Gonadotropin-positive pituitary tumors accompanied by ovarian tumors in aging female ERbeta-/- mice

Abstract

At 2 years of age, 100% (23/23) of ERbeta(-/-) female mice have developed large pituitary and ovarian tumors. The pituitary tumors are gonadotropin-positive and the ovarian tumors are sex cord (less differentiated) and granulosa cell tumors (differentiated and estrogen secreting). No male mice had pituitary tumors and no pituitary or ovarian tumors developed in ERalpha(-/-) mice or in ERalphabeta(-/-) double knockout mice. The tumors have high proliferation indices, are ERalpha-positive, ERbeta-negative, and express high levels of nuclear phospho-SMAD3. Mice with granulosa cell tumors also had hyperproliferative endometria. The cause of the pituitary tumors appeared to be excessive secretion of gonadotropin releasing hormone (GnRH) from the hypothalamus resulting from high expression of NPY. The ovarian phenotype is similar to that seen in mice where inhibin is ablated. The data indicate that ERbeta plays an important role in regulating GnRH secretion. We suggest that in the absence of ERbeta, the proliferative action of FSH/SMAD3 is unopposed and the high proliferation leads to the development of ovarian tumors. The absence of tumors in the ERalphabeta(-/-) mice suggests that tumor development requires the presence of ERalpha.

Fig. 1.

Gross morphology and histology of pituitaries of 2-year-old female WT mice and ERβ^−/− mice. Large pituitary tumors were found in 2-year-old ERβ^−/− female mice, whereas there were none in 2-year-old WT or in male mice (A). (B) A ventral view of the brain of an ERβ^−/− mouse with a macropituitary adenoma. Sections from WT male (D) and ERβ^−/− male (F) were apparently normal, containing mostly acidophilic cells. (C) In WT female mice, the anterior lobe contained mostly acidophilic cells with few mitotic and apoptotic cells. (E) Anterior pituitaries of ERβ^−/− female mice were markedly enlarged and contained mostly basophilic cells with clear mitotic figures. (Scale bars: 500 μm in C–F.)

Fig. 2.

Ovarian tumors in 2-year-old ERβ^−/− mice. Ovaries and uterine horns from WT mice appear to be of normal size and appearance (A). Bilateral ovarian tumors were present in ERβ^−/− mice (B). Normal histological structure of the ovary of a 2-year-old WT female mouse (C). (D) A higher magnification showing absence of mitotic cells. Example of a granulosa cell tumor in ERβ^−/− female mice (E). A higher magnification is shown (F), demonstrating evident mitotic cells (red arrow). (G) Example of an undifferentiated sex cord cell ovarian tumor in ERβ^−/− female mice. (H) A higher magnification showing evident mitotic cells and apoptotic cells (red arrow). (Scale bars: 0.5 mm in C, E, and G; 50 μm in D, F, and H.)

Fig. 3.

Immunohistological analysis of pituitary tumors. Serial sections of pituitary from WT (A, C, and E) and ERβ^−/− (B, D, and F) female mice were stained using gonadotrope marker (FSH/LH), and nongonadotrope markers (ACTH and prolactin). In ERβ^−/− female mice, specific staining shows that anterior pituitary tumor was of composed of gonadotropes, whereas the three markers were expressed equally in WT female mice. PTTG expression was high in ERβ^−/− female mice (H); in contrast, few PTTG-positive cells were found in WT female mice (G). (Scale bars: 100 μm.)

Fig. 4.

Overexpression of ERα and Ki67 in pituitary, uterus, and ovary of 2-year-old ERβ^−/− female mice. Pituitary (A and B), uterus (C and D), and ovary (I and J) of WT female mice expressed very low levels of ERα (A, C, and I) and Ki67 (B, D, and J). In contrast, high expression levels of ERα (E) and Ki67 (F) were seen in pituitaries of ERβ^−/− female mice. Granulosa cell ovarian tumors in ERβ^−/− female mice expressed high levels of ERα (M) and Ki67 (N). The uteri of these mice also expressed high levels of ERα (G) and Ki67 (H). In mice with undifferentiated sex cord tumors there was low level of ERα (K) and Ki67 (L) in uterus, but high expression of ERα (O) and Ki67 (P) in the ovary. (Scale bars: 50 μm.)

Fig. 5.

Ovarian tumors in ERβ^−/− mouse and endometrial hyperplasia. PhosphoSMAD2/3 was highly expressed in the granulosa cell tumors from 2-year-old ERβ^−/− mice (A). There was no positive staining for PhosphoSMAD1/5/8 in the granulosa cell tumors in ERβ^−/− mouse ovary (B). There was extreme endometrial hyperplasia with epithelial invasion of the stroma in ERβ^−/− mice at 18 months of age (C and D). (Scale bars: 50 μm.)

Fig. 6.

GnRH (LHRH)-expressing neurons in the hypothalami of 2-year-old ERβ^−/− female mice. The number of cells expressing GnRH (LHRH) in the medial septal nucleus (C) and medial preoptic nucleus (D) of ERβ^−/− female mice was higher than that seen in WT controls (A and B). The total number of GnRH (LHRH)-positive neurons in the medial septal nucleus and medial preoptic nucleus was counted in 10 sections from each mouse (E). There was a more than twofold increase in the number of GnRH (LHRH)-positive neurons in the hypothalami of ERβ^−/− female mice. (MS: medial septal; MPN: medial preoptic nucleus) (Scale bars: 50 μm.)

Fig. 7.

NPY expression in the hypothalami of ERβ^−/− mice. NPY expression in paraventricular nucleus (D), arcuate nucleus (E), and medial eminence (F) was higher in 2-year-old ERβ^−/− female mice than in WT female mice (A–C). (PVN, paraventricular nucleus; ARC, arcuate nucleus; ME, medial eminence) (Scale bars: 50 μm.)