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. 2018 Mar 19;159(5):2062–2074. doi: 10.1210/en.2018-00117

Figure 6.

Figure 6.

The effect of RA or SMAD inhibitors on Lhcgr expression relative to Lhcgr promoter methylation in cumulus cells of COCs. (A) The effect of SB431542 on the expression of Adh1, Adh5, Aldh1a1, Aldh1a7, and Cyp26b1 in cumulus cells of COCs. COCs were collected from ovaries of immature mice primed with eCG for 48 hours. COCs (n = 50) were cultured in DMEM/F12 medium containing sodium pyruvate (0.23 mM), bovine serum albumin (BSA; 3 mg/mL), IBMX (0.2 mM), 1% FCS, and estradiol 17β (10 μM) for 20 hours. SB431542: COCs were cultured for 20 hours with SB431542 (2 μM). Levels of mRNA were normalized to that of L19. The value of controls (i.e., COCs cultured without SB431542) was set as 1, and the data are presented as fold induction. (B) The effect of SB431542 on RARE-LacZ activity in COCs. Data are shown for activity of LacZ enzyme driven by RARE promoter in cumulus cells of COCs with or without 2 μM of SB431542 for 20 hours. Values are RARE-LacZ activity at 595 nm after the protein samples were incubated for 2 hours at 37°C with chlorophenol red-β-d-galactopyranoside. Values are given as mean ± standard error of the mean (SEM) of more than three replicates. (C) The effect of RA or SB431542 on the expression of Lhcgr in cumulus cells of COCs. COCs were collected from ovaries of immature mice primed with eCG for 48 hours. COCs (n = 50) were cultured in DMEM/F12 medium containing sodium pyruvate (0.23 mM), BSA (3 mg/mL), IBMX (0.2 mM), and estradiol 17β (10 μM) for 20 hours. RA: COCs were cultured for 20 hours with RA (5 μM). Levels of mRNA were normalized to that of L19. (D) The effect of RA or SB431542 on methylation of the Lhcgr promoter region in cumulus cells of COCs. COCs were collected from ovary of immature mice primed with eCG for 48 hours. COCs (n = 100) were cultured in DMEM/F12 medium containing sodium pyruvate (0.23 mM), BSA (3 mg/mL), IBMX (0.2 mM), and estradiol 17β (10 μM) for 20 hours. Sequence analysis was performed on more than five colonies for each treatment of three replicates. Values are given as mean ± SEM. SB431542: COCs were cultured for 20 hours with SB431542 (2 μM). RA: COCs were cultured for 20 hours with RA (5 μM). *P < 0.05 compared with control. C, control.