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Endocrinology Vol. 145, No. 10 4500-4506
Copyright © 2004 by The Endocrine Society

Estrogen Receptor ß Modulates Estradiol Induction of Progestin Receptor Immunoreactivity in Male, But Not in Female, Mouse Medial Preoptic Area

Andrea E. Kudwa, Jan-Åke Gustafsson and Emilie F. Rissman

Department of Biochemistry and Molecular Genetics (E.F.R.) and Neuroscience Graduate Program (A.E.K., E.F.R.), University of Virginia Medical School, Charlottesville, Virginia 22908; and Center for Biotechnology and Department of Medical Nutrition (J.-A.G.), Karolinska Institute, NOVUM S-141 86 Huddinge, Sweden

Address all correspondence and requests for reprints to: Emilie F. Rissman, P.O. Box 800733, University of Virginia, Medical School, Charlottesville, Virginia 22908. E-mail: rissman{at}virginia.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The medial preoptic area (mPOA) of the hypothalamus contains many neurons that express estrogen receptor {alpha} (ER) and/or ERß. We examined the distribution of these receptors and assessed responses to estradiol (E2) in the adult mouse mPOA. Gonadectomized adult male and female mice were killed, and brains were processed for immunocytochemistry for ER{alpha} and ERß. More ER{alpha} immunoreactive (-ir) than ERß-ir neurons were present in the mouse mPOA. Numbers of ER{alpha}-ir cells were equivalent between males and females, but males had significantly more ERß-ir neurons than females. Using breeders that were heterozygous for disrupted ER{alpha} and ERß genes, we produced offspring with varying numbers (0, 1, or 2) of functional and disrupted ER{alpha} and ERß genes. After gonadectomy, half the mice received E2 for 5 d before they were killed. Estradiol treatment, sex, and genotype each had independent effects on numbers of PR-ir neurons in the mPOA. In all cases, brains that lacked at least one functional copy of ER{alpha} had reduced PR-ir cell numbers. In gonadectomized, untreated mice, one functional copy of the ERß gene was correlated with the largest amount of PR-ir. After E2 treatment, both sexes had greatly enhanced numbers of PR-ir containing neurons. In females, maximal PR induction required the presence of at least one functional copy of ER{alpha}, whereas in males, at least a single copy of both functional ERß and ER{alpha} genes was needed for maximal PR-ir induction. We hypothesize that the two ERs have dependent and independent roles in sexual differentiation of neuroendocrine function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTROGEN HAS UBIQUITOUS actions in the central nervous system. The estrogen receptors, ER{alpha} and ERß, are nuclear transcription factors. The two receptors can either act independently as homodimers, or together as heterodimers, to bind estrogen-responsive elements and influence gene transcription (1). Functional differences between ER{alpha} and ERß have been noted in vitro (2, 3, 4) and in vivo (5, 6, 7). One well-documented action of ERs is the regulation of transcription of progestin receptor (PR) protein. ER{alpha} knockout (ER{alpha}KO) female mice exhibit low baseline levels of PR in brain; however, treatment with estradiol (E2) promotes significant induction of PR-immunoreactive (-ir) cells or PR mRNA in the medial preoptic area (mPOA) (8, 9). Moreover, double ER{alpha} and ERß knockout (ER{alpha}ßKO) females continue to display modest PR mRNA and protein in several brain sites in response to E2 (10, 11). ERßKO mice show alterations in PR induction after long-term E2 treatment. In males, the lack of ERß enhances PR-ir induction in several regions, but the opposite effect is noted in female brains (12). These observations suggest that ERß may act differently in males vs. females.

In the present study, first we asked whether ER{alpha}-ir and ERß-ir cell numbers were equivalent in the adult mouse male and female mPOA. In rats, the male mPOA exhibits more ERß immunoreactivity than is present in females (13). During development (embryonic d 17 to postnatal d 15), male BALB/c mice have more ERß mRNA in the anterior hypothalamus/POA than do females (14). In our study, we did not note any sex differences in ER{alpha}-ir cell number, but castrated males had nearly three times more ERß-ir cells than did ovariectomized females. Next we asked whether this sex difference in ERß had a functional significance. We used male and female knockout mice carrying varying combinations of disrupted and functional copies of the ER{alpha} or ERß genes. These mice were treated with E2 or nothing for 5 d after gonadectomy, and brains were analyzed for PR immunocytochemistry. We report here that E2-mediated PR induction in the mPOA is influenced by ER{alpha} gene in both males and females, whereas ERß plays a role in males only.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult male and female C57BL/6J mice (2–3 months of age) used in the first experiment were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice in the second experiment were produced using double heterozygous breeding pairs. Each breeder carried one normal (functional) and one disrupted copy of both the ER{alpha} and the ERß genes (ER{alpha}ßHET). This line was at least eight generations backcrossed into the C57BL/6J strain. Thus, on average these mice were 99.6% genetically identical with C57BL/6J mice. All offspring were screened by PCR amplification of tail DNA (15, 16). Mice were group housed by sex after weaning (18–20 d of age) on a 12-h light, 12-h dark cycle (lights off at 1900 h EDT) and maintained on Harlan Teklad (Indianapolis, IN) Global Diet 8604 and water ad libitum. In the first experiment, adult male (n = 11) and female (n = 9) mice were gonadectomized and then killed 5 d later. In the second study, both adult male (n = 94) and female (n = 116) offspring from the ER{alpha}ßHET cross were used (50–75 d of age). All animals were gonadectomized and 7 d later received a sc SILASTIC brand (Dow Corning, Midland, MI) capsule (3.17 mm outer diameter x 1.98 mm inner diameter; total length was 2.5 cm) containing 50 µg of E2 dissolved in 25 µl sesame oil, or an empty capsule. This E2 implant yields a high physiological concentration of estradiol in plasma (85–120 pg/ml; Rissman, E., unpublished data). All surgeries were conducted in mice under general anesthesia (100 mg/kg ketamine and 10 mg/kg xylazine injected ip), and all animals were killed 5 d after implantation. The University of Virginia Animal Care and Use Committee approved our procedures.

Immunocytochemistry
In the first study, mice were deeply anesthetized using sodium pentobarbitol and brains perfused with approximately 50 ml heparinized saline followed by 200 ml 4% paraformaldehyde in phosphate buffer (pH 7.4). In the second study, mice were anesthetized using halothane and brains were removed and fixed via 5% acrolein immersion as described previously (17). Different fixation methods were employed in the two studies to optimize immunoreactivity with the three different antibodies we used. In both experiments, brains were placed in 0.1 M phosphate buffer containing 30% sucrose for 24 h, and then each brain was frozen and serial coronal sections (30 µm) through the forebrain were collected on a cryostat. Consecutive sections were placed in four vials containing cryoprotectant and stored at –20 C.

In both experiments, brain sections were removed from cryoprotectant and processed for immunocytochemistry. All sections were first rinsed in 0.2 M Tris-buffered saline (TBS, 5 x 10 min), pH 7.8. These rinses were followed by a 30-min incubation in a 1% NaBH4 solution and a 10 min incubation in 0.3% H2O2, with three 10-min rinses in between. Sections were incubated at 4 C for 48 h in a primary antibody. In the first experiment, we used two antibodies directed against either ER{alpha} or ß. For ER{alpha} detection, we used a rat monoclonal antibody directed against ER{alpha} (1:1000, H222; generously provided by Abbott Labs, Chicago, IL). This antibody has been validated previously for use in mouse tissue (18, 19). For ERß detection, we used a polyclonal antibody directed against the C terminus of the mouse ERß protein (1:1000, Z8P; Zymed Laboratories, Inc., South San Francisco, CA). The specificity of this ERß antiserum has been demonstrated by Western blots, peptide preabsorbing, and immunocytochemistry (20, 21, 22, 23). Next, tissue was incubated for 1 h in a biotinylated goat antirabbit secondary antibody (Vector Laboratories, Burlingame, CA; 1:500 for ERß) or biotinylated donkey antirat secondary antibody (Vector Laboratories; 1:500 for ER{alpha}). After rinses, sections were incubated for 90 min in an avidin-biotin complex (1:1000; Vectastain Elite; Vector Laboratories). After additional 5-min TBS rinses, tissues were incubated for 5 min with a nickel-intensified diaminobenzidine (DAB) solution (0.25% nickel ammonium sulfate and 0.05% DAB) activated by 0.001% hydrogen peroxide. All sections were processed for ER{alpha} and ß in the same staining run.

In the second experiment, every fourth section was processed for immunocytochemical analysis of PRs using the same protocol described above. We used a monoclonal primary antibody directed against the hinge region of the PR (0.2 mg/ml; H-928, StressGen Biotechnologies Corp., Victoria, British Columbia, Canada) and a biotinylated horse antimouse secondary antibody (1:500; Vector Laboratories). The use of this primary antibody in mouse brain has been previously validated (9). The chromagen was visualized with nickel-intensified DAB as described above. The sections were processed in three runs, and within each run, identical incubation times and reagents were used. Each run also contained an equivalent cross section of brains from each group.

Image analysis and statistics
In the first study, we quantified ER{alpha} and ERß immunoreactivity two ways. We counted neurons that were immunoreactive throughout one side of the mPOA, and in addition we selected one best-matched section from each brain and counted cells on both sides of the mPOA. In the second experiment, we focused on the same region and used the best matched section to count PR-ir neurons. Computer-assisted analysis of steroid-receptor immunoreactivity was performed on an Olympus BX60 microscope fitted with a Photometrics charge-coupled device video camera connected to a Dell PC and a high-resolution Hitachi 21-in. monitor. Our image analysis program is Metamorph Image Analysis (version 4.5; Universal Imaging, West Chester, PA). The camera gain and black levels are automatically adjusted so unstained sections produce grayscale levels of about 5 U and the black standard produces grayscale levels of 254 U. The cell nuclei are selected based on an average pixel size. As illustrated previously (9), the area quantified was the medial preoptic nucleus (mPOA, 0.14 mm anterior to bregma; Fig. 30 from the Franklin and Paxinos atlas; Ref. 23). In this region, the two major fiber tracts that serve as landmarks are the optic tract, which is barely present at the base of the brain, and the anterior commisure, which is present at its greatest lateral extent. We intentionally excluded the region of the mPOA representing the anteroventral periventricular (AVPv) nucleus by avoiding counting immunoreactive cells located within four cell bodies distance from the ventricle.

In the first study, ER{alpha} and ERß-ir cell numbers were analyzed using two one-way ANOVAs with sex as the factor. We did this analysis on the average number of cells per section in the entire mPOA and also bilaterally for the selected best-matched sections. To assess differences in PR-ir we conducted a three-way ANOVA with sex, hormone treatment, and genotype as factors. To follow up on the effects of each ER on basal vs. E2-induced PR-ir, we performed identical analyses on brains that did and did not experience E2 treatment. To determine the contributions of ER{alpha} and ERß, we limited the analysis to untreated vs. E2-treated animals and conducted a two-way ANOVA wherein the two variables were functional ER{alpha} and ERß gene copy numbers. Differences among groups were further analyzed using Bonferroni’s multiple comparison, which adjusts significance levels for the number of comparisons made.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Male mPOA contains more ERß-ir than female mPOA
A robust sex difference in numbers of ERß-ir neurons was noted; male mPOA had more ERß-ir cells than females. The sex difference in ERß-ir was statistically significant when either the counts were averaged from the entire mPOA [F(1,20) = 4.85; P < 0.05] or if a single best match section was considered [F(1,20) = 15.44; P < 0.001; Figs. 1Go and 2Go]. There was no effect of sex on ER{alpha}-ir cell number within the mPOA [average count across the mPOA: F (1, 20) = 0.37; P = 0.55; best-matched section: F(1, 20) = 3.37; P = 0.08; Fig. 2Go]. Interestingly, although ER{alpha}-ir cell numbers did not differ between the sexes, there appeared to be a difference in the distribution of immunoreactive cells. As illustrated in Fig. 1Go, males had a more lateral distribution of ER{alpha}-ir neurons than did female mPOA.



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  		FIG. 1. Representative photomicrographs of ERß-ir (A–F) and ER{alpha}-ir (G, H) in the mPOA of ovariectomized female (A–C and G) and castrated male (D–F and H) C57BL/6J mice. ERß-ir is shown in three coronal planes (anterior to posterior) through the mPOA to depict distribution of the cells. As illustrated in G and H, male mPOA contains more ERß-ir cells than females. Overall, the numbers of ER{alpha}-ir cells in the mPOA are equivalent throughout the nuclei between males and females, but females appear to exhibit a more medial concentration of ER{alpha}-ir cells compared with a more even (medial to lateral) distribution in males. Scale bar, 100 µm.

 


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  		FIG. 2. Mean (±SEM) ER{alpha}-ir neurons (A) and ERß-ir cell numbers (B) in the mPOA of adult ovariectomized female (n = 9) and castrated male (n = 11) mice. All mice were gonadectomized 1 wk before they were killed. *, Significantly different from females (P < 0.001).

 
Estradiol, genotype, and sex all affect PR-ir cell numbers in mPOA
In mice with varying combinations of functional and disrupted ER{alpha} and ERß genes, each of the main factors (genotype, sex, and hormone treatment) had significant effects in our overall ANOVA. Estradiol treatment significantly enhanced PR-ir cell numbers [F(1,210) = 386.60, P < 0.001]. Genotype also affected PR-ir cell numbers [F(8,210) = 14.72, P < 0.001]. Sex differences were noted wherein females had more PR-ir neurons than did males [F(1, 210) =16.65, P < 0.001]. Interactions between sex and hormone treatment [F(1, 210) = 11.15, P < 0.001] and between genotype and hormone treatment were observed [F(8, 210) = 5.85, P < 0.001]. The sex-by-treatment effect was attributed to differences between E2-treated and untreated animals. In addition, E2-treated females had more PR-ir neurons than males treated in a similar manner (P < 0.05). Genotype and E2 treatment interactions were more complex, and are illustrated in Fig. 3Go. In general, mice that received E2 treatment but were lacking any functional copies of ER{alpha} had fewer PR-ir neurons than similarly treated mice of other genotypes.



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  		FIG. 3. Mean (± SEM) PR-ir neurons in mPOA. All mice were gonadectomized, and 1 wk later each received either a SILASTIC brand implant containing E2 or left empty. Mice were killed 5 d later. A, Data from male mice; B, females. Data from mice that did not receive any hormone treatment after gonadectomy are shown in white histograms and data from E2-treated mice are in black histograms. In both sexes E2 treatment significantly increased PR-ir cell numbers. Examining data from gonadectomized untreated mice only we observed the following: +, Significantly more PR-ir neurons than double KO mice. *, Significantly fewer PR-ir neurons than same-sex mice with two functional copies of ER{alpha} and one functional copy of ERß. Turning to data from E2-treated mice, we noted the following: A, In males, letters designates significant differences between genotypes. Histograms with the same letters do not differ from each other, but different letters indicate significant differences (P < 0.05). B, In females, letters designates significant differences between genotypes. Histograms with the same letters do not differ from each other, but different letters indicate significant differences (P < 0.05). Group sizes ranged from two to 10 individuals.

 
To further describe these effects, data from males and females were analyzed separately. Using two-way ANOVAs, we found significant effects of E2 treatment [F(1,94) = 156.91 for males and F(1,116) = 296.46 for females, P < 0.001], genotype [F(8,94) = 7.69 for males and F(8,116) = 11.25 for females, P < 0.001], and interactions [F(8,94) = 2.09 for males and F(8,116) = 6.14 for females, P < 0.05 at least]. The interesting differences were assessed by examination of PR-ir responses to E2 in mice lacking any copies of functional ER{alpha} (see Fig. 3Go). ER{alpha}KO females had less PR-ir neurons than females in most other genotypes, including females that lacked any functional ERß genes. Yet in general, ER{alpha}KO males only had significantly less PR-ir than males of other genotypes that had at least one functional copy of ERß.

Genotype affects basal numbers of PR-ir cells in brains of gonadectomized mice
When we examined data from gonadectomized mice that did not receive E2, we noted an effect of genotype on basal numbers of PR-ir neurons [F(8,98) = 4.29, P < 0.001]. There was no effect of sex [F (1,98) = 0.45], nor was an interaction between sex and genotype present [F (8,98) = 0.32]. Mice with two functional copies of the ER{alpha} gene and only one functional copy of ERß had the greatest basal numbers of PR-ir cells. This group was significantly different from ER{alpha}ßKO and ER{alpha}KO animals (P < 0.05; Fig. 3Go). The PR-ir cell numbers in mPOA of double heterozygous and ERßKO brains were also greater than the numbers of PR-ir cells noted in ER{alpha}ßKO brains (P < 0.05; Fig. 3Go).

To better describe the actions of the two ERs, we conducted a two-way ANOVA with number of functional copies of ER{alpha} and ERß as the two factors. Both ER{alpha} [F(2,98) = 10.50] and ERß [F(2,98) = 7.33] genes had significant effects on PR-ir cell numbers (P < 0.002 at least). No interaction was detected [F (4,98) = 0.80]. Brains lacking both functional copies of ER{alpha} had fewer PR-ir neurons than brains with one or two functional copies of the gene (P < 0.05; Fig. 4Go). In the case of ERß, brains that had one functional and one disrupted copy of this gene had the greatest amount of PR-ir neurons (P < 0.05).



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  		FIG. 4. A, Mean (± SEM) PR-ir cell numbers in the mPOA of adult female and male mice. All mice were gonadectomized and then killed 12 d later. To describe the requirement for ERß gene, we present data from genotypes with no functional copies of ERß gene and either two disrupted copies of ER{alpha} (n = 7 females; n = 10 males), one disrupted copy of ER{alpha} (n = 4 females; n = 2 males) or two functional copies of ER{alpha} gene (n = 7 females, n = 5 males). There are no sex differences in this data set. *, Significantly more PR-ir cells than in brains from ER{alpha}ßKO mice P < 0.05. B, Mean (± SEM) PR-ir cell numbers in the mPOA of adult female and male mice. All mice were gonadectomized, and 1 wk later received an E2 implant for 5 d before they were killed. Again to assess whether males and females have different requirements for the ERß gene, we present data from genotypes with no functional copies of ERß gene and either two disrupted copies of ER{alpha} (n = 9 females; n = 8 males), one disrupted copy of ER{alpha} (n = 9 females; n = 4 males) or two functional copies of ER{alpha} gene (n = 6 females; n = 6 males). A sex difference is noted in this data set with females having more PR-ir neurons than males. This sex difference is only seen when one or more copies of functional ER{alpha} are present. In males, two copies of functional ER{alpha} are needed to compensate for the lack of ERß. In females, if one copy of ER{alpha} is present PR-ir induction is maximized. *, Significantly different from mPOA in male ER{alpha}ßKO mice, but not from female ER{alpha}ßKO mice P < 0.05. **, Significantly different from all other groups.

 
After E2 treatment, the female mPOA has more PR-ir than male mPOA
To examine the roles of ER{alpha} and ERß in response to E2, we limited our analysis to E2-treated animals. A sex difference [F (1,112) = 21.09, P < 0.001] and effect of genotype [F(8,112) = 13.40, P < 0.001] were noted, and an interaction was detected [F(8,112) = 3.28, P < 0.0025]. Planned comparisons revealed that females had more PR-ir neurons than males (P < 0.05). Similar to the data from the untreated brains, the three groups that lacked any copies of functional ER{alpha} gene were responsible for the genotype effect. In these brains, significantly less PR-ir was induced as compared with brains from the other groups (P < 0.05). The interaction between sex and genotype was caused by a sex difference (females>males) in two of nine genotypes. When no functional copies of ERß were present, females with one or two copies of functional ER{alpha} had more PR-ir induction than males from the same genotypes (P < 0.05; Fig. 3Go).

Both ERs are required for maximal PR-ir induction by E2 treatment in male mPOA
To better characterize the roles of each ER in male vs. female brains, we conducted two-way ANOVAs using data from animals treated with E2. One ANOVA was conducted on data from males and the other with female data. In both cases, the two factors were numbers of functional ER{alpha} and ERß genes. In females, only ER{alpha} gene copy number affected PR-ir cell numbers in the mPOA [F(2,62) = 41.79, P < 0.001]. Female brains with one or two copies of ER{alpha} had significantly more PR-ir cells than brains lacking both functional copies of ER{alpha} (P < 0.05). Presence or absence of functional copies of ERß had no effect [F (2,62) = 1.40], nor was an interaction noted [F (4,62) = 1.16]. In contrast, PR-ir induction in males was affected by both ER{alpha} [F(2,50) = 14.56, P < 0.001] and ERß gene [F(2,50) = 5.10, P < 0.01] manipulation, but no interaction was revealed [F (4,50) = 1.86; Fig. 4Go]. Males required at least one functional copy of both ER{alpha} and ERß in order for E2 to induce maximal PR-ir (P < 0.05; Fig. 4Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These data illustrate both a sex difference in ERß-ir neurons in the adult mouse mPOA, and a potential sex difference in function of the two ERs. In the mPOA, adult males have more neurons containing ERß-ir than do females. It is important to note that we conducted this study in gonadectomized animals; thus, the data do not reflect differences in ERß that could be caused by differences in circulating concentrations of estradiol. In gonadectomized rats, a subregion of the mPOA, the AVPv, is also sexually dimorphic for ERß (24). However, the sex difference in the AVPv is reversed, with females having more ERß containing neurons than do males. This reversed sex difference is present from postnatal d 7–60. Interestingly, in this study no sex differences in ERß in the mPOA or the bed nucleus of the stria terminalis were noted (24). Adult gonad-intact male rats were reported to have more intense ERß-ir in the mPOA, bed nucleus of the stria terminalis, and a few other regions compared with females, but these findings were not quantified (13). Sex differences in ERß mRNA have also been reported in brains of BALB/c mouse embryos and pups (24). In the hypothalamic/preoptic region of embryonic d 17 through postnatal d 15 animals, male brains expressed more ERß mRNA than females. However, in contrast to findings presented here, no sex difference was present in gonad-intact adults (24). Furthermore, no adult sex difference in either ER{alpha} or ß was discerned in preoptic area tissue taken from rams and estrous ewes (25). Perhaps adult sex differences in these aforementioned studies were masked by high circulating E2 concentrations in the plasma because these animals were not gonadectomized. Regardless, these results, and our own, show that during several time points throughout development, male brains, and particularly the mPOA region, express more ERß than do females.

To determine whether this sex difference in ERß (males more than females) can be related to functional differences in the neuroendocrinology of males vs. females, we selected a well-characterized ER-dependent function, induction of PR, to assess the roles of ER{alpha} and ERß in the mPOA. As reported using different methods in rats, mice, and guinea pigs, there are more PR-ir cells in the mPOA (12, 26, 27, 28) and the periventricular preoptic subregion (27) in females than males after E2 treatment. In contrast, male guinea pigs express more PR-ir cells in the preoptic area after a single EB injection as compared with females (29). The unique aspect of the present study is that, by using animals with varying copy numbers of functional ER{alpha} and ERß, we determined which ER is required by each sex to elicit maximal E2mediated induction of PR. We found that induction of PR by E2 in the female mPOA only requires the ER{alpha} gene, and more specifically, only one functional copy is required for maximal induction. However, in male brains at least one copy of both ERs is essential for the maximal response to E2. Although more data are needed, we suggest that the sex difference in ERß observed in this same region may be related to a male-specific role for ERß in PR-ir induction.

Interestingly, although no sex differences in PR-ir were detected in brains of gonadectomized mice that were not treated with E2, we did observe effects of genotype. Because these differences were noted in the absence of adult E2 treatment, they likely reflect organizational actions of the two ERs during development. Baseline expression of PR-ir in adult mouse brain was influenced by both ERs. More specifically, at least one functional copy of ER{alpha} or ERß was correlated with higher basal PR-ir cell counts. In fact, mice with two functional copies of ER{alpha} and one copy of ERß had the largest number of PR-ir neurons in the mPOA. Although ERs have been shown to form both homo- and heterodimers in vitro (30, 31), these data may indicate a role of heterodimeric associations between ER{alpha} and ß during development, particularly in the absence of ligand.

Both ER{alpha} and ß are expressed in the rat (32) and mouse (33) mPOA, and E2 can modulate hypothalamic ER and PR colocalization in a region-specific manner in the female rat brain (34). Whereas nearly all of ERß-ir cells in the female rat periventricular preoptic subregion express ER{alpha}, only about 15% of the ER{alpha}-ir cells express ERß. After E2 treatment, ERß levels decrease and PR is induced (34). Also, the degree of colocalization of ERß and PR does not change after E2, which further supports our finding that PR induction in females occurs through ER{alpha}, independent of ERß. To our knowledge, the same detailed analysis of ER and PR colocalization described above has not been completed in the male rat or in mouse, but based on the findings of this study, we speculate that the percentage of cells containing both ERß and PR in the male mouse mPOA would increase after E2, and the pattern of colocalization in female mice would mimic that observed in the female rat.

Certainly PR induction by E2 plays a well-established role in regulation of female sexual behavior, and this is mediated largely by ER{alpha}. For example, in ERßKO female mice lordosis levels are equal to, or greater than, wild-type (WT) counterparts, and yet all lordosis activity is abolished upon disruption of ER{alpha} (11, 18). The ability of E2 to induce PR in the ventromedial nucleus of the female mouse is well correlated with the presence or absence of functional ER{alpha} gene (11). The mPOA is also important for the expression of female receptivity (35), and thus the roles of each ER in induction of PR in the female mPOA are consistent with the role of PR in feminine sexual behavior. Whether or not PR is also important for male reproduction is less well established. The initial report of male sexual behavior in PR knockout male mice suggested that they were slower to initiate masculine sexual behavior, but perhaps of more interest was the fact that after castration WT males persisted in the demonstration of masculine sexual behavior for significantly longer than did PRKO littermates (36). Thus, the gonad-independent regulation of masculine sexual behavior may involve PR, perhaps via a ligand-independent mechanism such as dopamine. Our data also support work in rats and lizards (37, 38) that show that androgen’s actions on male sexual behavior are enhanced when PR is functional. PRKO male mice also reportedly display better parental behavior than WT littermates (39). We suggest that studying interactions between PR and ERß may shed new light on the role of PR in male behaviors.

Based on the data presented here and by others, we propose that in male brain ERß is involved in both the development and display of adult social behavior. We have recently demonstrated that ERß plays an important role in the timing of male sexual behavior at puberty. Pubertal ERßKO males display their first ejaculation at a significantly older age than WT littermates (40). In addition, pubertal ERßKO males have a transient increase in both aggression and plasma testosterone compared with WT males (41). Yet, adult male sexual behavior is not different in WT and ERßKO males (Ref. 42 ; and Bodo, C., and E. Rissman, unpublished data). Because the mPOA is essential for the expression of male social behaviors (43), we hypothesize that ERß is in involved.

Interestingly the majority of GnRH neurons in rats, many of which reside in the mPOA, coexpress ERß (44). Castrated adult ERßKO mice have significantly higher plasma LH levels than WT counterparts (40). However, other aspects of GnRH function (e.g. cell numbers in the mPOA, peptide content and its ability to trigger LH) do not differ between male WT and ERßKO mice (40). Thus, another potential function of ERß in male mPOA could involve regulation of GnRH. When GT1–7 cells are treated with the phytoestrogen coumestrol, GnRH mRNA was suppressed, and this was reversed by cotreatment with a selective ERß antagonist (45). Because the sex difference in ERß in the mPOA is amplified during development, ERß may be important for the process of neural defeminization and/or masculinization of the LH surge system and/or behavior in males. Studies are presently underway in our lab to test the hypothesis that ERß mediates sexual differentiation of neuroendocrine function.


   Acknowledgments
 
We thank Savera Shetty and Aileen Wills for expert technical assistance. We also thank Dr. Alexander Kauffman for his input on the final draft of this manuscript.


   Footnotes
 
This work was supported by National Institutes of Health (NIH) Grants R01MH57759 and K02 MH01349 (to E.F.R.). A.E.K. was supported first by NIH Training Grant HD072323 and subsequently by an individual National Research Service Award Fellowship F31MH70092.

Abbreviations: AVPv, Anteroventral periventricular; DAB, diaminobenzidine; E2, estradiol; ER, estrogen receptor; ER{alpha}KO, ER{alpha} knockout; ir, immunoreactive; mPOA, medial preoptic area; PR, progestin receptor; WT, wild-type.

Received December 16, 2003.

Accepted for publication June 8, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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