Fig. 2

Involvement of MAPK3/1 in EGFR signaling and EGF-stimulated activin subunit expression. (A) Effects of U0126 (MEK1/2 inhibitor) on EGF-stimulated expression of inhbaa, inhbab and inhbb in cultured follicle cells. The cells were treated with EGF at 20 nM for 2 h. The expression values are the mean ± SEM (n = 3–4). ***P < 0.001 vs. respective control of each gene. The graph is representative of three independent experiments. (B) Time course of EGF-induced MAPK3/1 phosphorylation (p-MAPK3/1) in the follicle cells. (C) Blockade of EGF-induced MAPK3/1 phosphorylation by EGFR inhibitor AG1478. (D) Blockade of basal and EGF-induced MAPK3/1 phosphorylation by MEK1/2 inhibitor U0126. The follicle cells were pre-treated with the inhibitors for 30 min before treatment with EGF for 5 min. (E) Immunohistochemical staining for EGF-induced MAPK3/1 phosphorylation in intact follicles. The follicles were treated with (20 nM) or without EGF for 20 min before sampling for fixation, sectioning and IHC staining. No signal was detected in the control follicles without EGF treatment (a and b), whereas strong signal of MAPK3/1 phosphorylation was detected in the somatic follicle layer (arrow head) but not the oocyte (c and d). (F) Semi-quantitative measurement of immunohistological staining for p-MAPK3/1 in the follicle layer and oocyte compartments of intact follicles. MAPK3/1 phosphorylation level increased in follicle layer but not the oocyte in response to EGF treatment. The values are the mean ± SEM (n = 4). ***P < 0.001 vs. control.