FIGURE 4.

Effect of GDF9 treatment on gonadotropin subunit expression. A, LβT2 cells were serum-starved overnight and treated with increasing concentrations of GDF9 for 6 h. B, cells were serum-starved overnight and treated with either 2 μg of control antibody (IgG) or 2 μg of GDF9 IgG for 8 h. After 2 h of antibody treatment, cells were exposed to vehicle or 100 ng/ml GDF9 for 6 h. C, cells were serum-starved overnight and stimulated with 1 nm GnRH for 2 h, followed by 4 h without GnRH, or with vehicle. Cells were cotreated with GDF9 (10 ng/ml) for 6 h. D, primary pituitary cells from adult mice were pretreated with follistatin (500 ng/ml) overnight and treated with either vehicle or GDF9 (200 ng/ml) for 6 h. E, primary pituitary cells from ovariectomized female rats were pretreated with follistatin (500 ng/ml) overnight and treated with either vehicle, GDF9 (100 ng/ml), or activin (50 ng/ml; used as a positive control) for 6 h. Conditioned medium was harvested and sent to the Ligand Assay and Analysis Core facility at the University of Virginia for protein measurement. A and E, one-way ANOVA with Bonferroni post test corrections was used. B and C, two-way ANOVA with Bonferroni post test corrections was used. D, two-tailed Student's t test was used. ***, p < 0.001; **, p < 0.01; *, p < 0.05. The data shown are representative of three independent experiments. Error bars, S.E. A.U., arbitrary units.