Estrogen impairs glucocorticoid dependent negative feedback on the hypothalamic-pituitary-adrenal axis via estrogen receptor alpha within the hypothalamus.

Numerous studies have established a link between individuals with affective disorders and a dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis, most notably characterized by a reduced sensitivity to glucocorticoid negative (-) feedback. Furthermore there is a sex difference in the etiology of mood disorders with incidence in females being two to three times that of males, an association that may be a result of the influence of estradiol (E2) on HPA axis function. In these studies, we have examined the effect of E2 on glucocorticoid-mediated HPA axis (-) feedback during both the diurnal peak and the stress-induced rise in corticosterone (CORT). Young adult female Sprague-Dawley (SD) rats were ovariectomized (OVX) and 1 week later treated subcutaneous (s.c.) with oil or estradiol benzoate (EB) for 4 days. On the 4th day of treatment, animals were injected with a single dose of dexamethasone (DEX), or vehicle. EB treatment significantly increased the evening elevation in CORT and the stress-induced rise in CORT. In contrast, DEX treatment reduced the diurnal and stress induced rise in CORT and adrenocorticotropic hormone (ACTH), and this reduction was not apparent following co-treatment with EB. To determine a potential site of E2's action, female SD rats were OVX and 1 week later, wax pellets containing E2, the estrogen receptor beta (ERbeta) agonist diarylpropionitrile (DPN), or the estrogen receptor alpha (ERalpha) agonist propylpyrazoletriol (PPT), was implanted bilaterally and dorsal to the paraventricular nucleus of the hypothalamus (PVN). Seven days later, animals were injected s.c. with a single dose of DEX, or vehicle to test for glucocorticoid-dependent (-) feedback. Results show that E2 and PPT increased, while DPN decreased the diurnal peak and stress-induced CORT and ACTH levels as compared to controls. Furthermore, E2 and PPT impaired the ability of DEX to inhibit both the diurnal and the stress-induced rise in CORT and ACTH, whereas DPN had no effect. Neuronal activation was measured by c-fos mRNA expression within the PVN following restraint. E2 and PPT increased c-fos mRNA, and impaired the normal DEX suppression of neuronal activation in the PVN. Taken together, these data indicate that estradiol causes a dysregulation of HPA axis (-) feedback as evidenced by the inability of DEX to suppress diurnal and stress-induced CORT and ACTH secretion. Additionally, the ability of E2 to inhibit glucocorticoid (-) feedback occurs specifically via ERalpha acting at the level of the PVN.

Differential expression of estrogen receptor beta splice variants in rat brain: identification and characterization of a novel variant missing exon 4.

Estrogen receptor beta (ER-beta) mRNA is found in abundance in rat brain. The distribution of ER-beta mRNA in brain differs from that of ER-alpha suggesting they subserve different functions. ER-beta mRNA has been reported to be variably spliced, in contrast to ER-alpha, resulting in numerous isoforms that possess different functional properties. The present study was undertaken to determine whether the isoforms of ER-beta mRNA are differentially distributed in different brain regions. In order to assess the range of transcript forms expressed in various brain regions in the same assay, a micropunch dissection technique was combined with semiquantitative RT-PCR. The relative abundance of each ER-beta isoform (beta1>beta2>beta1delta3>beta2delta3) was similar in all ER-beta positive brain regions with the exception of the hippocampus, which contained low levels of most isoforms and a fifth ER-beta isoform, which we are calling ER-beta1delta4. Based on its sequence, ER-beta1delta4 encodes an ER-beta that is missing exon 4. Initial characterization studies of this showed that it did not bind estrogen, and that, unlike ER-beta1, it localized to the cytoplasm when expressed in cultured cells. The distribution of ER-beta1delta4 was different from that of the other isoforms in that it was expressed at high levels in the hippocampus, where the other isoforms were low, and that it was nearly undetectable in the brain regions that expressed the highest levels of the other ER-beta splice variants. These data suggest that a highly complex pattern of estrogen signaling can occur in a region specific manner in the rat brain.

Distribution of estrogen receptor-beta messenger ribonucleic acid in the male sheep hypothalamus.

As a first step in determining possible influences of the newly discovered estrogen receptor (ER)-beta on reproduction, we have localized mRNA for ER-beta within the male sheep hypothalamus using in situ hybridization and a rat ER-beta cRNA probe. Highest amounts of hybridization signal were observed in the preoptic area (POA), bed nucleus of the stria terminalis, paraventricular nucleus, and supraoptic nucleus. Relatively moderate amounts of hybridization signal were observed in the retrochiasmatic area (RCH), anterior hypothalamic area, dorsomedial hypothalamus, and lateral hypothalamus. Only a low level of hybridization signal was observed in the ventromedial hypothalamus, suprachiasmatic nucleus, and arcuate nucleus. The presence of ER-beta mRNA in several areas of the male sheep hypothalamus suggests multiple functions for this receptor. The distribution of ER-beta in the ovine hypothalamus was similar to that described for the rat, suggesting a high degree of functional conservation across species. A role for ER-beta in influencing reproduction is suggested by its presence in the POA and RCH, regions of the hypothalamus that control reproduction.

Ethanol exposure during the last week of gestation in the rat: inhibition of the prenatal testosterone surge in males without long-term alterations in sex behavior.

In utero ethanol exposure decreases the prenatal testosterone (T) surge in male rats. To determine the functional significance of this suppression, we measured sex behavior in adult litter representatives of pregnant rats that were administered a fortified liquid diet containing 35% ethanol-derived calories from day 15 of gestation through parturition. Control dams were pair-fed an isocaloric liquid diet with the ethanol calories replaced by sucrose. Results from the behavioral studies showed that gonadally intact fetal alcohol-exposed (FAE) males exhibited little masculine sex behavior in the first of four weekly sessions. However, their behavior in the subsequent three tests was indistinguishable from pair-fed controls. Lordosis quotients in the same males following castration and estrogen and progesterone treatment were under 10%. In castrated FAE females, no effects of prenatal ethanol exposure were observed in masculine behaviors following androgen replacement or feminine sex behaviors following estrogen and progesterone replacement. Additional studies measured the duration of prenatal ethanol exposure necessary to inhibit the prenatal T surge in order to determine whether the inhibition was due to a direct effect of the drug. Results revealed an inhibition of the surge in males exposed to ethanol from days 14 through 20 of pregnancy, days 14 through 16 of pregnancy, or days 17 through 20 of pregnancy. A normal surge of T was observed on days 18-19 of gestation in control fetuses. These findings indicate that ethanol does not have to be present in blood at the time of the surge to have an inhibitory effect. They also reveal that the surge can be inhibited with as little as 24-36 h of ethanol exposure prior to its normal appearance on day 18 of gestation. In spite of this inhibition of the prenatal T surge, the behavioral results indicate that normal masculinization and defeminization of sex behavior occurs in FAE males exposed to ethanol after the beginning of the period of differentiation of the hypothalamus and testes.

Alterations in the estrogen sensitivity of hypothalamic proenkephalin mRNA expression with age and prenatal exposure to alcohol.

Studies suggest that exposure to alcohol in utero causes reproductive and neuroendocrine deficits in adult female rats. The ventromedial nucleus of the hypothalamus (VMN) is an estrogen-sensitive brain region which is regarded as a primary locus for modulating female reproduction. Proenkephalin (PE) mRNA expression in the VMN is dramatically increased by estrogen and this elevation is thought to be involved in modulating female reproductive behavior and neuroendocrine function. To examine whether prenatal alcohol exposure has long-term effects on the ability of estrogen to influence hypothalamic PE mRNA levels, female rats at 2-3, 6-7 or 15-18 months of age, derived from alcohol- or control-fed dams, were studied. 7 days following ovariectomy, animals received either estrogen or sham treatment for 2 days prior to sacrifice. PE mRNA levels in the VMN and striatum were determined by in situ hybridization histochemistry. Film autoradiogram density, numbers of PE mRNA-expressing cells and exposed silver grains/cell were analyzed. Estrogen treatment increased hybridization density, the number of PE mRNA-expressing cells and PE mRNA (grains) level/cell in the VMN of normal adult female rats. In old rats, estrogen increased the number of PE mRNA-expressing cells without up-regulating PE mRNA grain density/cell. In fetal alcohol-exposed (FAE) female rats, the number of cells that expressed PE mRNA did not increase following estrogen treatment at any age. Elevation of grain density/cell following estrogen was observed in FAE animals but only at 7-8 months of age. Overall, these data indicate that the estrogen responsiveness of PE mRNA expression in the VMN declines with age and, furthermore, prenatal exposure to alcohol blunts estrogen's effects on PE mRNA expression in the adult VMN. These finding may help to explain the mechanisms underlying the loss of reproductive function observed in FAE females.

Alterations in kappa opioid receptor mRNA levels in the paraventricular nucleus of the hypothalamus by stress and sex steroids.

In this study, the levels of kappa opioid receptor (kOR) mRNA were determined using in situ hybridization following two types of stress (i.p. injection of hypertonic saline or novelty). In addition, we examined the possibility that estrogen or androgen would modify kOR mRNA. Gonadectomized male rats treated with estrogen or dihydrotestosterone were sacrificed 45 min after spending 15 min in a novel open field, or 60 min following hypertonic saline injection. Two-way ANOVA revealed that estrogen and novelty increased the levels of kOR mRNA in the ventral zone of the medial parvocellular part of the paraventricular nucleus (PVN), but not in the lateral parvocellular part of the PVN, claustrum, nucleus accumbens or the nucleus of the lateral olfactory tract. Furthermore, novelty increased kOR mRNA in gonadectomized (GDX) and GDX rats treated with dihydrotestosterone (DHTP), but not in sham-operated or estrogen-treated animals. Taken together, these data indicate that kOR mRNA levels are under estrogenic control and up-regulated in a stressor specific fashion.

Distribution of NMDAR1 receptor subunit mRNA and [125I]MK-801 binding in the hypothalamus of intact, castrate and castrate-DHTP treated male rats.

This study examines NMDAR1 receptor subunit mRNA expression and [125I]MK-801 binding in hypothalamic and limbic nuclei of intact, castrate and castrate-dihydrotestosterone propionate (DHTP)-treated male rats. In intact rats, the highest levels of NMDAR1 mRNA were observed in the supraoptic, suprachiasmatic, ventromedial and arcuate nuclei. Low levels of hybridization were observed in the bed nucleus of the stria terminalis, lateral preoptic area, lateral hypothalamic area and lateral septum. In castrated rats both NMDAR1 mRNA and [125I]MK-801 binding are significantly decreased in the lateral septum compared to castrate rats treated with DHTP, a non-aromatizable androgen. NMDAR1 mRNA was also significantly decreased in the supraoptic nucleus of castrate rats when compared to castrate rats treated with DHTP. These data suggest that androgens may modulate NMDA receptor function in some parts of the central nervous system.

Plasma beta-endorphin levels and childhood intussusception.

To determine whether childhood intestinal intussusception is associated with elevated plasma beta-endorphin levels, a series of patients was studied prospectively. Fourteen patients (age range between 3 months and 7 years) presented to two university pediatric emergency departments in Chicago with clinical symptoms and signs of intussusception. Venous blood (2cc) was withdrawn for plasma beta-endorphin determination, followed by barium enema. Plasma beta-endorphin levels were measured by radioimmunoassay. The mean beta-endorphin level of the 8 patients with barium enema proven intussusception was 14.1 +/- 12.0 pg/ml. Two of these patients presented with marked lethargy and had beta-endorphin levels of 7.5 and 21.2 pg/ml. The mean plasma beta-endorphin level of the 5 patients with negative barium enema studies was 18.1 +/- 10.0 pg/ml (P = 0.56). A sixth control patient had a plasma beta-endorphin level of 1569 pg/ml. In conclusion, childhood intestinal intussusception is not associated with elevated plasma beta-endorphin levels.

Plasma beta-endorphin concentrations and analgesia-muscle relaxation in the newborn infant supported by mechanical ventilation.

We evaluated the effects of pancuronium and opiates on plasma beta-endorphin concentrations in 25 infants supported by mechanical ventilation. Infants receiving opiate were randomly assigned to receive either fentanyl or morphine. There was no change in beta-endorphin concentrations after administration of pancuronium, whereas both fentanyl and morphine reduced beta-endorphin concentrations by approximately 60%.

Androgen inhibits the increases in hypothalamic corticotropin-releasing hormone (CRH) and CRH-immunoreactivity following gonadectomy.

To characterize the effect of androgens on the hypothalamo-pituitary-adrenal (HPA) axis we examined the regulation of corticotropin-releasing hormone (CRH) following gonadectomy and hormone replacement. Three-month-old male Fischer 344 (F344) rats were gonadectomized (GDX) or sham GDX. Control animals remained intact. Animals were sacrificed 1, 4, 7, 10, or 21 days following surgery. GDX rats had significantly elevated (p < 0.05) levels of hypothalamic CRH 21 days after surgery compared to intact and sham-operated rats. In a second study, 3-month-old male F344 rats were GDX and treated with the non-aromatizable androgen, dihydrotestosterone (DHT), using a Silastic capsule containing crystalline DHT propionate subcutaneously implanted in each animal's back. Control animals were GDX and sham-treated or left intact (INT). Three weeks following gonadectomy, CRH levels in the hypothalamus of GDX rats showed a significant increase (p < 0.05) compared to intact animals. DHT treatment, beginning at the time of gonadectomy prevented this increase. CRH or arginine vasopressin (AVP) immunoreactivity was examined using immunocytochemistry. The number of CRH-immunoreactive (IR) cells in the paraventricular nucleus (PVN) of GDX, DHT-treated animals was significantly decreased (p < 0.05) compared to GDX rats. No differences were seen between treatment groups in CRH-IR cell numbers in the bed nucleus of the stria terminalis or the central amygdaloid nucleus or in AVP-IR cell numbers in the PVN. These data demonstrate that long-term castration increases hypothalamic CRH content and CRH-IR cell numbers in the PVN by removal of an androgen-dependent repression.