Effect of estradiol on the ovarian surface epithelium in older mice

Abstract

The adult ovary is covered by a simple mesothelium that is an uncommitted phenotype with epithelial and stromal characteristics. The aging ovarian surface bears the morphological signature of repetitive ovulatory rupture and repair, a process always preceded by the localized production of high levels of estradiol. With time, surface invaginations and inclusion cysts become more common. Furthermore, older ovarian surface epithelium (OSE) exhibits cell layering and columnar-shaped cells suggesting focal metaplasia may occur with age. Approximately 90% of ovarian cancers arise from OSE. Common sites for the development of ovarian epithelial cancer are within inclusion cysts and in surface invaginations where OSE tends toward metaplasia. During transition to the cancerous state, OSE acquires complex epithelial characteristics aligned to Müllerian ductal epithelia, and loses its stromal characteristics.

The main hypothesis for this thesis is that periods of elevated ovarian estradiol contribute to aberrant differentiation of OSE causing it to acquire the phenotypic characteristics of Müllerian duct epithelia.

To test this hypothesis, 7-10 month old Swiss Webster mice were administered two subcutaneous injections of estradiol valerate (EV) given 14 days apart. Blood and ovarian tissue estradiol levels were measured by radioimmunoassay and showed a significant elevation in estradiol levels within 2 days of a single injection. Levels remained significantly elevated following second estradiol exposure, but the degree of elevation was approximately half that following initial exposure. Nevertheless, estradiol was found to accumulate in the blood and ovarian tissue over time. It was hypothesized that the older ovary may be more sensitive to estradiol's mitogenic effects, and that elevated estradiol levels in the ovary would promote morphologic alteration that included OSE hyperplasia, and metaplastic changes typical of differentiation toward the Müllerian phenotype. This was initially investigated with histology, BrdU immunohistochemistry and morphometry. Two days after EV treatment the ovary showed increases in surface area and volume. Ovarian constituent volumes revealed this response was primarily due to hypertrophy of OSE and possible OSE hyperplasia. Morphological analysis showed abundant evidence of metaplasia in OSE.

A second EV injection did not produce these changes. It was proposed this could be due to the ability of OSE to differentially regulate estrogen receptor (ER) expression, thereby limiting estradiol's effects on OSE. Histomorphometric alteration to OSE would likely involve cell-cell junctions and influence the expression of E-cadherin, a cell adhesion molecule and marker of OSE transition to Müllerian epithelia. Immunohistochemistry and immunofluorescence with confocal microscopy was used to show normal expression of Erα, ERβ and E-cadherin in older OSE and neighbouring oviduct. Expression of Erα and ERβ was predominately nuclear but both receptors also localized to cytoplasm, suggesting nuclear-cytoplasmic receptor shuttling. Differential expression of ER subtype and degree of colocalization appeared to differ with OSE cell shape, and between OSE and oviduct. Qualitative analysis suggested there was a large down-regulation of Erα in OSE and stroma, and ERβ in OSE after first EV injection. Strong Erα expression in OSE (but not stroma) and weak ERβ expression was evident following second estradiol exposure. Semi-quantitative analysis of ERβ protein expression by immunofluorescence profiling revealed an 11-fold decrease in ERβ expression had occurred after first EV injection (P<0.0001). In contrast E-cadherin expression increased after first and decreased following second estradiol exposure. These results suggest a role for estradiol in the aberrant differentiation of OSE.