Fig 2: TGF-ß promotes NPPC expression and maintains oocyte meiotic arrest.a–c MGCs were collected from prepubertal mice and cultured for 24 h in cell culture medium supplemented with 10 ng/ml TGFB1, 50 ng/ml TGFB2, 10 ng/ml TGFB3, TGF-ß (including 10 ng/ml TGFB1, 50 ng/ml TGFB2, 10 ng/ml TGFB3), 1 µmol/l SD208 (SD), and/or 10 ng/ml FSH. NPPC and/or NPR2 mRNA and protein levels were detected by qRT-PCR (a), western blotting (b) and a luciferase immunoassay (c), respectively. The blots shown are representative of three images captured. GAPDH was used as a loading control. d Effect of TGF-ß on oocyte meiotic resumption of COCs. MGCs were cultured in the medium supplemented with TGF-ß, SD208, and/or FSH for 24 h, and then COCs isolated from eCG-primed mice were cocultured with MGCs for 1, 2, 3, and 4 h. n = 30 for each treatment across all three replicates. **P ? 0.01 and ***P ? 0.001 compared with corresponding control. e Effect of TGF-ß on the meiotic resumption of oocytes within follicles. Large antral follicles isolated from eCG-primed mice were cultured in the medium supplemented with TGF-ß, and/or SD208 for 4 h. n = 30 for each treatment across all three replicates. Bars indicate the mean ± SEM of three independent replicates. Values not indicated by the same letter are significantly different (P < 0.05)

Fig 3: Selective Tgfbr2 depletion in granulosa cells impairs oocyte meiotic arrest, ovulation, and female fertility.a, b The detection of TGFBR2 knockout efficiency in MGCs from eCG-primed mice. The expression levels of TGFBR2 in MGCs were detected by immunofluorescence (a) and western blotting (b). c–e Effects of Tgfbr2 depletion on NPPC and NPR2 expressions. MGCs were isolated from eCG-primed mice. The gene and protein levels of NPPC and NPR2 were detected by qRT-PCR (c) and western blotting (d), respectively. MGCs isolated from Tgfbr2gc-/- mice were cultured for 24 h in cell medium without or with TGF-ß (including 10 ng/ml TGFB1, 50 ng/ml TGFB2, and 10 ng/ml TGFB3) to detect the effect of TGF-ß on Nppc mRNA levels (e). f, g Selective Tgfbr2 depletion in granulosa cells impairs oocyte meiotic arrest. A prophase-arrested oocyte (GV) within a large antral follicle of a WT ovary and an oocyte with metaphase I (MI) chromosomes within a large antral follicle of a Tgfbr2gc-/- ovary (f). Percentages of oocytes that had resumed meiosis, counted in serial sections of ovaries from eCG-primed mice (g). The small black boxes indicate the enlarged areas in the following images. n = 6. h–j Selective Tgfbr2 depletion in granulosa cells impairs ovulation and female fertility. Histology of ovarian sections from WT and Tgfbr2gc-/- ovaries at 48 h after hCG injection (h). CL: corpus luteum. The superovulation assay shows decreased ovulation in Tgfbr2gc-/- mice at 16 h after hCG injection (i). Comparison of the cumulative number of progeny per female WT and Tgfbr2gc-/- female mice over a 6-month period (j). n = 6. *P ? 0.05, **P ? 0.01, and ***P ? 0.001 compared with corresponding control. The images and blots shown are representative of three images captured in a, b, d, f, h. Scale bars: 200 µm in a, f, h. GAPDH was used as a loading control in b, d. Bars indicate the mean ± SEM of three independent replicates. Values not indicated by the same letter are significantly different (P < 0.05)