The E2/GPR30 pathway enhances the response of EGF signaling and accelerates cumulus expansion. COCs were cultured in the medium with 1 ng/mL EGF, 10 ng/mL EGF, 17β-E2, G1, G15 or ICI182780 addition for 16 h, and the cumulus expansion level was assessed. (A) Representative images of COCs isolated from ovaries (0 h) and cultured under different conditions for 16 h. (B) CEI of COCs cultured for 16 h in each group. (C) The different grades of COC culture for 16 h in each group. Data are represented as average±SEM. Different lowercase letters indicate significant differences (p<0.05). Three independent replicates were performed for this experiment.