Revisiting the neural role of estrogen receptor beta in male sexual behavior by conditional mutagenesis

Estradiol derived from neural aromatization of gonadal testosterone plays a key role in the perinatal organization of the neural circuitry underlying male sexual behavior. The aim of this study was to investigate the contribution of neural estrogen receptor (ER) β in estradiol-induced effects without interfering with its peripheral functions. For this purpose, male mice lacking ERβ in the nervous system were generated. Analyses of males in two consecutive tests with a time interval of two weeks showed an effect of experience, but not of genotype, on the latencies to the first mount, intromission, pelvic thrusting and ejaculation. Similarly, there was an effect of experience, but not of genotype, on the number of thrusts and mating length. Neural ERβ deletion had no effect on the ability of males to adopt a lordosis posture in response to male mounts, after castration and priming with estradiol and progesterone. Indeed, only low percentages of both genotypes exhibited a low lordosis quotient. It also did not affect their olfactory preference. Quantification of tyrosine hydroxylase- and kisspeptin-immunoreactive neurons in the preoptic area showed unaffected sexual dimorphism of both populations in mutants. By contrast, the number of androgen receptor- and ERα-immunoreactive cells was significantly increased in the bed nucleus of stria terminalis of mutant males.These data show that neural ERβ does not play a crucial role in the organization and activation of the neural circuitry underlying male sexual behavior. These discrepancies with the phenotype of global ERβ knockout models are discussed.     

雌激素受体α和β免疫反应在雄性和雌性棕色田鼠脑区分布的比较

单配制和多配制动物社会行为有差异,这些差异可能与雌激素受体类型有关（ERs）。虽然多配制大鼠和小鼠中枢神经雌激素受体α（ERα）和β（ERβ）免疫反应在大脑的分布已有报道,单配制雄性草原田鼠中枢神经ERα的分布也有报道,但单配制田鼠ERα和（或）ERβ在雌性和雄性分布差异未见报道。本研究对雄性和雌性棕色田鼠前脑区域ERα和ERβ免疫反应（IR）细胞数量进行比较。研究结果表明：（1）免疫反应主要分布在细胞核中。 （2）ERα-IR和ERβ-IR细胞广泛分布于整个雌性和雄性前脑区域,在许多脑区表达有重叠。然而,不同受体在雌雄不同脑核中的分布数量是不同的。（3）ERα 和ERβ的分布存在性别差异。例如,雌性ERα在视前核中部（MPN）,终纹床和（BNST）和杏仁内侧核（MeA）比雄性多,相反雄性ERβ在MPN和BNST比雌性多。这些研究结果可能为我们理解如何通过ERα和ERβ调节动物的社会行为,及雌性和雄性社会行为的差异提供一个重要的神经解剖学基础。     

Sexually dimorphic estrogen receptor α mRNA expression in the preoptic area and ventromedial hypothalamus of green anole lizards

Estradiol (E2) is important in activation of male reproductive behaviors, and masculinizes morphology of associated brain regions in a number of mammalian and avian species. In contrast, it is testosterone, rather than its metabolites, that is the most potent activator of male sexual behavior in green anole lizards. As in other vertebrate groups, however, E2 is critical for receptivity in females of this species. Aromatase, the enzyme which converts testosterone to E2, is more active in the male than female green anole brain, and appears to be actively regulated on a seasonal basis, suggesting some role for E2 in males. This study was designed to enhance our understanding of potential E2 actions by localizing and quantifying relative levels of estrogen receptor-alpha (ERα) mRNA in forebrain regions involved in masculine and feminine behaviors in anoles. These areas include the preoptic area (POA), ventromedial amygdala (AMY) and ventromedial hypothalamus (VMH). In situ hybridization was conducted in adult males and females collected during both breeding and non-breeding seasons. ERα mRNA was expressed in each brain region across sexes and seasons. However, expression was up to 3 times greater in the VMH compared to the POA and AMY. In the POA and VMH, expression was higher in females compared to males, independent of season. The increased receptor expression in females is consistent with E2 playing a larger role in female than male reproductive behaviors.     

Localization of estrogen receptor‐α and ‐βmRNA in brain areas controlling sexual behavior in Japanese quail

Two estrogen receptors (ERs), denoted ERα and ERβ, have been identified in humans and various animal species, including the Japanese quail. Estrogens play a key role in sexual differentiation and in activation of sexual behavior in Japanese quail. The distribution of ERα in the brain of male and female adult quail has previously been studied using immunohistochemistry, whereas in situ hybridization has been employed to study the distribution of ERβ mRNA in males only. In this article, we used in situ hybridization to study the distribution of mRNAs for both ERα and ERβ in brain areas controlling sexual behavior of Japanese quail. Our results show that both ERα mRNA and ERβ mRNA are localized in areas important for sexual behavior, such as the preoptic area and associated limbic areas, in both males and females. Moreover, we found differences in distribution of mRNA for the two receptors in these areas. The results of this article support previously reported data and provide novel data on localization of ER mRNAs in adult quail brain of both sexes. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

Auditory sexual difference in the large odorous frog Odorrana graminea

Acoustic communication is an important behavior in frog courtship. Male and female frogs of most species, except the concave-eared torrent frog Odorrana tormota, have largely similar audiograms. The large odorous frogs (Odorrana graminea) are sympatric with O. tormota, but have no ear canals. The difference in hearing between two sexes of the frog is unknown. We recorded auditory evoked near-field potentials and single-unit responses from the auditory midbrain (the torus semicircularis) to determine auditory frequency sensitivity and threshold. The results show that males have the upper frequency limit at 24 kHz and females have the upper limit at 16 kHz. The more sensitive frequency range is 3–15 kHz for males and 1–8 kHz for females. Males have the minimum threshold at 11 kHz (58 dB SPL), higher about 5 dB than that at 3 kHz for females. The best excitatory frequencies of single units are mostly between 3 and 5 kHz in females and at 7–8 kHz in males. The underlying mechanism of auditory sexual differences is discussed.     

Changes in Electroencephalogram Approximate Entropy Reflect Auditory Processing and Functional Complexity in Frogs

Brain systems engage in what are generally considered to be among the most complex forms of information processing. In the present study, we investigated the functional complexity of anuran auditory processing using the approximate entropy(Ap En) protocol for electroencephalogram(EEG) recordings from the forebrain and midbrain while male and female music frogs(Babina daunchina) listened to acoustic stimuli whose biological significance varied. The stimuli used were synthesized white noise(reflecting a novel signal), conspecific male advertisement calls with either high or low sexual attractiveness(reflecting sexual selection) and silence(reflecting a baseline). The results showed that 1) Ap En evoked by conspecific calls exceeded Ap En evoked by synthesized white noise in the left mesencephalon indicating this structure plays a critical role in processing acoustic signals with biological significance; 2) Ap En in the mesencephalon was significantly higher than for the telencephalon, consistent with the fact that the anuran midbrain contains a large well-organized auditory nucleus(torus semicircularis) while the forebrain does not; 3) for females Ap En in the mesencephalon was significantly different than that of males, suggesting that males and females process biological stimuli related to mate choice differently.     

Effects of postnatal estrogen manipulations on juvenile alloparental behavior

Sex- and species-specific patterns of estrogen receptor (ER)-α expression are established early in development, which may contribute to sexual differentiation of behavior and determine male social organization. The current study investigated the effects of ERα and ERβ activation during the second postnatal week on subsequent alloparental behavior and ERα expression in juvenile prairie voles. Male and female pups were treated daily with 17β-estradiol (E2, ERα/ERβ agonist), PPT (selective ERα agonist), DPN (selective ERβ agonist), or the oil vehicle on postnatal days (PD) 8–14. Alloparental behavior and ERα expression were examined at PD21. PPT treatment inhibited prosocial motivation in males and increased pup-directed aggression in both sexes. E2 and DPN had no apparent effect on behavior in either sex. PPT-treated males had increased ERα expression in the medial preoptic area (MPN), medial amygdala (MEApd) and bed nucleus of the stria terminalis (BSTpr). DPN treatment also increased ERα expression in males, but only in the BSTpr. Female ERα expression was unaffected by treatment. These results support the hypothesis that ERα activation in early life is associated with less prosocial patterns of central ERα expression and alloparental behavior in males. The lack of an effect of E2 on behavior suggests that ERβ may antagonize the effects of ERα on alloparental behavior. The results in DPN-treated males suggest that ERα in the MEApd, and not the BSTpr, may be a primary determinant of alloparental behavior in males.     

Changes in androgen receptor, estrogen receptor alpha, and sexual behavior with aging and testosterone in male rats

Reproductive aging in males is characterized by a diminution in sexual behavior beginning in middle age. We investigated the relationships among testosterone, androgen receptor (AR) and estrogen receptor alpha (ERα) cell numbers in the hypothalamus, and their relationship to sexual performance in male rats. Young (3 months) and middle-aged (12 months) rats were given sexual behavior tests, then castrated and implanted with vehicle or testosterone capsules. Rats were tested again for sexual behavior. Numbers of AR and ERα immunoreactive cells were counted in the anteroventral periventricular nucleus and the medial preoptic nucleus, and serum hormones were measured. Middle-aged intact rats had significant impairments of all sexual behavior measures compared to young males. After castration and testosterone implantation, sexual behaviors in middle-aged males were largely comparable to those in the young males. In the hypothalamus, AR cell density was significantly (5-fold) higher, and ERα cell density significantly (6-fold) lower, in testosterone- than vehicle-treated males, with no age differences. Thus, restoration of serum testosterone to comparable levels in young and middle-aged rats resulted in similar preoptic AR and ERα cell density concomitant with a reinstatement of most behaviors. These data suggest that age-related differences in sexual behavior cannot be due to absolute levels of testosterone, and further, the middle-aged brain retains the capacity to respond to exogenous testosterone with changes in hypothalamic AR and ERα expression. Our finding that testosterone replacement in aging males has profound effects on hypothalamic receptors and behavior has potential medical implications for the treatment of age-related hypogonadism in men.     

The Impact of Oxytocin Gene Knockout on Sexual Behavior and Gene Expression Related to Neuroendocrine Systems in the Brain of Female Mice

Social relations are built and maintained from the interaction among individuals. The oxytocin (OT), vasopressin (VP), estrogen, dopamine, and their receptors are involved in the modulation of sexual behavior in females. This study aimed to analyze the impact of OT gene knockout (OTKO) on sexual behavior and the gene expression of oxytocin (OTR), estrogen alpha (ERα), estrogen beta (ERβ), vasopressin (V_1aR), and dopamine (D₂R) receptors in the olfactory bulb (OB), prefrontal cortex (PFC), hippocampus (HPC), and hypothalamus (HPT), as well as in the synthesis of VP in the HPT of female mice. Wild-type (WT) littermates were used for comparisons. The _CDNAs were synthesized by polymerase chain reaction and the gene expression was calculated with the 2^?ΔΔCt formula. Our results showed that the absence of OT caused an increase in the frequency and duration of non-receptive postures and a decrease in receptive postures in the OTKO. OTKO females showed a significant decrease in the gene expression of OTR in the HPC, V_1aR in the HPT, and ERα and ERβ in the PFC. There was no significant difference in the gene expression of D₂R of OTKO. However, OTKO showed an increased gene expression of V_1aR in the HPC. There is no significant difference in VP mRNA synthesis in the HPT between OTKO and WT. Our findings demonstrate that the absence of OT leads to significant changes in the expression of the studied genes (OTR, ERα, ERβ, V_1aR), and these changes may contribute to the decreased sexual behavior observed in OTKO females.     

Estrogen receptor alpha is required in GABAergic,but not glutamatergic,neurons to masculinize behavior

Masculinization of the altricial rodent brain is driven by estrogen signaling during a perinatal critical period. Genetic deletion of estrogen receptor alpha (Esr1/ERα) results in altered hypothalamic-pituitary-gonadal (HPG) axis signaling and a dramatic reduction of male sexual and territorial behaviors. However, the role of ERα in masculinizing distinct classes of neurons remains unexplored. We deleted ERα in excitatory or inhibitory neurons using either a Vglut2 or Vgat driver and assessed male behaviors. We find that Vglut2-Cre;Esr1^lox/lox mutant males lack ERα in the ventrolateral region of the ventromedial hypothalamus (VMHvl) and posterior ventral portion of the medial amygdala (MePV). These mutants recapitulate the increased serum testosterone levels seen with constitutive ERα deletion, but have none of the behavioral deficits. In contrast, Vgat-Cre;Esr1^lox/lox males with substantial ERα deletion in inhibitory neurons, including those of the principal nucleus of the bed nucleus of the stria terminalis (BNSTpr), posterior dorsal MeA (MePD), and medial preoptic area (MPOA) have normal testosterone levels, but display alterations in mating and territorial behaviors. These mutants also show dysmasculinized expression of androgen receptor (AR) and estrogen receptor beta (Esr2). Our results demonstrate that ERα masculinizes GABAergic neurons that gate the display of male-typical behaviors.     

Sexual experience affects reproductive behavior and preoptic androgen receptors in male mice

Reproductive behavior in male rodents is made up of anticipatory and consummatory elements which are regulated in the brain by sensory systems, reward circuits and hormone signaling. Gonadal steroids play a key role in the regulation of male sexual behavior via steroid receptors in the hypothalamus and preoptic area. Typical patterns of male reproductive behavior have been characterized, however these are not fixed but are modulated by adult experience. We assessed the effects of repeated sexual experience on male reproductive behavior of C57BL/6 mice; including measures of olfactory investigation of females, mounting, intromission and ejaculation. The effects of sexual experience on the number of cells expressing either androgen receptor (AR) or estrogen receptor alpha (ERα) in the primary brain nuclei regulating male sexual behavior was also measured. Sexually experienced male mice engaged in less sniffing of females before initiating sexual behavior and exhibited shorter latencies to mount and intromit, increased frequency of intromission, and increased duration of intromission relative to mounting. No changes in numbers of ERα-positive cells were observed, however sexually experienced males had increased numbers of AR-positive cells in the medial preoptic area (MPOA); the primary regulatory nucleus for male sexual behavior. These results indicate that sexual experience results in a qualitative change in male reproductive behavior in mice that is associated with increased testosterone sensitivity in the MPOA and that this nucleus may play a key integrative role in mediating the effects of sexual experience on male behavior.     

Specific activation of estrogen receptor alpha and beta enhances male sexual behavior and neuroplasticity in male Japanese quail

Two subtypes of estrogen receptors (ER), ERα and ERβ, have been identified in humans and numerous vertebrates, including the Japanese quail. We investigated in this species the specific role(s) of each receptor in the activation of male sexual behavior and the underlying estrogen-dependent neural plasticity. Castrated male Japanese quail received empty (CX) or testosterone-filled (T) implants or were daily injected with the ER general agonist diethylstilbestrol (DES), the ERα-specific agonist PPT, the ERβ-specific agonist DPN or the vehicle, propylene glycol. Three days after receiving the first treatment, subjects were alternatively tested for appetitive (rhythmic cloacal sphincter movements, RCSM) and consummatory aspects (copulatory behavior) of male sexual behavior. 24 hours after the last behavioral testing, brains were collected and analyzed for aromatase expression and vasotocinergic innervation in the medial preoptic nucleus. The expression of RCSM was activated by T and to a lesser extent by DES and PPT but not by the ERβagonist DPN. In parallel, T fully restored the complete sequence of copulation, DES was partially active and the specific activation of ERα or ERβ only resulted in a very low frequency of mount attempts in few subjects. T increased the volume of the medial preoptic nucleus as measured by the dense cluster of aromatase-immunoreactive cells and the density of the vasotocinergic innervation within this nucleus. DES had only a weak action on vasotocinergic fibers and the two specific ER agonists did not affect these neural responses. Simultaneous activation of both receptors or treatments with higher doses may be required to fully activate sexual behavior and the associated neurochemical events.     

Paradoxical nature of estrogen agonist and antagonist binding in rat liver

Whereas Tamoxifen exerts potent antiestrogenic action in ER dependent breast cancer, it was largely without effect on rat liver gluconeogenesis which could be dramatically diminished by estrogens and androgens. Although estradiol was preferentially bound to an ER₄ component that coeluted with CBG from DE-52 columns, ³H-tamoxifen labelled the ER₃ moiety that was clearly distinct from transcortin. Similarly, testosterone was bound to the AR₄ entity but R-1881 was eluted in the AR₃ region. All these ER and AR populations were furthermore distinct from liver GR. These, for the first time, demonstrate polymorphic nature of AR and ER and suggest that agonist and antagonist actions may be expressed via separate populations of the receptor, contrary to the established, classical view that dictates competitive antagonism between them for the one and the same site.     

Sex differences in progesterone receptor immunoreactivity in neonatal mouse brain depend on estrogen receptor α expression

Around the time of birth, male rats express higher levels of progesterone receptors in the medial preoptic nucleus (MPN) than female rats, suggesting that the MPN may be differentially sensitive to maternal hormones in developing males and females. Preliminary evidence suggests that this sex difference depends on the activation of estrogen receptors around birth. To test whether estrogen receptor alpha (ERα) is involved, we compared progesterone receptor immunoreactivity (PRir) in the brains of male and female neonatal mice that lacked a functional ERα gene or were wild type for the disrupted gene. We demonstrate that males express much higher levels of PRir in the MPN and the ventromedial nucleus of the neonatal mouse brain than females, and that PRir expression is dependent on the expression of ERα in these regions. In contrast, PRir levels in neocortex are not altered by ERα gene disruption. The results of this study suggest that the induction of PR via ERα may render specific regions of the developing male brain more sensitive to progesterone than the developing female brain, and may thereby underlie sexual differentiation of these regions. © 2001 John Wiley & Sons, Inc. J Neurobiol 47: 176–182, 2001     

Role of GPER on proliferation,migration and invasion in ligand‐independent manner in human ovarian cancer cell line SKOV3

G protein‐coupled estrogen receptor (GPER) is identified as a critical estrogen receptor, in addition to the classical estrogen receptors ERα and ERβ. In ERα‐negative ovarian cancer cells, our previous studies have found that estrogen stimulated cell proliferation and metastasis via GPER. However, the ligand‐independent function of GPER in ovarian cancer cells is still not clear. Herein, we describe that GPER has a co‐expression with ERα and ERβ, which are first determined in SKOV3 ovarian cancer cell line. In the absence of estrogen, GPER depletion by specific siRNA inhibits the proliferation, migration and invasion of SKOV3 cells. Whereas abrogation of ERα or ERβ by specific antagonist MPP and PHTPP has the opposite effects for stimulation of cell growth. Markedly, GPER knockdown attenuates MPP or PHTPP‐induced cell proliferation, migration and invasion. Furthermore, GPER modulates protein expression of the cell cycle critical components, c‐fos and cyclin D1 and factors for cancer cell invasion and metastasis, matrix metalloproteinase 2 (MMP‐2) and MMP‐9. These findings establish that GPER ligand‐independently stimulates the proliferation, migration and invasion of SKOV3 cells. Knockdown of GPER attenuates the progression of ovarian cancer that caused by functional loss of ERα or ERβ. Targeting GPER provides new aspect as a potential therapeutic strategy in ovarian cancer. Copyright © 2015 John Wiley & Sons, Ltd.     

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 ERβ), work on alternate spliced forms of ERα, and the potential for membrane estrogen receptors, an animal with a null background for ERα function is invaluable for distinguishing biological responses of estrogens working via the ERα protein and those working via another ER protein. Data generated to date, and reviewed here, indicate that there are profound ramifications of the ERα 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 ERα⁻mice confirm that ERα is essential in females for normal regulation of the hypothalamic–pituitary gonadal axis. Second, ovariectomized female ERα⁻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 ERα⁻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 ERα protein on behavior and neuroendocrinology. The ERα⁻mouse can be used to ascertain the specific functions of ERα, to suggest functions for the other estrogen receptors, and to study indirect effects of ERα on behavior via actions on other receptors, neurotransmitters, and neuropeptides.     

Assessment of 11-β hydroxysteroid dehydrogenase (11-βHSD1) 4478T>G and tumor necrosis factor-α (TNF-α)-308G>A polymorphisms with obesity and insulin resistance in Asian Indians in North India

11-β hydroxysteroid dehydrogenase (11-βHSD1), tumor necrosis factor-α (TNF-α) and their role in obesity, regional adiposity and insulin resistance has been sparsely evaluated. We determined the polymorphic status of 11-βHSD1 4478T>G and TNF-α-308G>A in Asian Indians in north India. In this cross-sectional study (n = 498; 258 males, 240 females), association of genotypes (PCR–RFLP) of 11-βHSD1 and TNF-α were analyzed with obesity [BMI ≥ 25 kg/m², percentage body fat (%BF by DEXA); subcutaneous and intra-abdominal fat area (L_2–3 level by single slice MRI) in a sub sample] and insulin resistance. 46 percent subjects had generalized obesity, 55 % abdominal obesity and 23.8 % were insulin resistant. Frequencies (%) of [T/T] and [T/G] genotypes of 11-βHSD1 were 89.57 and 10.43 respectively. Homozygosity for 11-βHSD1 4478G/G was absent with no association with parameters of obesity and insulin resistance. Frequencies (%) of TNF-α [G] and [A] alleles were 88 and 12 respectively. Higher frequency of variant -308[A/A] was observed in females versus males (p = 0.01). Females with at least one single A allele of TNF-α-308G>A had significantly high %BF and total skinfold, whereas higher values of waist hip ratio, total cholesterol, triglycerides and VLDL were observed in males. Subjects with even a single A allele in TNF-α genotype showed higher subscapular skinfold predisposing them to truncal subcutaneous adiposity (p = 0.02). Our findings of association of TNF-α-308G>A variant in females with obesity indices suggests a gender-specific role of this polymorphism in obesity. High truncal subcutaneous adiposity is associated with A allele of TNF-α-308G>A in this population.