Pulsed-Field Gel Electrophoretic Analysis of High-Molecular Weight DNA Cleavage
As we have previously described for this model system, follicles were embedded in agarose plugs (50-^l total volume, 0.5% final) and immediately immersed in 10 ml of PFGE lysis buffer (100 mM EDTA, 1% N-lauroyl-sar-cosine) for overnight incubation at 37°C. Plugs were then removed, immersed in 1 ml of PFGE lysis buffer containing 50 ^g/ml of proteinase-K, and incubated at 50°C for an additional 12 h. After proteolytic digestion, plugs were preequilibrated in 0.5 X TBE (0.89 M Tris-HCl, 0.89 M boric acid, 2.5 mM EDTA) for at least 3 h and then subjected to PFGE using a clamped homogeneous electric field (CHEF) pulsed-field system (Bio-Rad Laboratories, Hercules, CA) for 19 h at 14°C and 6 V/cm, with a linear-switch interval ramp from 0.5 to 45 sec. Size standards included chromosomes from S. cerevisiae and DNA size standards provided by Bio-Rad. After PFGE, DNA was visualized by ethidium bromide staining and UV transillumination.
Analysis of Granulosa Cell Nuclei in Incubated Follicles
Follicles isolated from gonadotropin-primed ovaries were either immediately fixed (4% neutral-buffered paraformaldehyde) or were fixed after incubation without or with 150 mM KCl, as described above. Individual follicles were then embedded in paraffin, sectioned (6 ^m), and mounted on a glass microscope slide. Sections were then deparaffinized, rehydrated, and stained with the DNA-binding fluorescent dye, 4′,6′-diamidino-2-phenylindole (DAPI), at a final concentration of 0.4 ^g/ml in 1X Dul-becco PBS (pH 7.5) for 10 min at 20°C. The samples were then examined by UV-fluorescence microscopy for evidence of nuclear condensation (brightly fluorescent pyk-notic nuclei) associated with apoptosis.