Estrogens are important effectors of reproduction and are critical for upregulating female reproductive behavior or lordosis in females. In addition to the importance of transcriptional regulation of genes by 17?-estradiol-bound estrogen receptors (ER), extranuclear signal transduction cascades such as protein kinase A (PKA) are also important in regulating female sexual receptivity. GPR30 (G-protein coupled receptor 30), also known as GPER1, a putative membrane ER (mER), is a G protein-coupled receptor that binds 17?-estradiol with an affinity that is similar to that possessed by the classical nuclear ER and activates both PKA and extracellular-regulated kinase signaling pathways. The high expression of GPR30 in the ventromedial hypothalamus, a region important for lordosis behavior as well as kinase cascades activated by this receptor, led us to hypothesize that GPR30 may regulate lordosis behavior in female rodents.
Sex steroid action is critical to form sexually dimorphic nuclei, although it is not fully understood. We previously reported that masculinization of the principal nucleus of the bed nucleus of the stria terminalis (BNSTp), which is larger and has more neurons in males than in females, involves aromatized testosterone that acts via estrogen receptor-? (ER?), but not estrogen receptor-? (ER?). Here, we examined sex steroid action on the formation of the anteroventral periventricular nucleus (AVPV) that is larger and has more neurons in females. Morphometrical analysis of transgenic mice lacking aromatase, ER?, or ER? genes revealed that the volume and neuron number of the male AVPV were significantly increased by deletion of aromatase and ER? genes, but not the ER? gene. We further examined the AVPV and BNSTp of androgen receptor knockout (ARKO) mice. The volume and neuron number of the male BNSTp were smaller in ARKO mice than those in wild-type mice, while no significant effect of ARKO was found on the AVPV and female BNSTp. We also examined aromatase, ER?, and AR mRNA levels in the AVPV and BNSTp of wild-type and ARKO mice on embryonic day (ED) 18 and postnatal day (PD) 4. AR mRNA in the BNSTp and AVPV of wild-type mice was not expressed on ED18 and emerged on PD4. In the AVPV, the aromatase mRNA level was higher on ED18, although the ER? mRNA level was higher on PD4 without any effect of AR gene deletion. Aromatase and ER? mRNA levels in the male BNSTp were significantly increased on PD4 by AR gene deletion. These results suggest that estradiol signaling via ER? during the perinatal period and testosterone signaling via AR during the postnatal period are required for masculinization of the BNSTp, whereas the former is sufficient to defeminize the AVPV.
Maternal separation (MS) is an animal model mimicking the effects of early life stress on the development of emotional and social behaviors. Recent studies revealed that MS stress increased social anxiety levels in female mice and reduced peri-pubertal aggression in male mice. Estrogen receptor (ER) ? plays a pivotal role in the regulation of stress responses and anxiety-related and social behaviors. Behavioral studies using ER? knockout (?ERKO) mice reported increased social investigation and decreased social anxiety in ?ERKO females, and elevated aggression levels in ?ERKO males compared to wild-type (WT) mice. In the present study, using ?ERKO and WT mice, we examined whether ER? contributes to MS effects on anxiety and social behaviors. ?ERKO and WT mice were separated from their dam daily (4 h) from postnatal day 1-14 and control groups were left undisturbed. First, MS and ER? gene deletion individually increased anxiety-related behaviors in the open field test, but only in female mice. Anxiety levels were not further modified in ?ERKO female mice subjected to MS stress. Second, ?ERKO female mice showed higher levels of social investigation compared with WT in the social investigation test and long-term social preference test. However, MS greatly reduced social investigation duration and elevated number of stretched approaches in WT and ?ERKO females in the social investigation test, suggesting elevated levels of social anxiety in both genotypes. Third, peri-pubertal and adult ?ERKO male mice were more aggressive than WT mice as indicated by heightened aggression duration. On the other hand, MS significantly decreased aggression duration in both genotypes, but only in peri-pubertal male mice. Altogether, these results suggest that ?ERKO mice are sensitive to the adverse effects of MS stress on subsequent female and male social behaviors, which could then have overrode the ER? effects on female social anxiety and male aggression.
The opioid peptides, dynorphin (DYN) and enkephalin (L-ENK) are contained in the hippocampal mossy fiber pathway where they modulate synaptic plasticity. In rats, the levels of DYN and L-ENK immunoreactivity (-ir) are increased when estrogen levels are elevated (Torres-Reveron et al., 2008, 2009). Here, we used quantitative immunocytochemistry to examine whether opioid levels are similarly regulated in wildtype (WT) mice over the estrous cycle, and how these compared to males. Moreover, using estrogen receptor (ER) alpha and beta knock-out mice (AERKO and BERKO, respectively), the present study examined the role of ERs in rapid, membrane-initiated (6 h), or slower, nucleus-initiated (48 h) estradiol effects on mossy fiber opioid levels. Unlike rats, the levels of DYN and L-ENK-ir did not change over the estrous cycle. However, compared to males, females had higher levels of DYN-ir in CA3a and L-ENK-ir in CA3b. In WT and BERKO ovariectomized (OVX) mice, neither DYN- nor L-ENK-ir changed following 6 or 48 h estradiol benzoate (EB) administration. However, DYN-ir significantly increased 48 h after EB in the dentate gyrus (DG) and CA3b of AERKO mice only. These findings suggest that cyclic hormone levels regulate neither DYN nor L-ENK levels in the mouse mossy fiber pathway as they do in the rat. This may be due to species-specific differences in the mossy fiber pathway. However, in the mouse, DYN levels are regulated by exogenous EB in the absence of ER? possibly via an ER?-mediated pathway requiring new gene transcription.
Thyroid hormones influence both neuronal development and anxiety via the thyroid hormone receptors (TRs). The TRs are encoded by two different genes, TR? and TR?. The loss of TR?1 is implicated in increased anxiety in males, possibly via a hippocampal increase in GABAergic activity. We compared both social behaviors and two underlying and related non-social behaviors, state anxiety and responses to acoustic and tactile startle in the gonadally intact TR?1 knockout (?1KO) and TR? (?KO) male mice to their wild-type counterparts. For the first time, we show an opposing effect of the two TR isoforms, TR?1 and TR?, in the regulation of state anxiety, with ?1 knockout animals (?1KO) showing higher levels of anxiety and ?KO males showing less anxiety compared to respective wild-type mice. At odds with the increased anxiety in non-social environments, ?1KO males also show lower levels of responsiveness to acoustic and tactile startle stimuli. Consistent with the data that T4 is inhibitory to lordosis in female mice, we show subtly increased sex behavior in ?1KO male mice. These behaviors support the idea that TR?1 could be inhibitory to ER? driven transcription that ultimately impacts ER? driven behaviors such as lordosis. The behavioral phenotypes point to novel roles for the TRs, particularly in non-social behaviors such as state anxiety and startle.
Oestrogen receptor (ER)? plays important roles in the development and function of various neuronal systems through activation by its ligands, oestrogens. To visualise ER?-positive neurons, we generated transgenic (tg) mice expressing green fluorescent protein (GFP) under the control of the ER? promoter. In three independent tg lines, GFP-positive neurons were observed in areas previously reported to express ER? mRNA, including the lateral septum, bed nucleus of the stria terminalis, medial preoptic nucleus (MPO), hypothalamus, and amygdala. In these areas, GFP signals mostly overlapped with ER? immunoreactivity. GFP fluorescence was seen in neurites and cell bodies of neurons. In addition, the network and detailed structure of neurites were visible in dissociated and slice cultures of hypothalamic neurons. We examined the effect of oestrogen deprivation by ovariectomy on the structure of the GFP-positive neurons. The area of ER?-positive cell bodies in the bed nucleus of the stria terminalis and MPO was measured by capturing the GFP signal and was found to be significantly smaller in ovariectomy mice than in control mice. When neurons in the MPO were infected with an adeno-associated virus that expressed small hairpin RNA targeting the ER? gene, an apparent induction of GFP was observed in this area, suggesting a negative feedback mechanism in which ER? controls expression of the ER? gene itself. Thus, the ER? promoter-GFP tg mice will be useful to analyse the development and plastic changes of the structure of ER?-expressing neurons and oestrogen and its receptor-mediated neuronal responses.
In the sexually dimorphic anteroventral periventricular nucleus (AVPV) of the hypothalamus, females have a greater number of tyrosine hydroxylase-immunoreactive (TH-ir) and kisspeptin-immunoreactive (kisspeptin-ir) neurons than males. In this study, we used proteomics analysis and gene-deficient mice to identify proteins that regulate the number of TH-ir and kisspeptin-ir neurons in the AVPV. Analysis of protein expressions in the rat AVPV on postnatal day 1 (PD1; the early phase of sex differentiation) using two-dimensional fluorescence difference gel electrophoresis followed by MALDI-TOF-MS identified collapsin response mediator protein 4 (CRMP4) as a protein exhibiting sexually dimorphic expression. Interestingly, this sexually differential expressions of CRMP4 protein and mRNA in the AVPV was not detected on PD6. Prenatal testosterone exposure canceled the sexual difference in the expression of Crmp4 mRNA in the rat AVPV. Next, we used CRMP4-knockout (CRMP4-KO) mice to determine the in vivo function of CRMP4 in the AVPV. Crmp4 knockout did not change the number of kisspeptin-ir neurons in the adult AVPV in either sex. However, the number of TH-ir neurons was increased in the AVPV of adult female CRMP4-KO mice as compared with the adult female wild-type mice. During development, no significant difference in the number of TH-ir neurons was detected between sexes or genotypes on embryonic day 15, but a female-specific increase in TH-ir neurons was observed in CRMP4-KO mice on PD1, when the sex difference was not yet apparent in wild-type mice. These results indicate that CRMP4 regulates the number of TH-ir cell number in the female AVPV.
Testosterone is known to play an important role in the regulation of male-type sexual and aggressive behavior. As an aromatised metabolite of testosterone, estradiol-induced activation of estrogen receptor ? (ER?) may be crucial for the induction of these behaviors in male mice. However, the importance of ER? expressed in different nuclei for this facilitatory action of testosterone has not been determined. To investigate this issue, we generated an adeno-associated virus vector expressing a small hairpin RNA targeting ER? to site-specifically knockdown ER? expression. We stereotaxically injected either a control or ER? targeting vector into the medial amygdala, medial pre-optic area (MPOA), or ventromedial nucleus of the hypothalamus (VMN) in gonadally intact male mice. Two weeks after injection, all mice were tested biweekly for sexual and aggressive behavior, alternating between behavior tests each week. We found that suppressing ER? in the MPOA reduced sexual but not aggressive behavior, whereas in the VMN it reduced both behaviors. Knockdown of ER? in the medial amygdala did not alter either behavior. Additionally, it was found that ER? knockdown in the MPOA caused a parallel reduction in the number of neuronal nitric oxide synthase-expressing cells. Taken together, these results indicate that the testosterone facilitatory action on male sexual behavior requires the expression of ER? in both the MPOA and VMN, whereas the testosterone facilitatory action on aggression requires the expression of ER? in only the VMN.
Clinical evidence suggests that antiestrogens inhibit the development of androgen-insensitive prostate cancer. Here, we show that the estrogen receptor ? (ER?) mediates inhibition by the antiestrogen ICI 182,780 (ICI) and its enhancement by estrogen. ER? associated with gene promoters through the tumor-suppressing transcription factor KLF5 (Krüppel-like zinc finger transcription factor 5). ICI treatment increased the recruitment of the transcription coactivator CBP [CREB (adenosine 3,5-monophosphate response element-binding protein)-binding protein] to the promoter of FOXO1 through ER? and KLF5, which enhanced the transcription of FOXO1. The increase in FOXO1 abundance led to anoikis in prostate cancer cells, thereby suppressing tumor growth. In contrast, estrogen induced the formation of complexes containing ER?, KLF5, and the ubiquitin ligase WWP1 (WW domain containing E3 ubiquitin protein ligase 1), resulting in the ubiquitination and degradation of KLF5. The combined presence of KLF5 and ER? positively correlated with longer cancer-specific survival in prostate cancer patients. Our results demonstrate that estrogens and antiestrogens affect prostate tumor growth through ER?-mediated regulation of KLF5.
To investigate the effects of early life stress on the development of social behaviors in male mice, we examined behavioral responses toward same sex stimulus mice in the social investigation test and aggressive behaviors in peripubertal male mice exposed to maternal separation (MS) during the first 2 weeks of life. MS suppressed aggressive behaviors from 5-9 weeks of age, but had no effect on social investigative behaviors in the social investigation test. Investigation of neuroendocrine bases of behavioral effects of MS showed that MS reduced plasma testosterone levels and decreased arginine vasopressin and increased oxytocin immunoreactivity in the paraventricular nucleus of peripubertal males. These results collectively suggest that early life stress disrupts the development of male aggressive behaviors and associated neuroendocrine systems.
The principal nucleus of the bed nucleus of the stria terminalis (BNSTp) is a sexually dimorphic nucleus, and the male BNSTp is larger and has more neurons than the female BNSTp. To assess the roles of neuroestrogen synthesized from testicular androgen by brain aromatase in masculinization of the BNSTp, we performed morphometrical analyses of the adult BNSTp in aromatase knockout (ArKO), estrogen receptor-? knockout (?ERKO), and estrogen receptor-? knockout (?ERKO) mice and their respective wild-type littermates. In wild-type littermates, the BNSTp of males had a larger volume and greater numbers of neuronal and glial cells than did that of females. The volume and neuron number of the BNSTp in ArKO and ?ERKO males and glial cell number of the BNSTp in ?ERKO males were significantly smaller than those of wild-type male littermates, and they were not significantly different from those in female mice with either gene knockout. In contrast, there was no significant morphological difference in the BNSTp between ?ERKO and wild-type mice. Next, we examined the BNSTp of ArKO males subcutaneously injected with estradiol benzoate (EB) on postnatal days 1, 2, and 3 (1.5 ?g/day). EB-treated ArKO males had a significantly greater number of BNSTp neurons than did oil-treated ArKO males. The number of BNSTp neurons in EB-treated ArKO males was comparable to that in wild-type males. These findings suggested that masculinization of the BNSTp in mice involves the actions of neuroestrogen that was synthesized by aromatase and that this estrogen mostly binds to ER? during the postnatal period.
Estrogen receptor alpha (ERalpha) typically masculinizes male behavior, while low levels of ERalpha in the medial amygdala (MeA) and the bed nucleus of the stria terminalis (BST) are associated with high levels of male prosocial behavior. In the males of the highly social prairie vole (Microtus ochrogaster), increasing ERalpha in the MeA inhibited the expression of spontaneous alloparental behavior and produced a preference for novel females. To test for the effects of increased ERalpha in the BST, a viral vector was used to enhance ERalpha expression in the BST of adult male prairie voles. Following treatment, adult males were tested for alloparental behavior with 1-3-day-old pups, and for heterosexual social preference and affiliation. Treatment did not affect alloparental behavior as 73% of ERalpha-BST males and 62.5% of control males were alloparental. Increasing ERalpha in the BST affected heterosexual affiliation, with ERalpha-BST males spending significantly less total time in side-by-side contact with females relative to time spent with control males. ERalpha-BST males did not show a preference for either the familiar or novel female. These findings differed significantly from those reported in ERalpha-MeA enhanced males, where ERalpha inhibited alloparental behavior and produced a preference for a novel female. The findings from this study suggest two things: first, that increased ERalpha in the BST decreases social affiliation and second, that altering ERalpha in different regions of the social neural circuit differentially impacts the expression of social behavior.
Social recognition manifests itself in decreased investigation of a previously encountered individual. Estrogen receptor alpha (ERalpha) knock out mice show deficient social recognition and anxiety. These data show that the ERalpha is involved in these effects, but they do not say anything about the brain sites important for these effects. In this study, an shRNA encoded within an AAV viral vector directed against the ERalpha receptor gene (or containing luciferase control), was injected bilaterally into the posterodorsal amygdala (MePDA) or the ventromedial nucleus of the hypothalamus (VMN) of female rats. An 81% reduction of ERalpha expression in the MePDA eliminated social recognition. Moreover, this diminution of ERalpha in the MePDA reduced anxiety in the light/dark choice test. In contrast, social recognition was unaffected after ERalpha knockdown in the VMN while aggressiveness against the juvenile was enhanced. In conclusion, social recognition and anxiety in female rats are modulated by the ERalpha in the amygdala. Moreover, aggression against juveniles but not against adults could, at least partly, depend on the ERalpha in the VMN.
The display of copulatory behaviors usually requires the presence of a mate and is, therefore, preceded by a search for and approach to a potential partner. The intensity of approach behaviors is determined by a process labeled sexual incentive motivation. Although it is known that female sexual motivation depends on estrogens, their site of action within the brain is unknown. In the present experiment, we obtained data relevant to this issue. An shRNA encoded within an adeno-associated viral (AAV) vector directed against the estrogen receptor alpha (ERalpha) gene (or containing a nonsense base sequence as a control treatment) was injected bilaterally into the ventromedial nucleus of the hypothalamus (VMN) or the posterodorsal amygdala (MePDA) of female rats. After an 80% reduction of the number of ERalpha in the VMN, sexual incentive motivation was absent after treatment with estradiol and progesterone. Proceptivity and receptivity were also much reduced, while the number of rejections was enhanced. Suppression of the ERalpha in the MePDA lacked these effects. Likewise, the inactive control AAV vector failed to modify any behavior. Thus, the ERalpha in the VMN, but not in the MePDA, is important for proceptivity and receptivity as well as for sexual incentive motivation. These results show that ERalpha in the VMN is crucial for the entire sequence of behavioral events from the processes leading to the establishment of sexual contact until the accomplishment of copulatory behaviors.
In mammals, the formative environment for social and anxiety-related behaviors is the family unit; in the case of rodents, this is the litter and the mother-young bond. A deciding factor in this environment is the sex ratio of the litter and, in the case of mice lacking functional copies of gene(s), the ratio of the various genotypes in the litter. Both Sex and Genotype ratios of the litter affect the nature and quality of the individuals behavior later in adulthood, as well as metabolic activity in brain nuclei that underlie these behaviors. Mice were raised in litters reconstituted shortly after to birth to control for sex ratio and genotype ratio (wild type pups versus pups lacking a functional estrogen receptor alpha). In both males and females, the Sex and Genotype of siblings in the litter affected aggressive behaviors as well as patterns of metabolic activity in limbic nuclei in the social behavior network later in adulthood. Further, this pattern in males varied depending upon the Genotype of their brothers and sisters. Principal Components Analysis revealed two components comprised of several amygdalar and hypothalamic nuclei; the VMH showed strong correlations in both clusters, suggesting its pivotal nature in the organization of two neural networks.
The activity-dependent transcription factor zif268 is re-activated in sleep following hippocampal long-term potentiation (LTP). However, the activation of secondary genes, possibly involved in modifying local synaptic strengths and ultimately stabilizing memory traces during sleep, has not yet been studied. Here, we investigated changes in hippocampal and cortical gene expression at a time point subsequent to the previously reported initial zif268 re-activation during sleep. Rats underwent unilateral hippocampal LTP and were assigned to SLEEP or AWAKE groups. Eighty minutes after a long rapid-eye-movement sleep (REMS) episode (or an equivalent amount of time for awake group) animals had their hippocampi dissected and processed for gene microarray hybridization. Prefrontal and parietal cortices were also collected for qRT-PCR analysis. The microarray analysis identified 28 up-regulated genes in the hippocampus: 11 genes were enhanced in the LTPed hemisphere of sleep animals; 13 genes were enhanced after sleep, regardless of hemisphere; and 4 genes were enhanced in LTPed hemisphere, regardless of behavioral state. qRT-PCR analysis confirmed the up-regulation of aif-1 and sc-65 during sleep. Moreover, we observed a down-regulation of the purinergic receptor, P2Y4R in the LTP hemisphere of awake animals and a trend for the protein kinase, CaMKI to be up-regulated in the LTP hemisphere of sleep animals. In the prefrontal cortex, we showed a significant LTP-dependent down-regulation of gluR1 and spinophilin specifically during sleep. Zif268 was down-regulated in sleep regardless of the hemisphere. No changes in gene expression were observed in the parietal cortex. Our findings indicate that a set of synaptic plasticity-related genes have their expression modulated during sleep following LTP, which can reflect biochemical events associated with reshaping of synaptic connections in sleep following learning.
It has been shown that long-term estrogen treatment in gonadectomized female mice increases anxiety levels. On the other hand, a recent study has reported that estrogen may down-regulate the levels of anxiety by acting through estrogen receptor (ER) beta. In the present study, we investigated the role of ER-beta in the regulation of anxiety levels in female mice after long-term estrogen treatment. Gonadectomized ER-beta knockout (betaERKO) female mice and their wild type (betaWT) littermates were implanted several different doses (experiment 1: 2.0 microg/day, experiment 2: 1.0, 0.4, 0.2 or 0.1 microg/day) of an estradiol benzoate (EB) or placebo pellet. Ten days after pellet implant, behavioral tests commenced to measure the anxiety levels (experiment 1: light-dark transition test (LDT), experiment 2: LDT, elevated plus maze test (EPM) and social investigation test (SIT)). We found that, at higher-doses, long-term treatment of EB had anxiogenic effects in both betaWT and betaERKO mice as indicated by a decrease of the time spent in the light side and the number of transitions between two sides during LDT. In contrast, several behavioral measurements indicated that the lower-doses treatment of EB might reduce the anxiety levels possibly through ER-beta. Particularly, the anxiolytic effects of EB in the SIT were more pronounced in betaWT mice than betaERKO mice. Together, the findings in the present study suggest that estrogen may have both anxiolytic and anxiogenic effects in female mice, and that ER-beta gene disruption did not affect anxiogenic regulation by estrogen in female mice, but partially affected anxiolytic regulation.
Locus coeruleus (LC) is involved in the LHRH regulation by gonadal steroids. We investigated the expression of progesterone and estrogen receptors (PR; ER) in LC neurons of ERalpha (alphaERKO) or ERbeta (betaERKO) knockout mice, and their wild-type (alphaWT and betaWT). Immunocytochemical studies showed that LC expresses PR and both ERs, although ERbeta was more abundant. Estradiol benzoate (EB) decreased ERalpha-positive cells in WT and betaERKO mice, and progesterone caused a further reduction, whereas none of the steroids influenced ERbeta expression. ERbeta deletion increased ERalpha while ERalpha deletion did not alter ERbeta expression. In both WT mice, EB increased PR expression, which was diminished by progesterone. These steroid effects were also observed in alphaERKO animals but to a lesser extent, suggesting that ERalpha is partially responsible for the estrogenic induction of PR in LC. Steroid effects on PR in betaERKO mice were similar to those in the alphaERKO but to a lesser extent, probably because PR expression was already high in the oil-treated group. This expression seems to be specific of LC neurons, since it was not observed in other areas studied, the preoptic area and ventromedial nucleus of hypothalamus. These findings show that LC in mice expresses alphaER, betaER, and PR, and that a balance between them may be critical for the physiological control of reproductive function.
Eating is modulated by estradiol in females of many species and in women. To further investigate the estrogen receptor mechanism mediating this effect, ovariectomized rats and mice were treated with estradiol benzoate or the estrogen receptor-alpha (ER-alpha)-selective agonist PPT. PPT inhibited eating in rats much more rapidly than estradiol (approximately 2-6 h versus >24 h). In contrast, the latencies to vaginal estrus after PPT and estradiol were similar (>24 h). PPT also inhibited eating within a few hours in wild-type mice, but failed to inhibit eating in transgenic mice deficient in ER-alpha (ERalphaKO mice). PPT, but not estradiol, induced the expression of c-Fos in corticotrophin-releasing hormone (CRH)-expressing cells of the paraventricular nucleus (PVN) of the hypothalamus within 90-180 min in rats. Both PPT and estradiol reduced c-Fos expression in an ER-alpha-containing area of the nucleus of the solitary tract. The anomalously rapid eating-inhibitory effect of PPT suggests that PPTs neuropharmacological effect differs from estradiols, perhaps because PPT differentially activates membrane versus nuclear ER-alpha or because PPT activates non-ER-alpha membrane estrogen receptors in addition to ER-alpha. The failure of PPT to inhibit eating in ERalphaKO mice, however, indicates that ER-alpha is necessary for PPTs eating-inhibitory action and that any PPT-induced activation of non-ER-alpha estrogen receptors is not sufficient to inhibit eating. Finally, the rapid induction of c-Fos in CRH-expressing cells in the PVN by PPT suggests that PPT elicits a neural response that is similar to that elicited by stress or aversive emotional stimuli.
The developmental process of prolactin (PRL) cells in the fetal pituitary gland was studied in mice. Although PRL cells were hardly detectable in the pituitary gland of intact fetuses, a treatment with 17beta-estradiol (E(2)) in vitro induced a number of PRL cells that varied drastically in number depending on the stage of gestation with a peak at embryonic d 15. This effect was specific to E(2), with epidermal growth factor, insulin, and forskolin failing to induce PRL cells. Although both estrogen receptor (ER)alpha and ERbeta were expressed in the fetal pituitary gland, the results from ER knockout models showed that only ERalpha mediates E(2) action on PRL cells. A few PRL cells were observed in ERalpha-deficient mice as well as in their control littermates, suggesting that estrogen is not required for the phenotype determination of PRL cells. Unexpectedly, the effect of E(2) on the induction of PRL cells in vitro was diminished after embryonic d 15. Present results suggest that the exposure of fetal PRL cells to glucocorticoids (GCs) results in a reduction of sensitivity to E(2). The mechanism underlying the down-regulation of estrogen sensitivity by GCs was found not to be down-regulation of ER levels, induction of annexin 1, a GC-inducible inhibitor of PRL secretion, or a decrease in the number of PRL precursors by apoptosis. The effect of GCs appeared within 2 h and did not require a de novo protein synthesis. GCs are considered to be involved in the mechanisms of silencing pituitary PRL in gestation possibly through a novel mechanism.
The medial preoptic area has been shown to be intricately involved in many behaviors, including locomotion, sexual behavior, maternal care, and aggression. The gene encoding estrogen receptor-? (ER?) protein is expressed in preoptic area neurons, and a very dense immunoreactive field of ER? is found in the preoptic region. ER? knockout animals show deficits in maternal care and sexual behavior and fail to exhibit increases in these behaviors in response to systemic estradiol treatment. In the present study, we used viral-vector mediated RNA interference to silence ER? expression specifically in the preoptic area of female mice and measured a variety of behaviors, including social and sexual aggression, maternal care, and arousal activity. Suppression of ER? in the preoptic area almost completely abolished maternal care, significantly increasing the latency to pup retrieval and significantly reducing the time the moms spent nursing and licking the pups. Strikingly, maternal aggression toward a male intruder was not different between control and preoptic ER?-silenced mice, demonstrating the remarkably specific role of ER? in these neurons. Reduction of ER? expression in preoptic neurons significantly decreased sexual behavior in female mice and increased aggression toward both sexual partners and male intruders in a seminatural environment. Estrogen-dependent increases in arousal, measured by home cage activity, were not mediated by ER? expression in the preoptic neurons we targeted, as ER?-suppressed mice had increases similar to control mice. Thus, we have established that a specific gene in a specific group of neurons is required for a crucially important natural behavior.
Maternal separation (MS) stress is known to induce long-lasting alterations in emotional and anxiety-related behaviors, but effects on social behaviors are not well defined. The present study examined MS effects on female social behaviors in the social investigation (SIT) and social preference (SPT) tests, in addition to non-social behaviors in the open-field (OFT) and light-dark transition (LDT) tests in C57BL/6J mice. All females were tested as ovariectomized to eliminate confounding effects of endogenous estrogen during behavioral testing. Daily MS (3 hr) from postnatal day 1 to 14 did not affect anxiety levels in LDT, but were elevated in OFT with modified behavioral responses to the novel environment. Furthermore, MS altered social investigative behaviors and preference patterns toward unfamiliar stimulus mice in SIT and short- and long-term SPT paradigms. In SIT, MS reduced social investigation duration and increased number of stretched approaches towards both female and male unfamiliar stimulus mice, suggesting increased social anxiety levels in MS females. Similarly, MS heightened levels of social anxiety during short-term SPT but no MS effect on social preference was found. On the other hand, MS females displayed a distinctive preference for female stimuli, unlike control females, when tested for long-term SPT over a prolonged period of 5 days. Evaluation of FosB expression in the paraventricular nucleus, medial and central amygdala following stimulus exposure demonstrated greater number of FosB immunopositive cells in all three brain regions in MS females compared to control females. These results suggest that MS females might differ in neuroendocrine responses toward unfamiliar female and male opponents, which may be associated with modifications in social behaviors found in the present study. Taken together, this study provides new evidence that early life stress modifies female social behaviors by highlighting alterations in behavioral responses to situations involving social as well as non-social novelty.
Ovariectomized females were given an infusion in the medial preoptic area (MPOA) of a viral vector carrying either a shRNA directed against the estrogen receptor ? (ER?) or luciferase. The females were subjected to a test for sexual incentive motivation immediately followed by a test for receptivity and proceptive behaviors. Two weeks later they were tested in the light/dark choice procedure, and after another 2 weeks they were subjected to a test in a brightly lit open field. Finally, the females were given free access to a running wheel for 88h. The females were treated with estradiol benzoate (EB), 18 or 1.5?g/kg, in randomized order 52h before each test except the running wheel. In that experiment, they were given EB 48h after introduction into the wheel cage. They were given progesterone, 1mg/rat, about 4h before all tests, except the running wheel. The shRNA reduced the number of ER? with 83%. Females with few ER? in the MPOA showed increased lordosis quotient after the 1.5?g/kg dose of EB. There was no effect on proceptive behaviors or on rejections. When given the 18?g/kg EB dose, there was no difference between females with few preoptic ER? and controls. In the test for sexual incentive motivation, females with few preoptic ER? approached the castrated male incentive more than controls, regardless of EB dose. They also moved a shorter distance. In the light/dark choice test as well as in the open field, females with few ER? in the MPOA showed signs of reduced fear/anxiety, since they spent more time in the light part of the dark/light box and in the center of the open field. Finally, the data from the running wheel showed that females with few preoptic ER? failed to show enhanced activity after treatment with EB. These data show that the preoptic ER? inhibits lordosis in females with an intermediate level of receptivity while it fails to do so in fully receptive females. The ER? in the MPOA seems to be necessary for selective approach to a sexual incentive. Finally, activation of this receptor appears to have anxiogenic effects in the procedures employed here. A hypothesis for how all these actions of the preoptic ER? contributes to efficient reproductive behavior is outlined.
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