TUNEL and Propidium Iodide Labeling
The TUNEL procedure was used to detect DNA fragmentation observed in late stages of apoptosis. The terminal deoxynucleotidyl transferase enzyme is a DNA polymerase that catalyzes the transfer of a fluorescein isothiocyanate-conjugated dUTP nucleotide to a free 3′ hydroxyl group present in DNA strand breaks. Embryos were removed from culture medium (modified KSOM) and washed four times in 100-|xl drop of PBS (pH 7.4) containing 1 mg/ml PBS-PVP by transferring the embryos from drop to drop. Zona pellucida-intact embryos were fixed in a 100-|xl drop of 4% (w/v) paraformaldehyde in PBS pH 7.4 for 1 h at room temperature, washed twice in PBS-PVP, transferred to a poly-L-lysine coated slide, and allowed to dry for 24 h at room temperature. Embryos were then washed twice by dipping the slide in a Coplin jar containing PBS-PVP (2 min/ wash) and permeabilized in 0.5% (v/v) Triton X-100 containing 0.1% (w/ v) sodium citrate for 1 h at room temperature.
Positive controls were incubated with RQ1 RNase-free DNase (50 U/ml) at 37°C for 1 h. Embryos were washed in PBS-PVP and incubated with 50 |xl of TUNEL reaction mixture (containing fluorescein isothiocyanate-conjugated dUTP), and the enzyme terminal deoxynucleotidyl transferase (as prepared by the manufacturer) for 1 h at 37°C in the dark. Negative controls were incubated in the absence of terminal deoxynucleotidyl transferase. Embryos were then incubated with RNase A (50 |xg/ml) for 1 h at room temperature, followed by 0.5 |xg/ml of propidium iodide for 30 min at room temperature. Embryos were washed four times in PBS-PVP to remove excess propidium iodide. A coverslip was mounted with 16-|xl mounting medium containing Antifade as recommended by the manufacturer. Labeling was observed using a Zeiss Axioplan 2 fluorescence microscope with a dual filter. Each embryo was analyzed for total number of nuclei and number of TUNEL-labeled nuclei. Some embryos were also examined using a BioRad 1024ES laser scanning confocal microscope. For fluorescein, an ar-gon-ion laser adjusted to less than 560 nm was used, and for propidium iodide, a helium-neon laser adjusted to more than 560 nm was used. Images were obtained using a 30X objective, 10% power, and Z-steps of 0.5-1.0 |xm.