Figure 3

DHT inhibited in vitro FSH-induced granulosa cell proliferation through interfering cell cycle.

(A) Fifty μg of total protein form rat granulosa cells and human granulosa cells (hGC) were used to determine the specificity of the antibody to FSHR of rat granulosa cells (rGC) by immunoblotting (left panel). Ratio of FSHR positive cells were analyzed by quantifying the FSHR-positive cells by flow cytometry using a FACScan and the Cell Quest software. The white histograms are of isotype controls, whereas the orange overlays were of FSHR-positive cells (right panel). (B) Rat granulosa cells were treated with FSH and different doses of DHT or DHEA and after 72 hours, cell growth was determined by the MTT assay. The data are shown as means ± SD of 3 independent experiments. Granulosa cell growth was significantly suppressed by co-treatment with FSH and DHT (exceeding 25 uM) in comparison with FSH treatment only. (C) Rat granulosa cells were pretreated with DHT (50 μM), bicalutamide (50 μM), or insulin (500 nM) for one hour prior to FSH (10 ng/ml) treatment. After 24 hours, the cell cycle phase was determined by propidium iodide staining and FACScan analysis. Populations in the subG1, G1, S and G2/M phases are shaded M1 to M4, respectively. Representative cell cycle histograms of each group are shown. N = 3. (D) The percentage of cells in each cell cycle phase was analyzed and quantified by Cell Quest software and the data represent the mean ± SD of 3 independent experiments. The percentage of cells in the G2/M phase significantly decreased following treatment with FSH. Compared with cells treated with FSH alone, the number of cells in G2/M phase increased significantly following treatment with DHT and FSH. The percentage of cells in G2/M phase significantly decreased following treatment with DHT/FSH/Bicalutamide, in comparison with cells treated with DHT and FSH. Between-group comparisons were performed as indicated *P < 0.05.