On day 4 of culture, groups of epithelial cells and stromal cells were incubated with Oe 2 (10 −8)-containing medium for 48 hr. Uterine stromal cells were isolated and grown to confluence in 24-well plates separately. Uterine epithelial cells were isolated and grown to confluence on cell culture inserts (four to six inserts/group). Oestradiol primes uterine epithelial cells for stromal cell effects on epithelial TNF-α release. While epithelial monolayer integrity is directly influenced by Oe(2), TNF-alpha release in response to Oe(2) is dependent on the presence of stromal cells, indicating that paracrine communication is necessary for steroid regulation of some but not all cytokines. These studies indicate that Oe(2) has both direct and indirect effects on the uterine epithelium. In contrast, TGF-beta release by epithelial cells was not affected by Oe(2) when grown alone or in the presence of stromal cells. As determined by oestrogen receptor antagonist studies, Oe(2) primed epithelial cells for the action of the stromal paracrine factor(s). However, when epithelial cells were cocultured with stromal cells and treated with Oe(2), apical TNF-alpha release was significantly decreased, compared to cells not treated with hormone. While Oe(2) treatment of epithelial cells led to a significant decrease in TER, the amount of TNF-alpha released was not altered. Supernatants collected were assayed for transforming growth factor-beta (TGF-beta) and tumour necrosis factor-alpha (TNF-alpha) by bioassay and enzyme-linked immunosorbent assay, respectively. Epithelial cells grown alone or in coculture with stromal cells were treated with Oe(2). Transepithelial resistance (TER) was monitored with an EVOM voltohmmeter to determine monolayer polarity and integrity. Mouse uterine epithelial and stromal cells were isolated and cultured separately. This study was undertaken to determine the mode of Oe(2) action in regulating epithelial cell cytokine release in the uterus. A primary role for epithelial-stromal interactions has been established for mediating steroid hormone action in the uterus. Oe(2) can act directly on the epithelium via the epithelial oestrogen receptor (OR) or indirectly via the OR-positive underlying stroma. Oestradiol-17beta (Oe(2)) stimulates uterine epithelial cell proliferation and is critical for normal uterine differentiation and secretory function.
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