To analyze apoptosis in granulosa cells of whole follicles, healthy antral follicles between 700-800 ^m in diameter were isolated using nonenzymatic dissection, as detailed elsewhere. Briefly, the 8-10 largest follicles in each ovary were isolated with watchmaker’s forceps under a dissecting microscope and then cleaned of adherent stromal tissue and smaller follicles. Once isolated, follicles were sized for homogeneity and either snap-frozen immediately (Time 0, no incubation) or incubated under serum-free conditions for 24 h at 37°C. For these experiments, control groups of follicles were incubated in a modified (‘‘ion-deficient’’) RPMI medium lacking NaCl (normally 103 mM) and KCl (normally 5.4 mM), prepared in the NIEHS Cell Culture Media Core Facility, supplemented with 0.1% BSA, 2 mM L-glutamine, 100 U/ml penicillin, and 100 ^g/ml streptomycin sulfate.
For control cultures, mannitol was added to a final concentration of 300 mM to maintain osmolality at approximately 380 mOs. To assess the effects of the different cations on apoptosis, concentrated stock solutions of KCl, NaCl, or LiCl were added to the ion-deficient RPMI at a final concentration of 150 mM each (average osmolality of 375 ± 5 mOs). To examine the dose-response relationship of potassium concentration to DNA cleavage in granulosa cells, ion-deficient RPMI was supplemented with KCl so that final concentration of potassium reached 50, 100, or 150 mM with osmolality held constant by addition of the appropriate concentration of mannitol where needed (i.e., for 50 mM KCl, the medium also contained 200 mM mannitol; for 100 mM KCl, the medium also contained 100 mM mannitol; and for 150 mM KCl, no mannitol was added).