17 β-estradiol increases calcium content in fetal mouse parietal bones cultured in serum-free medium only at physiological concentrations

Using a bone organ culture system that shows mineralization in vitro, we investigated whether 17β-estradiol dose-dependently increases calcium content in cultured calvarial bones in serum-free medium. Fetal mouse parietal bones (3 × 3 mm) were cultured in phenol red-free BGJ medium containing phosphate (3–4 μmol/L), calcium (1–1.25 mmol/L) insulin (6μg/mL), and transferrin (6μg/mL) for 4–5 days. Under these culture conditions, the calcium content of the cultured bones (at dissection 34.0 t 4.6 μg/bone [mean ± SD], n = 50) increased by 15–20 μg during 4–5 days of culture. 17β-Estradiol increased the calcium content significantly at 10−12 to 10−11 mol/L, but not at lower (10−13 mol/L) or higher (10−10 to 10−9 mol/L) concentrations. 17α-Estradiol had no effect. The stimulatory effect of 17β-estradiol was completely inhibited by the antiestrogen agent ICI-182,780. The anabolic effect of 17β-estradiol was elicited not only in bones from females but also in those from males. 17β-Estradiol had no significant effect on 45Ca release from prelabeled parietal bones. Furthermore, light- and electron-microscopic examinations revealed that bone mineralization proceeded through formation of matrix vesicles, without any metastatic or dystrophic calcification. These in vitro findings suggest that 17β-estradiol elicits small, but reproducible, direct effects on calcium content in the parietal bones not only in female but also in male fetal mice at physiological-free E2 concentrations (10−12−10−11 mol/L), which is attainable in serum of normal human subjects. In contrast to in vivo studies, pharmacological doses of 17β-estradiol had no anabolic effect on parietal bones. The mechanism of such a biphasic effect of estrogens remains to be elucidated.