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. Author manuscript; available in PMC: 2021 Sep 1.
Published in final edited form as: Horm Behav. 2020 Aug 14;125:104827. doi: 10.1016/j.yhbeh.2020.104827

Key Role of Estrogen Receptor β in the organization of brain and behavior of the Japanese quail

Lucas Court 1, Laura Vandries 1, Jacques Balthazart 1, Charlotte A Cornil 1
PMCID: PMC7541764  NIHMSID: NIHMS1622357  PMID: 32735801

Abstract

Estrogens play a key role in the sexual differentiation of the brain and behavior. While early estrogen actions exert masculinizing effects on the male brain of rodents, a diametrically opposite effect is observed in birds where estrogens demasculinize the female brain. Yet, the two vertebrate classes express similar sex differences in the brain and behavior. Although ERα is thought to play a major role in these processes in rodents, the role of ERβ is still controversial. In birds, the identity of the estrogen receptor(s) underlying the demasculinization of the female brain remains unclear. The aim of the present study was thus to determine in Japanese quail the effects of specific agonists of ERα (propylpyrazole triol, PPT) and ERβ (diarylpropionitrile, DPN) administered at the beginning of the sensitive period (embryonic day 7, E7) on the sexual differentiation of male sexual behavior and on the density of vasotocin-immunoreactive (VT-ir) fibers, a known marker of the organizational action of estrogens on the quail brain. We demonstrate that estradiol benzoate and the ERβ agonist (DPN) demasculinize male sexual behavior and decrease the density of VT-ir fibers in the medial preoptic nucleus and the bed nucleus of the stria terminalis, while PPT has no effect on these measures. These results clearly indicate that ERβ, but not ERα, is involved in the estrogen-induced sexual differentiation of brain and sexual behavior in quail.

Keywords: sexual differentiation, estrogen receptor α, estrogen receptor β, vasotocin, PPT, DPN

INTRODUCTION

Estrogens play a key role in the sexual differentiation of the brain and behavior (Balthazart and Ball, 2012). In mammals and birds, the development of sex differences is determined by the genetic sex, which causes the differentiation of the gonads and enables the sex-specific patterns of hormonal secretion (McCarthy and Arnold, 2011). The brain is permanently organized by exposure to sex steroid hormones during critical periods of development. Organized circuits are later activated by adult hormonal secretions to ensure proper behavior. This dual action of steroids was initially described and is currently known as the organizational-activational hypothesis (Arnold, 2009). The origin and nature of the steroid(s) involved, the sex that is primarily differentiated under the action of steroids and the exact timing of the critical window of sensitivity to steroids depend on the species and the specific process considered, but the general process appears to be common to all species in these two classes of vertebrates. In rodents, males, but not females, are exposed during the perinatal period to testicular androgens which, depending on the process considered, act directly via activation of androgen receptors or indirectly following central aromatization into estrogens to masculinize and defeminize neural circuits (Morris et al., 2004). By contrast, in birds, estrogens presumably originating from the ovary demasculinize the brain and behavior of embryonic females, while males are not exposed to estrogens during the critical developmental period (Balthazart and Adkins-Regan, 2002; Balthazart, et al. 2009).

Classically, estrogens act through their nuclear estrogen receptors (ER), ERα and ERβ, which operate as ligand-activated transcription factors by interacting with specific DNA sequences, called estrogen response elements, or via protein–protein interaction with other transcription factors (Mangelsdorf et al., 1995). In turn, the differential expression of proteins affected by the action of estrogens influences distinct processes, such as neurogenesis, cell death, differential connectivity, cellular specification and epigenetic modifications, that lead to the establishment of enduring sex differences (McCarthy et al., 2017). Estrogens also act through membrane activated signaling (Ronnekleiv and Kelly, 2017) and evidence shows that membrane ERα plays a role in the organization of the rodent brain (Khbouz et al., 2019). Although ERα and ERβ have been proposed to both play a role in these processes in rodents (review in Kudwa et al., 2006, but see Naule et al., 2016), the identity of the receptor(s) underlying the demasculinization of the brain in female quail remains unclear.

In female quail, estrogens acting between embryonic day 7 (E7) and E12 (Adkins, 1975, 1979) permanently demasculinize the circuits underlying the expression of male sexual behavior, whereas masculinization of males does not seem to require prenatal hormonal exposure (for a review, see Bathazart et al., 2009). As a result, while testosterone activates the expression of male sexual behavior in adult males, it has no such effect in adult females (Adkins and Alder, 1972; Adkins, 1975; Balthazart et al., 1983). Demasculinization can be blocked by injecting female embryos with an aromatase inhibitor or an estrogen antagonist during the critical period (Adkins and Nock, 1976; Adkins, 1978; Balthazart et al., 1992; Cornil et al., 2011). By contrast, treating male embryos with estradiol leads to males that are unable to exhibit male copulatory behavior following adult testosterone treatment (Adkins 1975, 1978; Adkins-Regan et al. 1982; Schumacher et al. 1989; Balthazart et al., 1992; Halldin et al., 1999; Cornil et al., 2011).

Previous attempts to determine the specific role of ERα and ERβ on the organization of male sexual behavior of Japanese quail produced inconclusive results (Mattsson et al., 2008; Mattsson and Brunström, 2010, 2017) presumably because specific agonists for the two subtypes of receptors were administered too early (E3). Estrogens are also involved in the differentiation of the gonads in birds (Ayers et al., 2013) and therefore these treatments interfered with the normal development of the gonads and genital tracts. However, the neural circuits underlying sexual behavior presumably differentiates later during embryonic development and only marginal effects on this behavior were detected in the earlier studies. In addition, adult sexual behavior was tested in birds that were gonadally intact and not supplemented with exogenous testosterone. Although plasma concentration of testosterone were shown to be unaffected by the embryonic treatments in some of these studies (Mattsson and Brunström, 2010, 2017), it cannot be ruled out that changes in gonadal physiology obscured the organizational effects of the estrogen receptor agonists on brain organization, so that no definitive conclusion could be drawn regarding the identity of the receptor(s) mediating brain demasculinization (Mattsson et al., 2008a,b; Mattsson & Brunstrom 2010, 2017). In the present study, we addressed this same question by assessing the effects of specific agonists of ERα and ERβ (propylpyrazole triol [PPT] and diarylpropionitrile [DPN] respectively) administered at the beginning of the sensitive period to estrogens (E7) on the sexual differentiation of male copulatory behavior and on the density of vasotocin-immunoreactive (VT-ir) fibers, a known marker of the organizational action of estrogens on the quail brain (Panzica et al., 1998). We predicted that the in ovo treatment of male quail with PPT or/and DPN should 1) prevent the expression of male sexual behavior activated by testosterone in adulthood and 2) reduce the density of VT-ir fibers in three brain regions, the POM, the BST and the lateral septum (LS), that are characterized by a higher expression in males than in females after a similar testosterone treatment (Viglietti-Panzica et al., 1992; 2001; Panzica et al., 1998).

MATERIAL AND METHODS

Experimental animals

A total of 74 Japanese quail (Coturnix japonica) served as subjects and 17 gonadally intact, unfamiliar and sexually experienced females were used as stimuli. Fertilized eggs were obtained from our colony (Central Animal Facility from the University of Liège, agreement number: LA1610002). Experiments complied with the Belgian laws on the “Protection of experimental animals” and were approved by the Ethics Committee for the Use of Animals at the University of Liège (Protocol #1442).

In vivo treatments

Three hundred and twenty eggs were candled and selected for incubation at 37.5°C and 47% of relative humidity for 15 days (Egg Incubator, F.I.E.M. Ref. MG140/200). After 7 days of incubation, eggs were injected with 50μl of one of the four following solutions: pure propylene glycol (PG; Sigma, P4347), 17β-Estradiol 3-benzoate (EB; Sigma, E8515; 25μg/egg in PG), 1,3,5-Tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT; Sigma, H6036; 300μg/egg in PG) or 2,3-Bis(4-hydroxyphenyl) propionitrile (DPN; Sigma, H5915; 300μg/egg in PG). The two ER agonists were previously shown to display a high selectivity for the human ERα and ERβ (Stauffer et. al, 2000; Meyers et al., 2001). They have similarly been shown to produce in quail selective effects on reproductive organs (Mattsson et al., 2008a,b; 2017) as well as on the adult brain (Seredynski et al., 2015). Moreover, comparisons of the protein sequences of quail ERα (NP_001310118.1) and ERβ (AAC36463.2) with the equivalent sequences of human ERα (AAI28575.1) and ERβ (BAA24953.1) yielded 78% and 80% identity, respectively. These values increased to 94% and 88% when focusing on the ligand binding domains (aa310–547 of human ERα and aa 263–498 of human ERβ).

In ovo treatments were administered under sterile conditions with a 1mL syringe (Terumo, 300013) connected via a Filtropur S 0.45μm filter (Starstedt, 83.1826) to a 26G needle (Microlance, 304300). Injections were performed with the needle targeting the albumen through the shell at the apex of the egg. The hole was then sealed with a drop of melted paraffin. Twenty eggs were taken out of the incubator at a time, injected with a given solution and immediately placed back in the incubator in order to evenly distribute the treatments across time and over the four levels of the incubator.

On incubation day 15, eggs were gathered by treatment in stainless steel hatching baskets (38 X 36 X 9 cm) to avoid mixing the chicks that received different treatments. The humidity was increased to 60% to facilitate hatching. On incubation day 19, the newly hatched chicks were immediately marked with nail polish of different colors on the head, the back, and the legs and placed in a brooder (96 X 50 X 25 cm) at a temperature of 38–40°C. Temperature was gradually decreased over two weeks to reach room temperature (about 22°C). Constant light was provided for the full period and they had food and water available ad libitum.

Chicks were raised in mixed sex groups and gonadectomized at the age of 3 weeks before the onset of testosterone production and the activation of sexual behaviors (Ottinger and Brinkley, 1978, 1979; Panzica et al., 1987). Five groups of animals were then kept: males of the four treatments and a group of PG treated females (vehicle). At 5 weeks of age, all subjects were implanted with two 20 mm long Silastic™ capsules (Silclear® tubing, 0.0625” X 0.094”; Degania silicone, Ref: 20301502431) filled with crystalline testosterone (Sigma, B6500). All subjects were then, transferred to individual cages, maintained under a photoperiod stimulating long summer days (16 h light and 8 h dark) and provided with food and water ad libitum. A total of 74 subjects reached adulthood and were distributed into five groups as follows: 14 PG males, 12 PG females, 17 EB males, 16 PPT males and 15 DPN males. Subjects were weighed to the nearest gram and the cloacal gland area (length X width) was measured at eight weeks (three weeks after T implants were placed) after hatching.

Sexual behavior

At the age of 9 weeks, experimental animals were given seven copulatory tests over a period of 2 weeks. For each test the experimental subject was released in an arena (60 X 40 X 50cm) containing a sexually receptive female and the investigator who was blind to the previous endocrine treatment of the subjects quantified the stereotyped behavioral sequence of male copulatory behavior. The frequency and latency (time elapsed between the introduction of the male and the first occurrence of a behavior) of neck grabs, mount attempts (MA), mounts and cloacal contact movements (CCM; for more details, see Adkins and Adler, 1972; Hutchison, 1978) was scored in real time during 5 min for each of the seven tests. To avoid redundancy, only data for MA and CCM are presented here as they were similar to those of neck grabs and mounts respectively. Birds who did not show a given behavior were assigned a latency of 300 s (5 min) for statistical purposes.

Vasotocin-immunoreactive (VT-ir) fibers

Immunohistochemistry

One week after the last copulatory test, birds were killed by decapitation and the completeness of castration and presence of the subcutaneous Silastic™ implants were confirmed. Brains were collected and fixed with acrolein. Brains were soaked in 5% acrolein in phosphate-buffered saline (PBS 0.01M, pH 7.4) for 2.5 hours and rinsed twice in PBS for 30 min. Brains were then immersed in 30% sucrose until they sank, frozen on dry ice and stored at −80°C until further use. Seventy-three brains (as one female died before tissue was collected) were cryosectioned in four series of 30μm thick coronal slices from the level of the septopalliomesencephalic tract (TSM), which marks the rostral end of the POM, to the third nerve indicating the caudal end of the hypothalamus. One series of brain sections was immunostained for vasotocin ensuring that brains from each endocrine treatment were represented in each 24 well plate. Briefly, sections were first rinsed three times in 0.01M tris-buffered saline (TBS, pH 7.6) for 5 min each (same procedure applied for all following rinses). Sections were then incubated in 1% of sodium borohydride (NaBH4) for 15 minutes. After rinses, peroxidase activity was blocked with 1% of hydrogen peroxide (H2O2) at room temperature (RT) for 30 minutes in TBS containing 0.1% Triton X-100 (TBST) and 10% normal goat serum (NGS). Sections were then incubated with a rabbit anti-arginine vasopressin (AVP) primary antibody (1/5000, Merck Millipore, Ref: AB1565, Lot: 2860189) for two nights at 4°C. Sections were rinsed in TBS and incubated for 2 hours at RT with a goat anti-rabbit biotinylated secondary antibody (1/400, Jackson ImmunoResearch, Ref: 111–065-003, Lot: 124659). Sections were washed in TBS and incubated for one hour and a half in the ABC Kit peroxidase in TBST (Vector Laboratories, Ref: PK-6100, Lot: ZD1108) at RT, then rinsed in TBS. The localization of the peroxidase was finally visualized with the SG peroxidase substrate kit (15μl/mL TBS of chromogen and 24μl/mL TBS of H2O2;-Vector laboratories, Ref: SK-4700, Lot: ZD0630). Sections were finally rinsed in TBS, mounted on slides, dried overnight, left ten minutes in xylene and coverslipped using Eukitt (Sigma-Aldrich, Ref: 03989).

Antibody specificity

The specificity of the primary antibody was validated by a pre-adsorption test with [Arg8]-Vasotocin acetate salt (Sigma-Aldrich, Ref: V0130, Lot: 116F5865) using the brain of two adult male quail. Briefly, the primary antibody (1/5000) was incubated with three distinct concentrations of the peptide (0.1, 1 or 10 μg of peptide/mL of primary antibody) and 2% NGS in TBST pH 7.6 at 4°C for 6 hours. These media containing the primary antibody pre-adsorbed with increasing concentrations of the peptide were then used to immunostain three series of sections. A fourth series serving as control was incubated in parallel in the primary antibody that had not been pre-adsorbed (0 μg/mL of peptide/mL of primary antibody). The density of VT-immunoreactive signal progressively decreased with the increasing concentrations of peptide from 0 μg/mL to 0.1 μg/mL and to 1 μg/mL. Pre-adsorption with 10μg of peptide/mL of primary antibody completely eliminated all VT-ir cells and fibers in the caudal medial preoptic nucleus (POM), the bed nucleus of the stria terminalis (BST) and the lateral septal nucleus (LS) (Fig.1).

Figure 1:

Figure 1:

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Specificity of the immunostaining against vasotocin. Vasotocin immunoreactive fibers in the medial preoptic nucleus (POM), bed nucleus of the stria terminalis (BST) and lateral septum (LS) in control conditions (left column) and following pre-adsorption with 10μg of vasotocin per ml of primary antibody (right column). Magnification bar, 200μm.

Image analysis

Sections were examined with an Olympus BH-2 microscope at the magnification x40 and photographs were acquired with a Scion Corporation MTV-3 camera. The percentage of surface covered by VT-ir fibers was quantified in the POM, BST and LS as defined in a previous study (Taziaux et al., 2008). Briefly, four quantification fields located in the left and right caudal POM were photographed in the section where the anterior commissure (CA) reaches its largest extension (corresponding approximatively to plate A6.0 in the quail atlas; Bayle et al., 1974). For each hemisphere, the quantification field was first located in the corner formed by the ventral edge of the anterior commissure and the lateral edge of the third ventricle. The quantification field was then moved the height of one field ventrally along the third ventricle and the second quantification field was acquired at this location. As the dense cluster of stained fibers does not always extends along a strict vertical shape but rather assumes an oblique shape, the field was when needed adjusted to the center of the zone defined by the staining. Importantly, we made sure that the quantified fields did not include any magnocellular VT-ir cells and fibers from the periventricular region whose expression does not depend on endocrine conditions (Viglietti-Panzica et al., 1994).

In the BST, the left and right quantification fields were placed to cover the nucleus where it assumes a characteristic “V” shape. The first quantification field was centered on the nucleus, dorsally to the most lateral edge of the CA, approximatively under the lateral ventricle. The second quantification field was moved one field ventrally and centered on the rest of the nucleus. In the LS, the left and right quantification fields were placed to cover entirely the bulge of the nucleus into the lateral ventricle, at the level where the CA reaches its largest extension, i.e., in the same section used for the analysis of the POM.

The percentage of the area covered by VT immunoreactive fibers was determined by a semi-automatic method using the thresholding tool of ImageJ 1.50i (Schneider et al., 2012) where threshold was manually adjusted for each subject. The values obtained for each quantification field were averaged to obtain one single value for each brain region. BST was damaged in one control male and could therefore not be quantified. Data could be collected only on one side of the brain for POM and LS in one control male and for BST in two control males; the average percentage of surface covered by VT-ir fibers was based in these cases on values obtained in one side. For all analyses, the investigator was blind to the treatment of the birds.

Statistical analysis

The weight and cloacal gland area were analyzed by a one-way ANOVA with the treatments as independent factor, followed by Tukey post hoc tests when significant. Behavioral frequencies and latencies did not fulfill the assumptions required to apply parametric tests and were thus analyzed by non-parametric Kruskal-Wallis ANOVA to assess differences between treatments. A Bonferroni correction for multiple comparisons was applied taking into account the number of tests (Kruskal-Wallis performed on each data set), and the p values of each test were multiplied by the number of comparisons (corrected p or pcor in the rest of this paper). The percentage of sexually active males, defined as males showing at least one mount attempt or one cloacal contact movement during the sum of the seven tests, were compared between control or PPT males and all other groups by the Fisher exact probability test also followed by a Bonferroni correction. The areas covered by VT-ir structures were analyzed by Kruskal-Wallis ANOVA. Post-hoc comparisons were made by Dunn’s multiple comparisons tests. All analyses were performed with GraphPad Prism 6.0 for Windows 10. All effect sizes (Cohen d) were evaluated using calculators present on the websites https://www.psychometrica.de/effect_size.htm and http://www.campbellcollaboration.org/. Results were considered significant for p value <0.05 and were presented by means ± standard error of mean.

RESULTS

Body weight and cloacal gland size

Treatments did not affect the body mass of the birds (F4,69= 0.4005, p= 0.8077; d= 0.1587; Fig.2A), but significantly modified the cloacal gland area (F4,69= 4.524, p= 0.0026; d= −0.3481). Tukey post-hoc tests indicated that this effect resulted from a reduced cloacal size in females and EB treated males compared to control males (Both p<0.01; Fig.2B). PPT and DPN treated males did however not differ from control males (PPT, p = 0.0656; DPN, p = 0.2571) or females (PPT, p = 0.7235; DPN, p = 0.37).

Figure 2.

Figure 2.

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Body weight (A) and cloacal gland area (B) in adult male (M) and female (F) quail treated in ovo with estradiol benzoate (EB), the ERα agonist PPT, the ERβ agonist DPN or their vehicle (CTL) and chronically treated with testosterone in adulthood. ** = p< 0.01 vs. CTL males (Tukey post-hoc test following significant one way ANOVA).

Male sexual behavior

As expected, during the seven copulatory tests, females and EB-treated males never showed any male sexual behavior. Interestingly, only few DPN-treated males exhibited male sexual behavior, while most CTL and PPT-treated males exhibited the full copulatory sequence. The analysis of the percentage of birds who displayed at least one mount attempt (MA) during all seven tests revealed significant differences between CTL (n=14/14) or PPT (n=16/16) males on the one hand, and CTL females (n=1/12), EB males (n=1/17) or DPN males (n=3/15) on another hand (Fisher exact probability tests, all p<0.0001, pcor<0.001; Fig.3A). Similarly the percentage of CTL (n=12/14) or PPT (n=12/16) males that exhibited at least one cloacal contact movement (CCM) significantly differed from CTL females (n=0/12), EB males (n=0/17) and DPN males (n=1/15) (all p<0.001, pcor<0.001; Fig.2B). The percentage of CTL and PPT males showing MA or CCM did not differ between each other (p>0.9999 and p=0.6567 respectively, pcor>0.9999).

Figure 3.

Figure 3.

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Percentage of adult male (M) and female (F) quail that displayed at least one mount attempt (A) or one cloacal contact movement (B) after being treated in ovo with estradiol benzoate (EB), the ERα agonist PPT, the ERβ agonist DPN or their vehicle (CTL) and chronically treated with testosterone in adulthood. *** p<0.001 by Fisher’s exact probability test when compared to CTL- or PPT-treated males.

The analysis of the changes in time of the behavioral frequencies over the seven copulatory tests showed that as expected males injected in ovo with a control solution (PG) showed a gradual increase in the frequency of male sexual behavior associated with a decreased latency to copulate (Fig.4). MA was observed starting on the first test but the first few CCM only occurred during the second test. Males treated with the ERα agonist (PPT) showed a similar pattern of response. By contrast, females and EB-treated males did not show any such behavior. In addition, very few DPN-treated males exhibited a few rare MA and even less frequent CCM resulting in low copulatory frequencies and high latencies. The effect of treatments was compared between groups by separate Kruskal-Wallis tests for each behavioral test.

Figure 4.

Figure 4.

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Effect of an in ovo injection at embryonic day 7 with estradiol benzoate (EB), the ERα agonist PPT, the ERβ agonist DPN or their vehicle (CTL) in male (M) quail on the expression of adult male sexual behavior. The figure shows the frequencies (A-B) and latencies (C-D) to display mount attempts (MA; A,C) and cloacal contact movements (CCM; B,D) during the seven tests. Data for control females (F) are also shown. Data for each test were analyzed by Kruskal-Wallis ANOVA followed when significant by Dunn’s post hoc tests, whose results are indicated by letters in the graphs. a and b represent significant differences (p<0.05) between the CTL M or the PPT M groups and the EB M, DPN M and CTL F groups, c corresponds to differences between the PPT M and the EB M groups.

Separate Kruskal-Wallis tests for each test confirmed the existence of group differences in the MA frequency from test 1 to 7 (H≥40.40, p<0.0001, pcor<0.001, d≥ 2.113 in each case) and the CCM frequency from T4 to T7 (H≥19.26, p<0.0007, pcor<0.007, d≥ 1.066 in each case). In parallel, the latency to show sexual behavior after the introduction into the test arena was significantly different between groups from T1 to T7 for MA (H≥41.13, p<0.0001, pcor<0.001, d≥ 2.159 in each case) and from T4 to T7 for CCM (H≥19.01, p<0.0008, pcor<0.008, d≥ 1.055 in each case). Dunn’s post hoc tests revealed that these effects largely reflected significant differences between CTL females, EB- and DPN-treated males on the one hand and CTL- and PPT-treated males on the other hand (see Fig.4 for details).

Effect of in ovo treatment on the area covered by vasotocin-immunoractive (VT-ir) fibers

As expected, a lower VT immunoreactivity, as assessed by the percentage of area covered by VT-ir fibers, was observed in the POM and BST of females as compared to males, although this difference did not have the same magnitude as in previous studies (Viglietti-Panzica et al., 1992; Panzica et al., 1998) and there was a clear overlap between male and female data (Fig.5–6). This overlap was even more important in the LS. The area covered by VT-ir fibers did not seem to be affected by PPT, but was almost entirely suppressed by EB. Males injected with DPN displayed a major average suppression of VT-immunoreactivity, but the effect was associated with a substantial individual variation. In particular, one DPN male had retained a high VT immunoreactivity and clearly appeared as an outlier in this group, although his data were retained in statistical analyses (individual identified by a cross in Fig. 5). Interestingly, this male was also the only one of this group who showed cloacal contact movements during the behavioral tests, suggesting that for some reason (reduced sensitivity to estrogens or technical issue at the time of injection), he had escaped the effects of treatment and was as a result not demasculinized.

Figure 5.

Figure 5.

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Percentage of area covered by vasotocin-immunoreactive fibers in the medial preoptic nucleus (POM), the bed nucleus of the stria terminalis (BST) and the lateral septal nucleus (LS) of adult male (M) and female (F) quail treated in ovo with estradiol benzoate (EB), the ERα agonist PPT, the ERβ agonist DPN or their vehicle (CTL) and chronically treated with testosterone in adulthood. Data for each nucleus were analyzed by Kruskal-Wallis ANOVA followed when significant by Dunn’s post hoc tests whose results are indicated by letters in the graphs. a = p<0.05 vs. CTL M, PPT M, CTL F; b = p<0.05 vs. CTL M and PPT M; c = p<0.05 vs. all groups.

Figure 6.

Figure 6.

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Representative photomicrographs illustrating the effects of the embryonic treatments on the density of vasotocin-immunoreactive fibers in the medial preoptic nucleus (POM), the bed nucleus of the stria terminalis (BST) and the lateral septum (LS). Abbreviations: V3, third ventricle; CA, anterior commisure; FPL, lateral forebrain bundle; LV, lateral ventricle; CTL F, control females; CTL M, control males; DPN, ERβ agonist; EB, estradiol benzoate; PPT, ERα agonist. Scale bar represents 100 μm.

Kruskal-Wallis analyses confirmed the existence of significant differences between groups in each of the three nuclei (POM: H=46.45, p<0.0001, d=2.578; BST: H=48.43, p<0.0001, d=2.806; LS: H=44.61, p<0.0001, d=2.435). Dunn’s post hoc tests indicated that these effects largely originated from a significantly larger average surface covered by VT-ir fibers in control and PPT males compared to both EB and DPN treated males and in females (for details, see Fig.5). Despite a smaller average surface covered in females compared to control and PPT males in POM and BST, this difference was not significant.

DISCUSSION

The aim of this study was to identify the ER subtype that is mediating the organizational effect of estrogens on the expression of male sexual behavior and on the density of VT-ir fibers in different brain regions of Japanese quail. The results confirmed the demasculinizing action of EB on male sexual behavior (Adkins, 1975, 1978; Schumacher et al., 1989; Balthazart et al., 1992) and on the density of VT-ir fibers in the POM, BST and LS (Panzica et al., 1998). This demasculinizing effect was largely mimicked by the ERβ selective agonist (DPN), which prevented the expression of male sexual behavior and resulted in a strong reduction of the density of VT-ir fibers in the POM and the BST, but not the LS. By contrast, males treated with the ERα selective agonist (PPT) exhibited no alteration of their sexual behavior nor of the expression of VT in fibers, indicating that, as control males, they underwent a normal masculinization. Together, these results strongly suggest that ERβ, but not ERα, is involved in the sexual differentiation of the brain and behavior in quail.

In previous studies, the ERα selective agonist PPT mimicked the effects of estradiol on the size of the cloacal gland and on gonadal development (Mattsson et al., 2008b; see above), but did not alter the ability of males to show copulatory behavior. These data thus suggested that ERα is not implicated in the organizational effects of estrogens on male sexual behavior. However subsequent experiments Mattsson and Brunstöm (2010, 2017) showed that injection in quail eggs of another ERα selective agonist (16αLE2) alone (Mattsson and Brunstöm, 2010) or in combination with an ERβ agonist (WAY-200070) (Mattsson and Brunstöm, 2017), but not the ERβ agonist alone, slightly interferes with the development of male sexual behavior and marginally inhibits some but not all measures of the behavior. In one of their studies (Mattsson and Brunstöm, 2010), they suggested however that the effect of 16αLE2 was mediated via a cross-reaction with ERβ. The discrepancy between these and the present results is likely explained by differences in the timing of treatments. The process of sexual differentiation occurs at different periods of development for different tissues and responses. Gonadal differentiation occurs first under the direct genetic influence of the sex chromosomes and is essentially completed by embryonic day 4 or 5 (Ayers et al., 2013; Balthazart et al., 2009). In turn, the differential hormonal secretions occurring in males and females during quail embryogenesis (Scheib et al., 1985; Schumacher et al., 1989) organize the neural circuitry leading to stable sex differences in the adult brain and behavior. This process cannot obviously start before the differentiation of the gonads and it is complete by embryonic day 12. In the studies published by Mattsson and colleagues (Mattsson et al., 2008a,b; Mattsson and Brunstrom, 2010, 2017), eggs were injected at embryonic day 3, that is prior to the completion of gonadal differentiation and presumably before the opening of the sensitive period of brain organization by estrogens, which is thought to occur around day 7 (Adkins, 1975; 1979; Cornil et al., 2011). Treating embryos with ERα agonists (PPT or 16α-LE2) at E3 alters gonadal development of male quail, resulting in the formation of an ovary-like cortex in the left testis and in a size reduction of the right testis of male embryos (Mattsson et al., 2008a,b; Mattsson and Brunstrom, 2010; 2017). By contrast, an ERβ agonist (WAY200070 or DPN) administered at the same age does not seem to affect testis development unless it is combined with an ERα agonist (Mattsson and Brunstrom, 2011, 2017). It is thus likely that the altered gonadal development induced by early activation of ERα may have led to changes in the nature and/or the amount of hormones secreted by these male embryos and, as a result, may have indirectly interfered with brain sexual differentiation. Although their experiments provide important information regarding the identity of the receptor involved in gonadal and genital tract differentiation, they do not allow a definitive conclusion regarding the identity of the receptor(s) involved in the permanent programming of the brain and behavior. At any rate, these authors only detected very modest effects of the treatments they implemented on the display of adult male sexual behavior. By contrast, the present results were obtained after treating embryos at the beginning of the critical window of estrogen sensitivity of the brain when estrogenic treatments do no longer interfere with gonadal development. In addition, all adults were tested for behavior after being gonadectomized and supplemented with exogenous testosterone, so that any alteration of behavior could be assigned to a differential brain sensitivity to testosterone and not to a change in the steroid environment resulting in a differential activation of behavior. Therefore, the present results unequivocally identify ERβ as a major, if not the only, player in the organization of the neural circuits underlying the expression of male sexual behavior in Japanese quail.

The sex difference in the size of the cloacal gland is known to result from both a difference in adult circulating androgens and a developmental difference in exposure to estrogens. In adulthood, the growth of this gland tightly depends on the circulating concentration of androgens and the cloacal gland size is for this reason frequently used as a proxy providing an integrated measure of circulating testosterone and action (Sachs, 1967; Delville et al., 1984; Balthazart et al., 1990). During development, females are exposed to higher concentrations of estrogens (Schumacher et al., 1989). Manipulations of estrogen concentrations prior to E12 permanently affects the size of the gland: male embryos treated with EB prior to E12 develop a smaller cloacal gland than controls (Adkins, 1975, 1978; Schumacher et al., 1989; Balthazart et al., 1992; Mattsson and Brunstrom, 2010; Cornil et al., 2011), while blocking estrogen synthesis leads to females with a larger cloacal gland when treated with testosterone in adulthood (Balthazart et al., 1992; Cornil et al., 2011). The present results confirm the sex difference and the effect of embryonic EB on cloacal gland size. However, although PPT and DPN treated males presented a smaller cloacal gland than control males, their gland was still larger than the gland of females and as a result they did not statistically differ from control males or females. This is contrasting with the work of Mattsson and colleagues who consistently found an effect of ERα, but not ERβ, agonists administered at E3 on this structure (Mattsson et al, 2008b; Mattsson and Brunström, 2010, 2017). As discussed before, this discrepancy between their and our results likely reflects the difference in the timing of treatments as the altered differentiation of the testes induced by this precocious treatment likely resulted in altered gonadal secretions and thus it affected brain and/or cloacal gland development both during ontogeny and in adulthood. Indeed, as their animals were not castrated, it cannot be ruled out that the reduced cloacal gland size they observed in PPT treated males resulted from a modified testosterone secretion in adulthood. The fact that neither PPT nor DPN did significantly alter the cloacal gland size of males in our study suggests that activation of both receptors might be necessary for the sexual differentiation of this structure in response to estrogens. A treatment combining PPT and DPN would thus be required to mimic the effect of EB on cloacal gland size. Finally, this process might also require a higher dose than the dose used here to modify the organization of the brain. It is indeed known that higher concentrations of testosterone are necessary to promote full spermatogenesis in the testis than to activate behavior (Grunt and Young, 1953; Damassa et al., 1977). It is thus not impossible that the same applies for their organizational actions.

In order to confirm whether the effects of treatments on male sexual behavior resulted from a permanent programming of the brain, the brains of all subjects were immunostained for vasotocin as its expression in fibers of the POM, BST and LS is higher in male than female quail (Viglietti-Panzica et al., 1992, 1994, 2001) as a result of a differential exposure to embryonic estrogens (Panzica et al., 1998). In adulthood, vasotocin immunoreactivity in these fibers is increased by estrogens derived from the aromatization of testosterone (Viglietti-Panzica et al., 1994, 2001). The expression of vasotocin in these fibers is thus regulated by sex steroid hormones in a way that mirrors the regulation of sexual behavior and a causal link between vasotocin and male sexual behavior was demonstrated although mechanistic underpinnings of this regulation remains unclear (Castagna et al., 1998). For reasons that are not entirely clear, the results of the present immunohistochemical study were not as clear as the behavioral data. Individual results were highly variable resulting namely in an absence of sex difference in the density of VT-ir fibers in control birds even if a smaller average density of VT-ir fibers was present in the POM and BST of females compared to control males (Fig. 5–6). Yet, embryonic treatment of males with EB yielded the expected drastic reduction in the density of VT-ir fibers compared to controls in the three brain regions. This effect was mimicked to a large extent by the ERβ agonist DPN in the POM and the BST, but not in the LS. DPN males therefore ended up showing an intermediate average density of VT-ir fibers between the control females and the EB males indicating that these males were demasculinized, at least in part. In contrast, the ERα agonist (PPT) treated males displayed a similar average density of VT-ir fibers as control males. This conclusion fits in well with the fact that ERβ, but not ERα, seems to mediate the demasculinization of male copulatory behavior and ERβ is expressed in higher densities than ERα in these brain regions during the critical period of sensitivity to estrogens (Axelsson et al., 2007). Indeed, Although quantitative analysis of whole brains revealed that ERα expression is higher in the brain of females than in males between E9 and E12, the anatomical analysis of distribution of the mRNA coding for these two receptors only found ERβ expression in the POM, the BST and the tuberal hypothalamus at E9. At E17, both receptors were detected in the POM, the nucleus taeniae of the amygdala (TnA) and the ventromedial nucleus of the hypothalamus (VMN) of both sexes, where ERβ always appeared more densely expressed than ERα, although the intensity of the signal was not quantified. In the BST, only ERβ was detected (Axelsson et al., 2007). Therefore, although ERα may be more densely expressed than ERβ when considering the whole brain, ERβ seems to be more abundant in brain regions involved in the control of social behavior, and sexual behavior in particular.

Most of the evidence supporting a role of estrogens on the sexual differentiation of the mammalian brain was obtained in rats and ferrets in which blocking the synthesis or action of estrogens was shown to disrupt both masculinization and defeminization (Baum, 2003). In rats, some evidence points towards a role of ERα in these processes. Knocking down ERα expression by injection of antisense oligodeoxynucleotides in female pups was shown to prevent the defeminizing effect of neonatal testosterone on lordosis behavior (McCarthy et al., 1993). This observation was supported by the defeminization obtained following neonatal treatment of females with an agonist selective for ERα (ZK 281471, also known as 16-LE2), but not with an ERβ-specific agonist (ZK 281738, also known as 8-VE2), an effect that was paralleled by an increased size of the SDN-POA, a sexually dimorphic brain structure organized by estrogens (Patchev et al., 2004). Neonatal treatment with PPT or DPN however failed to mimic the defeminizing action of EB on lordosis behavior in another study, (Patisaul et al., 2009). Both ERα and ERβ are however present in the brain of developing rats (Yokosuka et al., 1997; Walker et al., 2009). In mice, pharmacological evidence confirmed the defeminizing action of estrogens on lordosis behavior (Kudwa et al., 2006). Studies of mice knock-out revealed that ERαKO mice show a profound deficit in male sexual behavior, while ERβKO males only exhibit subtle deficits in this behavior (Ogawa et al., 1999; Temple et al., 2001; Kudwa et al., 2005; Antal et al., 2012). However, ERβKO males were found to display some degree of lordosis behavior when primed with EB and progesterone suggesting a role for ERβ in the defeminization process (Kudwa et al 2005). This hypothesis is supported by the observation that females neonatally treated with DPN, but not PPT, show reduced lordosis behavior (Kudwa et al. 2006). A recent study of a mouse model where ERβ was specifically ablated from neural cells (Naulé et al., 2016) somehow contradicts these observations unless ERβ is expressed by glial cells, which would not be unprecedented (McCarthy et al., 2017). Importantly, it should be noted that these mouse models are constitutive knock-outs. Consequently, they are not well suited to distinguish between the developmental (organizing) and the adult (activating) actions of these receptor subtypes. Together, these observations thus suggest that there is not a single receptor subtype associated with the two processes involved in the sexual differentiation of the rodent brain.

As opposed to rodents, the present data clearly show that the demasculinization of the avian brain by estrogens is mediated almost exclusively by ERβ, but of course, we do not know really if and how this process relates to the masculinization and defeminization observed in mammals. Several mechanisms including differential neurogenesis, cell death or changes in connectivity have been described that explain the establishment of lifelong sex differences (McCarthy et al. 2017). The masculinization of the quail brain likely depends on similar processes. Whether the same endpoints (e.g., increased vasotocinergic innervation of the preoptic area in males) are organized by the same mechanisms in quail and in rodents is not known. The major difference between birds and rodents is that these processes occur by default (in absence of estrogens) in male birds, while they require exposure to this hormone in male rodents. However, the underlying mechanisms remain hypothetical and further studies will be necessary to bring new insights into this discussion.

Highlights.

  • Quail eggs were injected at embryonic day 7 with an ERα or ERβ agonist

  • The ERβ agonist DPN demasculinized copulatory behavior of adult males

  • DPN reduced the density of sexually dimorphic VT-ir fibers in the preoptic area

  • The ERα agonist PPT did not have any of these effects

  • Quail demascuilinization is mediated by ERβ with no major contribution of ERα

ACKNOWLEDGEMENTS

This research was supported by NIH/NIMH grant R01 MH50388. CAC is a F.R.S.-FNRS Research Associate. LC is a FRIA PhD student (F.R.S-FNRS). The authors would like to thank Léonore Fagot and Elena Sevrin for their technical assistance.

Footnotes

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