The comet assay protocol developed for analysis of DNA cleavage in single human sperm was followed with minor modifications. Briefly, after the 24-h incubation without or with experimental treatments, oocytes were treated with Tyrode solution for 30 sec at 20°C, followed by one wash with PBS. Immediately afterward, oocytes within each experimental treatment group were pooled (510 oocytes per pool) and mixed with 30 ^l of 1% low-melting point agarose (Boehringer-Mannheim) previously prepared in comet assay electrophoresis buffer (45 mM Tris-HCl, 45 mM boric acid, 1.25 mM EDTA, pH 10) and maintained at 37°C. The oocytes, in agarose, were carefully pipetted onto Superfrost-Plus slides and air-dried for 2-3 days at room temperature.
Slide-mounted oocytes in hardened agarose were then rehydrated and incubated in comet assay lysis buffer (2.5 M NaCl, 1% A-lauroylsarcosine, 10 mM EDTA, 10 mM Tris-HCl, 1% Triton X-100, 13.3 |xg/ ml proteinase K, pH 10) for 1 h at 20°C, followed by an additional 20-h incubation at 37°C. At the end of incubation, the gels, still attached to the slides and containing the oocytes, were transferred to a horizontal electrophoresis chamber containing comet assay electrophoresis buffer, equilibrated for 30 min at 20°C, and then subjected to electrophoresis at 25 V for 40 min. Afterward, slides were transferred to distilled water for 30 min, stained with eth-idium bromide (50 ^g/ml in PBS) for 40 min, and then gently washed once with distilled water. Excess water was blotted away, mounting medium was added, and slides were sealed with coverslips. The occurrence and pattern of DNA migration out of the permeabilized oocytes was viewed under ultraviolet (UV)-fluorescence microscopy.