ABSTRACT: Ovaries from cyclic sows were derived from a local slaughterhouse and transported to the laboratory at 27-33ºC within 2 hours after death. After removal of surrounding tissue, the ovaries were rinsed with water (27-33ºC) and submersed in PBS containing 100U/ml penicillin and 100µg/ml streptomycin (Gibco, Breda, The Netherlands) at 37ºC. Granulosa cells and oocytes were aspirated with a 26G needle from healthy, 3-6 mm follicles. Follicular health was determined according to previously described criteria (28). Harvested cells were pooled and the cell suspension was washed twice by centrifugation at room temperature in McCoys 5a medium with glutamine (Invitrogen, Breda, The Netherlands) supplemented with 100U/ml penicillin, 100µg/ml streptomycin, 0.1% BSA (Fluka, Zwijndrecht, The Netherlands), 2.5µg/ml transferrin and 4ng/ml sodium selenite. Oocytes were removed from the cell suspension by filtration through a 70µm cell sieve (Becton en Dickinson, Alphen a/d Rijn, The Netherlands). The number of viable GCs was estimated by trypan blue exclusion. Granulosa cells were cultured in serum free medium as previously described by Picton and colleagues (26). For co-culture experiments cumulus oocyte complexes (COC) were aspirated from 3-6mm follicles. Cell debris and COC were allowed to settle at the bottom of the tube and the supernatant was discarded. Cumulus oocyte complexes were washed in TL-HEPES and subsequently selected by morphological appearance (dark cytoplasm and at least 2 layers of cumulus cells). The COC were transferred to wash medium containing 0.1U/l FSH, 100mg/l long R3-IGF-1, 10mg/l insulin (all from Sigma Aldrich, Zwijndrecht, The Netherlands) and 100mg/l testosterone (Fluka, Zwijndrecht, The Netherlands). Granulosa cells, 7.5*106 per well of a 6 wells plate, and COC, 80-100, were co-cultured in 3.5ml medium per well of a 6 wells plate. Cells were cultured in a humidified atmosphere at 37ºC and 5% CO2 for 48 hrs.
