Preparation of Granulosa Cell Nuclear Protein Extracts and Plasmid DNA Degradation Assays
Granulosa cells were harvested from eCG-primed rat ovaries, and nuclei were isolated as described earlier (see Rat Granulosa Cell Nuclear Autodigestion Assays). Nuclear extracts were prepared by freeze-thawing isolated nuclei in TSN/CaCl2/MgCl2 buffer, followed by a 37°C incubation for 30 min. Debris, chromatin, and membranes were pelleted by ultracentrifugation (100 000 X g) for 30 min at 4°C, and the resultant supernatant containing nuclear proteins was collected and assessed for protein content.
To test for nuclease activity, the pUC18 plasmid (Stratagene, La Jolla, CA) was linearized with the SmaI restriction enzyme and added to tubes (1 ^g/reaction) containing 3 ^g of GC nuclear protein extract without or with KCl, NaCl, or LiCl (150 mM final) or 1 mM sodium aurothiomalate (SAM—apreviouslycharaeterizednuelease inhibitor) in a total volume of 10 ^l (in 50 mM Tris-HCl, 1 mM MgCl2, and 1 mM CaCl2). Samples were incubated for 1.5 h at 37°C, after which 20 ^g of proteinase-K (1 ^l of a 20 mg/ml stock) was added, followed by an additional incubation at 55°C for 1 h. Samples were then resolved through 1.0% agarose gels (1.5 h, 80 V), stained with ethidium bromide, and visualized by UV transillumination.