Granulosa Cells Inhibit the Resumption of Meiosis in Bovine Oocytes in Vitro1

The oocytes of cattle are not as sensitive as those of laboratory animals to purines, cAMP, or follicular extracts. To study the resumption of meiosis, a method is needed that is capable of inhibiting meiosis completely for a minimum of 24 h. This study was designed to evaluate interrelationships in granulosa-oocyte-cumulus complexes using fresh granulosa cells aspirated from small follicles (1−5 mm) in which the cumulus is normally firmly attached. Selected oocyte-cumulus complexes obtained from a slaughterhouse (n = 2,236) were co-incubated with one of the following: various concentrations of fresh granulosa cells in tissue cultures medium (TCM) 199 or bovine follicular fluid (BFF) either without or after one washing and/or freezing; resuspended granulosa cells previously cultured for 7 days; blood cells; or medium alone. Additionally, oocyte-cumulus complexes were embedded in agar cylinders before incubation with or without cells. The rate of maintenance of intact germinal vesicles (GV) in oocytes after 24 h ranged from 40−77% when 5−100 × 106 unwashed cells/ml BFF were used, compared to only 16% in oocytes cultured in BFF alone. The pattern was the same when washed cells were used (30−77%, using 5−100 × 106 cells/ml BFF), but they were not as effective as unwashed cells. With TCM-199 and the same five concentrations of cells (5, 10, 25, 50, and 100 × 106/ml), a similar inhibition was obtained with ≥ 25 but not with 5 (3%) or 10 (5%) × 106/ml. The mechanical separation of live cells from oocytes by agar resulted in intermediate inhibition (28% GV), possibly indicating that diffusion of a fresh product was implicated since complete BFF alone resulted in only 16% GV. Granulosa cells cultured for 7 days were also able to inhibit the resumption of meiosis when resuspended at a concentration of 25 × l06/ml (40% GV). Blood cells were ineffective (<5% GV) at concentrations of up to l09/ml, indicating that the capacity to inhibit oocyte maturation is specific to granulosa cells. Frozen-thawed lysed cells that were at the same concentration as the top portion of aspirated fluid gave results similar (15% GV) to those obtained with BRF alone, suggesting that the inhibitory activity of granulosa cells depends on their viability.