The individual ovarian follicles were dissected from the ovary under a stereoscopic microscope as previously described. In brief, healthy antral follicles (300^-50 im in diameter) from eight ovaries per group were frozen and used for biochemical assays. From each ovary, a pool of isolated follicles was frozen, and the results obtained from each pool were considered a single datum.
For immunohistochemical quantification of apoptosis, formalin-fixed tissue sections were processed for in situ localization of nuclei exhibiting DNA fragmentation by the TUNEL technique with an apoptosis detection kit (Apoptag plus peroxidase in situ Apoptosis detection kit; Chemicon International, Inc., Temecula, CA) as previously described. The 4-im-thick tissue sections were deparaffinized and digested for 15 min at room temperature with proteinase K (20 ig/ml; Gibco, Grand Island, NY). Endogenous peroxidase was quenched with 3% hydrogen peroxide in PBS. The labeling reaction was carried out by incubating tissue sections with buffer containing digoxygenin-20-deoxyuridine 5′-triphosphate prior to incubation with terminal deoxynucleotidyl transferase (TdT) for 1 h at room temperature.