Physiological responses of cultured bovine granulosa cells to elevated temperatures under low and high oxygen in the presence of different concentrations of melatonin.

Our understanding of the effects of temperature on granulosa cell (GC) physiology is primarily limited to in vitro studies conducted under atmospheric (∼20% O2 ) conditions. In the current series of factorial experiments we identify important effects of O2 level (i.e. 5% vs 20% O2 ) on GC viability and steroidogenesis, and go onto report effects of standard (37.5 °C) vs high (40.0 °C) temperatures under more physiologically representative (i.e. 5%) O2 levels in the presence of different levels of melatonin (0, 20, 200 and 2000 pg/ml); a potent free-radical scavenger and abundant molecule within the ovarian follicle. Cells aspirated from antral (4-6 mm) follicles were cultured in fibronectin-coated wells using serum-free M199 for up to 144 h. At 37.5 °C viable cell number was enhanced and luteinization reduced under 5 vs 20% O2 . Oxygen level interacted (P < 0.001) with time in culture to affect aromatase activity and cell estradiol (E2 ) production (pg/mL/105  cells). These decreased between 48 and 96 h for both O2 levels but increased again by 144 h for cells cultured under 5% but not 20% O2 . Progesterone (P4 ) concentration (ng/mL/105  cells) was greater (P < 0.001) under 20 vs 5% O2 at 96 and 144 h. Cell number increased (P < 0.01) with time in culture under 5% O2 irrespective of temperature. However, higher doses of melatonin increased viable cell number at 40.0 °C but reduced viable cell number at 37.5 °C (P = 0.004). Melatonin also reduced (P < 0.001) ROS generation at both O2 levels across all concentrations. E2 increased with time in culture at both temperatures under 5% O2 , however P4 declined between 96 and 144 h at 40.0 but not 37.5 °C. Furthermore, melatonin interacted (P < 0.001) with temperature in a dose dependent manner to increase P4 at 37.5 °C but to reduce P4 at 40.0 °C. Transcript expression for HSD3B1 paralleled temporal changes in P4 production, and those for HBA were greater at 5% than 20% O2 , suggesting that hemoglobin synthesis is responsive to changes in O2 level. In conclusion, 5% O2 enhances GC proliferation and reduces luteinization. Elevated temperatures under 5% O2 reduce GC proliferation and P4 production. Melatonin reduces ROS generation irrespective of O2 level and temperature, but interacts with temperature in a dose dependent manner to influence GC proliferation and luteinization.