Gonadotropin positive pituitary tumors accompanied by ovarian

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					Gonadotropin-positive pituitary tumors accompanied
by ovarian tumors in aging female ERβ−/− mice
Xiaotang Fana,b, Chiara Gabbia,c, Hyun-Jin Kimc, Guojun Chenga, Leif C. Anderssond, Margaret Warnera,c, and
Jan-Åke Gustafssona,c,1
 Division of Medical Nutrition, Department of Biosciences and Nutrition, Karolinska Institute, Novum 141 86, Sweden; bDepartment of Histology and
Embryology, Third Military Medical University, Chongqing 400038, China; cCenter for Nuclear Receptors and Cell Signaling, University of Houston, Houston,
TX 77204; and dDepartment of Pathology, Haartman Institute, University of Helsinki, FIN-00014 Helsinki, Finland

Contributed by Jan-Åke Gustafsson, February 19, 2010 (sent for review December 9, 2009)

At 2 years of age, 100% (23/23) of ERβ−/− female mice have devel-             dependent cell proliferation (22, 23). In many epithelial tissues
oped large pituitary and ovarian tumors. The pituitary tumors are             studied, ERα mediates the proliferative response to estrogen,
gonadotropin-positive and the ovarian tumors are sex cord (less               whereas ERβ represses proliferation and induces differentiation
differentiated) and granulosa cell tumors (differentiated and estro-          (24). We have shown that there is epithelial hyperproliferation and
gen secreting). No male mice had pituitary tumors and no pituitary            poor cellular differentiation in the prostate (25), colon (26), uterus
or ovarian tumors developed in ERα−/− mice or in ERαβ−/− double               (27), and mammary gland (28) of ERβ−/− mice. Furthermore,
knockout mice. The tumors have high proliferation indices, are ERα-           numerous clinical and in vitro studies suggest that imbalanced
positive, ERβ-negative, and express high levels of nuclear phospho-           ERα and ERβ expression is a common feature and could be a
SMAD3. Mice with granulosa cell tumors also had hyperprolifera-               critical step of estrogen-dependent tumor progression. These
tive endometria. The cause of the pituitary tumors appeared to be             include the better prognosis of breast cancers, which express ERβ
excessive secretion of gonadotropin releasing hormone (GnRH)                  (29–31); the protective role of ERβ in development of colon
from the hypothalamus resulting from high expression of NPY.                  cancer (32); the tumor suppressor role of ERβ in malignant mes-
The ovarian phenotype is similar to that seen in mice where inhibin
                                                                              othelioma (33); the proapoptotic role of the vitamin E metabolite
is ablated. The data indicate that ERβ plays an important role in
                                                                              tocotrienol, an ERβ ligand in breast cancer cells (34); and the
regulating GnRH secretion. We suggest that in the absence of ERβ,
                                                                              effect of ERβ agonist on expression of TMPRSS2-ERG (a marker
the proliferative action of FSH/SMAD3 is unopposed and the high
                                                                              of very aggressive prostate cancer) (35). Introduction of ERβ into
proliferation leads to the development of ovarian tumors. The
                                                                              cancer cell lines reduces their proliferation in cell culture as well as
absence of tumors in the ERαβ−/− mice suggests that tumor develop-
ment requires the presence of ERα.
                                                                              in s.c. implants in immune compromised mice (36).
                                                                                 In the present study, we report that ERβ−/− female mice spon-
                                                                              taneously develop pituitary and ovarian tumors. No tumors
                         |          |
estrogen receptor β (ERβ) pituitary gonadotropin releasing hormone
                                                                              developed in ERβ−/− male mice and none were detected in ERα−/−
       |                                   |
(GnRH) follicle stimulating hormone (FSH) TGFβ
                                                                              or double knockout ERαβ−/− female mice. The ovarian tumors
                                                                              seem to have an overactivity of the TGFβ signaling pathway.
T   he hypothalamic-pituitary-gonadal (HPG) axis regulates
    reproductive function (1). Estrogen plays numerous modu-
latory roles within all three components of the HPG axis including
                                                                              Pituitary and Ovarian Tumors in 2-Year-Old ERβ−/− Female Mice. In
hypothalamus, pituitary, and gonads and is necessary for main-                the present study, there were pituitary tumors in 23/23 of the female
tenance of proper function of the HPG axis (2). In mammals,                   ERβ−/− mice 20 to 24 months of age. Spontaneous tumorigenesis
steroid hormones produced in the gonads are responsible for                   was not observed in WT or ERα−/− age-matched male mice or in
negative feedback on the HPG axis. In the pituitary, estrogen via             ERα−/− or ERαβ−/− female mice (Table 1). Four of the 23 WT
ERα modulates release of luteinizing hormone (LH) and follicle-               female mice developed pituitary tumors. The absence of tumors in
stimulating hormone (FSH) (3–6).                                              both ERα−/− and ERαβ−/− mice suggests that tumor development
   Both estrogen receptors, ERα and ERβ (7, 8), are expressed in              requires the presence of ERα. In most cases, visual examination of
the HPG axis, but each receptor has a distinct tissue expression
                                                                              tumors revealed enlargement of the anterior lobe. In ERβ−/−female
pattern (9). Several studies have shown that in the rodent and
                                                                              mice, the pituitary tumors were large (diameter > 3 mm), whereas
human hypothalamus, the GnRH neurons express ERβ but not
                                                                              four pituitary tumors formed in WT female mice were small-size
ERα (10, 11). ERβ appears to regulate the amount of GnRH
                                                                              tumors (diameter <3 mm) (Fig. 1 A and B).
secreted and lack of ERβ might lead to hyperstimulation of the
                                                                                 Histological staining showed that pituitary sections from WT
pituitary (12, 13). In the anterior lobe of the pituitary, ERα is the
                                                                              (Fig. 1D) and ERβ−/− (Fig. 1F) males were apparently normal,
predominant receptor and expression of ERβ is very low (14–16).
                                                                              containing mostly acidophilic cells (including somatotropes and
In the ovary, ERα is the predominant receptor in interstitial and
                                                                                                                                                                             MEDICAL SCIENCES

                                                                              lactotropes), typical sinusoid vasculature features and the rare
thecal cells, whereas ERβ is localized in the granulosa cells of
                                                                              occurrence of either mitotic and apoptotic cells. Pituitary sections
growing follicles, which are the primary sites of FSH action and
                                                                              from aging WT female mice (Fig. 1C) showed enlarged anterior
estradiol synthesis (17, 18). Studies of ERα and ERβ knockout
                                                                              lobes containing mostly acidophilic cells with few mitotic and
mice have revealed that ERα, but not ERβ, is indispensable for the
                                                                              apoptotic cells. In pituitary sections from ERβ−/− female mice
negative-feedback effects of estradiol and thus maintenance of
                                                                              (Fig. 1E), the anterior pituitary was very enlarged with mostly
proper LH secretion from the pituitary. ERα−/− mice are charac-
terized by hemorrhagic follicles and infertility (19). ERβ-mediated
estradiol actions are vital to FSH-induced granulosa cell differ-
                                                                              Author contributions: X.F., M.W., and J.-Å.G. designed research; X.F., C.G., and H.-J.K.
entiation, and inactivation of ERβ leads to defects in follicular             performed research; C.G. and G.C. contributed new reagents/analytic tools; X.F., C.G.,
maturation at the antral stage (20, 21). Thus ERα and ERβ play                H.-J.K., G.C., L.C.A., M.W., and J.-Å.G. analyzed data; and X.F., H.-J.K., M.W., and J.-Å.G.
different roles in modulation of HPG function.                                wrote the paper.
   There is growing evidence that ERα and ERβ also affect growth              The authors declare no conflict of interest.
and differentiation: in cell lines ERβ negatively regulates ERα-              1
                                                                               To whom correspondence should be addressed. E-mail:                                                          PNAS | April 6, 2010 | vol. 107 | no. 14 | 6453–6458
Table 1. Pituitary tumors in 2-year-old ERβ−/− mice
                                            Pituitary tumors/animals

Genotype                           Female                              Male

WT                                   4/23                           0/12
ERβ−/−                              23/23                           0/17
ERα−/−                               0/6                           ND/ND
ERαβ−/−                              0/7                           ND/ND

basophilic cells. Both mitotic figures and apoptotic cells were
abundant in pituitary tumors.
  Ovarian tumors were found in ERβ−/− female mice with mac-
ropituitary tumors (Fig. 2B). No ovarian tumors and no hyper-
plasia were found in any of the WT female mice (Fig. 2A).
  The ovaries of WT female mice were of small size without
detectable mitotic cells (Fig. 2 C and D). In 2-year-old ERβ−/−
female mice, the commonest ovarian tumors were of granulosa
cell (Fig. 2 E and F) and undifferentiated sex cord cell types (Fig.
2 G and H) with abundant mitotic cells and apoptotic cells.

Immunohistological Analyses of Pituitary Tumors. To characterize
pituitary hormone expression in the tumors, histological analyses
were performed on pituitary tumors. We analyzed the expression

                                                                              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 magnifica-
                                                                              tion 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.)

                                                                              patterns of the markers of the mature pituitary gonadotropes (FSH/
                                                                              LH), corticotropes [adrenocorticotropic hormone (ACTH)] and
                                                                              lactotropes (prolactin) (Fig. 3 A–F). Immunohistochemical analysis
                                                                              of pituitary sections with specific markers revealed that 21/23 of
                                                                              pituitary tumors of ERβ−/− female mice contained mostly FSH/LH-
                                                                              positive cells (Table 2). In the very large pituitary tumors, tissue
                                                                              structure was markedly disorganized and immunoreactivity for
                                                                              pituitary hormones diminished. Two macropituitary tumors were
                                                                              positive for gonadotropes, as well as for corticotropes and thyro-
                                                                              tropes. Gonadotropes were observed only at the periphery of the
Fig. 1. Gross morphology and histology of pituitaries of 2-year-old female    tissue, with no staining detected in the core of the gland where the
WT mice and ERβ−/− mice. Large pituitary tumors were found in 2-year-old      structure was completely destroyed. Two macropituitary tumors were
ERβ−/− female mice, whereas there were none in 2-year-old WT or in male       negative for all three different markers. One small ERβ−/− pituitary
mice (A). (B) A ventral view of the brain of an ERβ−/− mouse with a macro-    tumor was positive for both FSH/LH and ACTH, whereas in the four
pituitary adenoma. Sections from WT male (D) and ERβ−/− male (F) were
apparently normal, containing mostly acidophilic cells. (C) In WT female
                                                                              pituitary tumors found in WT female mice, two tumors were positive
mice, the anterior lobe contained mostly acidophilic cells with few mitotic   for prolactin and two other tumors were positive for FSH/LH.
and apoptotic cells. (E) Anterior pituitaries of ERβ−/− female mice were         Pituitary tumor-transforming gene (PTTG) is known to induce
markedly enlarged and contained mostly basophilic cells with clear mitotic    angiogenesis during pituitary tumorigenesis. PTTG regulates
figures. (Scale bars: 500 μm in C–F.)                                          βFGF secretion and is under the control of estrogen (37). It is

6454 |                                                                                                Fan et al.
                                                                                Overexpression of ERα and Ki67 in Pituitary, Uterus, and Ovary of
                                                                                2-Year-Old ERβ−/− Female Mice. With the aim of evaluating the
                                                                                relationship between presence of ERα and cell proliferation, we
                                                                                checked ERα and Ki67 on sequential sections of pituitary tumors,
                                                                                uteri, and ovaries. In 2-year-old WT female mice, expression of both
                                                                                ERα and Ki67 was very low in pituitary glands (Fig. 4 A and B) and
                                                                                ovaries (Fig. 4 I and J). As expected, ERα expression was high in the
                                                                                uterus (Fig. 4C), but there were very few Ki67-positive cells (Fig.
                                                                                4D). In ERβ−/− female mice, expression of ERα and Ki67 was high
                                                                                in the anterior pituitary lobe and most of Ki67-positive cells local-
                                                                                ized in the ERα-positive regions (Fig. 4 E and F). In the uterus (Fig.
                                                                                4 G, H, K, and L) and ovary (Fig. 4 M–P), both ERα and Ki67 were
                                                                                highly expressed in ERβ−/− female mice with granulosa cell and
                                                                                undifferentiated sex cord cell ovarian tumors.

                                                                                TGFβ Signaling in ERβ−/− Mouse Ovaries. PhosphoSMAD2/3, a marker
                                                                                for the TGFβ pathway was highly expressed in the nuclei of ovarian
                                                                                tumor cells (Fig. 5A), although there was no positive staining for
                                                                                phosphoSMAD1/5/8, a marker for BMP activation (Fig.5B).

                                                                                Age at Which Ovarian Tumors Develop. Because of the high mitotic
                                                                                indices in the ovarian tumors in 2-year-old mice, we anticipated
                                                                                that these were very rapidly growing tumors and were initiated
                                                                                after the mice were one-year-old. We therefore examined female
                                                                                ERβ−/− mice at 15, 17, and 18 months of age to determine when the
                                                                                ovarian and pituitary tumors became evident. There were four
                                                                                mice in each age group, and all of these mice had ovarian and
                                                                                pituitary tumors but they were much smaller than those found in
                                                                                2-year-old mice. Ovarian hyperplasia was observed in ERβ−/−
                                                                                female mice with micropituitary tumors. All of the ovarian tumors
                                                                                were granulosa cell tumors, and they appeared to be secreting
                                                                                estrogen because there was hyperplasia and a high proliferation
                                                                                index in the endometrium of these mice. In some mice, there was
                                                                                extreme endometrial hyperplasia with epithelial invasion of the
Fig. 3. Immunohistological analysis of pituitary tumors. Serial sections of     stroma (Fig. 5 C and D). This observation is compatible with our
pituitary from WT (A, C, and E) and ERβ−/− (B, D, and F) female mice were       previous finding that in ERβ−/− mice the proliferative response of
stained using gonadotrope marker (FSH/LH), and nongonadotrope markers           the uterus to estrogen is exaggerated (27).
(ACTH and prolactin). In ERβ−/− female mice, specific staining shows that
anterior pituitary tumor was of composed of gonadotropes, whereas the           Overexpression of GnRH (LHRH-Positive) and NPY in the Hypothalamus
three markers were expressed equally in WT female mice. PTTG expression         of ERβ−/− Female Mice. Recent identification of ERβ within GnRH
was high in ERβ−/− female mice (H); in contrast, few PTTG-positive cells were   neurons of the rodent and human brain suggests that estrogens
found in WT female mice (G). (Scale bars: 100 μm.)                              may exert direct actions upon GnRH neurons through ERβ. Using
                                                                                immunohistochemistry to identify GnRH-positive cells, we found
                                                                                that the expression pattern of GnRH in the hypothalamus was
expressed at low levels in normal human tissues but is abundant in
                                                                                similar in ERβ−/− and WT female mouse brains. There was a
cancer cell lines and in pituitary tumors. Immunohistochemical                  twofold increase (Fig.6E) in the number of GnRH-positive cells in
staining revealed that PTTG was localized in the cytoplasm of cells             medial septal nucleus and medial preoptic nucleus of ERβ−/−
in the mouse pituitary tumors. In ERβ−/− female mice, the 11                    female mouse hypothalami (Fig. 6 C and D) compared to WT (Fig.
macropituitary tumors were strongly stained for PTTG, whereas                   6 A and B) female mouse hypothalami. There is evidence that
the nine micro pituitary tumors were less stained. No PTTG was                  estrogens can affect NPY synthesis in the arcuate nucleus (ARC)
detected in WT female mouse pituitaries (Fig. 3 G and H).                       and increase release of NPY in the paraventricular nucleus (PVN)
                                                                                through receptors localized in NPY neurons (38). NPY colo-
                                                                                calized with GnRH in neurons in the median eminence (ME),
                                                                                PVN, and ARC of the hypothalamus. NPY expression was higher
                                                                                in ERβ−/− (Fig. 7 D–F) female mice than in WT (Fig. 7 A–C)
                                                                                                                                                          MEDICAL SCIENCES

Table 2. Immunohistological analyses of pituitary tumors of 2-
year old ERβ−/− female mice                                                     female mice. These data suggest that NPY overexpression con-
       Pituitary tumor                              IHC analysis                tributes to a high level of GnRH in the hypothalamus of 2-year-old
                                                                                ERβ−/− female mice.
Number             Diameter             Pituitary hormone              PTTC
                                                                                ERα and ERβ are differently regulated in estrogen-sensitive tissues.
11                 >3    mm                    FSH                      +++
                                                                                In vitro studies have demonstrated that the ERα/ERβ ratio can
9                  <3    mm                    FSH                      ++
                                                                                affect cell proliferation (39). In agreement with this, loss of ERβ has
2                  >3    mm                     −                        −
                                                                                been found to increase susceptibility of tissues to estrogen-induced
1                  <3    mm                 FSH, ACTH                    −
                                                                                carcinogenesis in several animal models: ERβ deficiency enhances
                                                                                small intestinal tumorigenesis (40) and down-regulation of ERβ and
2                  <3 mm                     Prolactin                   −
                                                                                the coregulator SNURF/RNF4 genes contributes to testicular
2                  <3 mm                       FSH                       −
                                                                                tumorigenesis (41). The question still remains as to whether loss of

Fan et al.                                                                                             PNAS | April 6, 2010 | vol. 107 | no. 14 | 6455
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.)

ERβ alone can lead to spontaneous tumorigenesis. In the present                           pituitary hyperplasia. Moreover, a recent study reported one case of
study, we report that loss of ERβ leads to spontaneous tumori-                            gonadotrope macroadenoma and two cases of gonadotrope cell
genesis in both the pituitary and ovary in female mice 15 to 24                           hyperplasia in patients with Klinefelter syndrome probably due to
months of age. Because there is no spontaneous tumorigenesis in                           continuous stimulation of gonadotropes because of lack of andro-
ERα−/− or ERαβ−/− female mice, it appears that tumor develop-                             gen feedback (43). In the hypothalamus, there were more GnRH-
ment requires the presence of ERα. Our study showed that levels of                        positive neurons in 2-year-old ERβ−/− female mice than in age-
ERα in the pituitary, ovary, and uterus were significantly higher in                       matched WT mice suggesting that increased GnRH neurons in the
ERβ−/− mice than in WT mice. Staining of sequential sections                              hypothalamus may contribute to gonadotrope macroadenoma.
showed that high expression of ERα was accompanied by a high                                 Neuropeptide Y has been reported to be involved in the regu-
level of proliferation measured with Ki67-specific antibodies. We                          lation of GnRH neuronal function (44). In the rat, NPY stimulates
conclude that a change in ERα/ERβ ratio in favor of ERα can lead to                       GnRH release from the hypothalamus in the presence of estrogen,
spontaneous tumorigenesis in both the pituitary and ovary.                                whereas it inhibits GnRH during estrogen deprivation (45). In the
   Histological staining showed a markedly enlarged anterior                              present study, NPY expression in the PVN, ARC, and ME of
pituitary containing mostly basophilic cells in ERβ−/− female mice.                       hypothalamus was increased significantly compared to WT female
Immunohistochemical analysis with specific markers revealed that                           mice. This indicates that NPY overexpression in the hypothalamus
most of pituitary tumors in ERβ−/− female mice were FSH/LH                                of 2-year-old ERβ−/− female mice mediates GnRH release, leading
positive. Little is known about the molecular pathogenesis of                             to pituitary hyperplasia.
gonadotrope-specific pituitary tumors. Development of pituitary                               Recent identification of ERβ within GnRH neurons of the rodent
adenoma is considered to be a multistep process including genetic                         and human brains indicates that estrogens may exert direct actions
alterations and hormone-induced proliferation (42). The effect of                         upon GnRH neurons exclusively through ERβ. Loss of ERβ may
hormone stimulation on the formation of pituitary adenoma is                              also directly affect GnRH neurons, but further studies are needed to
supported by evidence that pregnancy and hypothyroidism lead to                           explore the biological significance of this pathway.

6456 |                                                                                                                    Fan et al.
                                                                                    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)
Fig. 5. Ovarian tumors in ERβ−/− mouse and endometrial hyperplasia.                 (Scale bars: 50 μm.)
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               ERβ in the uterus. The uterus is thought of as an ERα-regulated
extreme endometrial hyperplasia with epithelial invasion of the stroma in           tissue. We have shown an exaggerated proliferative response of
ERβ−/− mice at 18 months of age (C and D). (Scale bars: 50 μm.)                     the uterus to estrogen in our ERβ−/− mice (27).
                                                                                       There is some argument as to why the phenotype of the ERβ−/−
                                                                                    mice produced in the laboratory of Oliver Smithies (47) is different
   In ERβ−/− mice gonadotropin-positive pituitary tumors were                       from the phenotype of those produced by the Chambon Lab in
accompanied by ovarian sex cord tumors. They are characterized                      Strasbourg (48). The Strasbourg mice are reported to be com-
by high levels of proliferation and apoptosis. In the present study,                pletely normal except for infertility in the female mice. The
we found that in the sex cord ovarian tumors there is increased LH                  Smithies female mice are also infertile and, as reported in the
receptor and SMAD3 expression. This is similar to mice in which                     present study, have abnormalities in GnRH secretion. The big
inhibin has been inactivated (46). We conclude that in the absence                  difference between the two knockout strains is that in the Smithies
of ERβ, there is a defect in the inhibitory wing of the TGFβ sig-                   mouse, the neo cassette remains in the ERβ gene but has been
naling pathway and the proliferative actions of FSH/SMAD3                           removed in the Strasbourg mouse. So unless the Strasbourg mice
continue unopposed. In addition, high expression of ERα con-                        are infertile for a completely different reason than are the Smithies
tributes to the malignancy of the tumors by increasing estrogen-                    mice, it is difficult to understand why they do not develop the
stimulated proliferation.                                                           abnormalities, which we have described in the present study.
   The marked hyperplasia seen in the endometrium of the mice                          In conclusion, this study demonstrates a role for ERβ in pituitary
bearing granulosa cell tumors is supportive evidence for a role of                  and ovarian tumorigenesis in aging mice and indicates that ERβ
                                                                                    directly modulates proliferation in estrogen sensitive tissues. The
                                                                                    data are supportive evidence for other studies, which show that the
                                                                                    ERα/ERβ ratio is important for controlling cellular proliferation.
                                                                                    Further studies are needed to explore the mechanism behind the
                                                                                    increase in ERα expression when ERβ is inactivated because this is
                                                                                    a pattern seen in most cancers.
                                                                                    Materials and Methods
                                                                                    Animals and Tissue Preparation. ERβ−/− mice were generated as described (47).
                                                                                    Heterozygous mice were used for breeding. All animals were housed in the
                                                                                    Karolinska University Hospital animal facility (Huddinge, Sweden) in a con-
                                                                                    trolled environment on a 12-h light/12-h dark illumination schedule and fed a
                                                                                    standard pellet diet with water provided ad libitum. To obtain tissues, mice
                                                                                    were anesthetized deeply with CO2 and perfused with PBS followed by 4%
                                                                                    paraformaldehyde (in 0.1 M PBS, pH 7.4). Tissues were collected and postfixed
                                                                                    in the same fixative overnight at 4 °C. After fixation, tissues were processed
                                                                                    into either paraffin (6 μm) or frozen (30 μm) sections. All animal experiments
                                                                                    were approved by Stockholm’s Södra Försöksdjursetiska Nämnd.

                                                                                    Immunohistochemistry. Paraffin sections were deparaffinized in xylene,
                                                                                    rehydrated through graded alcohol, and processed for antigen retrieval by
                                                                                                                                                                         MEDICAL SCIENCES

                                                                                    boiling in 10 mM citrate buffer (pH 6.0) for 5 min. The sections were incubated in
                                                                                    0.5% H2O2 in PBS for 30 min at room temperature to quench endogenous
                                                                                    peroxidase and then were incubated in 0.5% Triton X-100 in PBS for 30 min. To
                                                                                    block nonspecific binding, sections were incubated in 3% BSA for 1 h at 4 °C.
                                                                                    Sections were then incubated with anti-phosphoSMAD 2/3 (Chemicon), anti-
                                                                                    phosphoSMAD 1/5/8 (Chemicon), anti-LHRH (GnRH) (Chemicon), anti-FSH/LH
                                                                                    (Chemicon), anti-ACTH (Chemicon), anti-PTTG (Zymed Lab), anti-prolactin
Fig. 6. GnRH (LHRH)-expressing neurons in the hypothalami of 2-year-old ERβ−/−      (DAKO), anti-ERα (Santa Cruz Biotechnology), anti-Ki67 (Novocastra), and
female mice. The number of cells expressing GnRH (LHRH) in the medial septal        anti-NPY (Peninsular Laboratories) in 1% BSA and 0.1% Triton X-100 over-
nucleus (C) and medial preoptic nucleus (D) of ERβ−/− female mice was higher than   night at room temperature. BSA replaced primary antibodies in negative
that seen in WT controls (A and B). The total number of GnRH (LHRH)-positive        controls. After washing, sections were incubated with the corresponding
neurons in the medial septal nucleus and medial preoptic nucleus was counted in     secondary biotinylated antibodies in 1:200 dilutions for 2 h at room temper-
10 sections from each mouse (E). There was a more than twofold increase in the      ature. The Vectastain ABC kit (Vector Laboratories) was used for the avidin-
number of GnRH (LHRH)-positive neurons in the hypothalami of ERβ−/− female          biotin complex (ABC) method according to the manufacturer’s instructions.
mice. (MS: medial septal; MPN: medial preoptic nucleus) (Scale bars: 50 μm.)        Peroxidase activity was visualized with 3, 3-diaminobenzidine (DAKO). The

Fan et al.                                                                                                     PNAS | April 6, 2010 | vol. 107 | no. 14 | 6457
sections were lightly counterstained with hematoxylin, dehydrated through                   20× magnification. GnRH-positive neurons were visualized with GnRH anti-
an ethanol series to xylene, and mounted.                                                   body and counted manually on the captured images. Estimates of the
                                                                                            number of GnRH-positive neurons were made based on the counts of the 10
Data Analysis. Stained brain sections (10–12 sections per area for each mouse)              images showing the highest number of GnRH-labeled neurons. Statistical
were examined under a light microscope, and images were captured under                      analysis was performed using Student’s t test.

 1. Gharib SD, Wierman ME, Shupnik MA, Chin WW (1990) Molecular biology of the              26. Wada-Hiraike O, et al. (2006) Role of estrogen receptor beta in colonic epithelium.
    pituitary gonadotropins. Endocr Rev 11:177–199.                                             Proc Natl Acad Sci USA 103:2959–2964.
 2. Petersen SL, Ottem EN, Carpenter CD (2003) Direct and indirect regulation of            27. Wada-Hiraike O, et al. (2006) Role of estrogen receptor beta in uterine stroma and
    gonadotropin-releasing hormone neurons by estradiol. Biol Reprod 69:1771–1778.              epithelium: Insights from estrogen receptor beta-/- mice. Proc Natl Acad Sci USA 103:
 3. Shupnik MA, Gharib SD, Chin WW (1988) Estrogen suppresses rat gonadotropin gene             18350–18355.
    transcription in vivo. Endocrinology 122:1842–1846.                                     28. Cheng G, Weihua Z, Warner M, Gustafsson JA (2004) Estrogen receptors ER alpha and
 4. Burger LL, Haisenleder DJ, Dalkin AC, Marshall JC (2004) Regulation of gonadotropin         ER beta in proliferation in the rodent mammary gland. Proc Natl Acad Sci USA 101:
    subunit gene transcription. J Mol Endocrinol 33:559–584.                                    3739–3746.
 5. Gharib SD, Wierman ME, Badger TM, Chin WW (1987) Sex steroid hormone regulation         29. Iwao K, Miyoshi Y, Egawa C, Ikeda N, Noguchi S (2000) Quantitative analysis of estrogen
    of follicle-stimulating hormone subunit messenger ribonucleic acid (mRNA) levels in         receptor-beta mRNA and its variants in human breast cancers. Int J Cancer 88:733–736.
    the rat. J Clin Invest 80:294–299.                                                      30. Shaaban AM, et al. (2003) Declining estrogen receptor-beta expression defines
 6. Shupnik MA, Gharib SD, Chin WW (1989) Divergent effects of estradiol on
                                                                                                malignant progression of human breast neoplasia. Am J Surg Pathol 27:1502–1512.
    gonadotropin gene transcription in pituitary fragments. Mol Endocrinol 3:474–480.
                                                                                            31. Roger P, et al. (2001) Decreased expression of estrogen receptor beta protein in
 7. Walter P, et al. (1985) Cloning of the human estrogen receptor cDNA. Proc Natl Acad
                                                                                                proliferative preinvasive mammary tumors. Cancer Res 61:2537–2541.
    Sci USA 82:7889–7893.
                                                                                            32. Mostafaie N, et al. (2009) Correlated downregulation of estrogen receptor beta and
 8. Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA (1996) Cloning of a
                                                                                                the circadian clock gene Per1 in human colorectal cancer. Mol Carcinog 48:642–647.
    novel receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 93:
                                                                                            33. Pinton G, et al. (2009) Estrogen receptor-beta affects the prognosis of human
                                                                                                malignant mesothelioma. Cancer Res 69:4598–4604.
 9. Hatoya S, et al. (2003) Expression of estrogen receptor alpha and beta genes in the
                                                                                            34. Comitato R, et al. (2009) A novel mechanism of natural vitamin E tocotrienol activity:
    mediobasal hypothalamus, pituitary and ovary during the canine estrous cycle.
                                                                                                Involvement of ERbeta signal transduction. Am J Physiol Endocrinol Metab 297:
    Neurosci Lett 347:131–135.
10. Hrabovszky E, et al. (2007) Gonadotropin-releasing hormone neurons express                  E427–E437.
    estrogen receptor-beta. J Clin Endocrinol Metab 92:2827–2830.                           35. Bonkhoff H, Berges R (2009) The evolving role of oestrogens and their receptors in
11. Skinner DCDL, Dufourny L (2005) Oestrogen receptor beta-immunoreactive neurones             the development and progression of prostate cancer. Eur Urol 55:533–542.
    in the ovine hypothalamus: Distribution and colocalisation with gonadotropin-           36. Hartman J, et al. (2009) Tumor repressive functions of estrogen receptor beta in
    releasing hormone. J Neuroendocrinol 17:29–39.                                              SW480 colon cancer cells. Cancer Res 69:6100–6106.
12. Merchenthaler I (2005) Estrogen stimulates galanin expression within luteinizing        37. Chesnokova V, Melmed S (2009) Pituitary tumour-transforming gene (PTTG) and
    hormone-releasing hormone-immunoreactive (LHRH-i) neurons via estrogen                      pituitary senescence. Horm Res 71 (Suppl 2):82–87.
    receptor-beta (ERbeta) in the female rat brain. Neuropeptides 39:341–343.               38. Hilke S, Holm L, Man K, Hökfelt T, Theodorsson E (2009) Rapid change of
13. Merchenthaler I, Hoffman GE, Lane MV (2005) Estrogen and estrogen receptor-beta             neuropeptide Y levels and gene-expression in the brain of ovariectomized mice after
    (ERbeta)-selective ligands induce galanin expression within gonadotropin hormone-           administration of 17beta-estradiol. Neuropeptides 43:327–332.
    releasing hormone-immunoreactive neurons in the female rat brain. Endocrinology         39. Matthews J, et al. (2006) Estrogen receptor (ER) beta modulates ERalpha-mediated
    146:2760–2765.                                                                              transcriptional activation by altering the recruitment of c-Fos and c-Jun to estrogen-
14. Kuiper GG, et al. (1997) Comparison of the ligand binding specificity and transcript         responsive promoters. Mol Endocrinol 20:534–543.
    tissue distribution of estrogen receptors alpha and beta. Endocrinology 138:863–870.    40. Weyant MJ, et al. (2001) Reciprocal expression of ERalpha and ERbeta is associated
15. Mitchner NA, Garlick C, Ben-Jonathan N (1998) Cellular distribution and gene                with estrogen-mediated modulation of intestinal tumorigenesis. Cancer Res 61:
    regulation of estrogen receptors alpha and beta in the rat pituitary gland.                 2547–2551.
    Endocrinology 139:3976–3983.                                                            41. Hirvonen-Santti SJ, et al. (2003) Down-regulation of estrogen receptor beta and
16. Scully KM, et al. (1997) Role of estrogen receptor-alpha in the anterior pituitary          transcriptional coregulator SNURF/RNF4 in testicular germ cell cancer. Eur Urol 44:
    gland. Mol Endocrinol 11:674–681.                                                           742–747, discussion 747.
17. Sar M, Welsch F (1999) Differential expression of estrogen receptor-beta and estrogen   42. Kastelan D, Korsic M (2007) High prevalence rate of pituitary incidentaloma: Is it
    receptor-alpha in the rat ovary. Endocrinology 140:963–971.                                 associated with the age-related decline of the sex hormones levels? Med Hypotheses
18. Pelletier G, Labrie C, Labrie F (2000) Localization of oestrogen receptor alpha,
    oestrogen receptor beta and androgen receptors in the rat reproductive organs.
                                                                                            43. Scheithauer BW, et al. (2005) The pituitary in klinefelter syndrome. Endocr Pathol 16:
    J Endocrinol 165:359–370.
19. Emmen JM, Korach KS (2003) Estrogen receptor knockout mice: Phenotypes in the
                                                                                            44. Gaikwad A, Biju KC, Muthal PL, Saha S, Subhedar N (2005) Role of neuropeptide Y in the
    female reproductive tract. Gynecol Endocrinol 17:169–176.
                                                                                                regulation of gonadotropin releasing hormone system in the forebrain of Clarias
20. Glidewell-Kenney C, et al. (2007) Nonclassical estrogen receptor alpha signaling
                                                                                                batrachus (Linn.): Immunocytochemistry and high performance liquid chromatography-
    mediates negative feedback in the female mouse reproductive axis. Proc Natl Acad Sci
                                                                                                electrospray ionization-mass spectrometric analysis. Neuroscience 133:267–279.
    USA 104:8173–8177.
                                                                                            45. Woller MJ, Campbell GT, Liu L, Steigerwalt RW, Blake CA (1993) Estrogen alters the
21. Drummond AE, Baillie AJ, Findlay JK (1999) Ovarian estrogen receptor alpha and beta
    mRNA expression: Impact of development and estrogen. Mol Cell Endocrinol 149:               effects of neuropeptide-Y on luteinizing hormone and follicle-stimulating hormone
    153–161.                                                                                    release in female rats at the level of the anterior pituitary gland. Endocrinology 133:
22. Liu MM, et al. (2002) Opposing action of estrogen receptors alpha and beta on cyclin        2675–2681.
    D1 gene expression. J Biol Chem 277:24353–24360.                                        46. Li Q, Graff JM, O’Connor AE, Loveland KL, Matzuk MM (2007) SMAD3 regulates
23. Matthews J, Gustafsson JA (2003) Estrogen signaling: A subtle balance between ER            gonadal tumorigenesis. Mol Endocrinol 21:2472–2486.
    alpha and ER beta. Mol Interv 3:281–292.                                                47. Krege JH, et al. (1998) Generation and reproductive phenotypes of mice lacking
24. Heldring N, et al. (2007) Estrogen receptors: How do they signal and what are their         estrogen receptor beta. Proc Natl Acad Sci USA 95:15677–15682.
    targets. Physiol Rev 87:905–931.                                                        48. Antal MC, Krust A, Chambon P, Mark M (2008) Sterility and absence of
25. Imamov O, et al. (2004) Estrogen receptor beta regulates epithelial cellular                histopathological defects in nonreproductive organs of a mouse ERbeta-null mutant.
    differentiation in the mouse ventral prostate. Proc Natl Acad Sci USA 101:9375–9380.        Proc Natl Acad Sci USA 105:2433–2438.

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