Fig. 1: LH/hCG promotes TGFB1 expression in cumulus cells.

Large follicles and COCs were isolated from eCG-primed mice. OOX cumulus cells were produced by microsurgically removing oocytes from the COCs. OCCs were collected from the ampullae of superovulated mice (at 13 h post-hCG treatment). a Immunofluorescence analysis of TGFB1 (green) in cumulus cells before and after hCG injection (at 13 h post-hCG treatment). (n = 3 independent experiments). Nuclei were counterstained by DAPI (blue). The small white box indicates the location of the enlarged area, as shown in the lower left corner. Scale bars represent 50 μm. b, c Comparison of steady-state mRNA (b) and protein (c) levels of TGFB1 in cumulus cells before and after hCG injection (at 13 h post-hCG treatment). (n = 5 independent experiments in (b) and n = 4 independent experiments in (c)). d, e The effects of LH and EGF on TGFB1 mRNA (d) and/or protein (e) expression in cumulus cells. Follicles were cultured in medium supplemented with LH (1 μg/ml) and/or AG1478 (1 μM) for 12 h, and COCs were cultured in medium supplemented with EGF (10 ng/ml) and/or AG1478 for 12 h. (n = 3–5 independent experiments). f, g The effects of EGF, oocytes, and ODPFs on TGFB1 mRNA (f) and protein (g) expression in cumulus cells. COCs or OOX cumulus cells were cultured in a medium supplemented with EGF, oocytes (3 oocytes/μl), GDF9 (500 ng/ml), BMP15 (500 ng/ml), FGF8B (FGF8, 100 ng/ml), and/or TGFB2 (50 ng/ml) for 12 h. ODPFs, GDF9 + BMP15 + FGF8B + TGFB2; AG, AG1478. (n = 3–5 independent experiments). β-actin was used as a loading control in (c), (e), and (g). Bars indicate the mean ± SD. Each data point represents a biologically independent experiment. Statistical analysis was performed by two-tailed unpaired Student’s t-test for two groups, and by one-way ANONA Tukey test for experiments involving more than two groups. ns, no significance (P ≥ 0.05). *P < 0.05, **P < 0.01, and ***P < 0.001.