Hypothalamic Estrogen Receptors Research Articles

Analysis of arsenic-modulated expression of hypothalamic estrogen receptor, thyroid receptor, and peroxisome proliferator-activated receptor gamma mRNA and simultaneous mitochondrial morphology and respiration rates in the mouse.

Arsenic has been identified as an environmental toxicant acting through various mechanisms, including the disruption of endocrine pathways. The present study assessed the ability of a single intraperitoneal injection of arsenic, to modify the mRNA expression levels of estrogen- and thyroid hormone receptors (ERα,β; TRα,β) and peroxisome proliferator-activated receptor gamma (PPARγ) in hypothalamic tissue homogenates of prepubertal mice in vivo. Mitochondrial respiration (MRR) was also measured, and the corresponding mitochondrial ultrastructure was analyzed. Results show that ERα,β, and TRα expression was significantly increased by arsenic, in all concentrations examined. In contrast, TRβ and PPARγ remained unaffected after arsenic injection. Arsenic-induced dose-dependent changes in state 4 mitochondrial respiration (St4). Mitochondrial morphology was affected by arsenic in that the 5 mg dose increased the size but decreased the number of mitochondria in agouti-related protein- (AgRP), while increasing the size without affecting the number of mitochondria in pro-opiomelanocortin (POMC) neurons. Arsenic also increased the size of the mitochondrial matrix per host mitochondrion. Complex analysis of dose-dependent response patterns between receptor mRNA, mitochondrial morphology, and mitochondrial respiration in the neuroendocrine hypothalamus suggests that instant arsenic effects on receptor mRNAs may not be directly reflected in St3-4 values, however, mitochondrial dynamics is affected, which predicts more pronounced effects in hypothalamus-regulated homeostatic processes after long-term arsenic exposure.

Estrogen receptor beta activity contributes to both tumor necrosis factor alpha expression in the hypothalamic paraventricular nucleus and the resistance to hypertension following angiotensin II in female mice

Sex differences in the sensitivity to hypertension and inflammatory processes are well characterized but insufficiently understood. In male mice, tumor necrosis factor alpha (TNFα) in the hypothalamic paraventricular nucleus (PVN) contributes to hypertension following slow-pressor angiotensin II (AngII) infusion. However, the role of PVN TNFα in the response to AngII in female mice is unknown. Using a combination of in situ hybridization, high-resolution electron microscopic immunohistochemistry, spatial-temporal gene silencing, and dihydroethidium microfluorography we investigated the influence of AngII on both blood pressure and PVN TNFα signaling in female mice. We found that chronic (14-day) infusion of AngII in female mice did not impact blood pressure, TNFα levels, the expression of the TNFα type 1 receptor (TNFR1), or the subcellular distribution of TNFR1 in the PVN. However, it was shown that blockade of estrogen receptor β (ERβ), a major hypothalamic estrogen receptor, was accompanied by both elevated PVN TNFα and hypertension following AngII. Further, AngII hypertension following ERβ blockade was attenuated by inhibiting PVN TNFα signaling by local TNFR1 silencing. It was also shown that ERβ blockade in isolated PVN-spinal cord projection neurons (i.e. sympathoexcitatory) heightened TNFα-induced production of NADPH oxidase (NOX2)-mediated reactive oxygen species, molecules that may play a key role in mediating the effect of TNFα in hypertension. These results indicate that ERβ contributes to the reduced sensitivity of female mice to hypothalamic inflammatory cytokine signaling and hypertension in response to AngII.

17-α-Estradiol Has Sex-Specific Effects on Neuroinflammation That Are Partly Reversed by Gonadectomy.

17-α-Estradiol (17aE2) treatment from 4 months of age extends life span in male mice and can reduce neuroinflammatory responses in the hypothalamus of 12-month-old males. Although 17aE2 improves longevity in males, female mice are unaffected, suggesting a sexually dimorphic pattern of life-span regulation. We tested whether the sex-specific effects of 17aE2 on neuroinflammatory responses are affected by gonadal removal and whether hypothalamic changes extend to other brain regions in old age. We show that sex-specific effects of 17aE2 on age-associated gliosis are brain region specific and are partially dependent on gonadectomy. 17aE2 treatment started at 4 months of age protected 25-month-old males from hypothalamic inflammation. Castration before 17aE2 exposure reduced the effect of 17aE2 on hypothalamic astrogliosis in males. In contrast, sex-specific inhibition of microgliosis generated by 17aE2 was not significantly affected by castration. In the hippocampus, gonadectomy influenced the severity of gliosis and the responsiveness to 17aE2 in a region-dependent manner. The male-specific effects of 17aE2 correlate with increases in hypothalamic estrogen receptor alpha expression, specifically in gonadally intact males, consistent with the idea that 17aE2 might act through this receptor. Our results indicate that neuroinflammatory responses to 17aE2 are partially controlled by the presence of sex-specific gonads. Loss of gonadal function and age-associated neuroinflammation could, therefore, influence late-life health and disease onset, leading to sexual dimorphism in both aging and in response to drugs that modify the pace of aging.

SAT-587 Molecular Markers of Beige Adipose Following ESR1 Knockdown in the Mediobasal Hypothalamus of Adult Female Rhesus Monkeys

Our studies in female marmoset monkeys show that the ablation of ovarian estradiol (E2) production fails to alter energy homeostasis or body fat accumulation. Peripheral E2 may therefore not play a crucial role in metabolic regulation in female primates. shRNA-mediated knockdown of ESR1 expression in the hypothalamic ventromedial nucleus (VMN) in adult female rodents, however, induces obesity and suggests ESR1 is a hypothalamic target for E2 regulation of energy homeostasis, and likely mediates thermogenesis in brown/beige adipose depots. In female primates, including humans, the hypothalamic estrogen receptor mediating metabolic regulation is unknown. To test the hypothesis that ESR1 mediates female primate regulation of energy homeostasis, 11 ovary intact, adult female rhesus macaques, pair housed with female peers, received five 12µl MRI-guided MBH infusions into the rostral-to-caudal extent of both right and left VMN. Each infusion comprised a gadolinium contrast agent and ~3–4 x 1010 adeno-associated virus 8 (AAV8) particles containing either an shRNA specific for ESR1 (n=6, ERaKD) or scrambled shRNA (n=5, control). Mid-surgery MRI scans identified targeting accuracy. ~ 1.5 yrs following AAV8 infusion, pronounced gain in BMI enabled conversion of 83% of ERaKD females to overweight/obese compared to 20% of controls (p=0.08). Percent increase in BMI remained intermittently greater (p<0.05) than controls thereafter. Adipose depots were harvested at necropsy ~2.5–3 yrs following treatment. Total RNA was isolated using the Qiagen AllPrep DNA/RNA/miRNA Universal kit. RNA was reverse transcribed with High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). All quantitative real-time PCR (qRT-PCR) were performed on a StepOnePlus System using Power SYBR Green master mix (Applied Biosystems). Primer sequences were designed using NCBI Primer-Blast. Expression of TATA-box binding protein (TBP) was used as the internal control housekeeping gene. The relative expression of target genes was measured using the comparative cycle threshold (Ct) method with results expressed as target mRNA expression relative to TBP using the formula 2^-delta Ct. Upper body beige adipose represents an organ system in primates, including humans, involved in thermogenesis. Axillary beige adipose depots in ERaKD females, however, did not exhibit significantly diminished gene expression for selected markers of beige adipocytes, including PAT2, CD137 and C/EBPβ, compared to control females. More crucially, thermogenically relevant UCP1 expression also did not differ between ERaKD females and controls. Taken together, these results suggest that knockdown of VMN ESR1 in adult female monkeys, while inducing modest weight gain after 1.5 years, may not markedly alter beige adipose gene expression of initially selected thermogenically relevant genes.

Sexual dimorphism in hypothalamic inflammation in the offspring of dams exposed to a diet rich in high fat and branched-chain amino acids.

Branched-chain amino acid (BCAAs: leucine, isoleucine, and valine) contribute to the development of obesity-associated insulin resistance in the context of consumption of a high-fat diet (HFD) in humans and rodents. Maternal diet is a major determinant of offspring health, and there is strong evidence that maternal HFD alters hypothalamic developmental programming and disrupts offspring energy homeostasis in rodents. In this study, we exposed pregnant and lactating C57BL/6JB female mice to either HFD, HFD with supplemented BCAA (HFD+BCAA), or standard diet (SC), and we studied offspring metabolic phenotypes. Both maternal HFD and HFD supplemented with BCAA had similar effect rendering the offspring metabolic imbalance and impairing their ability to cope with HFD when challenged during aging. The metabolic effects of HFD challenge were more profound in females, worsening female offspring ability to cope with an HFD challenge by activating hypothalamic inflammation in aging. Moreover, the sex differences in hypothalamic estrogen receptor α (ER-α) expression levels were lost in female offspring upon HFD challenge, supporting a link between ER-α levels and hypothalamic inflammation in offspring and highlighting the programming potential of hypothalamic inflammatory responses and maternal nutrition.

ERα and GnRH co-localize in the hypothalamic neurons of the South American plains vizcacha, Lagostomus maximus (Rodentia, Caviomorpha).

Gonadotropin-releasing hormone (GnRH) is the key regulator of the hypothalamic-pituitary-gonadal axis. Estradiol (E2) affects GnRH synthesis and delivery. Hypothalamic estrogen receptors (ER) modulate GnRH expression acting as transcription factors. The South American plains vizcacha, Lagostomus maximus, is able to ovulate up to 800 oocytes per reproductive cycle, and shows continuous folliculogenesis with pre-ovulatory follicle formation and an ovulatory event at mid-gestation. The aim of this work was to analyze the hypothalamic expression of ER in the vizcacha at different gestational time-points, and its relationship with GnRH expression, serum luteinizing hormone (LH) and E2. The hormonal pattern of mid-gestating vizcachas was comparable to ovulating-females with significant increases in GnRH, LH and E2. Hypothalamic protein and mRNA expression of ERα varied during pregnancy with a significant increase at mid-gestation whereas ERβ mRNA expression did not show significant variations. Hypothalamic immunolocalization of ERα was observed in neurons of the diagonal band of Brocca, medial preoptic area (mPOA), periventricular, suprachiasmatic, supraoptic (SON), ventromedial, and arcuate nuclei, and medial eminence, with a similar distribution throughout gestation. In addition, all GnRH neurons of the mPOA and SON showed ERα expression with no differences across the reproductive status. The correlation between GnRH and ERα at mid-gestation, and their co-localization in the hypothalamic neurons of the vizcacha, provides novel information compared with other mammals suggesting a direct action of estrogen as part of a differential reproductive strategy to assure GnRH synthesis during pregnancy.

The role of hypothalamic estrogen receptors in metabolic regulation.

Estrogens regulate key features of metabolism, including food intake, body weight, energy expenditure, insulin sensitivity, leptin sensitivity, and body fat distribution. There are two 'classical' estrogen receptors (ERs): estrogen receptor alpha (ERS1) and estrogen receptor beta (ERS2). Human and murine data indicate ERS1 contributes to metabolic regulation more so than ESR2. For example, there are human inactivating mutations of ERS1 which recapitulate aspects of the metabolic syndrome in both men and women. Much of our understanding of the metabolic roles of ERS1 was initially uncovered in estrogen receptor α-null mice (ERS1(-/-)); these mice display aspects of the metabolic syndrome, including increased body weight, increased visceral fat deposition and dysregulated glucose intolerance. Recent data further implicate ERS1 in specific tissues and neuronal populations as being critical for regulating food intake, energy expenditure, body fat distribution and adipose tissue function. This review will focus predominantly on the role of hypothalamic ERs and their critical role in regulating all aspects of energy homeostasis and metabolism.

Complete alleviation of treatment refractory primary SUNCT syndrome with clomiphene citrate (a medicinal deep brain hypothalamic modulator)

To report the first ever case of primary short-lasting unilateral neuralgiform headache attacks (SUNCT) syndrome completely responsive to clomiphene citrate. Case report. SUNCT is a primary headache disorder marked by frequent attacks of one-sided headache with cranial autonomic associated symptoms. When SUNCT is deemed medicinally treatment refractory, it can cause tremendous patient-related disability. Surgical treatment options are available including hypothalamic deep brain stimulation, occipital nerve stimulator placement or arterial decompression surgery, but these procedures carry significant morbidity. A patient presented with a 10 month complaint of multiple, daily short-lasting, right-sided headaches each lasting from 60 to 120 seconds in duration and occurring from 100 to 200 times per day. The head pain was associated with ipsilateral eyelid ptosis and conjunctival injection. The patient was diagnosed with SUNCT but was unresponsive to multiple recognized medicinal treatments. He had complete alleviation of his attacks with clomiphene citrate, a synthetic, non-steroidal, ovulatory stimulant that directly binds to hypothalamic estrogen receptors. The clomiphene was tolerated without any adverse events. A putative mechanism of action for clomiphene in the prevention of SUNCT will be presented. Clomiphene citrate is a unique treatment for SUNCT and appears to be very safe and effective.

An Unusual Cause of Inguinal Hernia in a Male Patient: Endometriosis

Endometriosis is an ectopic endometrium, commonplaced in the female. But its occurence in the man is mysterious. In the literature two cases of endometriosis in men have occured following surgery.1 The present case is the first reported arising in the inguinal region, near the ductus deference. Forty-nine years old man was presented to hospital with left inguinal hernia. He was operated from that area for 3 times, before. Furthermore he has unexplained infertility. His physical examination revealed normal external genitelia. He underwent surgical operation for hernia. In the operation, a mass was discovered beside the spermatic cord and sent to our laboratory. Macroscopically the mass was 8×7×6 cm in diameters (Fig. 1). In dissection cystic cavity was seen and beside it, ductus deferens was identified. Cyst wall was containing small lumens that was thought as small vessels. In microscopic examination, cyst was lined with columnar epithelial cells and simple tubular invaginations showing the same type of cell lining, and cellular stroma, with typical features of endometrial mucosa. In addition, there was smooth muscle proliferation and some endometrial glands and stroma in these areas (Fig. 2). The pathological diagnosis was endometriosis. Chromosom analysis was normal (46, XY). The patient had an uneventful postoperative course and was discharged on the seven postoperative day. The patient decided on regularly follow-up in the out-patient department. Fig. 1 Uterus-like mass beside the deferent ductus, holded area in the figure. Fig. 2 High power field of endometriotic area (H&E stain, ×200). Three theories of the pathogenesis of endometriosis have been proposed in the female: transplantation, coelomic metaplasia, and embryologic cell rests.2,3 Transplantation theory is obviously impossible in the male because of the absence of a source of menstruel material.1 The coelomic metaplasia could occur secondary to inflammatory and hormonal influences. This theory could explain endometriosis in women with Mullerian agenesis, who have an absent uterus or the occasional presence of endometriosis in men.4 Third theory, also known as induction theory, is based on induction of the embryologic cell rests. Between the utricle and the appendices testes, cell rests may persist. These mullerian cell rests would be expected to lie along the course of the ejaculatory and deferent ducts.1 In this case, endometriosis was diagnosed along the ductus deferens; so this theory is very likely for this case. This patient has used a drug named of Fertilin, for sperm maturation and activation. The drug consists of clomiphen citrate that shows antiestrogenic affect by binding estrogen receptors in hypothalamus and hypophysis.5 It is a contrary event for the cases in the literature that were associated with prolonged estrogen therapy which appearing to be necessary for the development of endometriosis in the male. But also it is surprising not to have seen this phenomenon more frequently in men with prostatic carcinoma treated with estrogens. So, in our case additional factor must have been for the development of the lesion. In conclusion, endometriosis within an inguinal hernia is an exceptionally rare event; most surgeons are unlikely to see one. All previously reported elderly cases underwent estrogen therapy for prostatic carcinoma, contrary, our patient had antiestrogenic therapy for infertility.

Sexually dimorphic expression of hypothalamic estrogen receptors α and β and Kiss1 in neonatal male and female rats.

Release of gonadotropins in adult rodents is sex specific and dependent upon kisspeptin (Kiss1) neurons. This crucial pathway within the hypothalamic-pituitary-gonadal (HPG) axis is profoundly influenced by neonatal estrogens, which induce a male-like phenotype. Classically, estrogen activity is mediated via the estrogen receptors α and β (ERα and ERβ), but the relative roles each plays in organizing the sex-specific ontogeny of kisspeptin signaling pathways remain unresolved. Thus, the present study used in situ hybridization histochemistry (ISHH) to map the temporal and sexually dimorphic neonatal mRNA expression profiles of ERα, ERβ, and Kiss1 in the anterioventral periventricular nucleus (AVPV), medial preoptic area (MPOA), ventromedial nucleus (VMN), and arcuate nucleus (ARC), all regions critical for kisspeptin regulation of gonadotropin secretion. In general, females had higher levels of ERα, in all regions examined, a sex difference that persisted until postnatal day (PND) 19 except in the ARC. Males had significantly more ERβ expression in the AVPV at birth, but this sex difference was lost and then re-emerged on PND 19, with females having more than males. VMN ERβ levels were higher in females until PND 19. Kiss1 was not detectable until PND 11 in the anterior hypothalamus, but expression levels were equivalent at birth in the ARC. By PND 2, ARC ERα and Kiss1 levels were abundant, sexually dimorphic (higher in females), and, respectively, showed a U- and a bell-shaped pattern with age. Sex differences in ARC Kiss1 expression provide evidence that Kiss1 may play a role in the sexual dimorphic organization of the neonatal brain. These detailed profiles of neonatal Kiss1 and ERs mRNA levels will help elucidate the relative roles each plays in the sex-specific, estrogen-dependent organization of gonadotropin signaling pathways.

Elevated estrogen receptor expression in hypothalamic preoptic area decreased by electroacupuncture in ovariectomized rats

In the present study, effects of electroacupuncture (EA) on estrogen receptor alpha (ERα) and beta (ERβ) mRNA and protein expression in the hypothalamus of ovariectomized (OVX) rats were detected by quantitative real-time reverse transcription PCR (qRT-PCR) and western blot analysis. Gonadotropin-releasing hormone (GnRH) release and GnRH mRNA level in hypothalamic preoptic area (POA) were evaluated by push–pull perfusion and qRT-PCR. Our results showed that elevated mRNA and protein expression of ERα and ERβ in hypothalamus were restrained following EA treatment in OVX rats. EA treatment also inhibited GnRH release and GnRH mRNA levels in OVX rats. These results provide novel evidence that EA treatment down regulates the expression of hypothalamic estrogen receptors (ERs), thus restores the response of GnRH neurons to estrogen depression, and partially resets the negative feedback of estrogen to hypothalamus–pituitary–ovary axis (HPOA) in OVX rats, which may be a critical mechanism for EA on female reproductive disorders.

The effect of diabetes and centrally administered insulin on anterior hypothalamic estrogen receptor α immunoreactivity

Diabetic rats have characteristic reproductive deficits. Peripheral and central (intra-cerebro-ventricular [ICV]) insulin restores the reproductive phenotype to control levels. In this experiment, we evaluated a possible defect in steroid feedback by evaluating hypothalamic estrogen receptor (ER) alpha availability using ERalpha immunocytochemistry. Animals were ovariectomised and given estradiol and progesterone. Diabetic and control animals were given ICV insulin or saline. Nuclear and/or cytoplasmic ERalpha immunoreactivity was evaluated in the paraventricular nucleus (PVN) and the organum vasculosum laminae terminalis (OVLT). In the PVN, nuclear immunoreactivity was increased among diabetic, ICV insulin-treated animals. Diabetic saline-treated and nondiabetic animals had similar ERalpha immunoreactivity. The OVLT had lower numbers of immunoreactive neurons compared to the PVN; no differences among the treatment groups were found in the OVLT. Central insulin treatment increased the number of PVN nuclear ERalpha immunoreactive neurons among diabetic animals. However, there was no reduction in ERalpha when comparing saline-treated diabetic animals to non-diabetic rats, suggesting that decreases in hypothalamic ERalpha in the regions studied do not account for diabetes-induced reproductive deficits.

Clomiphene Citrate as a New Treatment for SUNCT: Hormonal Manipulation for Hypothalamic‐Influenced Trigeminal Autonomic Cephalalgias

We present a patient with treatment refractory short-lasting unilateral neuralgiform headache attacks with conjunctival injection and tearing (SUNCT) who was found to have low levels of serum testosterone supporting the hypothalamic connection to this trigeminal autonomic cephalalgia. Clomiphene citrate therapy induced a significant elevation of testosterone levels (by its effect on hypothalamic estrogen receptors) and led to a dramatic reduction in SUNCT attacks. Hormonal manipulation may be a treatment strategy for hypothalamic-influenced trigeminal autonomic cephalalgias.

Morphological Studies of Prolactin-Secreting Cells in Estrogen Receptor α and Estrogen Receptor β Knockout Mice

Estrogens play a major role in the regulation of prolactin (PRL) secretion through activation of pituitary and hypothalamic estrogen receptors (ERs). In order to evaluate the relative role of ERα and ERβ in the control of PRL density in the pituitary gland, we performed immunocytochemical localization of PRL and ERs in pituitaries of wild-type (WT), ERα knockout (KO) and ERβKO mice. In WT and ERβKO anterior pituitaries, the vast majority of secretory cells contained ERα immunoreactivity, while no ERα immunostaining could be found in ERαKO pituitaries. No ERβ immunoreactivity could be detected in pituitaries of WT, ERαKO or ERβKO mice. At the light microscopic level, a large number of cells staining for PRL were present in pituitaries of female WT, while in female ERαKO pituitaries, the density of PRL cells was much lower. In WT male pituitaries, the density of PRL cells was lower than observed in female WT, while PRL staining was markedly decreased in male ERαKO as compared to male WT. In ERβKO mice of both sexes, the results were identical to those observed in WT animals. At the electron microscopic level, in WT mice of both sexes, type 1 PRL cells exhibited a well-developed Golgi apparatus and a large number of strongly stained large mature and immature secretory granules. Type 2 PRL cells were also present in the pituitary. Type 2 PRL cells contain small poorly labelled granules. In ERαKO mice of both sexes, type 1 PRL cells were atrophied with poorly developed Golgi apparatus, and no type 2 PRL cells could be observed. In ERαKO pituitaries, typical gonadectomy cells were found. No ultrastructural changes were observed in PRL cells of ERβKO mice. The present data strongly suggest that the positive regulation of PRL expression at the pituitary level by estrogens is mediated by ERα and does not involve ERβ activation.

Exposure of newborn male and female rats to environmental estrogens: delayed and sustained hyperprolactinemia and alterations in estrogen receptor expression.

Environmental estrogens (xenoestrogens) are synthetic compounds that are abundant in the environment and mimic natural estrogens. The estrogenicity of two such compounds, bisphenol A (BPA) and octylphenol (OP), during development of the neuroendocrine system was investigated. The objective was to compare the effects of neonatal exposure to BPA, OP, and diethylstilbestrol (DES), a potent synthetic estrogen, on prepubertal serum PRL levels and estrogen receptor (ER) expression in the anterior pituitary and medial basal hypothalamus. Receptor expression in the uterus and prostate, two peripheral estrogen-responsive tissues, was also examined. Newborn male and female Fischer 344 rats were s.c. injected on days 1-5 after birth with corn oil (control), BPA and OP (100 or 500 microg/day), or DES (5 microg/day). Rats were bled on days 15, 20, and 25 and on the day of death (day 30), and serum PRL was analyzed by RIA. Relative expressions of ERalpha and ERbeta were determined by RT-PCR. BPA and OP induced delayed, but progressive, increases in serum PRL levels, up to 3-fold above control levels, in both males and females. The low dose of either compound was equally or more effective as the high dose in eliciting and sustaining elevated serum PRL levels, namely hyperprolactinemia. In contrast, the DES treatment resulted in a transient rise in serum PRL levels. BPA, OP, and, to a lesser extent, DES increased the expression of both ERalpha and ERbeta in the anterior pituitary of males, but not females, whereas the hypothalamic ERs were less responsive to these compounds. DES treatment caused down-regulation of ERalpha expression in the uterus and up-regulation of ERbeta in the prostate, whereas BPA or OP was without effect. In conclusion, exposure of newborn rats of either sex to environmental estrogens results in delayed and sustained hyperprolactinemia and differential alterations in ER expression in the hypothalamus and pituitary. DES appears to target the lower reproductive tract more effectively than the neuroendocrine system.

Exposure of Newborn Male and Female Rats to Environmental Estrogens: Delayed and Sustained Hyperprolactinemia and Alterations in Estrogen Receptor Expression

Environmental estrogens (xenoestrogens) are synthetic compounds that are abundant in the environment and mimic natural estrogens. The estrogenicity of two such compounds, bisphenol A (BPA) and octylphenol (OP), during development of the neuroendocrine system was investigated. The objective was to compare the effects of neonatal exposure to BPA, OP, and diethylstilbestrol (DES), a potent synthetic estrogen, on prepubertal serum PRL levels and estrogen receptor (ER) expression in the anterior pituitary and medial basal hypothalamus. Receptor expression in the uterus and prostate, two peripheral estrogen-responsive tissues, was also examined. Newborn male and female Fischer 344 rats were sc injected on days 1–5 after birth with corn oil (control), BPA and OP (100 or 500 μg/day), or DES (5 μg/day). Rats were bled on days 15, 20, and 25 and on the day of death (day 30), and serum PRL was analyzed by RIA. Relative expressions of ERα and ERβ were determined by RT-PCR. BPA and OP induced delayed, but progressive, increases in serum PRL levels, up to 3-fold above control levels, in both males and females. The low dose of either compound was equally or more effective as the high dose in eliciting and sustaining elevated serum PRL levels, namely hyperprolactinemia. In contrast, the DES treatment resulted in a transient rise in serum PRL levels. BPA, OP, and, to a lesser extent, DES increased the expression of both ERα and ERβ in the anterior pituitary of males, but not females, whereas the hypothalamic ERs were less responsive to these compounds. DES treatment caused down-regulation of ERα expression in the uterus and up-regulation of ERβ in the prostate, whereas BPA or OP was without effect. In conclusion, exposure of newborn rats of either sex to environmental estrogens results in delayed and sustained hyperprolactinemia and differential alterations in ER expression in the hypothalamus and pituitary. DES appears to target the lower reproductive tract more effectively than the neuroendocrine system.

Maternal High n-6 Polyunsaturated Fatty Acid Intake during Pregnancy Increases Voluntary Alcohol Intake and Hypothalamic Estrogen Receptor Alpha and Beta Levels among Female Offspring

Identification of nongenetic biological factors that predispose to alcohol abuse is central to attempts to prevent alcoholism. Since an exposure to estradiol in utero increases voluntary alcohol intake in adulthood, we investigated whether an increase in pregnancy estradiol levels, caused by feeding pregnant mice a high-fat corn oil diet, also influences voluntary alcohol intake among female offspring. In addition, the effect on estrogen receptor α (ER-α) and ER-β protein levels in the brain using Western blot assay, was determined. Pregnant CD-1 mice were kept on a high n-6 polyunsaturated fatty acid (PUFA; 43% calories from corn oil) or low n-6 PUFA diet (16% calories from corn oil) throughout gestation, and switched to a Purina laboratory chow after the pups were born. When 4 months of age, the female offspring were given a choice between 5% alcohol and tap water. The offspring of high n-6 PUFA mothers voluntarily consumed more alcohol than the offspring of low n-6 PUFA mothers. ER-α and ER-β protein levels in the hypothalamus were 1.5- and 2-fold higher, respectively, in the female offspring of high n-6 PUFA mothers than in the low n-6 PUFA offspring. No significant changes in the protein levels of ER-α and ER-β were seen in the frontal brain. Our findings indicate that a maternal exposure to a high n-6 PUFA diet during pregnancy increases alcohol intake among female offspring. This behavioral change, together with previously observed increase in aggressiveness and reduction in depressive-like behavior in these offspring, may be linked to an increase in the hypothalamic ER-α and ER-β levels.

Evidence for sexual dimorphism of estrogen receptors in hypothalamus and thymus of neonatal and immature Wistar rats

It is hypothesized that the rat thymus is sexually differentiated. To begin testing this, we used the 5-day-old rat, whose hypothalamus is sexually differentiated during this period. Cytosolic estrogen receptors (ER) were measured in cytosols prepared from the brain, thymus and uterus of Wistar rats at 5, 18 and 30 days post-partum. ER concentrations were significantly higher in hypothalamus in cytosols of female 5-day-old rats, and this difference had disappeared by day 18. The pattern in thymus was identical to that observed in hypothalamus, suggesting the presence in the thymus of the aromatase system that converts androgen to estrogen, and that estrogen-mediated sexual differentiation of thymus might be proceeding at 5 days. Unlike the case for hypothalamus, no experimental model exists at present for testing functional sexual differentiation in thymus. Therefore we tested the effects of aromatase inhibitors on estrogen receptor activity in thymus well after the five-day period, and before atrophy of the thymus has commenced. Male and female rats were implanted at 15 days of age with SILASTIC implants containing 5 mg of estradiol or with 25 mg of the aromatase inhibitor 4-hydroxyandrostenedione (4-OHA) and cytosolic ER prepared at 30 days and activity measured. Administration of estradiol resulted in failure to detect available receptor, suggesting that the binding components measured were ER. After 4-OHA administration, ER concentrations were significantly increased in cytosols from male but not female hypothalamus and thymus. There is therefore a basis for exploring further the hypothesis that rat thymus is sexually differentiated.

Lordosis-enhancing medial preoptic area lesions do not alter hypothalamic estrogen receptor- or progestin receptor-immunoreactivity in prepubertal female guinea pigs

Female guinea pigs rarely display adult-typical lordosis responses to ovarian steroid hormones until 40–50 days of age. Behavioral hyporesponsiveness in prepubertal females may be due, in part, to deficiencies in hypothalamic estrogen receptors and/or estradiol-induced progestin receptors. This study was designed to test the hypothesis that bilateral medial preoptic area (MPOA) lesions, which enhance the display of progesterone-facilitated lordosis in juvenile females, increase levels of hypothalamic estrogen receptors and/or estradiol-induced progestin receptors. Hartley guinea pigs were ovariectomized at 11–12 days of age and at 14–15 days of age received bilateral electrolytic or sham lesions aimed at the MPOA. At approximately 3 weeks of age, lesioned and sham-lesioned animals were either tested for the display of progesterone-facilitated lordosis or perfused, and their hypothalamic tissue processed for estrogen receptor- or estradiol-induced progestin receptor-immunostaining. Although a significantly higher percentage of MPOA-lesioned than sham-lesioned guinea pigs displayed progesterone-facilitated lordosis (85.7% vs. 5.8%, respectively, p<0.05), there were no significant lesion-related differences in the number or staining intensity of cells containing estrogen receptor- or estradiol-induced progestin receptor-immunoreactivity in the ventrolateral hypothalamus or arcuate nucleus. These data do not support the hypothesis that the enhanced display of progesterone-facilitated lordosis in prepubertal guinea pigs following MPOA lesions is due to increased hypothalamic concentrations of estrogen receptors or estradiol-induced progestin receptors.

Estrogen and progesterone receptor expression in neuroendocrine and related neurons of the pubertal female monkey hypothalamus.

Expression of hypothalamic estrogen receptors (ER) and progesterone receptors (PR) is barely evident in prepubertal monkeys but is prominent in adults. To investigate whether adult patterns of ER and PR expression are established in mid-pubertal female cynomolgus monkeys, we labeled neuroendocrine (NEU) neurons by microinjection of retrograde tracer into the median eminence, and then identified ER and PR by specific immunostaining in separate sets of hypothalamic sections. ER and PR appeared in the cytoplasm and nuclei of cells identified exclusively as neurons, and retrograde tracer remained clearly visible in the cytoplasm of NEU neurons after immunostaining. Numbers of NEU and related neurons expressing ER or PR were quantified in principal hypothalamic regions. In the supraoptic nucleus, almost all neurons analyzed (n = 580) contained ER (94%) with many also NEU (73% ER + NEU), while lesser amounts of the neurons examined (n = 214) expressed PR (75%) and were NEU (53% PR + NEU). In the paraventricular nucleus, most of the neurons analyzed (n = 302) contained ER (90% ER; 54% ER + NEU), but few of the neurons studied (n = 269) contained PR (34% PR; 19% PR + NEU). In the periventricular zone, nearly all neurons examined (n = 795) contained ER (95% ER; 48% ER + NEU), but fewer of those studied (n = 298) exhibited PR (79% PR; 47% PR + NEU). In the arcuate-periventricular zone, all neurons examined (n = 542) contained ER (100%) but few were NEU (4% ER + NEU), while nearly all neurons studied (n = 418) contained PR (95%), some of which were NEU (21% PR + NEU). Neurons expressing ER were also prevalent in areas without NEU labeling, including the diagonal band of Broca, medial preoptic area, and mammillary bodies, but were less common in the septum and dorsomedial hypothalamus. Likewise, neuronal PR expression was seen frequently in the mammillary bodies, but occurred less often in the diagonal band of Broca, medial preoptic area, and dorsomedial hypothalamus. Neurons in the suprachiasmatic nucleus and lateral hypothalamic area lacked retrograde labeling. These results identify the principal sites and subsets of NEU and related neurons which express ER and PR in the mid-pubertal female monkey hypothalamus. They appear to correlate well with known populations of steroid-sensitive NEU neurons present in these areas in adults. The data also suggest that functional patterns of ER and PR expression arise upon reactivation of the hypothalamic-pituitary-gonadal axis at puberty. The degrees of receptor expression and of nuclear translocation most likely reflect peripubertal changes in the levels of gonadal steroids. Taken together, these results provide important insights into the mechanisms and development of neuroendocrine control during the pubertal period in primates.