Progestin Receptor Induction and Sexual Behavior by Estradiol Treatment in Male and Female Rats

Sex differences in the induction of cytosolic progestin receptors (CPR) by estrogen priming were correlated with the sex differences in behavioral responses. We evaluated the temporal relationship between CPR in several brain regions and pituitary and the time-course of 17β-estradiol (E₂) activation of female sexual behavior in gonadectomized male and female rats implanted with subcutaneous E₂ Silastic capsules for 6 h, 24 h and 48 h. Both CPR levels and mating behavior increase monotonically with the time of E₂ exposure. Induction of CPR was observed in the periventricular region of the preoptic area (PVPOA), arcuate nucleus (ARC), ventromedial nuclei (VMN) and pituitary in both sexes. A small induction of CPR was found in parietal cortex. The VMN in female rats showed a significant E₂-induced CPR increase at all times of exposure, while in male rats this induction was only significant after 24 h. Significant sex differences in absolute CPR levels and E₂-induced receptors were found in the following structures: VMN, 18 h after 6 h of E₂ treatment and after 24 h and 48 h of continuous E₂ exposure; PVPOA, only after 48 h of continuous E₂ exposure; ARC at 24 h and 48 h; and pituitary after all E₂ treatment. Mating behavior was tested under two conditions: E₂ alone (2 h after removal of E₂ capsules) and E₂+progesterone (2 h after a progesterone injection given 10 min after concluding the first test). Receptivity was first observed after 24 h E₂ exposure in female rats, whereas in male rats a small response appeared only after 48 h of E₂ exposure. After progesterone priming, the time of E₂ exposure necessary for expression of female sexual behavior was reduced to 6 h in females and 24 h in males. The appearance of mating behavior appears to follow that of inducible CPR in the VMN in both sexes. In addition, the CPR levels associated with the first receptivity either by male rats (16.6 fmol/mg protein) or female rats (15.3 fmol/mg protein) are very similar suggesting the presence of a threshold level controlling the expression of feminine sexual behavior. It is likely that inhibitory neural input plays a role in determining the threshold level of E₂-induced CPR, which is sufficient to trigger lordosis behavior.     

The mTOR and canonical Wnt signaling pathways mediate the mnemonic effects of progesterone in the dorsal hippocampus

Although much is known about the neural mechanisms responsible for the mnemonic effects of 17β‐estradiol (E₂), very little is understood about the mechanisms through which progesterone (P₄) regulates memory. We previously showed that intrahippocampal infusion of P₄ in ovariectomized female mice enhances object recognition (OR) memory consolidation in a manner dependent on activation of dorsal hippocampal ERK and mTOR signaling. However, the role of specific progesterone receptors (PRs) in mediating the effects of progesterone on memory consolidation and hippocampal cell signaling are unknown. Therefore, the goals of this study were to investigate the roles of membrane‐associated and intracellular PRs in mediating hippocampal memory consolidation, and identify downstream cell signaling pathways activated by PRs. Membrane‐associated PRs were targeted using bovine serum albumin‐conjugated progesterone (BSA‐P), and intracellular PRs (PR‐A, PR‐B) were targeted using the intracellular PR agonist R5020. Immediately after OR training, ovariectomized mice received bilateral dorsal hippocampal infusion of vehicle, P₄, BSA‐P, or R5020. OR memory consolidation was enhanced by P₄, BSA‐P, and R5020. However, only P₄ and BSA‐P activated ERK and mTOR signaling. Furthermore, dorsal hippocampal infusion of the ERK inhibitor U0126 blocked the memory‐enhancing effects of BSA‐P, but not R5020. The intracellular PR antagonist RU486 blocked the memory‐enhancing effects of R5020, but not BSA‐P. Interestingly, P₄ robustly activated canonical Wnt signaling in the dorsal hippocampus, which is consistent with our recent findings that canonical Wnt signaling is necessary for OR memory consolidation. R5020, but not BSA‐P, also elicited a modest increase in canonical Wnt signaling. Collectively, these data suggest that activation of ERK signaling is necessary for membrane‐associated PRs to enhance OR, and indicate a role for canonical Wnt signaling in the memory‐enhancing effects of intracellular PRs. This study provides the first evidence that membrane and intracellular PRs may employ different molecular mechanisms to enhance hippocampal memory. © 2014 Wiley Periodicals, Inc.     

Immunohistochemical expression of aromatase and estrogen,androgen and progesterone receptors in normal and neoplastic human meningeal cells

Evidence suggests that sex hormones may play a role in the tumorigenesis of meningiomas, and studies have demonstrated the expression of hormone receptors in these tumors. Aromatase expression has been detected in several normal tissues, including neurons in the CNS, and tumor tissues. We aim to assess the expression of aromatase (ARO) and of progesterone receptor (PR), estrogen receptor (ER) and androgen receptor (AR) in both normal and neoplastic meningeal cells. A cross‐sectional study was conducted with 126 patients diagnosed with meningioma (97 women and 29 men; mean age, 53.6 years) submitted to neurosurgery at Hospital São José, Complexo Hospitalar Santa Casa de Porto Alegre, southern Brazil. Control sections of normal meningeal cells, 19 patients, were obtained by evaluating the arachnoid tissue present in the arachnoid cyst resected material. Immunohistochemistry was applied to assess ARO, PR, ER and AR. Aromatase expression was detected in 100% of the control patients and in 0% of the patients with meningioma. ER was present in 24.6% of the meningiomas and in 0% of the controls, AR in 18.3% of the meningiomas and in 0% of the controls, and PR in 60.3% of the meningiomas and in 47.4% of the controls. A positive association was observed between the presence of AR and ER (OR 3.7; P = 0.01) in meningiomas. There were no significant differences in the presence of hormone receptors between meningioma histological subtypes. PR expression in women with meningioma was significantly higher than that found in men (OR 2.3; P = 0.08). Behavior pattern differences observed between aromatase expression, present in normal tissues and absent in meningiomas, and estrogen and androgen hormone receptors, absent in normal tissues and present in meningiomas, suggest that there is heterogeneity in modulation by sex steroids in the development of these tumors.     

Increased estrogen receptor binding in amygdala correlates with facilitation of feminine sexual behavior induced by olfactory bulbectomy

In this experiment we tested the hypothesis that the potentiation of feminine sexual behavior following olfactory bulb removal in female rats would be associated with increased ovarian steroid receptor binding in brain. Ovariectomized adult female rats underwent either bilateral olfactory bulb removal (BOB) or a sham operation and were exposed to 100% estradiol (E₂) in silastic capsules for 4 or 6 h. Following behavior testing, either cell nuclear estrogen receptor levels were measured in amygdala, hypothalamus, preoptic area and pituitary, or cytosol progestin receptors levels were determined in cortex, preoptic area and hypothalamus. After 6 h E₂ exposure, BOB females showed increasing lordosis responding with increasing progesterone (P) doses, at levels significantly higher than those of sham-operated rats. After 4 h E₂ exposure bulbectomized rats showed both a facilitation of lordosis and elevated estrogen receptor levels in amygdala. Sham-operates showed neither response to the 4 h E₂ stimulus. For both BOB and sham groups, progestin receptors were induced after 6 h E₂ exposure, but were uninduced after 4 h E₂ exposure. Additional rats were exposed to 5% E₂ for 24 or 48 h, followed by P. Lordosis was potentiated in BOB rats at 24 h; sham-operates showed high levels of lordosis only after 48 h of 5% E₂ exposure. Proceptivity was enhanced in BOB rats after both 24 and 48h of 5% E₂ exposure. In contrast, proceptivity was rarely observed in sham-operates, or in either group after 4 or 6 h E₂ exposure. We propose that increased estrogen receptor binding in the amygdala may provide a biochemical basis for the increased estrogen sensitiity found in olfactory bulbectomized female rats.     

Psychosocial correlates of progesterone receptors in breast cancer

Background: The diagnosis of cancer may lead to psychological distress with anxiety and depression as the most prevalent symptoms. Several investigators have found a correlation between psychosocial factors and tumor levels of estrogen receptors and progesterone receptors (PRs) while others have not. The aim of this study was to investigate demographic characteristics and severity of depression and anxiety as expressed by the Hospital Anxiety and Depression (HAD) scale of patients with high or low PR expression in breast cancers. Methods: Two hundred and seventy‐eight patients with primary breast cancer were divided into two subgroups according to PRs expressed in breast cancers. Results: The subgroup of patients with PR‐negative breast cancers expressed depression, as measured by the HAD scale, to a smaller degree (4.7±4.1) than the subgroup of patients with PR‐positive breast cancers (5.8±4.1). The difference was rather small but still statistically significant (t=2.1, df=236.7, P<.05). In contrast, we did not observe any correlation between anxiety and PR status. Differences between the subgroups according to family history of mental disorders were observed (χ²=4.7, df=1, P<.05). In the subgroup of patients with PR‐negative breast cancers; 13% of patients had a family history of mental disorders compared with 23% of patients with PR‐positive breast cancers. Conclusions: Depression expressed by patients with primary breast cancers could be influenced by the PR status of the tumors; however, other factors such as cancer treatment and family history of mental disorders could also be important. Depression and Anxiety, 2009. © 2008 Wiley‐Liss, Inc.     

Involvement of adenosine and glutamate receptors in the induction of c-fos in the striatum by haloperidol

The psychostimulant drugs amphetamine and cocaine induce the expression of immediate early genes, such as c-fos, in the striatum via D₁ dopamine receptor activation. This occurs primarily in the striato-nigral neurons. Conversely, neuroleptic drugs, such as haloperidol, which block D₂-type dopamine receptors, induce c-fos expression in striatal neurons projecting to the globus pallidus. In order to gain insight into the neurochemical substrates of neuroleptic-induced c-fos expression, we examined the effects of adenosine A₂ and N-methyl-D-aspartate (NMDA) receptor antagonists as well as inhibition of nitric oxide synthase, on haloperidol-induced Fos immunoreactivity in the striatum. While blockade of D₁ receptors had no effect on haloperidol-induced Fos expression, adenosine A₂ receptor antagonists decreased the number of neurons in the striatum expressing haloperidol-induced Fos by half. NMDA receptor antagonists also potently blocked the induction of Fos immunoreactivity by haloperidol, while inhibition of nitric oxide synthase activity had no effect. These results indicate that in the presence of a dopamine D₂ antagonist, Fos expression in striato-pallidal neurons is mediated in part through activation of A₂ receptors by adenosine, and via NMDA receptor activation by glutamate. © 1996 Wiley-Liss, Inc.     

Down-Regulation of Estrogen Receptor Immunoreactivity by 17β-estradiol in the Guinea Pig Forebrain

Low amplitude pulses of estradiol-17β (E₂-17β) are more effective than large single bolus injections or constant exposure to E₂-17β in inducing progesterone-facilitated sex behavior in female rats and guinea pigs. The present study examined whether the increased responsiveness to E₂-17β is due to an increase in the number of estrogen receptors in the estrogen receptor rich areas of the hypothalamus and amygdala. Initial studies examined the rapid effects (20 min) of a high dose of E₂-17β (50 μg) on estrogen receptor immunostaining using either the H222 antibody or the ER 21 antiserum. ER 21 immunostaining was not affected by the E₂-17β treatment suggesting that it binds to both occupied and unoccupied estrogen receptors. Therefore the ER 21 antiserum was used to characterize the regulation of estrogen receptor immunoreactivity (ER-IR) by E₂-17β. ER-IR was examined for 48 h and serum E₂-17β for 24 h following a 2 μg s.c. injection of E₂-17β (a dose similar to that used in multiple pulse paradigms). Serum E₂-17β peaked 15 to 30 min following the injection and returned to baseline values by 1 h. In all but one area maximal suppression of ER-IR occurred at 12 h. In summary, 1) decreases in estrogen receptor immunoreactivity following E₂-17β are consistent with studies in which estrogen receptors were assayed by binding assays and estrogen receptor mRNA was determined by in situ hybridization; 2) the ER 21 antiserum is able to detect both occupied and unoccupied estrogen receptors and 3) H222 immunoreactivity is influenced by the presence of E₂-17β, so that the level of H222-IR is a reflection of ligand/receptor binding dynamics. The data suggest that up-regulation of estrogen receptors does not account for the increase in behavioral sensitivity which is observed following multiple pulses of E₂-17β.     

Effects of repeated 9 and 30-day exposure to extremely low-frequency electromagnetic fields on social recognition behavior and estrogen receptors expression in olfactory bulb of Wistar female rats

Objective: We investigated the short- and long-term effects of extremely low-frequency electromagnetic fields (EMF) on social recognition behavior and expression of α- and β-estrogen receptors (ER).

Methods: Rats were exposed to 60-Hz electromagnetic fields for 9 or 30 days and tested for social recognition behavior. Immunohistochemistry and western blot assays were performed to evaluate α- and β-ER expression in the olfactory bulb of intact, ovariectomized (OVX), and ovariectomized+estradiol (E2) replacement (OVX+E2).

Results: Ovariectomization showed impairment of social recognition after 9 days of EMF exposure and a complete recovery after E2 replacement and so did those after 30 days. Short EMF exposure increased expression of β-ER in intact, but not in the others. Longer exposure produced a decrease in intact but an increase in OVX and OVX+E2.

Discussion: Our findings suggest a significant role for β-estrogen receptors and a lack of effect for α-estrogen receptors on a social recognition task.

Abbreviations: EMF: extremely low frequency electromagnetic fields; ERs: estrogen receptors; OB: olfactory bulb; OVX: ovariectomized; OVX + E₂: ovariectomized + estradiol replacement; IEI: interexposure interval; β-ER: beta estrogen receptor; E₂: replacement of estradiol; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; WB: Western blot; PBS: phosphate-buffer saline; PB: phosphate-buffer     

Nicotinic acetylcholine receptors regulate type 1 inositol 1,4,5‐trisphosphate receptor expression via calmodulin kinase IV activation

Type 1 inositol 1,4,5‐trisphosphate receptors (IP₃R‐1) are among the important calcium channels regulating intracellular Ca²⁺ concentration in the central nervous system. In a previous study, we showed that drugs of abuse, such as cocaine, methamphetamine, and ethanol, induced IP₃R‐1 upregulation via the calcium signal transduction pathway in psychological dependence. Although nicotine, a major component in tobacco smoke, participates in psychological and/or physical dependence, it has not yet been clarified how nicotine alters IP₃R‐1 expression. The present study, therefore, seeks to clarify the mechanism bgy which nicotine modifies IP₃R‐1 expression by using mouse cerebral cortical neurons in primary culture. Nicotine induced dose‐ and time‐dependent upregulation of IP₃R‐1 protein following its mRNA increase, and the latter was significantly suppressed by a nonselective nicotinic acetylcholine receptors (nAChR) antagonist, mecamylamine. Both cFos and phosphorylated‐cJun (p‐cJun) were immediately increased in the nucleus, together with an increase of calmodulin kinase (CaMK) IV but not CaMKII expression after nicotine exposure. A nonselective inhibitor of CaMKs, KN‐93, and a calcium chelating regent, BAPTA‐AM, completely suppressed the expression of cFos and p‐cJun in the nucleus as well as the nicotine‐induced IP₃R‐1 upregulation. These results indicate that nAChR activation by nicotine upregulates IP₃R‐1 via increase of activator protein‐1, which is a cFos and cJun dimmer, in the nucleus, with activation of Ca²⁺ signaling transduction processes. © 2014 Wiley Periodicals, Inc.     

Neural steroid hormone receptors

(1) Cell nuclear and cytoplasmic receptors for estrogens, androgens, and glucocorticoids have been identified in brains and pituitary glands of vertebrates. With respect to topography, estradiol (E₂) receptors are localized primarily in the hypophysiotrophic area and amygdala; 5-α-dihydrotestosterone (DHT) receptors are found in hypothalamus and limbic regions in smaller amounts and more uniformly distributed than those for estradiol; and corticosterone receptors are found in the hippocampal formation, septum, entorhinal cortex and amygdala. (2) Where information is available, mainly for estrogen receptors, their neural topography shows a remarkable constancy among vertebrates. The neural topography of estrogen and glucocorticoid receptors of rat and rhesus monkey will be compared. (3) A complicating factor in the study of androgens interacting with the brain is the conversion of testosterone (T) in neural tissue to both estrogenic and androgenic metabolites. Two of the products, E₂ and DHT, are recovered attached to cell nuclear receptors in the rat brain, whereas only DHT and T itself are found in pituitary cell nuclei. Evidence from other laboratories suggests that interactions of E₂ and DHT or T with intracellular receptors each subserve different behavioral and neuroendocrine functions in the rat. (4) The topography of estrogen receptors in the rat brain provides an excellent opportunity for studying estrogen action on brain chemistry. Estrogen effects on monoamine oxidase, choline acetylase, and glucose-6-phosphate dehydrogenase activity will be described. The overall importance of the action of steroid hormones on gene expression will be briefly discussed.     

Nicotine and 17β-estradiol produce an antidepressant-like effect in female ovariectomized rats

Nicotine and estrogen may influence depressive behavior in women. In this study, we sought to determine whether nicotine (NIC), alone and in combination with 17β-estradiol (E₂), might influence depressive characteristics of female rats in the forced swim test (FST). Ovariectomized adult female Sprague-Dawley rats (n = 8/group) received subcutaneous (s.c.) injections of nicotine, E₂ or the combination (NIC: 0.2 mg/kg, bw and E₂: 10 μg) before the FST. Controls received saline and/or corn oil. Locomotor activity was also assessed. Acute administration of nicotine significantly reduced immobility in the FST. Acute administration of E₂ also decreased immobility. The combination of nicotine (0.2 mg/kg) and E₂ (10 μg) significantly reduced immobility; however, this reduction was not greater than either agent administered alone. No differences in locomotor activity were detected among the treatment groups. Acute administration of nicotine and E₂, alone and in combination, significantly decreased immobility of ovx rats in the FST, suggesting an antidepressant-like effect of these two agents in this model. However, the combination of E₂ and nicotine did not have any further reduction, indicating a lack of an additive or synergistic effect. Results from this study suggest that agents that target nicotinic acetylcholine receptors and estrogen receptors may be beneficial in alleviating depressive symptoms in women during periods of low estrogen levels.     

Novel effects of estradiol and estrogen receptor α and β on cognitive function

Estrogen influences the development of memory function in humans and rodents and can modulate memory in adults. In these studies we examined the role of the estrogen receptors α (ERα) and β (ERβ) in mediating performance on a hippocampal-dependent, hormone-sensitive task, inhibitory avoidance (IA). Ovariectomized (OVX) estrogen receptor-α-knockout (ERαKO) mice displayed impaired performance on the IA task and OVX heterozygotic (HET) mice exhibited performance that was intermediate between ERαKO and wild-type (WT) mice. Impaired performance by ERαKO mice was rescued by E₂ treatment. The ER antagonist, tamoxifen, did not block enhancement of retention by E₂ suggesting that E₂ mediated modulation of memory is not caused by known genomic receptor mechanisms. In contrast to ERαKO mice, IA performance by OVX estrogen receptor-β-knockout (ERβKO) mice was not compromised. The results indicate an important role for ERα, relative to ERβ, in the establishment of cognitive function and suggest that E₂ modulates memory function via a novel estrogenic mechanism.     

Co-localization of estrogen and angiotensin receptors within subfornical organ neurons

A double-staining immunocytochemical study was done in ovariectomized (OVX) female rats that were either treated with 17β-estradiol (E₂) (OVX+E₂) to produce an approximate circulating level of 30 pg/ml plasma, or not-treated with E₂ (OVX), to investigate the distribution of subfornical organ (SFO) neurons that contained estrogen receptors (ER), and to determine whether these neurons also contained the angiotensin II AT₁-receptor (AT₁R). Neurons that contained either ER-like immunoreactivity only, AT₁R-like immunoreactivity only, or both ER and AT₁R immunoreactivity were found throughout the extent of the SFO in both the OVX+E₂ and OVX rats. However, some regional differences were apparent in both groups of female rats. Neurons containing the ER were predominantly found in the peripheral regions of the SFO, near large blood vessels and the ependymal layer of the third ventricle. A number of lightly stained ER containing neurons was also observed scattered throughout the central core region of the SFO. OVX only animals were found to have a larger number of ER containing neurons in the SFO compared to the E₂ treated animals. Neurons containing AT₁R were also found throughout the SFO, but without a distinct distribution pattern in either group of rats, although there were more neurons that exhibited AT₁R immunoreactivity in the OVX animals. Finally, a distinct group of SFO neurons was found that exhibited both ER and AT₁R immunoreactivity in both groups of animals, although a larger number of these double labelled neurons was found in the OVX animal. Most of these neurons were also found along the peripheral border of the SFO in close proximity to blood vessels and the ventricular lining. These data have demonstrated the co-existence of ER and AT₁R in SFO neurons of the female rat, and suggest that circulating level of E₂ alter the expression of both the ER and AT₁R in these neurons. In addition, these data suggest that E₂ may alter the physiological responses of SFO neurons to angiotensin II by down regulating the number of AT₁R.     

WIN55,212-2, a cannabinoid receptor agonist, protects against nigrostriatal cell loss in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease

Parkinson’s disease (PD) is characterized by the progressive loss of nigrostriatal dopamine neurons leading to motor disturbances and cognitive impairment. Current pharmacotherapies relieve PD symptoms temporarily but fail to prevent or slow down the disease progression. In this study, we investigated the molecular mechanisms by which the non‐selective cannabinoid receptor agonist WIN55,212‐2 (WIN) protects mouse nigrostriatal neurons from 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐induced neurotoxicity and neuroinflammation. Stereological analyses showed that chronic treatment with WIN (4 mg/kg, intraperitoneal), initiated 24 h after MPTP administration, protected against MPTP‐induced loss of tyrosine hydroxylase‐positive neurons in the substantia nigra pars compacta independently of CB₁ cannabinoid receptor activation. The neuroprotective effect of WIN was accompanied by increased dopamine and 3,4‐dihydroxyphenylacetic acid levels in the substantia nigra pars compacta and dorsal striatum of MPTP‐treated mice. At 3 days post‐MPTP, we found significant microglial activation and up‐regulation of CB₂ cannabinoid receptors in the ventral midbrain. Treatment with WIN or the CB₂ receptor agonist JWH015 (4 mg/kg, intraperitoneal) reduced MPTP‐induced microglial activation, whereas genetic ablation of CB₂ receptors exacerbated MPTP systemic toxicity. Furthermore, chronic WIN reversed MPTP‐associated motor deficits, as revealed by the analysis of forepaw step width and percentage of faults using the inverted grid test. In conclusion, our data indicate that agonism at CB₂ cannabinoid receptors protects against MPTP‐induced nigrostriatal degeneration by inhibiting microglial activation/infiltration and suggest that CB₂ receptors represent a new therapeutic target to slow the degenerative process occurring in PD.     

Estrogen receptors in the medial basal hypothalamus of the rat following complete hypothalamic deafferentation

This study was designed to investigate the effect of complete hypothalamic deafferentation (CHD) on the estrogen receptor (ER) concentration in the MBH as well as LH and FSH secretion. Adult female rats underwent CHD using a Halasz-Pupp knife. Sham CHD and intact animals served as controls. Five days after CHD all the rats were ovariectomized and 2 days later they were decapitated and trunk blood collected and the plasma analysed for LH and FSH by radioimmunoassay (RIA). The brains were rapidly removed and the MBH and preoptic area (POA) were dissected. Brain tissues were homogenized in 2.0 ml of phosphate buffer, centrifuged and the supernatant (cytosol) withdrawn. The cytosols were then incubated at 0–4°C for 3 h with [³H]E₂ or [³H]E₂ + unlabeled E₂. Bound and free receptor was separated using 5–30% sucrose gradient centrifugation, Sephadex LH-20 column or hydroxylapatite receptor assays. CHD resulted in a significant (P < 0.05) reduction in the concentration of ER in the MBH when compared with controls. ER concentration in the POA of the CHD group was not significantly different from the control group. CHD also resulted in a significant (P < 0.005) reduction in the plasma concentration of LH and FSH when compared with the controls. These data suggest that the estrogen receptors in the MBH are influenced by the connections with extrahypothalamic regions and that the effect of hypothalamic deafferentation on gonadotropin secretion may be in part secondary to this reduction of receptors in the MBH.