Tissues were then incubated for 30 min with a peroxidase-conjugated anti-digoxygenin monoclonal antibody, and apoptotic cells were visualized as positively immunostained structures after reaction with diaminobenzidine. Negative controls included TdT omission. Sections were counterstained with hematoxylin. The number of apoptotic cells was determined by counting labeled cells from follicles in 400X microscopic fields (three sections per ovary; five ovaries) and expressed as the apoptotic cell mean per field.
DNA Isolation and Fragmentation Analysis
Cellular DNA was extracted from 10 healthy antral follicles per ovary. Follicles were incubated for 24 h under serum-free conditions at 37°C in 500 il of Dulbecco modified Eagle medium F12 (1:1) containing 10 mM Hepes, supplemented with fungizone (250 ig/ml), and gentamicin (10 mg/ml ) (five ovaries per group) and gassed with 95%O2:5% CO2 at the start of culture. This model has the advantage of keeping the integrity of the follicle. In addition, the incubation in serum-free conditions for 24 h allows an exhibition of the typical apoptotic DNA ladder: the presence of internucleosomal fragments of 180-bp multiples.