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We have developed an estrogen bioassay using the Ishikawa human endometrial adenocarcinoma cell line growing in 96-well microtiter plates. Alkaline phosphatase enzyme activity (AlkP) in these cells is markedly stimulated by estrogens, and this enzyme can be easily quantified in situ using a chromogenic substrate. These cells are very sensitive to estrogens; estradiol induces AlkP at levels as low as 10(-12) M. Antiestrogens completely block the action of estradiol. Various estrogens stimulate AlkP with potencies comparable to those achieved in vivo. The induction of AlkP is specific for estrogens; no other type of steroid, including androgens, progestins, mineralocorticoids, or glucocorticoids produce this effect. The stimulation of AlkP in Ishikawa cells is specific for estrogens, is highly reproducible and sensitive, and permits large numbers of samples to be assayed with ease. We have used this assay to investigate the estrogenic action of the adrenal delta 5-3 beta-hydroxysteroids. While pregnenolone is inactive, dehydroepiandrosterone and its sulfate ester induce AlkP slightly. However, the C19 steroid, 5-androstene-3 beta, 17 beta-diol is considerably more estrogenic in this assay, although it stimulates Ishikawa AlkP with a potency of 1/30,000 that of estradiol. The stimulation by 5-androstene-3 beta,17 beta-diol is inhibited by antiestrogens, but it is not blocked by the delta 5-3 beta-hydroxysteroid isomerase/dehydrogenase inhibitor, cyanoketone, or by the aromatase inhibitor, 4-hydroxy-androstenedione. Thus, neither conversion to a delta 4-3-ketone nor aromatization is required for the action of this unusual estrogen.

Estradiol-17beta dehydrogenase activity in proliferative human endometrium (average of 1.5 nmole of estrone formed from estradiol/mg protein/h) was stimulated as much as as 6-fold during incubations of tissue slices in culture medium containing progesterone. Stimulation was already detectable at 7 h and the highest activity values were reached at 48-72 h of incubation in the presence of excess progesterone. Maximal stimulation was achieved with concentrations of the hormone of 0.25 mug/ml or higher. At concentrations approximately equal to midluteal plasma levels (20 ng/ml) more than 50% of the maximal response was observed. Norgestrel (17alpha-ethynyl-18-methyl-19-nortestosterone) was also effective in inducing enzymatic activity. The similarity of the effects obtained with progesterone (a possible substrate for estradiol dehydrogenase) and the synthetic progestin indicates that the stimulation of enzymatic activity was not due to substrate induction. Addition of estradiol to the culture medium had no influence on the activity of the enzyme. The induction of estradiol dehydrogenase by progesterone was inhibited by puromycin or actinomycin D. These observations indicate that progestational agents increase the rate of de novo synthesis of the enzyme. Stimulation of endometrial estradiol dehydrogenase was also observed after 2-3 day oral administration of medroxyprogesterone acetate to women in the follicular phase. In contrast, the enzymatic activity in endometrium obtained from women taking estrogens was found to be as low as in normal proliferative tissue. These in vitro and in vivo results point to progesterone as the agent responsible for the 10-fold increase in endometrial estradiol dehydrogenase activity observed during the luteal phase in menstruating women. Data obtained from superfusion studies of estrogen dynamics in endometrium indicate that changes in enzyme concentrations may play a physiologic role in the regulation of tissue levels of estradiol.

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Estradiol dehydrogenase (E 2 -DH) and20

In vitro decidualization of stromal cells isolated from human proliferative endometrium and cultured in RPMI-1640 containing 2% charcoal-treated fetal bovine serum and 0.1 U/ml insulin was achieved by adding to the medium cAMP derivatives [(Bu)2cAMP (db-cAMP) and 8-bromo-cAMP] or forskolin. PRL production under these conditions was demonstrated by documenting the synthesis of PRL mRNA (approximately 1.1 kilobase), the output of immunoprecipitable [35S]methionine-labeled PRL migrating as a single 23-kilodalton band during gel electrophoresis, and the time- and concentration-dependent secretion of PRL into the medium, measured by RIA (maximal on days 4-5 using 0.5 mM db-cAMP). Medroxyprogesterone acetate (1 microM) enhanced (1.7- to 2.5-fold) the effect of db-cAMP, 8-bromo-cAMP, or forskolin on PRL production, as evaluated by Western blotting analysis. Further evidence for a participation of db-cAMP in the decidualization process was provided by its ability to induce immunocytochemically detectable heat shock protein-27, insulin-like growth factor-binding protein-1, desmin, and laminin, all compounds produced by human decidual cells, but not expressed by stromal cells. The induction of PRL by cAMP may be a key step in the process of differentiation of fibroblast-like stromal cells to the decidual phenotype, as it has been previously reported by this laboratory that, under similar culture conditions, PRL itself is capable of inducing the production of heat shock protein-27, insulin-like growth factor-binding protein-1, desmin, and laminin in stromal cells isolated from proliferative endometrium.