Differential Response of Individual Uterine Cell Types from Immature Rats Treated with Estradiol*

Abstract

The separation of viable epithelial, stromal, and myometrial cells from immature rat uteri is described. The integrity of the separated cells was established by their ability to grow in culture and to exclude trypan blue and by transmission and scanning microscopy. Epithelial preparations contain insignificant contamination by other cells. Stromal fractions are estimated to contain 10% epithelial contamination. Myometrial fractions probably contain significant contamination by stroma, but the amount is difficult to quantify. The three cell types separated from untreated rats or from rats treated with 17β- estradiol (E₂) differ from each other physiologically. Control epithelial cells incorporated more tritiated cytidine per jug DNA than stromal or myometrial cells. 17β-Estradiol enhanced incorporation of tritiated cytidine into RNA of epithelial cells but not stromal or myometrial cells. RNA to DNA ratios of cells from untreated rats were 1.0 (epithelial), 0.5 (stromal), and 0.6 (myometrial). RNA to DNA ratios rose ∼30% in all cell types after E₂. Estrogen receptor (ER) concentrations, determined by Scatchard analysis under exchange conditions, also differed in the three cell types. Nuclear ER concentrations (as measured in crude nuclear fractions) were significantly higher (P < 0.01) in epithelial and stromal cell fractions from untreated animals than in the myometrial fraction, i.e. 5.5, 3.8, and 1.2 fmol/μg DNA, respectively. The chief effect of E₂ administration was a significant increase (P < 0.05) in cytosol ER of all cell types. Epithelial cells had the highest cytosol ER concentration, increasing from 0.5 to 3.1 fmolμg DNA 48 h after E₂ administration. Although the myometrial cells had the lowest ER concentration, they contained 84-89% of the entire uterine ER by virtue of the preponderance of myometrium in the whole uterus. The differing responses of epithelial and stromal cells, therefore, are diluted or masked by the ER content of the myometrium in assays of whole uterus. Although the total number of ER sites in the cytosol and nucleus (16,900-32,100 sites/cell) in separated stromal cells at various times after E₂ stimulation was similar to ER concentrations (20,000-30,000 sites/cell) previously reported for the whole uterus, the partition of ER between cytosol and nucleus (10:90) was different, favoring the nucleus even before E₂ administration. ER began to appear in increased amounts in the cytosol only 24 h post E₂ administration. When tritiated E₂ was given in vivo and its specific binding was assessed in the uterine cells subsequently separated, ER partition between cytosol and nucleus was also heavily in favor of the nucleus. However, when whole uterus was assayed, the partition of ER between cytosol and nucleus (90:10) was exactly the opposite. Possible reasons for this unusual finding are discussed. (Endocrinology106: 1634, 1980)