Effects of estradiol on DNA content of engineered tissues. DNA levels were measured using the Hoechst dye assay in cells that were cultured with 0 nmol/L estradiol (black bars) or 10nmol/L estradiol (gray bars, n = 3). A, In the absence of estradiol, there were no significant differences in DNA content between the stromal only (S), epithelial only (E), or cocultured (SE) tissue. In contrast, estradiol had a mitogenic effect on the epithelial cells leading to higher DNA content compared with these cells under 0 nmol/L estradiol conditions as well as compared with estradiol-treated S and SE tissues. B, Conditioned medium was used to look at potential paracrine factors in the estradiol-treated cultures. The addition of epithelial cell conditioned medium to the stromal tissues (S-Ecm) had no significant effect on DNA levels compared to any other conditions. The addition of stromal conditioned medium to the epithelial tissues (E-Scm) resulted in a decrease in DNA content, similar to that of the SE cocultures. * denotes P < .05 compared to epithelial cells cultured with 10 nmol/L estradiol.