Figure 2

1,25D ₃ enhanced testosterone-induced aromatase tyrosine phosphorylation and 17β-estradiol secretion during 18–24 h. (A) Rat ovarian granulosa cells were treated with vehicle, testosterone, testosterone plus 1,25D₃ or 1,25D₃ alone for 24 h (n = 4–5 in each group). Aromatase was immunoprecipitated before being subjected to SDS-PAGE for resolution. The resultant blot was probed using both phosphotyrosine and aromatase antibodies. The expression of aromatase tyrosine phosphorylation (denoted by pTyr-Arom) was normalized to the corresponding aromatase expression and is represented as a percentage relative to the vehicle-treated control. (B) The left panel is a positive control that was both immunoprecipitated and immunoblotted with an anti-aromatase antibody. The lysate was also immunoprecipitated with the anti-aromatase antibody or IgG and immunoblotted with an anti-phosphotyrosine antibody, as shown in the right panel. A weak band in the IgG lane is as a negative control. (C) Cells were treated with testosterone alone or testosterone plus 1,25D₃ as indicated for various time periods (n = 4–5 in each group). Each pTyr-Arom level was normalized using the corresponding aromatase level and is represented as a percentage relative to the vehicle-treated control. (D) After treatment for various time periods as indicated, the 17β-estradiol production levels were measured by radioimmunoassay. (E) After treatment with testosterone alone or testosterone plus 1,25D₃ for 18 h, the culture medium was removed and then replaced with fresh medium. Cells were continuously treated for another 6 h (18–24 h span). After the final 6 h, medium was collected and the 17β-estradiol secretion levels were measured by radioimmunoassay (n = 6 in each group). The values represent the means ± SEM. #, p < 0.05; ##, p < 0.005 for comparison of the two treatment groups. The values listed within column indicate the means.