Sonoko Ogawa

Roles of Estrogen Receptor-α Gene Expression in Reproduction-Related Behaviors in Female Mice1

The role of gene expression of the estrogen receptor-␣ form (ER␣) in the regulation of female reproductive behavior was investigated in estrogen receptor knockout (ERKO) mice, deficient specifically for the ER␣, but not the ER␤, gene. Estrogen-or estrogen-plus progesterone-treated gonadectomized ERKO mice did not show any lordosis response. Detailed behavioral analysis revealed that ERKO females were also deficient in sexual behavioral interactions preceding the lordosis response. They were extremely rejective toward attempted mounts by stud male mice, which could not show any intromissions. During resident-intruder aggression tests, gonadally intact ERKO females were more aggressive toward female intruder mice than wild-type (WT) mice. Gonadectomy did not influence the levels of aggressive behavior, and their genotype differences when mice were tested both before and after gonadectomy. However, when mice were tested after gonadectomy for the first time, very few ERKO mice showed aggression. In contrast to aggression, male-type sexual behavior shown by resident mice toward female intruder mice during aggression tests was not different between ERKO and WT mice and was completely abolished after gonadectomy of the resident mice. Finally, it was also found that ERKO females showed greatly reduced levels of parental behavior toward newborn pups placed in their home cage. These changes in parental behavior were not influenced by gonad-ectomy. ERKO females retrieved significantly fewer numbers of pups with longer latencies compared with wild-type (WT) or heterozygous (HZ) littermates when they were tested as gonadally intact or 20 – 65 days after gonadectomy. In addition, during parental behavior tests, a significantly higher percentage of ERKO mice exhibited infanticide compared with WT and HZ mice, which rarely showed infanticide. Taken together, these findings suggest that ER␣ gene expression plays a key role in female mice, not only for sexual behavior but also for other interrelated behaviors, such as parental and aggressive behaviors. In addition, persistence of genotype differences in parental and aggressive behavior after gonadectomy indicates that ER␣ activation during neural developmental processes may also be involved in the regulation of these behaviors. I T IS WELL established that the ovarian steroid, estrogen, regulates female reproduction and lordosis behavior by binding to intracellular estrogen receptors (ER) in the brain. This genomic action of estrogen is assumed to be mediated not only through the classical form of ER (now termed ER␣), but also possibly through the second form of ER, ER␤, which was recently cloned (1, 2). Recent studies have shown that …

Silencing of estrogen receptor α in the ventromedial nucleus of hypothalamus leads to metabolic syndrome

Estrogen receptor α (ERα) plays a pivotal role in the regulation of food intake and energy expenditure by estrogens. Although it is well documented that a disruption of ERα signaling in ERα knockout (ERKO) mice leads to an obese phenotype, the sites of estrogen action and mechanisms underlying this phenomenon are still largely unknown. In the present study, we exploited RNA interference mediated by adeno-associated viral vectors to achieve focused silencing of ERα in the ventromedial nucleus of the hypothalamus, a key center of energy homeostasis. After suppression of ERα expression in this nucleus, female mice and rats developed a phenotype characteristic for metabolic syndrome and marked by obesity, hyperphagia, impaired tolerance to glucose, and reduced energy expenditure. This phenotype persisted despite normal ERα levels elsewhere in the brain. Although an increase in food intake preceded weight gain, our data suggest that a leading factor of obesity in this model is likely a decline in energy expenditure with all three major constituents being affected, including voluntary activity, basal metabolic rate, and diet-induced thermogenesis. Together, these findings indicate that ERα in the ventromedial nucleus of the hypothalamus neurons plays an essential role in the control of energy balance and the maintenance of normal body weight.

Deficient pheromone responses in mice lacking a cluster of vomeronasal receptor genes

The mammalian vomeronasal organ (VNO), a part of the olfactory system, detects pheromones—chemical signals that modulate social and reproductive behaviours. But the molecular receptors in the VNO that detect these chemosensory stimuli remain undefined. Candidate pheromone receptors are encoded by two distinct and complex superfamilies of genes, V1r and V2r (refs 3 and 4), which code for receptors with seven transmembrane domains. These genes are selectively expressed in sensory neurons of the VNO. However, there is at present no functional evidence for a role of these genes in pheromone responses. Here, using chromosome engineering technology, we delete in the germ line of mice a ∼600-kilobase genomic region that contains a cluster of 16 intact V1r genes. These genes comprise two of the 12 described V1r gene families, and represent ∼12% of the V1r repertoire. The mutant mice display deficits in a subset of VNO-dependent behaviours: the expression of male sexual behaviour and maternal aggression is substantially altered. Electrophysiologically, the epithelium of the VNO of such mice does not respond detectably to specific pheromonal ligands. The behavioural impairment and chemosensory deficit support a role of V1r receptors as pheromone receptors.

An estrogen-dependent four-gene micronet regulating social recognition: A study with oxytocin and estrogen receptor-α and -β knockout mice

Estrogens control many physiological and behavioral processes, some of which are connected to reproduction. These include sexual and other social behaviors. Here we implicate four gene products in a micronet required for mammalian social recognition, through which an individual learns to recognize other individuals. Female mice whose genes for the neuropeptide oxytocin (OT) or the estrogen receptor (ER)-β or ER-α had been selectively “knocked out” were deficient specifically in social recognition and social anxiety. There was a remarkable parallelism among results from three separate gene knockouts. The data strongly suggest the involvement in social recognition of the four genes coding for ER-α, ER-β, OT, and the OT receptor. We thus propose here a four-gene micronet, which links hypothalamic and limbic forebrain neurons in the estrogen control over the OT regulation of social recognition. In our model, estrogens act on the OT system at two levels: through ER-β, they regulate the production of OT in the hypothalamic paraventricular nucleus, and through ER-α, they drive the transcription of the OT receptor in the amygdala. The proper operation of a social recognition mechanism allows for the expression of appropriate social behaviors, aggressive or affiliative.

Deficits in E2-Dependent Control of Feeding, Weight Gain, and Cholecystokinin Satiation in ER-α Null Mice

To test the role of gene expression of the classical ER (ER alpha) in the inhibitory effects of E on food intake and body weight, we ovariectomized and administered E2 benzoate (75 pg/d) or vehicle to wild-type (WT) mice and mice with a null mutation of ER alpha (alpha ERKO). Mice were ovariectomized at age 9 wk, at which time there was no significant effect of genotype on food intake or body weight. During an 18-d test after recovery from ovariectomy, vehicle-treated WT mice increased daily food intake and gained more body weight than E2-treated WT mice, whereas food intake and body weight gain were not different in E2- and vehicle-treated alpha ERKO mice. Carcass analysis revealed parallel changes in body lipid content, but not water or protein content. Because an increase in the potency of the peripheral cholecystokinin (CCK) satiation-signaling system mediates part of E2s influence on feeding in rats, the influence of ip injections of 250 microg of the selective CCK(A) receptor antagonist devazepide was then tested. Devazepide increased 3-h food intake in E2-treated WT mice, but was ineffective in both groups of alpha ERKO mice. Furthermore, ip injections of 4 microg/kg CCK-8 increased the number of cells expressing c-Fos immunoreactivity in the nuclei of the solitary tract of E2-treated WT mice more than it did in vehicle-treated WT mice, whereas E2 had no such effect in alpha ERKO mice. Thus, ER alpha is necessary for normal responsivity of food intake, body weight, adiposity, and the peripheral CCK satiation-signaling system to E2 in mice, and ER beta is not sufficient for any of these effects. This is the first demonstration that ER alpha gene expression is involved in the estrogenic control of feeding behavior and weight regulation of female mice.

Role of adrenal steroid mineralocorticoid and glucocorticoid receptors in long-term potentiation in the CA1 field of hippocampal slices.

We previously demonstrated in the dentate gyrus (DG) of anesthetized and freely behaving rats that both acute as well as chronic administration of corticosterone produces a suppression in long-term potentiation (LTP). In subsequent studies we showed, again in the DG, that activation of the two types of adrenal steroid receptors (mineralocorticoid (MR) and glucocorticoid (GR)) produce biphasic effects on synaptic plasticity; activation of MR produces an enhancement while activation of GR produces a suppression in LTP. In a separate study, we further demonstrated in rats administered the specific GR agonist RU 28362 that high-frequency stimulation, which normally produces LTP, instead produced long-term depression (LTD) in these animals. In the present study we investigated the effects of MR and GR activation by adrenal steroids on synaptic plasticity of the hippocampal CA1 field, but we studied this ex vivo, in a slice preparation. The results indicate that, as in our studies in the DG, adrenal steroids produce biphasic effects: in ADX rats, aldosterone (a specific MR agonist) enhanced while RU 28362 suppressed synaptic plasticity. Unlike the in vivo preparation, however, rarely was LTD observed in the animals receiving RU 28362. Also, ADX itself did not produce noticeable effects on synaptic plasticity. The present results are in agreement with previous studies showing that elevations in corticosterone or an acute episode of experimentally induced stress in vivo causes a suppression in LTP in the hippocampal CA1 field, in vitro.

Anxiety and fear behaviors in adult male and female C57BL/6 mice are modulated by maternal separation.

This study investigated the effects of maternal separation in C57BL/6 male and female mice during infancy on later adult fear and anxiety behaviors. Additionally, we observed the maternal behavior of the dams to examine aspects of maternal care that may be modulated by daily bouts of separation. In males, mice that experienced maternal separation during the neonatal period displayed significantly higher levels of anxiety and fear behavior, as measured by the open field test and elevated plus maze, compared to control, standard facility reared males. In females, however, maternal separation reduced anxiety and fear behavior in the open field test, but only when the females were in the diestrous phase of their estrous cycle. The 30-min daily observation of the dams revealed that the separation did not significantly alter the frequency of the maternal care provided by the dam at the time point measured. These results indicate that the emotionality of adult male and female mice can be modulated by maternal separation. However, this effect is dependent on the sex of the offspring and the phase of the estrous cycle of the female.

RNAi-mediated silencing of estrogen receptor α in the ventromedial nucleus of hypothalamus abolishes female sexual behaviors

Estrogen receptor α (ERα) plays a major role in the regulation of neuroendocrine functions and behaviors by estrogens. Although the generation of ERα knockout mice advanced our knowledge of ERα functions, gene deletion using this method is global and potentially confounded by developmental consequences. To achieve a site-specific knockdown of ERα in the normally developed adult brain, we have generated an adeno-associated virus vector expressing a small hairpin RNA targeting ERα. After bilateral injection of this vector into the hypothalamic ventromedial nucleus in ovariectomized female mice, expression levels of ERα as well as the estrogen-inducible progesterone receptor were profoundly reduced despite the continued presence of this receptor elsewhere in the brain. Functionally, silencing of ERα in the ventromedial nucleus abolished female proceptive and receptive sexual behaviors while enhancing rejection behavior. These results provide evidence that adeno-associated virus-mediated long-term knockdown of genes can be used to delineate their effects on complex behaviors in discrete brain regions.

Estrogen receptor-β regulates transcript levels for oxytocin and arginine vasopressin in the hypothalamic paraventricular nucleus of male mice

Estrogen receptor (ER)-beta, unlike ER-alpha, is localized in the hypothalamic paraventricular nucleus (PVN) which also contains neuropeptide synthesizing neurons, such as oxytocin (OT), arginine vasopressin (AVP) and corticotropin-releasing hormone (CRH). Although it is known that some ER-beta containing neurons co-express OT and AVP, but not CRH, in the PVN, it is not yet determined whether ER-beta activation may indeed play a role in estrogenic regulation on syntheses of these neuropeptides. In the present study, we tested this hypothesis by comparing the effects of estrogen on the levels of OT, AVP and CRH messenger RNA (mRNA) between ER-beta knockout (betaERKO) and wild type (WT) control male mice. Mice were gonadectomized and implanted with either a pellet containing estradiol benzoate (2.5-5.0 microg/day) or a placebo pellet for 21 days. In situ hybridization histochemistry revealed that estrogen treatment resulted in a significant increase in OT transcripts (151.6+/-6.0%) and a decrease in AVP transcripts (77.8+/-5.2%) in the PVN of WT mice, compared to the placebo control group. This estrogenic regulation of OT and AVP mRNA levels in the PVN was completely abolished in betaERKO mice. Similar genotype differences in the effects of estrogen on the numbers of OT- and AVP-containing cells were found in immunocytochemical studies performed in a separate set of mice. On the other hand, the expression of CRH mRNA in the PVN was not affected by estrogen treatment in either WT or betaERKO mice. Furthermore, estrogen did not cause any changes in the levels of OT or AVP mRNA, regardless of genotype, in the supraoptic nucleus where, unlike in rats, ER-beta containing neurons are rarely found in mice. Finally, estrogen significantly increased OT mRNA levels in both betaERKO and WT in the medial preoptic area, which contains both ER-alpha and ER-beta. These results suggest that ER-beta activation may play a critical role in estrogenic regulation of OT and AVP gene expression in the PVN.

Genotype/Age Interactions on Aggressive Behavior in Gonadally Intact Estrogen Receptor β Knockout (βERKO) Male Mice

Abstract Estrogen, as an aromatized metabolite of testosterone, has a facilitatory effect on male aggressive behavior in mice. Two subtypes of estrogen receptors, α (ER-α) and β (ER-β), in the brain are known to bind estrogen. Previous studies revealed that the lack of ER-α gene severely reduced the induction of male aggressive behavior. In contrast, mice that lacked the ER-β gene tended to be more aggressive than wild type (WT) control mice, although the behavioral effects of ER-β gene disruption were dependent on their social experience. These findings lead us to hypothesize that estrogen may facilitate aggression via ER-α whereas it may inhibit aggression via ER-β. In the present study, we further investigated the role of ER-β in the regulation of aggressive behavior by examining developmental changes starting at the time of first onset, around the age of puberty. Aggressive behaviors of ER-β gene knockout (βERKO) mice were examined in three different age groups, puberty, young-adult, and adult. Each mouse was tested every other day for three times in a resident–intruder paradigm against olfactory bulbectomized intruder mice and their trunk blood was collected for measurements of serum testosterone after the completion of the study. Overall, βERKO mice were significantly more aggressive than WT. These genotype differences were more pronounced in puberty and young adult age groups, but not apparent in the adult age group, in which βERKO mice were less aggressive than those in two younger age groups. Serum testosterone levels of βERKO mice were significantly higher than those of WT mice only in the pubertal age group, but not in young adult (when βERKO mice were still significantly more aggressive than WT mice) and adult (when no genotype differences in aggression were found) age groups. These results suggest that ER-β mediated actions of gonadal steroids may more profoundly be involved in the inhibitory regulation of aggressive behavior in pubertal and young adult mice.