Search

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 17 beta-estradiol (E2) differ from each other physiologically. Control epithelial cells incorporated more tritiated cytidine per microgram DNA than stromal or myometrial cells. 17 beta-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 approximately 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 less than 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/microgram DNA, respectively. The chief effect of E2 administration was a significant increase (P less than 0.05) in cytosol ER of all cell types. Epithelial cells had the highest cytosol ER concentration, increasing from 0.5 to 3.1 fmol/microgram 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 proponderance 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.

A high-affinity, low-capacity estradiol-binding molecule (RE) has been demonstrated in the basal zone trophoblast (BZT) of the pregnant rat. On day 11 of pregnancy (day 0 = first sperm-positive day) RE is present in BZT cytosol, where it has a ka of 1.2 X 10(6)M-1 sec-1, t1/2 = 12.7 min, at 20 C. The Kd, under similar conditions, consists of 2 components, 1.3 X 10(-4) sec-1, t1/2 = 90 min, and 5.9 X 10(-5) sec-1, t1/2 = 196 min. When one uses the faster component, the equilibrium constant, Kd, obtained from kd/ka is 1.1 X 10(-10)M, in close agreement with that obtained from Scatchard analysis of specific estradiol (E2) binding at 20 C. On day 11 there were approximately 12,000 sites/cell in BZT cytosol. Scatchard analysis of nuclear RE on day 11 indicated a Kd of 1.85 X 10(-10)M and approximately 21,000 sites/nucleus, but, in day 15 BZT, nuclear RE was undetectable. Neither cytosol nor nuclei prepared from placental labyrinthine zone (LZT) tissue (fetal placenta) showed evidence of high-affinity, low-capacity E2 binding. Sucrose density gradient analysis on 5-20% linear gradients showed the cytosol RE to be approximately 4S whether in high or low-salt conditions. When measured against binding by 3H-labeled estradiol (*E2), the cytosol BTZ RE was competed for strongly (80-90%) by estrone, estriol, diethylstilbestrol, and estradiol-17alpha at 200 times excess. Nafoxidine-HCl, also at 200X excess, competed to approximately 50%. Corticosterone, progesterone, testosterone, dehydroepiandrosterone, and pregnenolone did not compete. The hormone specificity of nuclear BZT RE was similar to that of the comparable cytosol RE with the exception that nafoxidine did not compete. This was probably due to differences in kinetics, nafoxidine requiring a longer time to reach equilibrium than the other estrogens. The size of the nuclear RE by sucrose density gradient analysis was approximately 2S by KCl extraction (which was inefficient) or 4S by trypsin extraction. We conclude that the BZT of the day-11 rat placenta contains an estrogen-binding molecule with many of the attributes of a true receptor.