Emilie F. Rissman

A Model System for Study of Sex Chromosome Effects on Sexually Dimorphic Neural and Behavioral Traits

We tested the hypothesis that genes encoded on the sex chromosomes play a direct role in sexual differentiation of brain and behavior. We used mice in which the testis-determining gene (Sry) was moved from the Y chromosome to an autosome (by deletion ofSry from the Y and subsequent insertion of anSry transgene onto an autosome), so that the determination of testis development occurred independently of the complement of X or Y chromosomes. We compared XX and XY mice with ovaries (females) and XX and XY mice with testes (males). These comparisons allowed us to assess the effect of sex chromosome complement (XX vs XY) independent of gonadal status (testes vs ovaries) on sexually dimorphic neural and behavioral phenotypes. The phenotypes included measures of male copulatory behavior, social exploration behavior, and sexually dimorphic neuroanatomical structures in the septum, hypothalamus, and lumbar spinal cord. Most of the sexually dimorphic phenotypes correlated with the presence of ovaries or testes and therefore reflect the hormonal output of the gonads. We found, however, that both male and female mice with XY sex chromosomes were more masculine than XX mice in the density of vasopressin-immunoreactive fibers in the lateral septum. Moreover, two male groups differing only in the form of their Sry gene showed differences in behavior. The results show that sex chromosome genes contribute directly to the development of a sex difference in the brain.

Distribution of estrogen receptor-beta-like immunoreactivity in rat forebrain.

The data presented here are the first to describe the distribution of estrogen receptor-beta (ER beta)-like immunoreactivity in brain tissue. We employed an affinity purified rabbit antiserum made against a portion of the C-terminal of the ER beta protein. The majority of ER beta-like immunoreactive (ER beta-ir) neurons were found in the following regions: lateral septum, bed nucleus of the stria terminalis, paraventricular nucleus, supraoptic nucleus, medial amygdala, the dentate gyrus and the CA1 and CA2 fields of the hippocampus. A few ER beta-ir neurons were noted in the anterior hypothalamus, periventricular nucleus, medial preoptic area, and in the arcuate nucleus. All of the immunoreactivity appeared nuclear in its subcellular distribution, with the exception of the cells in the lateral septum, CA1 and CA2. In these areas immunoreactivity was noted throughout the perikarya and in cell processes. The data suggest that ER beta mediates estrogens actions in a subset of hypothalamic and limbic neurons.

Disruption of estrogen receptor β gene impairs spatial learning in female mice

Here we provide the first evidence, to our knowledge, that estradiol (E2) affects learning and memory via the newly discovered estrogen receptor β (ERβ). In this study, ERβ knockout (ERβKO) and wild-type littermates were tested for spatial learning in the Morris water maze after ovariectomy, appropriate control treatment, or one of two physiological doses of E2. Regardless of treatment, all wild-type females displayed significant learning. However, ERβKOs given the low dose of E2 were delayed in learning acquisition, and ERβKOs administered the higher dose of E2 failed to learn the task. These data show that ERβ is required for optimal spatial learning and may have implications for hormone replacement therapy in women.

Masculine Sexual Behavior Is Disrupted in Male and Female Mice Lacking a Functional Estrogen Receptor α Gene

Masculine sexual behavior is regulated by testosterone (T). However, T can be metabolized to form estrogens or other androgens, which then activate their own receptors. We used knockout mice lacking a functional estrogen receptor alpha (ER alpha) gene to test the hypothesis that, following aromatization, T acts via the ER alpha to activate normal masculine sexual behavior. After gonadectomy and T replacement, wild-type (WT) male and female mice displayed masculine behavior. However, given the same T treatment, little masculine behavior was displayed by mice of either sex that lack a normal copy of the ER alpha gene. In particular, the latency to display masculine sex behavior and the number of mount attempts per trial were significantly reduced in the ER alpha- mice compared to WT littermates (P < 0.05). In addition, we found that in both sexes, ER alpha- mice have a smaller cluster of androgen receptor immunoreactivity in the bed nucleus of the stria terminalis. Using adult ER alpha- mice we were unable to determine whether these genotypic differences are due to organizational or activational effects. However, it is clear that the ER alpha plays a key role in the expression of masculine sexual behavior and in the regulation of androgen receptors in a neuronal cell population involved in the display of motivated behaviors.

Sex Chromosome Complement and Gonadal Sex Influence Aggressive and Parental Behaviors in Mice

Across human cultures and mammalian species, sex differences can be found in the expression of aggression and parental nurturing behaviors: males are typically more aggressive and less parental than females. These sex differences are primarily attributed to steroid hormone differences during development and/or adulthood, especially the higher levels of androgens experienced by males, which are caused ultimately by the presence of the testis-determining gene Sry on the Y chromosome. The potential for sex differences arising from the different complements of sex-linked genes in male and female cells has received little research attention. To directly test the hypothesis that social behaviors are influenced by differences in sex chromosome complement other than Sry, we used a transgenic mouse model in which gonadal sex and sex chromosome complement are uncoupled. We find that latency to exhibit aggression and one form of parental behavior, pup retrieval, can be influenced by both gonadal sex and sex chromosome complement. For both behaviors, females but not males with XX sex chromosomes differ from XY. We also measured vasopressin immunoreactivity in the lateral septum, which was higher in gonadal males than females, but also differed according to sex chromosome complement. These results imply that a gene(s) on the sex chromosomes (other than Sry) affects sex differences in brain and behavior. Identifying the specific X and/or Y genes involved will increase our understanding of normal and abnormal aggression and parental behavior, including behavioral abnormalities associated with mental illness.

Gestational exposure to bisphenol a produces transgenerational changes in behaviors and gene expression.

Bisphenol A (BPA) is a plasticizer and an endocrine-disrupting chemical. It is present in a variety of products used daily including food containers, paper, and dental sealants and is now widely detected in human urine and blood. Exposure to BPA during development may affect brain organization and behavior, perhaps as a consequence of its actions as a steroid hormone agonist/antagonist and/or an epigenetic modifier. Here we show that BPA produces transgenerational alterations in genes and behavior. Female mice received phytoestrogen-free chow with or without BPA before mating and throughout gestation. Plasma levels of BPA in supplemented dams were in a range similar to those measured in humans. Juveniles in the first generation exposed to BPA in utero displayed fewer social interactions as compared with control mice, whereas in later generations (F(2) and F(4)), the effect of BPA was to increase these social interactions. Brains from embryos (embryonic d 18.5) exposed to BPA had lower gene transcript levels for several estrogen receptors, oxytocin, and vasopressin as compared with controls; decreased vasopressin mRNA persisted into the F(4) generation, at which time oxytocin was also reduced but only in males. Thus, exposure to a low dose of BPA, only during gestation, has immediate and long-lasting, transgenerational effects on mRNA in brain and social behaviors. Heritable effects of an endocrine-disrupting chemical have implications for complex neurological diseases and highlight the importance of considering gene-environment interactions in the etiology of complex disease.

Estrogen Receptor Function as Revealed by Knockout Studies: Neuroendocrine and Behavioral Aspects☆

Estrogens are an important class of steroid hormones, involved in the development of brain, skeletal, and soft tissues. These hormones influence adult behaviors, endocrine state, and a host of other physiological functions. Given the recent cloning of a second estrogen receptor (ER) cDNA (the ER beta), work on alternate spliced forms of ER alpha, and the potential for membrane estrogen receptors, an animal with a null background for ER alpha function is invaluable for distinguishing biological responses of estrogens working via the ER alpha protein and those working via another ER protein. Data generated to date, and reviewed here, indicate that there are profound ramifications of the ER alpha disruption on behavior and neuroendocrine function. First, data on plasma levels of estradiol (E2), testosterone (T), and luteinizing hormone (LH) in wild-type (WT) versus ER alpha- mice confirm that ER alpha is essential in females for normal regulation of the hypothalamic-pituitary gonadal axis. Second, ovariectomized female ER alpha- mice do not display sexual receptivity when treated with a hormonal regime of estrogen and progesterone that induces receptivity in WT littermates. Finally, male sexual behaviors are disrupted in ER alpha- animals. Given decades of data on these topics our findings may seem self-evident. However, these data represent the most direct test currently possible of the specific role of the ER alpha protein on behavior and neuroendocrinology. The ER alpha- mouse can be used to ascertain the specific functions of ER alpha, to suggest functions for the other estrogen receptors, and to study indirect effects of ER alpha on behavior via actions on other receptors, neurotransmitters, and neuropeptides.

Lack of functional estrogen receptor β influences anxiety behavior and serotonin content in female mice

Estrogen has been linked to the modulation of anxiety in females. Here we report results of anxiety tests conducted in female estrogen receptor beta (ERbeta) knockout (ERbetaKO) and wild-type (WT) mice. Ovariectomized (OVX) mice treated with chronic estradiol (E2) replacement did not behave differently on the elevated plus-maze when compared with OVX mice that did not experience hormone replacement. However, a genotype difference was noted; WT females were more likely to explore the distal portion of the open arm of the maze than ERbetaKO littermates. In addition, ERbetaKO female mice had significantly lower serotonin (5-HT) content than WT littermates in several brain regions including: the bed nucleus of the stria terminalis, preoptic area, and hippocampus. A similar trend was noted in the dorsal raphe nucleus. Dopamine content was reduced within the caudate putamen in ERbetaKO mice as compared to brains from WT animals. Thus, in the absence of functional ERbeta, regardless of the presence or absence of circulating E2 in plasma, female mice exhibited enhanced anxiety and decreased concentrations of 5-HT or dopamine in several brain regions. We hypothesize that ERbeta is required during development to modulate the effects of estrogen on anxiety and catecholamine concentrations in female mouse brains.

Neurobiology of escalated aggression and violence.

Psychopathological violence in criminals and intense aggression in fruit flies and rodents are studied with novel behavioral, neurobiological, and genetic approaches that characterize the escalation from adaptive aggression to violence. One goal is to delineate the type of aggressive behavior and its escalation with greater precision; second, the prefrontal cortex (PFC) and brainstem structures emerge as pivotal nodes in the limbic circuitry mediating escalated aggressive behavior. The neurochemical and molecular work focuses on the genes that enable invertebrate aggression in males and females and genes that are expressed or suppressed as a result of aggressive experiences in mammals. The fruitless gene, immediate early genes in discrete serotonin neurons, or sex chromosome genes identify sexually differentiated mechanisms for escalated aggression. Male, but not female, fruit flies establish hierarchical relationships in fights and learn from previous fighting experiences. By manipulating either the fruitless or transformer genes in the brains of male or female flies, patterns of aggression can be switched with males using female patterns and vice versa. Work with Sts or Sry genes suggests so far that other genes on the X chromosomes may have a more critical role in female mouse aggression. New data from feral rats point to the regulatory influences on mesocortical serotonin circuits in highly aggressive animals via feedback to autoreceptors and via GABAergic and glutamatergic inputs. Imaging data lead to the hypothesis that antisocial, violent, and psychopathic behavior may in part be attributable to impairments in some of the brain structures (dorsal and ventral PFC, amygdala, and angular gyrus) subserving moral cognition and emotion.

Roles of estrogen receptors α and β in differentiation of mouse sexual behavior

Abstract Sex differences in brain and behavior are ubiquitous in sexually reproducing species. Developmental differences in circulating concentrations of gonadal steroids underlie many sexual dimorphisms. During the late embryonic and early perinatal periods, the testes produce androgens, thus, male brains are exposed to testosterone, and in situ testosterone is aromatized to estradiol. In contrast, females are not exposed to high concentrations of testosterone or estradiol until puberty. In many species, neural sex differences and sexually dimorphic behaviors in adults are initiated primarily by estradiol exposure during early development. In brain, estradiol activates two independent processes: masculinization of neural circuits and networks that are essential for expression of male-typical adult behaviors, and defeminization, the loss of the ability to display adult female-typical behaviors. Here, data for the roles of each of the known estrogen receptors (estrogen receptor α and estrogen receptor β) in these two processes are reviewed. Based on work done primarily in knockout mouse models, separate roles for the two estrogen receptors are suggested. Estrogen receptor α is primarily involved in masculinization, while estrogen receptor β has a major role in defeminization of sexual behaviors. In sum, estradiol can have selective effects on distinct behavioral processes via selective interactions with its two receptors, estrogen receptor α and estrogen receptor β.