In Situ DNA 3′-End-Labeling Analysis
At the end of the incubation period without or with the experimental treatments, oocytes were transferred into Ty-rode solution for 30 sec at 37°C to remove the zona pel-lucida, washed quickly in PBS, and then immediately fixed for 30 min in neutral-buffered 1% (wt/vol) paraformaldehyde prepared in PBS containing 0.1 mg/ml polyvinyl alcohol (average molecular weight of 30 000-70 000). After fixation, oocytes were washed once more with PBS, transferred to Superfrost-Plus slides (Fisher Scientific, Pittsburgh, PA) in small drops (10 oocytes/10 ^l drop), and air-dried. Slides were heated at 65°C for 4 h and then stored at 4°C until processed for in situ DNA 3′-end-labeling analysis (ISEL) as detailed elsewhere, with slight modifications.
Briefly, slide-mounted oocytes were heated at 65°C for 30 min, immediately rehydrated through a graded ethanol series (absolute, 90%, 80%, and 70% ethanol; 20 sec each) to sterile water, and then treated with proteinase K (10 g/ml) at 37°C for 30 min, followed by two washes in sterile water. To block for nonspecific binding, slide-mounted oocytes were preincubated with 3% (wt/vol) BSA for 30 min at 20°C and then pre-equilibrated with 1X terminal deoxynucleotidyl transferase (TdT) reaction buffer (Boehringer-Mannheim) for 20 min at 20°C. The TdT-me-diated labeling reaction of DNA 3′ ends was performed by incubating the slide-mounted oocytes in the presence of 1.25 U/^l TdT enzyme (Boehringer-Mannheim; with TdT reaction buffer and CoCl2 supplied with the enzyme) and 50 pM fluorescein-labeled dUTP (Boehringer-Mannheim) at 37°C for 15 min in the dark. After 3′-end labeling, the slides were placed in 1X-concentrated TE buffer (10 mM Tris-HCl, 100 mM EDTA, pH 8) to stop the reaction and then rinsed several times with sterile water. Excess water was blotted away, mounting medium was added, and the slides were sealed with coverslips. The occurrence of DNA cleavage was assessed by fluorescence microscopy using a fluorescein filter.