Novel approaches to quantify estradiol-induced loss of ERβ1 protein in older mouse ovarian surface epithelium: new tools to assess the role of ER protein subtypes in predisposing to ovarian epithelial cancer?

Loss of estrogen receptor-beta (ERβ) occurs in ovarian epithelial cancer (OEC), a cancer of mainly older women. OEC is linked epidemiologically to hormone replacement therapy, predominantly with estrogen-only formulations. This study introduces a novel, non-biased method to quantify levels of estradiol-induced loss of ERβ1 protein, and defines, for the first time, normal OSE expression patterns for ERα and ERβ1 with advancing age. Older (7-10 months) Swiss Webster mice were injected with estradiol valerate (EV) while age-matched diestrous controls received oil. Mice were culled after 48 h, and blood and one ovary were frozen for estradiol RIA. Contralateral ovaries were paraffin-embedded for immunohistochemistry. Subsets of serial sections, triple-labeled with immunofluroescent tags, were imaged with confocal microscopy to provide optimal visualization of ER protein subtype expression in OSE. Immunofluorescence emission profiles distinct to ERβ1 in OSE were standardized and quantified in control mice then compared to profiles from EV-exposed mice. Estradiol levels were significantly elevated in EV-treated mice, both in blood (p < 0.0001) and ovarian tissue (p < 0.001), resulting in 11-fold reduction in OSE expression of ERβ1 protein (p < 0.0001). In aging OSE, expression patterns of both ER subtypes varied within cells and with cell shape. ER co-localization appeared predominantly cytoplasmic and was infrequent in columnar compared to cuboidal-shaped OSE cells. Immunofluorescence emission profiling and multiple-label immunofluorescent tagging of ER using confocal microscopy, provides sharp definition of ER locus enabling concurrent qualitative and quantitative analysis of ER protein. It offers significant potential for assessing ER protein subtype status in predisposition to OEC.