Cell-Free Caspase Activity Assays
Using assays identical to those recently reported, caspase-1 activity was assessed by monitoring the conversion of pro-IL1p (33 kDa) to the active cytokine (17 kDa) after incubation of pro-IL1p with 1 ^g of active caspase-1 in the absence or presence of 150 mM KCl, NaCl, or LiCl. Caspase-3 activity was measured by analyzing the extent of cleavage of actin present in heat-inactivated crude protein extracts (50 ^g/reaction) prepared from eCG-primed rat ovarian granulosa cells after a 30-min incubation at 37°C with 1 ^g of active caspase-3 in the absence or presence of 150 mM KCl, NaCl, or LiCl. The extent of cleavage of pro-IL1p or actin was determined by immunoblot analysis by the enhanced chemiluminescence system (Amersham).
Rat Granulosa Cell Nuclear Autodigestion Assays
For each experimental replicate, nuclei were isolated as previously described from approximately 1.4 X 107 granulosa cells (six eCG-primed ovaries) in 1 ml of TSN buffer (10 mM Tris-HCl, pH 7.4); 25 mM NaCl; and 0.34 M sucrose) by four strokes with a tight-fitting pestle in a Kontes glass homogenizer, followed by centrifugation at 800 X g for 10 min at 4°C. The crude cytoplasmic extract (supernatant) was removed, and the crude nuclear pellet was gently resuspended in 1.4 ml of TSN. Aliquots of the resuspended nuclei (100 ^l) were dispensed into 1.5-ml tubes and then preincubated without or with 150 mM KCl, NaCl, or LiCl. An equal volume (110 ^l) of 10 mM Tris-HCl (pH 7.4) or CaCl2/MgCl2 (10 mM each in 10 mM Tris-HCl, pH 7.4; 5 mM each final for the autodigestion assay) was then added to the nuclei suspensions. Incubations for granulosa cell nuclear autodigestion were carried out at 37°C for 30 min, after which nuclei were pelleted by centrifugation (5 min at 4°C; 800 X g). The supernatants were removed and discarded, and the pellets were snap-frozen for radiolabeling and CAGE. Nuclei not incubated served as Time 0 data points (i.e., background DNA cleavage) for the assays.