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 (H₂O₂), 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 H₂O₂ could also be detected accurately after alkali treatment for 1-20 min. In human spermatozoa, DNA damage induced by MMS and H₂O₂ 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.

Figure 1

Results of comet assay using mouse spermatozoa treated in vitro with 50 and 100 µg ml⁻¹ MMS. At each dose of MMS, three independent experiments were performed with one mouse per experiment. Treatment with NaOH was carried out to unwind the spermatozoal DNA for 1, 5, 10 and 20 min before electrophoresis. Data are expressed as mean±SD. Bars not sharing the common letters (a–d) differ significantly (P<0.05) within a dose of MMS. MMS, methyl methanesulfonate.

Figure 2

Comets of mouse spermatozoa assayed according to the A/N protocol. Alkaline DNA unwinding time was set at 1 min (a, solvent control; a′, 100 µg ml⁻¹ MMS) and 20 min (b, solvent control; b′, 100 µg ml⁻¹ MMS). Scale bars=50 µm. MMS, methyl methanesulfonate.

Figure 3

Results of comet assay using mouse spermatozoa treated in vitro with 12.5 and 25.0 µg ml⁻¹ MMS. At each dose of MMS, four independent experiments were performed with one mouse in each experiment. Treatment with NaOH was carried out to unwind the spermatozoal DNA for 1 and 20 min before electrophoresis. Data are expressed as mean±SD. Bars not sharing the common letters (a–c) differ significantly (P<0.05) within the alkaline DNA unwinding time. MMS, methyl methanesulfonate.

Figure 4

Results of Comet assay using mouse spermatozoa treated in vitro with 25–100 µmol l⁻¹ H₂O₂. At each dose of H₂O₂, three independent experiments were performed with one mouse in each experiment. Treatment with NaOH was carried out to unwind the spermatozoal DNA for 1, 5, 10 and 20 min before electrophoresis. Data are expressed as mean±SD. Bars not sharing the common letters (a–c) differ significantly (P<0.05) within the alkaline DNA unwinding time. H₂O₂, hydrogen peroxide.

Figure 5

Results of comet assay using human spermatozoa treated in vitro with (a) 50–200 µg ml⁻¹ MMS and (b) 0.01–1 mmol l⁻¹ H₂O₂. Three independent experiments were performed using a fresh semen sample for each experiment. Treatment with NaOH was carried out to unwind the spermatozoal DNA for 1 min before electrophoresis. Data are expressed as mean±SD. Bars not sharing the common letters (a–d) differ significantly (P<0.05). H₂O₂, hydrogen peroxide; MMS, methyl methanesulfonate.