Human Endometrial Cells in Primary Tissue Culture: Estrogen Interactions and Modulation of Cell Proliferation*

Abstract

Human endometrium, obtained from premenopausal women after hysterectomy, was dissociated into cells and placed in culture in order to examine estrogen responsiveness in a model in vitro system. Experiments which utilized cell proliferation as an index of responsiveness spanned at least 7 days and were performed in Dulbecco's modified Eagle's medium supplemented with 10% steroid-depleted serum from castrate-adrenalectomized calves. The addition of 2 × 1(10⁻⁷ M 17β-estradiol (E₂) or diethylstilbestrol (DES) resulted in a 2- to 5-fold optimal acceleration in the rate of proliferation, with stimulation first being detected at 2-4 days. Ten-fold higher or lower concentrations of E₂ or DES resulted in little stimulation, and high (2 × 10⁻⁵ M) concentrations of either estrogen stopped cell proliferation. The addition of E₂ or DES (2 × 10⁻⁸−2 × 10⁻⁶ M) to cultures of a putative nontarget cell line (Detroit- 98) derived from human sternum failed to elicit any increase in the rate of proliferation, although high E₂ and DES concentrations (2 × 10⁻⁵ M) also terminated proliferation in this cell line. The direct interaction of [³H]E₂ with endometrial cell cultures was studied. Cells were found to convert E₂ to estrone (ca. 50% conversion by 2 days). In addition, serum in the culture medium was found to reduce the amount of steroid that has access to the cells. The rate of E₂ entry into cells was temperature dependent and resembled a second order rate process (dependent on both ligand concentration and cell number). The rate of entry was not saturable, suggesting that entrance occurs by passive diffusion without the participation of a carrier. Steroid exit from cells was also temperature dependent and was characterized as a first order rate process having two components. At 37 C, exit was rapid and the rate constant (k₋₁) for the major (slow) component was 6 × 10⁻⁵ sec"1, a value consistent with the exit of receptor-associated estradiol. Of the 19 uterine cultures assessed for growth stimulation by added estrogen, 63% showed enhanced cell proliferation, while 37% showed no change in growth rate. Hormone specificity was examined further in five cell strains showing a proliferative response to DES. Of these five strains, three showed some enhancement in rate of proliferation in response to testosterone, two were stimulated by dihydrotestosterone, two were stimulated by dexamethasone, but none was stimulated by 17α-estradiol. These studies document the in vitro responsiveness of human endometrial cells to estrogens and other steroid hormones and suggest that this in vitro cell culture system will be useful in studying the multihormonal regulation of growth and function in human endometrium.