Subsequently, ovaries were excised, cleared of adhering fat, weighed, and fixed either in 10% neutral buffered formalin for immunohistochemical detection of prohibitin, PCNA, or P450scc or for in situ terminal deoxyn-ucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) of ap-optotic cells nuclei (n = 5 rats/group). In addition, granulosa cells from each group (n = 10 rats/group) of animals were harvested by follicle puncture as previously described, washed, and resuspended in 10 mM Hepes buffer (pH 7.4) containing 1 mM EGTA and 2 mM PMSF. Cells were processed for Western blot analyses.
Porcine germinal vesicle (GV)-stage oocytes were collected from slaughterhouse ovaries and were matured and fertilized in vitro as described previously. Briefly, the oocyte-cumulus complexes (OCCs) were aspirated from ovaries, washed, and matured for 22 h at 39°C and 5% CO2 serum-free modified tissue-culture medium (TCM) 199 (Gibco, Grand Island, NY) supplemented with 3.05 mM glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 10 ng/ml of epidermal growth factor, 0.5 |xg/ml of FSH, 0.5 |xg/ml of LH, 0.1% polyvinyl alcohol (w/v), 75 |xg/ml of penicillin G, and 50 |xg/ml of streptomycin sulfate.