Reproduction Abstracts

Previous studies have assessed the effects of melatonin on cultured granulosa cells under 5% CO₂ in air, and focused on its antioxidant properties. Effects of melatonin under more physiological oxygen tensions are not known. Here we cultured granulosa cells from antral follicles (4–6 mm diameter) in fibronectin-coated 12-well plates (at 600 000 cell/ml) in TCM199, with melatonin added at one of four concentrations (0, 20, 200, 2000 pg/ml) under either i) low oxygen (5% O₂, 5% CO₂) or ii) in air (with 5% CO₂) for 48, 96 and 144 h. Cell viability (using trypan blue and crystal violet) and reactive oxygen species (ROS) generation (using nitroblue tetrazolium) were assessed. Mean number of viable cells was greater (P=0.04) under 5% O₂ than air (658 000 vs 610 000 cells). Cell number was further increased (P<0.001) with added melatonin under both 5% O₂ and air. ROS generation was similar under both 5% O₂ and air, but was reduced (P=0.002) with the inclusion of melatonin (at all levels). Oxygen tension interacted (P<0.001) with time in culture to affect media E2 concentration. E2 (pg/10⁵ cells) under air vs 5% O₂ =4218 vs 1851 at 48 h; 858 vs 669 at 96 h; and 838 vs 2009 at 144 h. P4 (ng/10⁵ cells) increased (P<0.001) between 48 and 96 h of culture, and was greater (P<0.001) at these time points under air than under low O₂ (176.5 vs 100.0). E2:P4 ratio fell between 48 and 96 h under air and remained low to 144 h (0.092, 0.005 and 0.007), whereas E2:P4 ratio under low O₂ initially fell and then increased (0.060, 0.007 and 0.020) (P<0.001). Cell number and viability is enhanced, luteinisation reduced and steroidogenesis altered under low O₂. Melatonin enhances cell viability irrespective of oxygen tension, but has no effect on steroidogenesis.