Figure 2. EGF-like growth factors stimulated by LH/hCG, induced the accumulation of GPR30 protein in COCs. PMSG-primed female mice were injected with hCG, and the ovaries were collected at 0, 2, 4, 8, and 16 h. The mRNA levels of AREG (A), EREG (B) and BTC (C) were measured using RT-qPCR. To further conform the role of EGF-like growth factors in LH/hCG–induced GPR30 accumulation in COCs, AREG (100 nM), EREG (100 nM), BTC (100 nM) and EGF (10 ng/mL) were added to the medium and COCs were cultured in vitro. GPR30 mRNA levels was measured using RT-qPCR (D), the protein levels were detected using western blot (E) and the bands were quantified using gray scanning (F). Data are represented as fold induction relative to the unstimulated control (0 h or con). Bars are presented as average±SEM. Different lowercase letters indicate significant differences between groups (p<0.05). Three independent replicates were performed for each experiment.