Effect of GM-CSF on cumulus cell in in vitro matured COC. (Left Graphs) COC were matured in SOF medium alone or supplemented with 1, 10 and 100 nM of GM-CSF. Parameters from cumulus cells derived from oocytes in the immature state and matured in TCM were used as positive control and not included in the statistical analysis. (Right Graphs) COC were matured in SOF medium supplemented with GM-CSF, LY294002 (10 or 100 μM) or DMSO. (A, B) Cumulus cell expansion was evaluated subjectively using the cumulus expansion index (CEI). (C, D) Cell viability was evaluated using Trypan Blue staining. (E, F) Cell number was determined using hemocytometer. (−−−) Corresponds with the lower limit for cumulus expansion (CEI ≥ 0.5). (*) Indicate significant differences between treatments of 0, 1 and 100 ng/ml of GM-CSF. *** P < 0.001, ** P < 0.01 and *P < 0.05. (#) Indicate significant differences compared with control ### P< 0.001, ## P< 0.01, # P < 0.05.