, the comet assay A/N protocol has been reported for murine spermatozoa. [5][6][7] In most cases, the alkaline DNA unwinding time is set at 20 min or more. To our knowledge, there is little information on the optimal time of alkali treatment to detect sperm DNA damage with the A/N protocol. In the present study, the optimal time of the alkali treatment was determined for mouse spermatozoa by the comet assay with A/N protocol.

Characterization of chromosomal damage accumulated in freeze-dried mouse spermatozoa preserved under ambient and heat stress conditions

“…Instead of standard alkaline comet assay, alkaline comet assay with "A/N protocol" (i.e., alkaline DNA unwinding followed by electrophoresis under neutral condition) (30) was carried out according to the procedure as described previously (36). Freeze-dried sperm samples were rehydrated by adding 50 to 70 μl distilled water to each glass ampoule immediately after breaking the ampoule neck.…”