Fig 2: Effects of inhibition of estrogen signaling in vivo. (A) Ovaries of fulvestrant (Ful) or vehicle (Ctrl)-injected mice with or without eCG treatment were sectioned and stained with H & E. Scale bar, 200 and 100 µm for left and right panels, respectively. A small or a large antral follicle in each section is shown in the right panels. (B) The number of secondary and antral follicles of each ovary was counted (n = 3). (C) Morphology of a uterus isolated from a fulvestrant (Ful) or vehicle (Ctrl)-injected mouse. Scale bar, 5 mm. Ctrl (+ eCG), n = 3; Ctrl (-eCG), n = 6; Ful (-eCG), n = 8. (D) MGCs were isolated from fulvestrant (Ful) or vehicle (Ctrl)-injected mice and FOXL2 expression was examined by western blotting (upper panel). Band intensities were quantified (lower panel) (n = 3). Different letters (a and b) denote significant differences (p < 0.05).

Fig 3: Effects of BMP15 and GDF9 signals on FOXL2 expression in MGCs. (A) MGCs were cultured with or without recombinant BMP15/GDF9 (ODPFs) and E2 for 24 h (n = 4). (B) MGCs cultured with both oocytes and E2 were treated with or without an inhibitor of ALK5, SB431542 for 24 h (n = 4). (C) Comparison of the expression levels of FOXL2 protein between MGCs of Bmp15-/-/Gdf9+/- mice (DM) and those of littermate control Bmp15+/-/Gdf9+/- mice (Ctrl) (n = 3 each). The expression of FOXL2 protein was examined by western blotting (upper panel) and band intensities were quantified (lower panel). Asterisks or different letters (a and b) denote significant differences (p < 0.05).

Fig 4: Effects of culture on the expression levels of transcriptional targets of FOXL2 in MGCs. Freshly isolated MGCs (Fresh) and MGCs cultured for 24, 48, and 72 h were examined for the expression kinetics of (A) Cyp19a1, (B) Fst, and (C) Sox9 mRNAs by qPCR (n = 3). (D) The expression of SOX9 protein was examined by western blotting. Different letters (a, b, and c) denote significant differences (p < 0.05).

Fig 5: Effects of oocyte-derived signals and 17ß-estradiol on the expression of FOXL2 in MGCs in vitro. MGCs were cultured with or without oocytes (2 oocytes/µl) and/or 17ß-estradiol (E2) for 24 h. (A) The expression levels of FOXL2 protein were examined by western blotting (upper panel). The relative intensities of protein bands were quantified (lower panel) (n = 3). (B) The expression levels of Foxl2 mRNA were examined by qPCR (n = 4). The expression levels of (C) Star and (D) Cyp11a1 mRNAs were examined by qPCR (n = 3). Different letters (a, b and c) denote significant differences (p < 0.05).