Thyroid Papillary Microtumor: Validation of the (Updated) Porto Proposal Assessing Sex Hormone Receptor Expression and Mutational BRAF Gene Status.

Given the high incidence and excellent prognosis of many papillary thyroid microcarcinomas, the Porto proposal uses the designation papillary microtumor (PMT) for papillary microcarcinomas (PMCs) without risk factors to minimize overtreatment and patients' stress. To validate Porto proposal criteria, we examined a series of 190 PMC series, also studying sex hormone receptors and BRAF mutation. Our updated Porto proposal (uPp) reclassifies as PMT incidental PMCs found at thyroidectomy lacking the following criteria: (a) detected under the age of 19 years; (b) with multiple tumors measuring >1 cm adding up all diameters; and (c) with aggressive morphologic features (extrathyroidal extension, angioinvasion, tall, and/or hobnail cells). PMCs not fulfilling uPp criteria were considered "true" PMCs. A total of 102 PMCs were subclassified as PMT, 88 as PMC, with no age or sex differences between subgroups. Total thyroidectomy and iodine-131 therapy were significantly more common in PMC. After a median follow-up of 9.6 years, lymph node metastases, distant metastases, and mortality were only found in the PMC subgroup. No subgroup differences were found in calcifications or desmoplasia. Expression of estrogen receptor-α and estrogen receptor-ß, progesterone receptor, and androgen receptor was higher in PMC than in nontumorous thyroid tissue. BRAF mutations were detected in 44.7% of PMC, with no differences between subgroups. In surgical specimens, the uPp is a safe pathology tool to identify those PMC with extremely low malignant potential. This terminology could reduce psychological stress associated with cancer diagnosis, avoid overtreatment, and be incorporated into daily pathologic practice.

Supplementation with phytoestrogens and insoluble fibers reduces intestinal carcinogenesis and restores ER-ß expression in Apc-driven colorectal carcinogenesis.

Supplementation with phytoestrogens and insoluble fibers has been reported to reduce duodenal polyps in colectomized familial adenomatous polyposis patients, with a mechanism involving, at least in part, upregulation of estrogen receptor-ß subtype, whose expression is lowered during intestinal tumorigenesis. These data suggest a protective effect also in the colon, the main target organ for tumorigenesis in familial adenomatous polyposis and a major cancer type in non-familial (sporadic) cancers. Therefore, we tested whether a similar preparation might reduce tumorigenesis in the colon of Pirc rats (F344/NTac-Apc) mutated in the Apc gene and thus, like familial adenomatous polyposis patients, spontaneously developing multiple tumors in the colon. We first demonstrate that estrogen receptor-ß expression in Pirc rat colon is significantly down-regulated compared to age-matched wt rats. Then, Pirc rats aged 1 month were treated for 3 months with Adipol (Adi), a patented preparation containing phytoestrogens and insoluble fibers. Colon tumorigenesis was significantly reduced by Adi treatment (colon tumors/rat were 5.3 ± 0.8 and 2.9 ± 0.3, Mucin Depleted Foci/rat 127 ± 6.6 and 97.1 ± 8.6 in Controls and Adi-treated rats, respectively, means ± SE, P < 0.01). The treatment also normalized colon proliferation pattern along the crypt and significantly increased apoptosis in colon tumors. Estrogen receptor-ß expression was increased by Adi treatment, especially in the tumors. These positive effects suggest that Adipol may be exploited as a chemopreventive agent to reduce cancer risk in familial adenomatous polyposis patients and to postpone prophylactic colectomy. Moreover, given the similarities between familial adenomatous polyposis and sporadic colorectal cancer, it might also be used as chemopreventive agent in colorectal cancer patients at risk.

FSH receptor binding inhibitor influences estrogen production, receptor expression and signal pathway during in vitro maturation of sheep COCs.

Follicle-stimulating hormone (FSH) promotes secretion of follicle fluid and follicle development. FSH acts via cognate FSH receptor (FSHR). It remains unknown whether the supplement of FSH-receptor binding inhibitor (FRBI) into the in vitro maturation (IVM)medium influence the estrogen receptor expression and signal pathway of oocytes in sheep. The present study aimed to investigate FRBI effects on inositol trisphosphate (IP3) of oocytes and protein kinase A (PKA) of sheep granulosa cells, further to elucidate the signal pathway of FRBI effects. Cumulus-oocyte complexes (COCs) were recovered from antral follicles. COCs were cultured for 24 h in the IVM medium supplemented with varying concentrations of FRBI (0, 10, 20, 30 and 40 µg/mL) and FSH (10IU/mL). ELISA was used to measure the concentrations of estradiol (E2) and IP3 in the IVM medium. Western blotting was utilized to detect protein expression of ERß of COCs and protein kinase A (PKA) of granulosa cells. The results showed IP3 concentrations of FRBI-3 and FRBI-4 groups were less than that of CG and FSH groups at 22 h and 24 h (P < 0.05). PKA levels of FRBI-3 and FRBI-4 groups were significantly less than that of CG and FSH group (P < 0.05 or P < 0.01). Expression levels of ERß mRNA and protein of FRBI-treated groups were gradually decreased in comparison to CG and FSH group. The minimum value was detected in the FRBI-4 group. ERß protein level of the FRBI-4 group was significantly less than that of FSH group (P < 0.05). E2 concentrations of FRBI-treated groups were elevated as compared to CG, with the highest increment of FRBI-2 group (P < 0.05). Our results revealed a higher dose of FRBI reduced IP3 production. FRBI could suppress slightly expression levels of ERß mRNA and protein of COCs and PKA of granulosa cells, additionally increased E2 production of sheep COCs.

Increased dietary levels of α-linoleic acid inhibit mammary tumor growth and metastasis.

OBJECTIVE: The aim of this study was to determine whether α-linolenic acid (ALA ω-3 fatty acid) enriched diet affects growth parameters when applied to a syngeneic model of mammary carcinoma. MATERIALS AND METHODS: BALB/c mice were divided and fed with: 1) a chia oil diet, rich in ALA or 2) a corn oil diet, rich in linoleic acid (LA ω-6 fatty acid). Mice were subcutaneously inoculated with a tumor cell line LM3, derived from a murine mammary adenocarcinoma. RESULTS: After 35 days, tumor incidence, weight, volume and metastasis number were lower in the ALA-fed mice, while tumor latency time was higher, and the release of pro-tumor metabolites derived from ω-6 fatty acids decreased in the tumor. Compared to the control group, a lower number of mitosis, a higher number of apoptotic bodies and higher T-lymphocyte infiltration were consistently observed in the ALA group. An ALA-rich diet decreased the estrogen receptor (ER) α expression, a recognized breast cancer promotor while showing an opposite effect on ERß in tumor lysates. CONCLUSION: These data support the anticancer effect of an ALA-enriched diet, which might be used as a dietary strategy in breast cancer prevention.

Period 1 and estrogen receptor-beta are downregulated in Chinese colon cancers.

To investigate whether Period 1 (PER1) and Estrogen receptor-beta (ER2) are associated with occurrence and development of Chinese colorectal cancers. By using RT-quantitative PCR, tissue microarray (TMA) and immunohistochemistry, we detected mRNA levels and protein levels of PER1 and ER2 in the cancerous tissues and paired normal adjacent tissues in patients with colorectal cancer. Survival analyses were performed by the Kaplan-Meier method utilizing log-rank test and univariate and multivariate Cox proportional modeling to measure 5-year disease-free survival (DFS) and overall survival (OS). Real-time PCR showed that, the delta Ct value (tumor tissue vs. normal mucosa) of PER1 or ER2 is 8.51 ± 2.81 vs. 7.34 ± 2.08 or 12.39 ± 2.43 vs. 9.76 ± 1.75, expression of PER1 and ER2 decreased significantly in tumor tissues compared with noncancerous mucosas of patients with or without metastasis (both of P values <0.001). Spearman test revealed that PER1 and ER2 were significantly down-regulated in cancerous tissues (r=0.283; P<0.001) which was also confirmed by immunohistochemistry of specimens from 203 colon cancer patients in a TMA format. The reduction of PER1 was associated with gender and distant metastasis (P=0.037 and P<0.001, respectively) whereas the decline of ER2 was associated with age (P=0.043) by analyzing the clinical data. However, we were not capable of detecting any association between PER1 level or ER2 level and overall survival (OS) or disease free survival (DFS). It is the first observation of correlated reduction of PER1 and ER2 in Chinese colon cancers, and they do play a certain role in colorectal cancer.

Expression status of mRNA for sex hormone receptors in human dental pulp cells and the response to sex hormones in the cells.

OBJECTIVES: Sex hormone receptors are reported to be present in human dental pulp (HDP) cells. The purpose of this study was to examine the biological significance of oestrogen and androgen receptors (ER and AR, respectively) in HDP cells. DESIGN: We isolated HDP cells expressing ER- and AR-mRNAs and investigated the expression status of the receptors and the response to sex hormones in the cells. RESULTS: HDP cells expressing ER- and/or AR-mRNAs had the ability to form alizarin red S-positive nodules in which calcium and phosphorus were deposited in vitro and to differentiate into odontoblasts-like cells and dentine-like tissue in vivo. Individual clones isolated from HDP cells exhibited a different expression pattern of mRNA for ER and AR. Some clones expressed ERα- and/or ERß-mRNAs and the others coexpressed ER- and AR-mRNAs. Using the Ingenuity software, we found that 17ß-estradiol (E2) and dihydrotestosterone (DHT) could act directly on HDP cells through ER-or androgen signalling-mediated mechanisms. E2 or DHT stimulated the mRNA expression for genes related to odontogenesis of dentine-containing teeth and odontoblast differentiation, suggesting that ER and AR in HDP cells may be involved in dentinogenesis. CONCLUSIONS: Our findings provide new insights into the biological significance of sex hormone receptors in HDP cells.

Effect of isoflavone on balloon catheter-induced neointimal hyperplasia in ovariectomised rabbit carotid artery.

BACKGROUND: This study was designed to investigate the effects of phytoestrogen isoflavone on balloon catheter-induced hyperplasia of carotid artery. METHODS: Forty-eight female New Zealand rabbits were randomly divided into four groups: control (balloon-induced carotid artery injury only); ovariectomy control (ovariectomy and carotid artery injury), oestrogen (ovariectomy, carotid artery injury and nilestriol, 5mg/kg daily for 28 days), and isoflavone (ovariectomy, carotid artery injury and isoflavone 120 mg/kg daily for 28 days). The arterial wall thickness was assessed by coloured ultrasonography, and the oestrogen-α and oestrogen-ß receptors in the abdominal aorta were measured by Western blotting. RESULTS: The medial layer thickness in the isoflavone group was less than in the ovariectomy control group (0.28±0.03 vs. 0.35±0.04 mm, p<0.01), and the intimal/medial layer (I/M) ratio is the isoflavone group was also less than in the ovariectomy control group (16.85±3.79 vs. 48.94±8.92, p<0.01). There was no statistically significant difference in the medial layer thickness or I/M ratio between the isoflavone and the oestrogen groups. The optical density of the oestrogen-α receptors in the isoflavone group (0.317±0.002) was less than in the oestrogen (0.633±0.002) or ovariectomy control group (0.590±0.001, p<0.01). The optical density of the oestrogen-ß receptors in the isoflavone group (1.350±0.002) and the ovariectomy control group (1.2033±0.002) was less than in the oestrogen group (1.7699±0.003, p<0.01). CONCLUSIONS: Isoflavone therapy in the ovariectomised rabbit model attenuated balloon catheter-induced intimal and medial layer hyperplasia in the carotid arteries. Down-regulation of the oestrogen-α receptors may be involved in the hyperplasia-preventative effect.

Identification of estrogen receptor dimer selective ligands reveals growth-inhibitory effects on cells that co-express ERα and ERß.

Estrogens play essential roles in the progression of mammary and prostatic diseases. The transcriptional effects of estrogens are transduced by two estrogen receptors, ERα and ERß, which elicit opposing roles in regulating proliferation: ERα is proliferative while ERß is anti-proliferative. Exogenous expression of ERß in ERα-positive cancer cell lines inhibits cell proliferation in response to estrogen and reduces xenografted tumor growth in vivo, suggesting that ERß might oppose ERα's proliferative effects via formation of ERα/ß heterodimers. Despite biochemical and cellular evidence of ERα/ß heterodimer formation in cells co-expressing both receptors, the biological roles of the ERα/ß heterodimer remain to be elucidated. Here we report the identification of two phytoestrogens that selectively activate ERα/ß heterodimers at specific concentrations using a cell-based, two-step high throughput small molecule screen for ER transcriptional activity and ER dimer selectivity. Using ERα/ß heterodimer-selective ligands at defined concentrations, we demonstrate that ERα/ß heterodimers are growth inhibitory in breast and prostate cells which co-express the two ER isoforms. Furthermore, using Automated Quantitative Analysis (AQUA) to examine nuclear expression of ERα and ERß in human breast tissue microarrays, we demonstrate that ERα and ERß are co-expressed in the same cells in breast tumors. The co-expression of ERα and ERß in the same cells supports the possibility of ERα/ß heterodimer formation at physio- and pathological conditions, further suggesting that targeting ERα/ß heterodimers might be a novel therapeutic approach to the treatment of cancers which co-express ERα and ERß.

Differential binding with ERá and ERâ of the phytoestrogen-rich plant Pueraria mirifica

Variations in the estrogenic activity of the phytoestrogen-rich plant, Pueraria mirifica, were determined with yeast estrogen screen (YES) consisting of human estrogen receptors (hER) hERá and hERâ and human transcriptional intermediary factor 2 (hTIF2) or human steroid receptor coactivator 1 (hSRC1), respectively, together with the â-galactosidase expression cassette. Relative estrogenic potency was expressed by determining the â-galactosidase activity (EC50) of the tuber extracts in relation to 17â-estradiol. Twenty-four and 22 of the plant tuber ethanolic extracts interacted with hERá and hERâ, respectively, with a higher relative estrogenic potency with hERâ than with hERá. Antiestrogenic activity of the plant extracts was also determined by incubation of plant extracts with 17â-estradiol prior to YES assay. The plant extracts tested exhibited antiestrogenic activity. Both the estrogenic and the antiestrogenic activity of the tuber extracts were metabolically activated with the rat liver S9-fraction prior to the assay indicating the positive influence of liver enzymes. Correlation analysis between estrogenic potency and the five major isoflavonoid contents within the previously HPLC-analyzed tuberous samples namely puerarin, daidzin, genistin, daidzein, and genistein revealed a negative result.

Differential binding with ERalpha and ERbeta of the phytoestrogen-rich plant Pueraria mirifica.

Variations in the estrogenic activity of the phytoestrogen-rich plant, Pueraria mirifica, were determined with yeast estrogen screen (YES) consisting of human estrogen receptors (hER) hERalpha and hERbeta and human transcriptional intermediary factor 2 (hTIF2) or human steroid receptor coactivator 1 (hSRC1), respectively, together with the beta-galactosidase expression cassette. Relative estrogenic potency was expressed by determining the beta-galactosidase activity (EC(50)) of the tuber extracts in relation to 17beta-estradiol. Twenty-four and 22 of the plant tuber ethanolic extracts interacted with hERalpha and hERbeta, respectively, with a higher relative estrogenic potency with hERbeta than with hERalpha. Antiestrogenic activity of the plant extracts was also determined by incubation of plant extracts with 17beta-estradiol prior to YES assay. The plant extracts tested exhibited antiestrogenic activity. Both the estrogenic and the antiestrogenic activity of the tuber extracts were metabolically activated with the rat liver S9-fraction prior to the assay indicating the positive influence of liver enzymes. Correlation analysis between estrogenic potency and the five major isoflavonoid contents within the previously HPLC-analyzed tuberous samples namely puerarin, daidzin, genistin, daidzein, and genistein revealed a negative result.

[Effect of ferulic acid on proliferation and mechanism in human breast cancer cells].

OBJECTIVE: To investigate phytoestrogenic effects of ferulic acid in ER-positive T47D and ER-negative MDA-MB231 cells in culture. METHOD: T47D and MDA-MB231 human breast cancer cells were treated with ferulic acid and examined cell proliferation by means of MTT assay. Cell cycle distribution, ERalpha and ERbeta expression were treated by flow cytometer. The pS2 mRNA expressions were detected by real-time fluorescence quantitative PCR. RESULT: The proliferations were enhanced significantly by treatment with ferulic acid on T47D cells and the proliferation effects were inhibited by adding Faslodex (1 x 10(-8) mol x L(-1)). However, there was no significant difference on the proliferation in MDA-MB-231 cells compared with solvent control group by both treatment with ferulic acid and co-treatment with Faslodex (1 x 10(-8) mol x L(-1)). Ferulic acid stimulated the amount of T47D cells in phase S and proliferation index increased significantly. The effects were inhibited by treatment with Faslodex (1 x 10(-8) mol x L(-1)), and the amount of cells in phase S and proliferation index decreased, the amount of cells in G0/G1 phase increased, cell cycle of T47D was arrested in G0/G1 phase. Ferulic acid up-regulated pS2 mRNA expressions and increased the level of ERalpha protein expression in T47D cells. Ferulic acid did not show remarkable effect to the level of ERbeta protein expression in T47D cells. CONCLUSION: Ferulic acid possessed phytoestrogenic effect by up-regulating pS2 gene expression and the receptor subtype of ERalpha.

A sexually dimorphic distribution pattern of the novel estrogen receptor G-protein-coupled receptor 30 in some brain areas of the hamster.

The isolation of the G-protein-coupled receptor 30 (GPR30), an orphan membrane receptor unrelated to nuclear estrogen receptors (ERs), has become a key factor towards the unraveling of rapid estrogen action. This membrane receptor together with cellular signaling intermediaries, i.e., extracellular signal-dependent kinases 1 and 2, may promote neuronal proliferation and differentiation activities. In the present study, an evident gene expression pattern of GPR30 characterized postnatal 7 (young) and 60 (adult) days of age hamsters as shown by its heterogeneous mRNA distribution in hypothalamic, amygdalar and cerebellar areas of both sexes. In particular, most of the brain areas considered in the adult hamster plus only the amygdala and cerebellum of young animals behaved in a sexually dimorphic fashion. This similar pattern was also detected for the ERalpha and beta, as shown by the latter receptor prevailing in young and adult females, while the former predominated in young females. Even for the two kinases, a sexually dimorphic distribution was featured above all for young hamsters. Overall, the findings of the present study established a distinct expression pattern of the novel ER (GPR30) that may operate differently in some brain areas of the hamster and this may provide interesting insights regarding its probable neuroprotective role during the execution of some hibernating states, which are typical of our rodent model.

Development of a screening assay for ligands to the estrogen receptor based on magnetic microparticles and LC-MS.

A high throughput screening assay for the identification of ligands to pharmacologically significant receptors was developed based on magnetic particles containing immobilized receptors followed by liquid chromatography-mass spectrometry (LC-MS). This assay is suitable for the screening of complex mixtures such as botanical extracts. For proof-of-principle, estrogen receptor-alpha (ER-alpha) and ER-beta were immobilized on magnetic particles functionalized with aldehyde or carboxylic acid groups. Alternatively, biotinylated ER was immobilized onto streptavidin-derivatized magnetic particles. The ER that was immobilized using the streptavidin-biotin chemistry showed higher activity than that immobilized on aldehyde or carboxylic acid functionalized magnetic particles. Immobilized ER was incubated with extracts of Trifolium pratense L. (red clover) or Humulus lupulus L. (hops). As a control for non-specific binding, each botanical extract was incubated with magnetic particles containing no ER. After magnetic separation of the particles containing bound ligands from the unbound components in the extract, the particles were washed, ligands were released using methanol, and then the ligands were identified using LC-MS. The estrogens genistein and daidzein were identified in the red clover extract, and the estrogen 8-prenylnaringenin was identified in the hop extract. These screening results are consistent with those obtained using previous screening approaches.

[Effects of combined acupuncture and medicine therapy on the breast tissue and expressions of estrogen receptor subgroups in the rat of hyperplasia of mammary glands].

OBJECTIVE: To study on the inhibitory action of combined acupuncture and medicine therapy on the model rat of hyperplasia of mammary glands and the mechanism. METHODS: The model rat of hyperplasia of mammary glands were prepared. After modelling, they were randomly divided into an acupuncture group, a Chinese drug group and a combined acupuncture and drug group, a control group and a model group. Except both the control group and the model group, other 4 groups were treated respectively with acupuncture, Chinese drug, combined acupuncture and Chinese drug, and Premormine, once each day, 9 sessions constituting one course. After treatment of 3 courses (30 days), changes of the breast tissue form were observed, and the diameter and the area of the acina cavity were determined and expressions of estrogen receptor subgroups (ERalpha and ERbeta) were detected with immunohistochamical methods. RESULTS: The diameter and the area of the acina cavity were increased in the model group as compared with those in the normal group (both P < 0.01), and in the treatment group they were decreased as compared with those in the model group (P < 0.05 or P < 0.01)); both acupuncture and Chinese drug could up-regulate the expression of ERbeta and down-regulate the expression of ERalpha. CONCLUSION: Both acupuncture and moxibustion, and Chinese medicine have inhibitory action on hyperplasia of mammary glands in the rat, with the strongest inhibitory action of the combined acupuncture and medicine treatment which is basically close to the level of Premormine. The mechanism is possily related with the up-regulation of ERbeta expression and down-regulation of ERalpha expression.

Estradiol and phytoestrogens differently influence the rodent postmenopausal mammary gland.

OBJECTIVE: In the following study, we investigated treatment-related changes in mammary gland histomorphology and structure after the administration of soy to adult virgin ovariectomized (OVX) female rats. Additionally, mammary receptor regulation was extensively evaluated by immunohistochemical analysis, and tissue proliferative activity analyzed by cell nuclear proliferating antigen expression (Ki67). DESIGN: OVX rats were treated, for 6 weeks, with either the vehicle, the soy extract (SSE 100 mg/kg/d PO), or 17beta-estradiol (0.5 mg/kg/d PO); a sham control group (SHAM) was also included in the study. When killed, mammary glands were collected and subsequently processed for light microscopy or immunohistochemistry. Immunoreactivity was quantified by a scoring system that took into account both the percentage of positive cells and the intensity of the staining. RESULTS: The 17beta-estradiol--treated rats had stimulated mammary glands compared with OVX rats, with an average lobulo-alveolar development not different from the SHAM controls. Only a partial regression of the glandular atrophy was observed in OVX rats receiving 100 mg/kg/d SSE, with a histological appearance between that of the OVX and SHAM controls. No significant changes were observed among experimental groups in the median ERalpha scores of the epithelial compartment (score of 3 in all groups); in the stromal compartment, a tendency toward decreased expression was seen with 17beta-estradiol rats compared with OVX controls (scores of 2 and 5, respectively). A significant reduction in ERbeta immunostaining was observed in the mammary glands of SSE-treated rats, in both epithelium and stroma (scores of 4 and 3, respectively), compared with those of OVX controls (score of 8 in both compartments). The ERbeta receptor status was not significantly affected by 17beta-estradiol. Compared with OVX rats (score of 1), PR expression was up-regulated by 17beta-estradiol (score of 6), whereas an ovariectomy-like pattern was observed after the administration of SSE (score of 0). Ki67 immunoreactivity in the epithelium and stroma was increased by the administration of 17beta-estradiol (scores of 4 and 5, respectively) and was unchanged after SSE treatment (scores of 0 and 2, respectively), compared with OVX controls (scores of 1 and 2, respectively). CONCLUSIONS: The differences observed in the histological pattern, hormonal receptor status regulation, and Ki67 modulation suggest a different role for phytoestrogens and 17beta-estradiol in postmenopausal rodent mammary glands.

Effects of 8-prenylnaringenin on the hypothalamo-pituitary-uterine axis in rats after 3-month treatment.

Phytoestrogens are increasingly consumed in artificially high doses as herbal preparations and nutritional supplements. The flavanone 8-prenylnaringenin (8PN) is a potent phytoestrogen, but its benefits and risks after long-term application are poorly identified. Therefore, we tested two doses of 8PN and 17beta-estradiol-3-benzoate (E2B) (effective doses: 6.8 and 68.4 mg/kg body weight (BW) of 8PN, and 0.17 and 0.7 mg/kg BW of 17beta-estradiol (E2)) and compared their effects on uterine weight, pituitary hormones (LH, FSH and prolactin) and the expression of estrogen-regulated genes and of estrogen receptor (ER)alpha and ERbeta in the hypothalamus, pituitary and uterus. Both doses of E2 and the high dose of 8PN suppressed serum LH and FSH, and stimulated serum prolactin levels, uterine weight, and progesterone receptor, insulin-like growth factor I and complement protein C3 mRNA transcripts. In the preoptic and the mediobasal areas of the hypothalamus, all treatments had negligible effects on ERalpha and ERbeta and gonadotropin-releasing hormone (GnRH) receptor gene expression, while ERbeta and GnRH receptor transcripts in the anterior pituitary were reduced under both E2 doses and the high 8PN dose. The mRNA concentrations of the LHalpha and -beta subunits in the pituitary were suppressed by E2 and 8PN. In summary, 8PN had very similar though milder effects than E2 on all tested parameters. Inhibition of climacteric complaints by E2 takes place in the hypothalamus, where it inhibits the overactive GnRH pulse generator. Hence, 8PN may be used to inhibit climacteric symptoms effectively. Human pharmacologic studies will show whether the stimulatory effect on the uterus that was found in the present animal model would require the concomitant administration of progestins to prevent endometrial overstimulation.