Differential expression of claudin family proteins in mouse ovarian serous papillary epithelial adenoma in aging FSH receptor-deficient mutants

Abstract

Ovarian cancer is a deadly disease with long latency. To understand the consequences of loss of follicle-stimulating hormone receptor (FSH-R) signaling and to explore why the atrophic and anovulatory ovaries of follitropin receptor knockout (FORKO) mice develop different types of ovarian tumors, including serous papillary epithelial adenoma later in life, we used mRNA expression profiling to gain a comprehensive view of misregulated genes. Using real-time quantitative reverse transcription-polymerase chain reaction, protein analysis, and cellular localization, we show, for the first time, in vivo evidence that, in the absence of FSH-R signaling, claudin-3, claudin-4, and claudin-11 are selectively upregulated, whereas claudin-1 decreases in ovarian surface epithelium and tumors in comparison to wild type. In vitro experiments using a mouse ovarian surface epithelial cell line derived from wild-type females reveal direct hormonal influence on claudin proteins. Although recent studies suggest that cell junction proteins are differentially expressed in ovarian tumors in women, the etiology of such changes remains unclear. Our results suggest an altered hormonal environment resulting from FSH-R loss as a cause of early changes in tight junction proteins that predispose the ovary to late-onset tumors that occur with aging. More importantly, this study identifies claudin-11 overexpression in mouse ovarian serous cystadenoma.

Figure 1

Evidence for the development of serous epithelial tumors in FORKO mice. Ovarian histopathology of WT and FORKO mice at 12 months of age. Normal ovary at 12 months of age in wild-type mice containing antral follicles and corpora lutea (A and C). Appearance of serous tumors in FORKO mice at 12 months of age (B and D). The appearance of serous tumors was characterized by a tall, columnar, ciliated epithelial cell lining and a clear serous fluid filling the cystic space. Note that the remnant of the ovary is small, with few identifiable follicular structures compared with the tumor. (C) and (D) are high-power magnifications of (A) and (B), respectively.

Figure 2

Quantitative analysis of claudin expression. Q-PCR was performed for each of the indicated claudins (A, claudin-1; B, claudin-3; C, claudin-4; D, claudin-11) to confirm microarray results. mRNA expression is normalized to wild-type controls. To identify potential gonadotropin-responsive gene products, ovaries were collected from sexually immature 24-day-old wild-type and FORKO females 24 hours after administering 5 IU of eCG, an FSH-R agonist. This treatment is designed to activate FSH-responsive ovarian cells. Fold change values of eCG-treated +/+ mice are compared with vehicle-treated wild-type mice. WT, wild-type vehicle-treated; FORKO, follitropin receptor knockout with vehicle; WT + eCG, wild-type mice administered eCG; FORKO + eCG, FORKO mice administered eCG.

Figure 3

Circulating sex steroid hormones and aging in female mice. (A) Plasma levels of steroid hormones were measured by specific radioimmunoassays at 3 and 12 months of age. Testosterone (n = 11–15 mice) and estradiol (n = 12) ratios are shown depicting androgen dominance in FORKOs. (B) Plasma levels of progesterone (ng/ml) measured at 3 and 12 months (n = 8). Asterisks indicate significant differences from wild-type mice (P < .05).

Figure 4

Evidence for quantitative alterations in proteins associated with maintaining cell polarity in the ovary. A representative Western blot image of (A) claudin-1, (B) claudin-3, (C) claudin-4, and (D) claudin-11 in 8-month-old ovaries of wild-type and FORKO mice (before tumors appear) is shown. (E) To further understand the ontogeny of claudin-11 expression, claudin-11 expression was compared in age-matched, wild-type, and FORKO mice at 24 days, 3 months, 6 months, and 12 months. A representative image is shown here. Lanes 1, 3, 5, and 7 are protein extracts from wild-type mice. Lanes 2, 4, 6, and 8 are protein extracts from FORKO mice. Protein levels were determined by densitometry and corrected for protein loading using β-actin levels. Data are expressed as the percentage of wildtype expression from three different experiments. Asterisks indicate significant differences from wild-type mice (P < .05).

Figure 5

Cellular localization of TJ proteins in normal and tumor-prone mouse ovaries. Immunofluorescence analysis of claudin-1, claudin-3, claudin-4, and claudin-11 staining in 8-month-old ovaries of different wild-type (A–D), FORKO (E–H), and serous papillary epithelial adenoma (I–L) samples. In the ovaries, the predominant source of claudin expression is the epithelial cell population (arrows). Data are expressed as the percentage of wild-type expression. Asterisks indicate significant differences from wild-type mice (P < .05). Representative sections from different animals are shown. Nuclear staining revealed by propidium iodide is shown in red. Bars = 10 µm.

Figure 6

Potential candidates (mechanisms) for altering adhering and TJ proteins in mouse ovarian epithelial cells. The ID-8 epithelial cell line from normal mice (MOSEC) was starved of all serum and growth factors overnight before treatment with different concentrations of hormones, namely, highly purified hFSH (10 and 200 ng/ml) and hLH (10 and 200 ng/ml), 17β estradiol (10^-6 and 10^-9 M), testosterone (10^-6 and 10^-9 M), and progesterone (10^-6 and 10^-9 M) for 24 hours. Treatment of ID-8 cells with 200 ng/ml FSH induced a significant decrease in claudin-11 (P < .05). Although LH increased claudin-3, testosterone increased claudin-3, claudin-4, and claudin-11 levels. Data are expressed as the percentage of vehicle-treated control cells. Asterisks indicate significant differences (P < .05).

Figure 7

Schematic representation of hormonal imbalances leading to alterations affecting epithelial cell-cell interactions in the ovary. (A) Normal ovary: the presence of FSH-R ensures normal cyclicity of hormones and regulation of cell junction status in the OSE. (B) In the FORKO mouse model, the lack of beneficial effects of FSH-R signaling induces major perturbations in hormones that alter signal transduction pathway(s) and the internal milieu of the ovary, leading to alterations in OSE in mutants. Although we have not investigated different pathways, signaling through TGFβ, Wnt, and MAPK, which have been previously shown to be involved in regulating various cell junctions, is also depicted. Here we implicate that hormonal imbalances result in increases in claudin-3, claudin-4, and claudin-11, whose effects are transmitted downstream, causing ovarian surface epithelial cells to acquire the capacity to survive and migrate inside the ovary. Subsequently, serous papillary epithelial adenomas develop in aging FORKO mice.