Skip to main content
NCBI home page
Endocrinology logoLink to Endocrinology
. 2013 Sep 10;154(10):3836–3846. doi: 10.1210/en.2013-1239

Differential Control of Sex Differences in Estrogen Receptor α in the Bed Nucleus of the Stria Terminalis and Anteroventral Periventricular Nucleus

D A Kelly 1,✉, M M Varnum 1, A A Krentzel 1, S Krug 1, N G Forger 1
PMCID: PMC3776875  PMID: 24025225

Abstract

The principal nucleus of the bed nucleus of the stria terminalis (BNSTp) and anteroventral periventricular nucleus of the hypothalamus (AVPV) are sexually dimorphic, hormone-sensitive forebrain regions. Here we report a profound sex difference in estrogen receptor-α (ERα) immunoreactivity (IR) in the BNSTp, with robust ERα IR in females and the near absence of labeling in males. This sex difference is due to the suppression of ERα IR by testicular hormones in adulthood: it was not present at birth and was not altered by neonatal treatment of females with estradiol; gonadectomy of adult males increased ERα IR to that of females, whereas gonadectomy of adult females had no effect. Treating gonadally intact males with an aromatase inhibitor partially feminized ERα IR in the BNSTp, suggesting that testicular suppression required aromatization. By contrast, in AVPV we found a modest sex difference in ERα IR that was relatively insensitive to steroid manipulations in adulthood. ERα IR in AVPV was, however, masculinized in females treated with estradiol at birth, suggesting that the sex difference is due to organizational effects of estrogens. The difference in ERα IR in the BNSTp of males and females appears to be at least in part due to greater expression of mRNA of the ERα gene (Esr1) in females. The sex difference in message is smaller than the difference in immunoreactivity, however, suggesting that posttranscriptional mechanisms also contribute to the pronounced suppression of ERα IR and presumably to functions mediated by ERα in the male BNSTp.

The bed nucleus of the stria terminalis (BNST) is a forebrain region involved in male sexual behavior, the processing of socially relevant olfactory cues, and the modulation of stress and anxiety (1–7). The principal nucleus of the BNST (BNSTp) is larger in adult males than in females of several species, including mice, rats, guinea pigs, and humans (8–12). In mice and rats, this sex difference emerges postnatally and is due to differential cell death (12–14). BNSTp volume and cell number are equivalent in both sexes at birth (13, 14), but the rate of developmental cell death is greater in females than in males, and sex differences in volume and cell number appear at the end of the first postnatal week (13–15). In mice, treating females on the day of birth with either T or estradiol benzoate (EB) masculinizes BNSTp cell number (16). Moreover, BNSTp volume and cell number are female-like in male mice with a targeted deletion of the estrogen receptor (ER)-α gene, whereas deletion of ERβ has no effect (17). Thus, sexual dimorphism of the BNST is due to suppression of cell death in males by estrogenic metabolites of T acting through ERα.

The BNSTp remains steroid sensitive in adulthood, expressing androgen receptors, ERα, and ERβ (18–23) as well as the aromatase enzyme (24–26). However, almost all studies that have examined ER immunoreactivity (IR) or mRNA levels in the BNSTp used only one sex (often ovariectomized females) or restricted analyses to early development (eg, Reference 23, 27). The only study we are aware of to have examined sex differences in ERα IR within the BNST of adult gonadally intact mice noted greater ERα IR in females but did not quantify (28). In a preliminary study, we were therefore surprised to observe an unusually large sex difference in ERα IR in gonadally intact adult C57BL/6 mice [29 (abstract)]. We found robust staining in all females examined (killed without regard to the stage of the estrous cycle) and the near absence of staining in all males. In fact, it was possible to reliably assign sex with a naked-eye inspection of sections immunoreacted for ERα. Sex differences in the expression of neural ERs have been described in other brain regions (28, 30–34), but the magnitude of this difference warranted further characterization, given the role of the BNSTp in sexually dimorphic, hormone-sensitive functions.

Moreover, in the same animals examined for ERα IR in the BNSTp, adjacent ERα-positive regions such as the preoptic area and anteroventral periventricular nucleus (AVPV) did not appear to display a prominent difference in labeling [29 (abstract)], suggesting very different regulation among regions. The AVPV is a small cell group located at the rostral extreme of the third ventricle involved in estrogen-positive feedback and control of the gonadotrophic surge (35, 36). Like the BNSTp, the AVPV is dimorphic due to sexually differentiated cell death. However, in this case the rate of cell death is greater in females, and volume and cell number are lower in adult males (12, 37, 38). Both ERα and ERβ have been implicated in sexual differentiation of the mouse AVPV (39, 40).

To test the hypothesis that ERα IR is differentially controlled in the BNSTp and AVPV of mice, we systematically quantified ERα expression in these regions under different hormonal conditions. We find sex differences favoring females in both regions, albeit of very different magnitude. We also find that the sex difference in ERα IR in AVPV is due primarily to organizational effects of steroids and appears relatively insensitive to changes in circulating steroids in adulthood. By contrast, ERα IR in the BNSTp of adult males is profoundly suppressed by circulating gonadal steroids.

Materials and Methods

Animals

Wild-type C57BL/6 mice were obtained from Jackson Labs or our breeding colony and housed in a temperature- (22°C) and light-controlled (14 hour light, 10 hour dark) facility. All procedures were approved by the Institutional Animal Care and Use Committee at the University of Massachusetts (Amherst, Massachusetts).

Experiment 1: Sex differences in adulthood and effects of neonatal estradiol

Female pups were injected sc on the day of birth [postnatal day (P) 1] with 20 μg of estradiol benzoate (Sigma-Aldrich) in 25 μL vehicle (90% peanut oil, 10% dimethylsulfoxide); control males and females received vehicle alone (n = 6 per group). This dose of EB previously masculinized BNSTp cell number in mice (16). Animals were killed and their brains collected between P60 and P65. Trunk blood was collected and assayed for serum estradiol via a competitive ELISA assay (Calbiotech Inc) by the Center for Research in Reproduction at the University of Virginia. Brains were postfixed in 5% acrolein in 0.1 M phosphate buffer at room temperature overnight and stored in 30% sucrose until sectioning into 3 series at 30 μm in the coronal plane. One series was processed for ERα IR.

Immunohistochemistry

Free-floating sections were processed as reported elsewhere (15) with the primary ERα antibody C1355 (1:20 000 dilution; Upstate Biotechnology). All sections within each experiment were processed in a single run. C1355 is a purified rabbit polyclonal antibody generated against the final 14 C-terminal amino acids of the rat ERα protein. This antibody has been extensively validated in neural tissue by preabsorption and Western blotting controls, and staining distribution is consistent with that seen using other ERα antibodies or by in situ hybridization (22, 41–43). Staining was abolished when the primary antibody was omitted. ERα IR was visualized with diaminobenzidine and nickel (diaminobenzidine peroxidase substrate kit; Vector Laboratories) and sections were counterstained with thionin.

Quantification of ERα IR

All measurements in this and subsequent experiments were made on slides coded to conceal sex and/or treatment group. ERα IR was quantified in the encapsulated region of the BNSTp and the AVPV. The BNSTp was identified based on its position dorsal to the anterior commissure using the lateral ventricles and surrounding white matter tracts (stria terminalis, fornix, stria medularis, and internal capsule) as landmarks [Reference 44 (Figures 32–33)]. AVPV was identified based on cell density in the thionin counterstain and the position of this nucleus just lateral to the rostral extreme of the third ventricle [corresponding to approximately Figure 29 (44)]. We first quantified ERα IR by stereological cell counts performed as previously (12) using the optical dissector method and StereoInvestigator software (MicroBrightfield). Outlines of the BNST and AVPV were traced in each section. Labeled cells were counted throughout each nucleus using a sampling grid of 45 × 45 μm and a counting frame of 18 × 18 μm. This provided an unbiased estimate of the total number of ERα IR cells in the nucleus but was quite time intensive. We therefore established an automated thresholding protocol that could more efficiently estimate total label within each nucleus.

Gray-scale images of the BNST and AVPV were captured and analyzed in Image J 1.43u (National Institutes of Health). The encapsulated BNSTp and AVPV were outlined bilaterally in each section and cross-sectional area was measured. ERα IR was then quantified in the 2 largest cross-sectional areas for each nucleus using a maximum entropy thresholding algorithm (45) with threshold set to capture only clearly labeled cells. Two measures were recorded. The first was total area of each nucleus covered by immunoreactivity, which we refer to throughout as total ERα IR. This measure closely corresponded to the pattern of results obtained from stereological cell counts and is depicted in the figures below. Because the size of each nucleus is known to vary by sex, we also calculated density of ERα IR, defined as the area covered by immunoreactivity divided by the cross-sectional area of the nucleus. These 2 measures were used in all subsequent experiments. The pattern of results for total ERα IR and density of ERα IR were the same in most cases. Exceptions to this are noted below and density of ERα IR analyses are provided as Supplemental Data, published on The Endocrine Society's Journals Online web site at http://endo.endojournals.org. In a few cases, tearing or other tissue artifacts prevented accurate measurements in one or the other brain region; the final number of animals per group is indicated in the figure legends.

Experiment 2: Activational effects of gonadal steroids

To examine the role of circulating gonadal steroids on sex differences in ERα IR, adult male and female mice underwent gonadectomy or sham surgery at 60–65 days of age under isoflurane inhalation anesthesia (n = 6 per group). Mice were killed 3 weeks after surgery, and brains were collected and processed for ERα IR in the BNSTp and AVPV as in experiment 1. To test whether the lack of ERα IR in gonadally intact males was dependent on antibody concentration, additional sections from intact and gonadectomized males were immunolabeled as described above with a range of concentrations of the primary antibody (1:20 000, 1:10 000, and 1:2000).

In addition, a small number of mice (60–65 d of age) were used in a pilot study examining the time course of changes in ERα IR in the BNSTp after castration of males or androgen treatment of females. Males were gonadectomized and brains collected 24 hours, 48 hours, or 7 days after gonadectomy (n = 2 at each time point). Gonadally intact females were given 2 sc injections of 500 μg testosterone propionate (0.1 mL of a 5 mg/mL solution in sesame oil) 24 hours apart; brains were collected 48 hours after the first injection. The sham-operated males and females killed 48 hours or 3 weeks after surgery served as controls. Brains were collected, sectioned, and processed for ERα IR as before and labeling in the BNSTp was compared across groups.

Experiment 3: Role of androgen and estrogen receptors

Based on the time-course study described above, 48 hours appeared sufficient for the up-regulation of ERα IR in gonadectomized males. We used that time point to determine whether testicular steroids act via androgen or estrogen receptors to regulate ERα IR. Adult males 60–65 days of age (n = 6 per group) were given sham surgeries and injected sc with the androgen receptor antagonist flutamide (50 mg/kg in 0.1 mL sesame oil; Sigma-Aldrich), the aromatase inhibitor letrozole (10 μg/mL suspended in 0.3% hydroxypropyl cellulose in PBS; Sigma-Aldrich), or vehicle injections alone. Gonadectomized males treated with vehicle served as an additional control. Dosages of flutamide and letrozole used were based on previous studies (46–48). Because flutamide and letrozole require different vehicles, each animal received 2 injections: both aqueous and oil vehicles, flutamide and aqueous vehicle, or letrozole and oil vehicle. Injections were given immediately after surgery and 24 hours later, and all animals were killed 48 hours after the surgery. Brains were collected and processed for ERα IR as above.

Experiment 4: Ontogeny of the ERα IR sex difference in the BNSTp

Estrogenic metabolites of T masculinize the BNSTp of newborn mice (16), and this action requires ERα (17). To examine when the sex difference in ERα IR emerges, brains were collected from both sexes at P1, P6, P20, or in adulthood (P60-P65; n = 6 per group). Tissue was fixed as described above and sectioned into 2 series in the coronal plane at 40 μm (P1 and P6) or 3 series at 30 μm (P20 and adult). Forty-micrometer sections were used for neonatal brains because thinner sections tended to fall apart during staining. One series was processed and analyzed for ERα IR, as described above. The cross-sectional area of the BNSTp was determined in each section in which ERα IR was analyzed (ie, the 2 largest sections through the nucleus); we report the sum of these measures as overall area of the BNSTp.

Statistical analysis

Data from experiments 1 and 3 were analyzed using 1-way ANOVAs. Data from experiments 2 and 4 were analyzed with 2-way ANOVAs, with sex and gonadal status or sex and age as factors, respectively. Planned comparisons were made using Fisher's least significant difference only after significant main effects or interactions. Means are expressed ± SEM and P < .05 was considered significant.

Results

Experiment 1: Sex differences in adulthood and effects of neonatal estradiol

ERα IR in the adult BNSTp was markedly reduced in males compared with females (Figure 1). Visual inspection indicated this was due primarily to a large reduction in the number of labeled cells in males and stereological cell counts throughout the BNSTp confirmed this observation. We found significant differences in the number of cells positive for ERα IR among males, females, and females treated with EB on the day of birth (F2,15 = 43.45, P < .0001). There were many more ERα IR cells in the BNSTp of females than of males (P < .0001; Figure 1A) and neonatally EB-treated females had more ERα-positive cells than control females (P < .01) or males (P < .0001; Figure 1A). We saw the same pattern using automated thresholding to quantify total ERα IR in the BNSTp (F2,15 = 63.45, P < .0001): this measure was more than 20 times larger in females than in males (P < .0001) and larger in EB-treated females than control females (P < .01; Figure 1, B and C). A similar pattern was seen for the density of ERα IR in the BNSTp, except that the density of ERα IR did not differ between control females and females treated with EB neonatally (Supplemental Data). Thus, the increase in labeling in EB-treated females compared with control females (Figure 1) likely reflects the greater overall size of the BNSTp in estrogen-treated females (16).

Figure 1.

Figure 1.

Open in a new tab

ERα IR in the BNSTp in control males, control females, and females treated with EB on the day of birth (female + EB). Immunoreactivity was quantified by either stereological cell counts (A) or thresholding to measure total ERα IR (B). Both methods (A and B) show ERα IR is much greater in females than in males and EB-treated females have greater ERα IR than either control group (n = 6 for all groups). C, Photomicrographs of coronal sections through the BNSTp illustrate ERα IR in each treatment group. Scale bar, 250 μm. f, fornix; ic, internal capsule; sm, stria medularis; st, stria terminalis.

The AVPV also displayed a sex difference in ERα IR (F2,14 = 7.74, P < .01), with approximately twice as many ERα-positive cells in females as in males based on stereological cell counts (P < .01; Figure 2). In contrast to the BNSTp, EB treatment of females on the day of birth masculinized (lowered) ERα cell number compared with vehicle-treated females (P < .01; Figure 2A). There was no difference in the number of ERα IR-positive cells in the AVPV of males and EB-treated females. The same pattern of results was seen for total ERα IR. An ANOVA indicated significant group differences (F2,15 = 19.27, P < .0001). Total ERα IR was greater in females compared with males (P < .0001; Figure 2B), and this measure did not differ between males and females treated neonatally with EB. The same pattern of results was seen for the density of ERα IR in AVPV (Supplemental Data).

Figure 2.

Figure 2.

Open in a new tab

ERα IR in the AVPV in control males, control females, and females treated with EB on the day of birth (female + EB). Immunoreactivity was quantified by either stereological cell counts (A) or thresholding to measure total ERα IR (B). Both methods show ERα IR is greater in females than in males and EB-treated females are similar to males on this measure (n = 6 for all groups except n = 5 for males in A). C, Photomicrographs of coronal sections through AVPV illustrate ERα IR in each treatment group. Scale bar, 250 μm. 3V, third ventricle.

Serum estradiol was variable and mean levels did not differ significantly among groups (male: 17.35 ± 4.81 pg/mL; female: 22.78 ± 7.19 pg/mL; female + EB: 18.41 ± 5.67 pg/mL). This is consistent with previous findings that plasma estradiol in male and female C57BL/6 mice does not differ when females are sampled without respect to the estrous cycle (49).

Experiment 2: Activational effects of gonadal steroids

The results of experiment 1 identified a profound sex difference in BNSTp ERα IR that was not reversed by neonatal treatment with estradiol. To test the effect of gonadal steroids circulating in adulthood, adult males and females were examined 3 weeks after gonadectomy or sham surgery. We found a main effect of sex (F1,20 = 5.35, P < .05), a main effect of gonadectomy (F1,20 = 6.74, P < .02), and a sex-by-gonadectomy interaction (F1,20 = 8.99, P < .01) on total ERα IR in the BNSTp (Figure 3A). Intact males again had markedly lower total ERα IR compared with females (P < .002). Gonadectomy did not affect total ERα IR in the BNSTp of females but increased it more than 100-fold in males (P < .001). Total ERα IR in the BNSTp of castrated males was not significantly different from that in the intact or gonadectomized females (Figure 3A). A 10-fold increase in antibody concentration did not affect total ERα IR in either the sham-operated or gonadally intact males (not shown), suggesting that the lack of ERα staining in the male BNSTp is not due to insufficient primary antibody concentration. The same pattern of results was seen for the density of ERα IR in the BNSTp (Supplemental Data).

Figure 3.

Figure 3.

Open in a new tab

ERα IR in the mouse BNSTp and AVPV in gonadectomized and sham-operated males and females. A, Gonadectomy markedly increases ERα IR in the BNSTp of males but has no effect on females (n = 6 for all groups). B, Coronal sections through the BNSTp illustrate ERα IR in each treatment group. C, In the AVPV, gonadectomy has no effect on ERα IR in either sex (n = 5 for males, females, and gonadectomized females; n = 4 for gonadectomized males). D, Coronal sections through the AVPV illustrate ERα IR in each treatment group. Medial is to the right in each image, with the third ventricle at its right edge. Scale bars, 250 μm. Gdx, gonadectomized; sm, stria medularis; st, stria terminalis.

In the AVPV, females again had more total ERα IR than males (F1,15 = 14.6, P < .002, main effect of sex), but there was no effect of gonadectomy (F1,15 = 0.06, P > .8) or sex-by-gonadectomy interaction (F1,15 = 0.51, P > .4) on this measure (Figure 3C). The same pattern of results was seen for the density of ERα IR in AVPV (Supplemental Data).

The regulation of ERα IR in the BNSTp appears to be relatively rapid because labeling markedly increased 24 hours after the removal of testicular steroids by castration of adult males (Figure 4). Although only 2 animals per time point were examined, the results were striking: ERα IR in males increased manyfold 24 or 48 hours after castration and appeared equivalent to that in males 3 weeks after castration. Similarly, ERα IR was markedly reduced 48 hours after testosterone propionate injections in females (Figure 4).

Figure 4.

Figure 4.

Open in a new tab

ERα IR in coronal sections through the BNSTp of adult male and female mice illustrating short- and long-term effects of gonadal steroids. A and B, Sham-operated male and female mice killed 48 hours after surgery. C, Sham-operated female mouse treated with T for 48 hours prior to the animals being killed; ERα IR is suppressed. D–F, Gonadectomized male mice killed 24 hours, 48 hours, or 3 weeks after castration. Male gonadectomy causes up-regulation of ERα within 24 hours. Scale bar, 250 μm. Gdx, gonadectomized; sm, stria medularis; st, stria terminalis.

Experiment 3: Role of androgen and estrogen receptors

To determine whether testicular steroids act through androgen or estrogen receptors to regulate ERα IR, intact males were treated with the androgen receptor antagonist flutamide or the aromatase inhibitor letrozole. We detected significant differences among groups in total ERα IR in the BNSTp (F4,23 = 45.70, P < .0001). As expected, total ERα IR in intact males was considerably less than in females (P < .0001) and males that had been castrated 48 hours prior to the time the animals were killed did not differ from females (Figure 5A). Flutamide treatment had no effect on total ERα IR, whereas letrozole increased this measure relative to vehicle-treated, gonadally intact males (P < .0005). This suggests that aromatization plays a role in ERα suppression in the male BNSTp. However, total ERα IR in letrozole-treated males remained lower than in females (P < .0001). In a follow-up experiment, males treated with both flutamide and letrozole did not have greater total ERα IR than males treated with letrozole alone (data not shown), suggesting that estrogen and androgen receptors do not act synergistically to suppress ERα in the male BNSTp. Density of ERα IR in the BNSTp showed the same patterns of results (Supplemental Data).

Figure 5.

Figure 5.

Open in a new tab

Effect of an androgen receptor antagonist (flutamide) or aromatase inhibitor (letrozole) on ERα IR in the BNSTp (A) or AVPV (B) of male mice. Labeling in sham-operated males given vehicle injections (male) is compared with that in sham-operated males treated with flutamide (Flut), sham-operated males treated with letrozole (Let), gonadectomized males (Gdx), and sham-operated, vehicle-injected females (female). A, Aromatase inhibition increases ERα IR in the BNSTp (n = 6 for male, Flut and Let groups; n = 5 for Gdx and female groups). B, There is no effect of either aromatase inhibition or androgen receptor blockade on ERα IR in the AVPV (n = 5 for Flut, Let, and female groups; n = 4 for male and Gdx groups).

In the AVPV, total ERα IR was significantly different among groups in the overall ANOVA (F4,18 = 4.30, P < .02), but none of the experimental treatments significantly altered total ERα IR in males. Consistent with the results of experiment 2, females had greater total ERα IR than males (Figure 5B). However, gonadectomized males did not differ from intact males, and neither flutamide nor letrozole influenced total ERα IR in the AVPV (Figure 5B). Density of ERα IR did not differ among groups in the AVPV (Supplemental Data).

Experiment 4: Ontogeny of the ERα IR sex difference in the BNSTp

Volume and cell number of the BNSTp are masculinized by treating females with estradiol or T on the day of birth, although sex differences in these measures do not emerge until the end of the first postnatal week (14–16). To determine when the sex difference in ERα IR arises, we examined overall area (sum of the cross-sectional areas of the sections examined for ERα IR), total ERα IR, and density of ERα IR in the BNSTp of males and females at P1, P6, P20, and P60. We found main effects of sex (F1,37 = 35.09, P < .0001) and age (F3,37 = 133.34, P < .0001) and a sex-by-age interaction (F3,37 = 13.95, P < .0001) on area of the BNSTp. There was no sex difference in BNSTp size at birth. A significant sex difference was evident on P6 (P < .05) and increased with age (P < .0001 on P20 and P < .0005 on P60; Figure 6A).

Figure 6.

Figure 6.

Open in a new tab

A, Cross-sectional area of the BNSTp in male and female mice from P1 to P60. There is no significant difference between males and females in either measure on P1. A sex difference emerges by P6 and increases with age. B, Both sexes express equivalent ERα IR at birth. Females continue to express similar levels at all time points examined. Males exhibit a reduction at P6 and profound reduction at P60 (n = 6 for all groups in both figures except n = 5 for P6 males and n = 4 for P1 males).

ERα IR was present in the BNSTp of both sexes at all ages examined. We found a main effect of sex (F1,37 = 10.78, P < .005), no main effect of age, but a sex-by-age interaction (F3,37 = 9.13, P < .0001) on total ERα IR. At birth this measure was high and did not differ between males and females (Figure 6B). Levels of ERα IR in females remained high throughout postnatal development. ERα IR was lower in males than in females at P6 (P < .005), but there was no sex difference at P20 (Figure 6B). Among the adults in this study, our measure of ERα IR was again more than 20 times higher in females than in males (P < .0001; Figure 6B). The same pattern of results was found for the density of ERα IR (Supplemental Data).

In situ hybridization for Esr1 mRNA in the BNSTp

While this study was in progress, Xu et al (50) reported a sex difference in mRNA for the ERα gene (Esr1) in the hypothalamus and BNST of adult, gonadally intact mice. Quantification suggested the sex difference in the BNSTp was relatively modest (<2-fold). To confirm, we examined Esr1 mRNA expression in the BNSTp using in situ hybridization in males and females (n = 3 per group; see Supplemental Methods for details). Although the number of animals examined was small, we reasoned that if the sex difference in mRNA approached the large fold differences seen with immunocytochemistry, we would detect it even with small numbers of animals. Our results are similar to those of Xu et al (50). Females had higher levels of Esr1 in the BNSTp than males (P < .05; Supplemental Figure 1), although the difference (1.9 fold) was modest compared with that seen with immunocytochemistry. Results for density of Esr1 label were similar (2.1 fold).

Discussion

Females have more ERα IR than males in the AVPV and the BNSTp of adult mice, but the magnitude and cause of the dimorphism differs in each region. In the AVPV, there is an approximately 2-fold sex difference in ERα IR that is relatively insensitive to adult hormone manipulations and decreased by neonatal estradiol treatment of females. In the BNSTp, ERα IR is nearly completely absent in males due to the action of testicular steroids in adulthood. At a minimum, this indicates different regulation of the ERα protein in the AVPV and BNSTp. The magnitude of the sex difference in the BNSTp also suggests that under normal conditions (ie, in gonadally intact animals), this region may exhibit a profound sex difference in sensitivity to circulating estrogens.

Using several quantification methods, we find that ERα IR in the BNSTp is 7–20 times greater in females than in males with no overlap between the sexes. The only previous report of an ERα IR sex difference in the brain of similar magnitude that we are aware of was in voles: in the medial amygdala of an Illinois subpopulation of prairie voles, females had approximately 15 times more ERα IR than males (32). The size of the ERα IR sex difference reported here is also reminiscent of the striking sex difference in progesterone receptor IR in the preoptic area of neonatal rats (male ≫ female) (51). We did not quantify ERα IR in brain regions other than the BNSTp and AVPV, but visual inspection suggests the large sex difference in the BNST is not present in other regions. For example, in males with a near total absence of ERα IR in the BNSTp, there was nonetheless appreciable staining in the AVPV and the ventromedial nucleus of the hypothalamus. In the POA, males appeared to have fewer ERα IR cells than females but not dramatically so.

Given the relatively intense investigation of the BNST in mice and rats, it is surprising that a sex difference of this size has not been noted before. However, several observations are consistent with our results. Brown et al (28) reported subjectively lower ERα IR in the preoptic area and BNST region of male than of female C57BL/6 and 129SvEv mice, and Agarwal et al (52) described ERα IR in the BNSTp of male mice as low in a mixed strain (129Sv/J × C57BL/6; females were not examined). In rats, the predominant rodent model for sexual differentiation, there have been no studies directly comparing ERα in the BNSTp of adult males and females as far as we are aware.

The sex difference in ERα IR in the BNSTp is at least in part due to a sex difference in gene expression. Xu et al (50) used microarrays to identify mRNAs with sexually dimorphic expression within a microdissection of the brain that included the hypothalamus and BNST. Among the genes identified, Esr1 was found to have higher expression in females, a result validated with in situ hybridization (50). Photomicrographs of the in situ hybridization suggest substantially higher Esr1 expression in the BNSTp of females compared with males (Supplemental Figure 1 in Reference 50), although quantitation by PCR suggested a more modest difference (less than 2-fold; Supplemental Figure 2 in Reference 50). Based on in situ hybridization in a small number of animals, we also found moderately higher expression of Esr1 in the BNSTp of females than of gonadally intact males. These findings confirm a sex difference in Esr1 mRNA but suggest that posttranscriptional mechanisms amplify the difference in gene expression to produce the very marked differences seen with immunolabeling. These could include, for example, greater stability of the Esr1 mRNA and/or ERα protein in females than in males.

Given the apparent difference in the magnitude of the sex difference in ERα seen between immunocytochemistry and in situ hybridization, other possibilities should be considered. For example, some steroid receptor antibodies are sensitive to the binding state of the receptor (53); if so, differences in ERα IR could be an artifact of ligand binding. The C1355 antibody used here has been used in a wide range of studies and in multiple species, including all previous studies on sex differences in ERα in the mouse brain of which we are aware (28, 33, 34). Greco et al (42) concluded that the C1355 antibody recognizes both ligand-bound and unbound ERα in the rat brain, based on the finding that the number of ERα-positive cells was unchanged in all brain regions examined, including the BNSTp, 20 minutes after injecting a high dose of estradiol. In the current study, depleting circulating estrogen levels by gonadectomy did not alter ERα IR in the BNSTp of female mice. Moreover, gonadally intact males with profound suppression of ERα IR in the BNSTp nonetheless exhibited considerable staining in other regions. However, as in other species, the BNSTp of mice expresses high levels of aromatase (24–26), which could generate unusually high local estrogen levels. Thus, although it seems unlikely, it is difficult to completely rule out the possibility of ligand binding affecting antibody labeling in a species-, brain region-, and estrogen level-specific manner.

Development of the sex difference in ERα IR in the BNSTp

The near absence of ERα IR in the adult male mouse BNSTp is noteworthy because this receptor is required for the masculinization of BNSTp morphology in neonatal males (16, 17). Male mice are exposed to elevated T produced by their testes perinatally (54–56), and given the relatively rapid suppression of ERα IR by testicular steroids in adulthood, one might expect that ERα IR would also be suppressed in the BNSTp of neonatal males. However, we found equivalent ERα IR in the BNSTp of both sexes at birth. There was a decrease in ERα IR in male mice at P6, but levels rebounded and were again equivalent to those of females at P20. Pang and Tang (55) found elevated T in male mice on P1, P3, and P5. It is possible that this neonatal exposure is responsible for the transient suppression of ERα IR seen in P6 males.

Different mechanisms regulating ERα in the AVPV and BNSTp

Gonadectomy, androgen receptor inhibition, or inhibition of the aromatase enzyme did not alter total ERα IR in the AVPV of adult mice. Similarly, in male rats, the number of ERα IR cells in AVPV was not significantly altered by a large dose of estradiol benzoate administered 24 hours prior to the time the animals were killed (57). By contrast, neonatal estradiol treatment masculinized (decreased) ERα IR in AVPV. Treating neonatal female rats or mice with estradiol increases developmental cell death and ultimately reduces cell density in the AVPV (12, 58, 59). Thus, the decrease seen here in ERα IR in the AVPV of adult females that were treated with EB neonatally may reflect reduced neuron number in this region. Taken together, these findings suggest that neonatal hormone exposure results in a more estrogen-sensitive AVPV in adult females that may also be immune to changes in hormonal status, such as those that occur over the estrous cycle.

In the BNSTp, by contrast, ERα IR was dynamically regulated by gonadal steroids in adulthood. ERα IR in the BNSTp of males increased to female-like levels 24–48 hours after castration, indicating that males retain the ability to express high levels of the receptor, but circulating testicular hormones suppress it. ERα IR was also suppressed in the BNSTp of adult females given exogenous T, suggesting that the ability to respond to T is present in both sexes. Because the dose of T used here was high, we cannot rule out the possibility of subtle sex differences in hormone sensitivity, but taken together, the bulk of the sex difference in ERα IR within the BNSTp appears to be due to activational rather than organizational effects of steroids.

Circulating estrogens are unlikely to be responsible for the suppression of ERα IR in the BNSTp because there was no significant difference in serum estradiol levels between control males and females. In addition, removing circulating estrogens from females via gonadectomy had no effect on ERα IR in the BNSTp. It is possible that aromatization of circulating T produces high local concentrations of estradiol in the BNSTp that in turn suppresses ERα in males. In partial support of this idea, gonadally intact males treated with the aromatase inhibitor letrozole showed an increase of ERα IR, although not to female-like levels. Similarly, T acts to decrease ERα IR in the preoptic area of mice and rats after its conversion to an estrogen (52, 60).

Although the mouse BNSTp expresses both ERα and ERβ, expression of ERβ in AVPV is low or absent (23, 34, 40, 61). When coexpressed within a single cell, ERα and ERβ can form heterodimers (62) and alter each other's transcriptional activity (63–65). There does not seem to be a sex difference in the expression of ERβ in the adult mouse BNSTp or AVPV (34, 61; Kelly, D.A., and S. Krug, unpublished data), suggesting that differences in the interaction of ERα and ERβ are likely to stem from differences in ERα expression within these regions. Our findings therefore suggest that given an acute exposure to an estrogen, the response of the BNSTp in females would incorporate actions from both receptors, but the response in males would tend to favor actions mediated by ERβ.

Lower ERα expression in the preoptic area of newborn male rats compared with females correlates with increased methylation of the Esr1 promoter region in males (66), and developmental changes in ERα in the cortex of rats also reflect changes in DNA methylation (67). Thus, differences in DNA methylation may underlie the sex differences in ERα expression in the AVPV and BNSTp. If so, this suggests the interesting possibility that the epigenetic marks are relatively stable in one region (the AVPV) but quite plastic in another (the BNSTp).

Acknowledgments

We are grateful to Jill McCutcheon and Lynn Bengston for their excellent technical assistance and Geert De Vries, Matthew Paul, and 3 anonymous reviewers for their comments that greatly improved the manuscript.

This work was supported by National Institutes of Health Grant R01068482 (to N.G.F.).

Disclosure Summary: The authors have nothing to disclose.

Footnotes

Abbreviations:
AVPV
anteroventral periventricular nucleus of the hypothalamus
BNST
bed nucleus of the stria terminalis
BNSTp
principal BNST
EB
estradiol benzoate
ER
estrogen receptor
IR
immunoreactivity
P
postnatal day.

References

  • 1. Emery DE, Sachs BD. Copulatory behavior in male rats with lesions in the bed nucleus of the stria terminalis. Physiol Behav. 1976;17:803–806 [DOI] [PubMed] [Google Scholar]
  • 2. Walker DL, Toufexis DJ, Davis M. Role of the bed nucleus of the stria terminalis versus the amygdala in fear, stress, and anxiety. Eur J Pharmacol. 2003;463:199–216 [DOI] [PubMed] [Google Scholar]
  • 3. Hammack SE, Richey KJ, Watkins LR, Maier SF. Chemical lesion of the bed nucleus of the stria terminalis blocks the behavioral consequences of uncontrollable stress. Behav Neurosci. 2004;118:443–448 [DOI] [PubMed] [Google Scholar]
  • 4. Bangasser DA, Santollo J, Shors TJ. The bed nucleus of the stria terminalis is critically involved in enhancing associative learning after stressful experience. Behav Neurosci. 2005;119:1459–1466 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5. Toufexis D. Region- and sex-specific modulation of anxiety behaviours in the rat. J Neuroendocrinol. 2007;19:461–473 [DOI] [PubMed] [Google Scholar]
  • 6. Choi DC, Furay AR, Evanson NK, et al. The role of the posterior medial bed nucleus of the stria terminalis in modulating hypothalamic-pituitary-adrenocortical axis responsiveness to acute and chronic stress. Psychoneuroendocrinology. 2008;33:659–669 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7. Been LE, Petrulis A. Lesions of the posterior bed nucleus of the stria terminalis eliminate opposite-sex odor preference and delay copulation in male Syrian hamsters: role of odor volatility and sexual experience. Eur J Neurosci. 2010;32:483–493 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8. Hines M, Davis FC, Coquelin A, Goy RW, Gorski RA. Sexually dimorphic regions in the medial preoptic area and the bed nucleus of the stria terminalis of the guinea pig brain: a description and an investigation of their relationship to gonadal steroids in adulthood. J Neurosci. 1985;5:40–47 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9. Hines M, Allen LS, Gorski RA. Sex differences in subregions of the medial nucleus of the amygdala and the bed nucleus of the stria terminalis of the rat. Brain Res. 1992;579:321–326 [DOI] [PubMed] [Google Scholar]
  • 10. Guillamon A, Segovia S, del Abril A. Early effects of gonadal steroids on the neuron number in the medial posterior region and the lateral division of the bed nucleus of the stria terminalis in the rat. Dev Brain Res. 1988;44:281–290 [DOI] [PubMed] [Google Scholar]
  • 11. Allen LS, Gorski RA. Sex difference in the bed nucleus of the stria terminalis of the human brain. J Comp Neurol. 1990;302:697–706 [DOI] [PubMed] [Google Scholar]
  • 12. Forger NG, Rosen GJ, Waters EM, Jacob D, Simerly RB, De Vries GJ. Deletion of Bax eliminates sex differences in the mouse forebrain. Proc Natl Acad Sci USA. 2004;101:13666–13671 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13. Chung WC, Swaab DF, De Vries GJ. Apoptosis during sexual differentiation of the bed nucleus of the stria terminalis in the rat brain. J Neurobiol. 2000;43:234–243 [PubMed] [Google Scholar]
  • 14. Gotsiridze T, Kang N, Jacob D, Forger NG. Development of sex differences in the principal nucleus of the bed nucleus of the stria terminalis of mice: role of Bax-dependent cell death. Dev Neurobiol. 2007;67:355–362 [DOI] [PubMed] [Google Scholar]
  • 15. Ahern TH, Krug S, Carr AV, et al. Cell death atlas of the postnatal mouse ventral forebrain and hypothalamus: effects of age and sex. J Comp Neurol. 2013;521(11):2551–2569 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16. Hisasue S, Seney ML, Immerman E, Forger NG. Control of cell number in the bed nucleus of the stria terminalis of mice: role of testosterone metabolites and estrogen receptor subtypes. J Sex Med. 2010;7:1401–1409 [DOI] [PubMed] [Google Scholar]
  • 17. Tsukahara S, Tsuda MC, Kurihara R, et al. Effects of aromatase or estrogen receptor gene deletion on masculinization of the principal nucleus of the bed nucleus of the stria terminalis of mice. Neuroendocrinology. 2011;94:137–147 [DOI] [PubMed] [Google Scholar]
  • 18. Roselli CE, Handa RJ, Resko JA. Quantitative distribution of nuclear androgen receptors in microdissected areas of the rat brain. Neuroendocrinology. 1989;49:449–453 [DOI] [PubMed] [Google Scholar]
  • 19. Lu S-F, McKenna SE, Cologer-Clifford A, Nau EA, Simon N. Androgen receptor in mouse brain: sex differences and similarities in autoregulation. Endocrinology. 1998;139:1594–1601 [DOI] [PubMed] [Google Scholar]
  • 20. Shughrue PJ, Scrimo PJ, Merchenthaler I. Evidence for the colocalization of estrogen receptor-β mRNA and estrogen receptor-α immunoreactivity in neurons of the rat forebrain. Endocrinology. 1998;39:5267–5270 [DOI] [PubMed] [Google Scholar]
  • 21. Chakraborty TR, Ng L, Gore AC. Age-related changes in estrogen receptor β in rat hypothalamus: a quantitative analysis. Endocrinology. 2003;144:4164–4171 [DOI] [PubMed] [Google Scholar]
  • 22. Chakraborty TR, Hof PR, Ng L, Gore AC. Stereologic analysis of estrogen receptor α (ERα) expression in rat hypothalamus and its regulation by aging and estrogen. J Comp Neurol. 2003;466:409–421 [DOI] [PubMed] [Google Scholar]
  • 23. Mitra SW, Hoskin E, Yudkovitz J, et al. Immunolocalization of estrogen receptor β in the mouse brain: comparison with estrogen receptor α. Endocrinology. 2003;144:2055–2067 [DOI] [PubMed] [Google Scholar]
  • 24. Roselli CE, Horton LE, Resko JA. Distribution and regulation of aromatase activity in the rat hypothalamus and limbic system. Endocrinology. 1985;117:2471–2477 [DOI] [PubMed] [Google Scholar]
  • 25. Foidart A, Harada N, Balthazart J. Aromatase-immunoreactive cells are present in mouse brain areas that are known to express high levels of aromatase activity. Cell Tissue Res. 1995;280:561–574 [DOI] [PubMed] [Google Scholar]
  • 26. Wu MV, Manoli DS, Fraser EJ, et al. Estrogen masculinizes neural pathways and sex-specific behaviors. Cell. 2009;139:61–72 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27. Merchenthaler I, Lane MV, Numan S, Dellovade TL. Distribution of estrogen receptor α and β in the mouse central nervous system: in vivo autoradiographic and immunocytochemical analyses. J Comp Neurol. 2004;473:270–291 [DOI] [PubMed] [Google Scholar]
  • 28. Brown A, Mani S, Tobet S. The preoptic area/anterior hypothalamus of different strains of mice: sex differences and development. Dev Brain Res. 1999;115:171–182 [DOI] [PubMed] [Google Scholar]
  • 29. Kelly DA, Varnum MM, Forger NG. A striking sex difference in estrogen receptor α immunoreactivity in the mouse BNSTp due to suppression by testicular steroids. Paper presented at: Program of the Annual Meeting of the Society for Neuroscience; 2011; Washington, D.C. Number 499.16. http://bit.ly/ZgoVA6 Accessed April 22, 2013 [Google Scholar]
  • 30. Hnatczuk OC, Lisciotto CA, DonCarlos LL, Carter CS, Morrell JI. Estrogen receptor immunoreactivity in specific brain areas of the prairie vole (Microtus ochrogaster) is altered by sexual receptivity and genetic sex. J Neuroendocrinol. 1994;6:89–100 [DOI] [PubMed] [Google Scholar]
  • 31. Yokosuka M, Okamura H, Hayashi S. Postnatal development and sex difference in neurons containing estrogen receptor-α immunoreactivity in the preoptic brain, the diencephalon, and the amygdala in the rat. J Comp Neurol. 1997;389:81–93 [DOI] [PubMed] [Google Scholar]
  • 32. Cushing BS, Razzoli M, Murphy AZ, Epperson PM, Le WW, Hoffman GE. Intraspecific variation in estrogen receptor α and the expression of male sociosexual behavior in two populations of prairie voles. Brain Res. 2004;1016:247–254 [DOI] [PubMed] [Google Scholar]
  • 33. Vida B, Hrabovszky E, Kalamantianos T, Coen CW, Liposits Z, Kallo I. Oestrogen receptor α and β immunoreactive cells in the suprachiasmatic nucleus of mice: distribution, sex differences and regulation by gonadal hormones. J Neuroendocrinol. 2008;20:1270–1277 [DOI] [PubMed] [Google Scholar]
  • 34. Chakraborty TR, Rajendren G, Gore AC. Expression of estrogen receptor (α) in the anteroventral periventricular nucleus. Exp Biol Med. 2005;230:49–56 [DOI] [PubMed] [Google Scholar]
  • 35. Wiegand SJ, Terasawa E. Discrete lesions reveal functional heterogeneity of suprachiasmatic structures in regulation of gonadotropin secretion in the female rat. Neuroendocrinology. 1982;34:395–404 [DOI] [PubMed] [Google Scholar]
  • 36. Gu GB, Simerly RB. Projections of the sexually dimorphic anteroventral periventricular nucleus in the female rat. J Comp Neurol. 1997;384:142–164 [PubMed] [Google Scholar]
  • 37. Tsukahara S, Kakeyama M, Toyofuku Y. Sex differences in the level of Bcl-2 family proteins and caspase-3 activation in the sexually dimorphic nuclei of the preoptic area in postnatal rats. J Neurobiol. 2006;66:1411–1419 [DOI] [PubMed] [Google Scholar]
  • 38. Sumida H, Nishizuka M, Kano Y, Arai Y. Sex differences in the anteroventral periventricular nucleus of the preoptic area and in the related effects of androgen in prenatal rats. Neurosci Lett. 1993;151:41–44 [DOI] [PubMed] [Google Scholar]
  • 39. Simerly RB, Zee MC, Pendleton JW, Lubahn DB, Korach KS. Estrogen receptor-dependent sexual differentiation of dopaminergic neurons in the preoptic region of the mouse. Proc Natl Acad Sci USA. 1997;94:14077–14082 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40. Bodo C, Kudwa AE, Rissman EF. Both estrogen receptor-α and –β are required for sexual differentiation of the anteroventral peniventricular area in mice. Endocrinology. 2006;147:415–420 [DOI] [PubMed] [Google Scholar]
  • 41. Moffatt CA, Rissman EF, Shupnik MA, Blaustein JD. Induction of progestin receptors by estradiol in the forebrain of estrogen receptor-α gene-disrupted mice. J Neurosci. 1998;18:9556–9563 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42. Greco B, Allegretto E, Tetel M. Coexpression of ER β with ER α and progestin receptor proteins in the female rat forebrain: effects of estradiol treatment. Endocrinology. 2001;142:5172–5181 [DOI] [PubMed] [Google Scholar]
  • 43. Schreihofer DA. Estrogen receptor-α (ERα), but not ERβ, modulates estrogen stimulation of the ERα-truncated variant, TERP-1. Endocrinology. 2002;143:4196–4202 [DOI] [PubMed] [Google Scholar]
  • 44. Franklin KBI, Paxinos G. The Mouse Brain in Stereotaxic Coordinates. 3rd ed. New York: Academic Press; 2007 [Google Scholar]
  • 45. Wong AKC, Sahoo PK. A gray-level threshold selection method based on maximum entropy principle. IEEE Trans Syst Man Cybern. 1989;19:866–871 [Google Scholar]
  • 46. Lund TD, Munson DJ, Haldy ME, Handa RJ. Dihydrotestosterone may inhibit hypothalamo-pituitary-adrenal activity by acting through estrogen receptor in the male mouse. Neurosci Lett. 2004;365:43–47 [DOI] [PubMed] [Google Scholar]
  • 47. Park JH, Bonthuis P, Ding A, Rais S, Rissman EF. Androgen- and estrogen-independent regulation of copulatory behavior following castration in male B6D2F1 mice. Horm Behav. 2009;56:254–263 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48. Carroll JC, Rosario ER, Kreimer E, et al. Sex differences in β-amyloid accumulation in 3xTg-AD mice: role of neonatal sex steroid exposure. Brain Res. 2010;1366:233–245 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49. Gorin-Meyer RE, Wiren KM, Tanchuck MA, Long SL, Yoneyama N, Finn DA. Sex differences in the effect of finasteride on acute ethanol withdrawal severity in C57BL/6J and DBA/2J mice. Neuroscience. 2007;146:1302–1315 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50. Xu X, Coats JK, Yang CF, et al. Modular genetic control of sexually dimorphic behaviors. Cell. [Erratum (2012) 148:1066–1067] 2012;148:596–607 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51. Wagner CK, Nakayama AY, De Vries GJ. Potential role of maternal progesterone in the sexual differentiation of the brain. Endocrinology. 1998;139:3658–3661 [DOI] [PubMed] [Google Scholar]
  • 52. Agarwal VP, Sinton CM, Liang CL, Fisher C, German DC, Simpson ER. Upregulation of estrogen receptors in the forebrain of aromatase knockout (ArKO) mice. Mol Cell Endocrinol. 2000;162:9–16 [DOI] [PubMed] [Google Scholar]
  • 53. Blaustein JD. Estrogen receptor immunoreactivity in rat brain: rapid effects of estradiol injection. Endocrinology. 1993;132:1218–1224 [DOI] [PubMed] [Google Scholar]
  • 54. Pointis G, Latreille MT, Cedard L. Gonado-pituitary relationships in the fetal mouse at various times during sexual differentiation. J Endocrinol. 1980;86:483–488 [DOI] [PubMed] [Google Scholar]
  • 55. Pang SF, Tang F. Sex differences in the serum concentrations of testosterone in mice and hamsters during their critical periods of neural sexual differentiation. J Endocrinol. 1984;100:7–11 [DOI] [PubMed] [Google Scholar]
  • 56. Motelica-Heino I, Castanier M, Corbier P, Edwards DA, Roffi J. Testosterone levels in plasma and testes of neonatal mice. J Steroid Biochem. 1988;31:283–286 [DOI] [PubMed] [Google Scholar]
  • 57. Yamada S, Noguchi D, Ito H, Yamanouchi K. Sex and regional differences in decrease of estrogen receptor α-immunoreactive cells by estrogen in rat hypothalamus and midbrain. Neuroci Lett. 2009;463:135–139 [DOI] [PubMed] [Google Scholar]
  • 58. Arai Y, Sekine Y, Murakami S. Estrogen and apoptosis in the developing sexually dimorphic preoptic area in female rats. Neurosci Res. 1996;25:403–407 [DOI] [PubMed] [Google Scholar]
  • 59. Waters EM, Simerly RB. Estrogen induces caspase-dependent cell death during hypothalamic development. J Neurosci. 2009;29:9714–9718 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 60. Clancy AN, Michael RP. Effects of testosterone and aromatase inhibition on estrogen receptor-like immunoreactivity in male rat brain. Neuroendocrinology. 1994;59:552–560 [DOI] [PubMed] [Google Scholar]
  • 61. Milner TA, Thompson LI, Wang G, et al. Distribution of estrogen receptor β containing cells in the brains of bacterial artificial chromosome transgenic mice. Brain Res. 2010;1351:74–96 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 62. Pettersson K, Grandien K, Kuiper GG, Gustafsson JA. Mouse estrogen receptor β forms estrogen response element-binding heterodimers with estrogen receptor α. Mol Endocrinol. 1997;11:1486–1496 [DOI] [PubMed] [Google Scholar]
  • 63. Hall JM, McDonnell DP. The estrogen receptor β-isoform (ERβ) of the human estrogen receptor modulates ERα transcriptional activity and is a key regulator of the cellular response to estrogens and antiestrogens. Endocrinology. 1999;140:5566–5578 [DOI] [PubMed] [Google Scholar]
  • 64. Matthews J, Gustafsson JA. Estrogen signaling: a subtle balance between ER α and ER β. Mol Interv. 2003;3:281–292 [DOI] [PubMed] [Google Scholar]
  • 65. Weiser MJ, Foradori CD, Handa RJ. Estrogen receptor β in the brain: from form to function. Brain Res Rev. 2008;57:309–320 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 66. Kurian JR, Olesen KM, Auger AP. Sex differences in epigenetic regulation of the estrogen receptor—a promoter within the developing preoptic area. Endocrinology. 2010;151:2297–2305 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 67. Westberry JM, Trout AL, Wilson ME. Epigenetic regulation of estrogen receptor-α gene expression in the mouse cortex during early postnatal development. Endocrinology. 2010;151:731–740 [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Endocrinology are provided here courtesy of The Endocrine Society

RESOURCES