Both Estrogen Receptor- and - Are Required for Sexual Differentiation of the Anteroventral Periventricular Area in Mice
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《内分泌学杂志》
Department of Biochemistry and Molecular Genetics (E.F.R.) and Program in Neuroscience (C.B., A.E.K., E.F.R.), University of Virginia, Charlottesville, Virginia 22908
Abstract
Sexual dimorphisms in the hypothalamus are mediated in several cases by local aromatization of androgens to estrogens during the perinatal period. In this series of experiments, the contributions of the two estrogen receptors (ERs), ER and ER, to the differentiation of the sexually dimorphic subpopulation of dopaminergic neurons in the anteroventral periventricular area (AVPV) was examined. In the first experiment, numbers of tyrosine hydroxylase (TH) immunoreactive (-ir) AVPV neurons in ER knockout and wild-type (WT) mice of both sexes were measured. In the second experiment, the average number of TH-ir neurons in the medial portion of the AVPV in ER knockout, ER knockout, double-ER knockout, and WT mice of both sexes was calculated. In both experiments TH-ir cell numbers were sexually dimorphic as expected, with female individuals of all genotypes exhibiting more TH-ir neurons than WT males. Interestingly the average number of TH-ir neurons in all knockout males was significantly higher than in WT male littermates. In fact, TH-ir cell numbers in all knockout males were equivalent to females. In a final experiment, C57BL/6J female mice were treated during the first 3 postnatal days with either estradiol, or a specific agonist for one of the two ERs. Additional male and female pups received vehicle injections. Treatments with estradiol or either ER-specific agonist significantly reduced the number of TH-ir AVPV neurons in female brains. Our data demonstrate that both ER and ER are involved in the sexual differentiation of the AVPV in mice.
Introduction
REGULATION OF THE hypothalamic-pituitary-gonadal (HPG) axis is sexually dimorphic. Specifically, positive feedback of estradiol (E2) on LH secretion by the pituitary occurs only in female individuals and constitutes a requirement for the preovulatory gonadotropin surge (1). Estradiol has been postulated to exert this effect both at the level of pituitary gonadotrophs (2) and by modification of the GnRH secretion pattern from the hypothalamus (reviewed in Ref.1). In rodents, the anteroventral periventricular area (AVPV) of the hypothalamus contains a sexually dimorphic subpopulation of dopaminergic neurons, with a larger number present in females, compared with males (3). Furthermore, these cells have been shown to project to GnRH neurons, and both selective lesions (4) and local administration of estrogen antagonists (5) can prevent the normal display of the preovulatory LH surge in experimental animals. This suggests that this area is important for the positive feedback effect of estradiol on gonadotropin release.
The estrogen receptor (ER)- is widely distributed in the mouse brain (6, 7), including the AVPV. We recently described a role for ER in the defeminization of reproductive behavior in mice (8). It is not known whether this role is restricted to the differentiation of those neural circuits responsible for the display of female-like mating behavior or, alternatively, whether ER is involved in the differentiation of other sexually dimorphic functions known to differentiate under the influence of gonadal steroids during development.
The purpose of this study was to test the hypothesis that ER plays a role in the sexual differentiation of the subpopulation of dopaminergic neurons in the AVPV. Two strategies were used: mice with and without functional ER genes and administration of selective ER-specific agonists to neonatal mice. In adulthood we determined the average number of tyrosine hydroxylase (TH)-positive cells in the AVPV. Although Simerly et al. (9) previously described an incompletely masculinized phenotype in the TH-immunoreactive (ir) neurons in the AVPV of ER knockout (ERKO) male mice and postulated a role for ER in the process based on these results, cross-regulation between both types of ERs has been suggested to take place in other hypothalamic areas (10, 11), raising the possibility that ER may interact with ER during development to reduce the number of dopaminergic neurons in adult males.
Materials and Methods
Animals
For experiments 1 and 2, subjects were generated from breeding pairs in which either both the dams and sires were heterozygous for disruptions of only the ER gene (experiment 1) or both ER and ER genes (experiment 2) (12, 13). The breeders had undergone at least eight generations of backcrosses into the C57BL/6J inbred mouse strain. Genotypes were determined by PCR amplification of tail DNA (12, 13). In the third experiment, subjects were C57BL/6J mice born in our laboratory. The subjects were weaned at 18–20 d of age and group housed by sex on a 12-h light, 12-h dark cycle (lights off at 1900 h EDT) with mouse chow (Harlan Teklad 7912, Indianapolis, IN) and water ad libitum.
Experiment 1.
The subjects were generated from heterozygous breeding pairs carrying one functional and one disrupted copy of the ER gene. When the mice were between 50 and 80 d of age, they were killed and brains were collected. For this study, six females of each genotype and nine males of each genotype were used for a total of 30 mice.
Experiment 2.
The mice were generated from double-heterozygous breeding pairs, with each breeder carrying one functional and one disrupted copy of both the ER and ER genes. The subjects were gonadectomized after reaching puberty (at least 50 d of age). At the time of surgery, each mouse received a sc estradiol implant (50 μg 17-estradiol dissolved in 25 μl sesame oil placed in a SILASTIC brand capsule 3.18 mm outer diameter x 1.98 mm inner diameter; Dow Corning, Midland, MI). These implants yield a steady estradiol concentration of 85–120 pg/ml in plasma (Rissman, E., unpublished data). Brain tissue was collected 5 d after gonadectomy. Group sizes ranged from 4–7, and a total of 46 animals were used.
Experiment 3.
Pups were generated from C57BL/6J breeding pairs from our animal facility. On postnatal days (PN) 1–3 (the actual day of birth designated as PN1), neonates were injected sc with 0.01 ml of vehicle [dimethylsulfoxide (DMSO)] containing the ER-selective agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN; 0.001 mg per 0.01ml; Tocris Cookson Inc., Ellisville, MO), the ER-selective agonist, 4,4',4''-[4-propyl(1H)pyrazole-1,3,5-triyl] Tris-phenol (PPT; 0.003 mg per 0.01ml; Tocris Cookson), E2 (0.1 μg/0.01 ml; Sigma, St. Louis, MO), or no drug (vehicle control). DPN has a 70-fold greater relative binding and a 170-fold larger relative potency in transcriptional assays for ER than ER (14), whereas PPT is selective for ER with a 400-fold preference for this isoform and minimal binding to ER (15). Doses were selected based on previous studies and were within the range used effectively in rodents (16, 17, 18, 19). Because it is not possible to individually mark pups at these young ages, all members of the same litter received the same treatment; at least three litters were used for each treatment. Whereas this may not be the ideal design, given the individual variability between pups based both on genes and in uterine and maternal environments, we do not abide by the view that every litter should contribute only one individual to studies of this kind. Moreover, we do not believe that use of only one pup per litter is in keeping with modern animal husbandry and care practices. The subjects were weaned at 18–20 d of age, group housed by sex until gonadectomy (40–60 d of age), and then individually housed for the rest of the experiment. Each animal received an estradiol benzoate (EB) injection (0.5 μg per 0.05 ml sesame oil) 45 h before a progesterone (P) injection (400 μg per 0.03 ml sesame oil) and was killed 3–5 h after the progesterone injection was given. The subjects were between 75 and 95 d of age at the time of brain collection. Each group contained eight subjects for a total of 40 mice used.
Tissue collection and immunocytochemistry
All the experimental subjects were deeply anesthetized with Halothane inhalant and killed for tissue collection. All animal care was conducted in accordance with the National Institutes of Health Animal Care and Use Guidelines and with the approval of the University of Virginia Animal Care Committee. Brains were quickly removed from the skull and fixed in cold 4% acrolein for 4 h. After completion of the fixation step, the tissue was cryoprotected in 30% sucrose, and brains were frozen and stored at –20 C. Brains were sectioned coronally using a cryostat set to 30 μm, the sections were collected in antifreeze (containing Tris-buffered saline, sucrose, polyvinylpyrrolidone-40, and ethylene glycol) and stored again at –20 C until they were processed for immunocytochemistry. All sections (experiment 1), every fourth section (experiment 2), and every other section (experiment 3) were processed to identify TH-positive neurons in the AVPV. The sections were pretreated and then incubated in primary antisera (1:5000; mouse monoclonal from Incstar/Diasorin, Stillwater, MN) overnight at room temperature. The tissue was rinsed and incubated in biotinylated secondary antisera (1:500; horse antimouse IgG; Vector Laboratories, Burlingame, CA) for 90 min. The tissue was finally rinsed and treated with avidin-biotin complex (1:1000; Vector Laboratories). Immunoreactivity was visualized with nickel-intensified diaminobenzidine activated by 0.1% hydrogen peroxide. The primary antiserum had been previously used for detection of TH-positive cells in mouse brain tissue (20). We conducted control experiments with this antiserum, and no specific staining was noticed when either the primary or secondary antibodies were not included in the staining run.
Data analysis
The tissue sections were mounted on gel-coated glass slides and coded to make the operator blind to the sex/genotype of each subject. The sections were visualized using a BX60 microscope (Olympus, Tokyo, Japan) fitted with a charge-coupled device video camera (Photometrics, Tucson, AZ). In experiment 1, the total number of nucleated cells exhibiting positive staining in the area identified as anteroventral periventricular nucleus [0.50–0.02 mm anterior to Bregma, Figs. 27–31, Franklin and Paxinos Mouse Brain Atlas (21)] on both sides of the third ventricle was calculated. In experiments 2 and 3, only sections located approximately 0.26 mm anterior to Bregma (Fig. 29, Franklin and Paxinos Mouse Brain Atlas) were considered. In those cases in which no section matching the described area could be identified, the counting was performed in the immediately adjacent sections (Fig. 28 or 30, Franklin and Paxinos Mouse Brain Atlas), and if that was not possible, the subject was eliminated from the experiment. In all cases, sections were identified using the specific shape of the third ventricle, anterior commissure, and optic tract as landmarks, and counting was performed using image analysis software (MetaMorph, version 4.5; Molecular Devices, West Chester, PA).
Statistical analysis
Data from experiment 1 were analyzed using a two-way ANOVA with sex and genotype as the independent factors. Data from experiment 2 were analyzed using a three-way ANOVA with sex and presence or absence of functional copies of ER and/or ER as the independent factors. Data from male and female individuals were also analyzed separately by a two-way ANOVA with functional copy of ER and ER as the independent factors. The data from experiment 3 were analyzed using a one-way ANOVA with treatment group as the independent factor. In all cases, post hoc comparisons were performed using the Newman-Keuls multiple-comparison test.
Results
TH-ir sex difference in the AVPV depends on functional ER gene
In experiment 1, a significant interaction between genotype and sex was identified [F (1, 29) = 7.19; P < 0.02]. No effects of sex or genotype were found [F (1, 29) = 2.93 for sex and 2.22 for genotype]. The interaction was caused by the wild-type (WT) male group, which had fewer TH-ir neurons than any other group; WT females, ER knockout (ERKO) females and ERKO males (Figs. 1 and 2).
Deletion of either or both ERs affects TH-ir cell numbers in AVPV
In experiment 2, main effects of both ER [F (1, 38) = 16.99; P < 0.001] and ER [F (1, 38) = 4.63; P < 0.05] were identified, but no effect of sex [F (1, 38) = 1.63] or interaction between any of the factors was observed [F (1, 38) = 2.63, 3.14, 1.64]. When the data were analyzed for males only, absence of functional ER [F (1, 17) = 15.14; P < 0.005] and ER [F (1, 17) = 6.21; P < 0.05] had a significant effect on TH-ir neurons. The absence of neither of the receptors was significant in females [F (1, 21) = 3.18, 0.16]. Post hoc analysis showed that the average number of TH-ir cells in the WT males was significantly lower than in all the other male groups (P < 0.05; Fig. 3). The values for the knockout males were no different from females in the same genotypic group in any case. A tendency to exhibit higher TH-ir cell counts was observed in individuals from both sexes belonging to the double-knockout group, compared with the other knockout groups.
Neonatal treatment with E2 or ER-specific agonists reduced TH-ir cell number in females
In the final experiment, a main effect of the neonatal treatment was observed [F (4, 39) = 7.58; P < 0.01]. Post hoc analysis revealed a sex difference in the medial portion of the AVPV with female subjects treated with vehicle on PN1–3 having more TH-ir neurons than males subjected to the same treatment. Females treated on PN1–3 with either E2 or PPT had TH-ir cell numbers equivalent to control males and significantly lower than those of vehicle-treated females (P < 0.05). Females treated with the ER-specific agonist (DPN) had TH-ir cell numbers intermediate to and significantly different from both vehicle-treated males and females (P < 0.05; Fig. 4)
Discussion
The present results demonstrate that a functional ER is required for normal differentiation of the sexually dimorphic subpopulation of dopaminergic neurons in the AVPV. Furthermore, our data from knockouts for ER and double knockouts, as well as the perinatal treatment with PPT, the ER-specific agonist, confirms the previously characterized role for ER in the sexual differentiation of this area (9). Taken together the data show that permanent disruption of either ER isoform resulted in the female-like TH-ir cell phenotype in the AVPV. As predicted by the data from knockout mice, selective activation of ERs in neonatal females produced more male-like TH-ir phenotypes in the AVPV. Specifically, treatment with the ER selective agonist, PPT, matched the effect of estradiol. The ER-specific agonist, DPN, had only an intermediate effect; TH-ir cell counts in the AVPV were between those in control males and females. Given that these agonists have different specificities, that we only used a single dose, and that treatment lasted only 3 d, it is possible that a higher dose and/or longer administration of DPN would have resulted in male-like TH-ir neuron numbers in female brains. Based on the present results, we propose that during development estradiol in the male brain acts on both ERs to masculinize this important region. Thus, ER is involved in the differentiation of the HPG axis in mice, in addition to its previously characterized role in the defeminization of mating behavior (8).
After the original reports describing the expression of ER, but not ER, in GnRH-containing neurons in the rat (22, 23), evidence has accumulated that suggest a participation of ER in the regulation of their function, including phosphorylation of the cAMP response element binding protein (24) and the induction of galanin expression (25). The present set of data shows that, in addition to these acute actions on GnRH neurons in the adult, ER is also involved in the differentiation of one of their afferent inputs (1). Taken together, these results suggest that ER may regulate the function of the HPG axis in rodents by influencing GnRH neurons at multiple levels. If this were in fact true, ERKO individuals should exhibit specific deficits in the HPG axis function. ERKO female mice are known to exhibit subnormal fertility (12), and behavioral sexual maturity is delayed in ERKO males (26). These deficits may be explained by a disruption in endocrine physiology among other causes. However, confirmation of this hypothesis will require a more detailed characterization of neuroendocrine reflexes ERKO individuals.
Our results on TH-ir cell numbers in ERKO individuals confirm and extend previous work (9). In our study the phenotypic reversal of the ERKO males is more complete than reported earlier (9). One possible explanation for this divergence is a technical difference; the earlier study considered the full rostrocaudal extent of the AVPV (9), whereas our data from ERKO individuals is restricted to a single coronal section per subject in the middle portion of the nucleus. Increased size of the area in females relative to males has been described previously in several species of rodents (27), raising the possibility that the high number of TH-ir neurons in females previously reported may be explained by the inclusion of additional sections not present in males. However, this explanation seems unlikely because we failed to find any sex difference in the rostrocaudal extent of the AVPV in experiment 1, in which all coronal sections spanning the area were considered.
Another difference between the two studies is the hormonal status of the experimental subjects. Simerly et al. (9) used gonad-intact animals, whereas we standardized the levels of circulating steroids. In experiment 2, gonadectomized animals received equivalent E2 treatments. This reduced potential variation because it is well known that adult ERKO females have 10-fold higher E2 levels in plasma, compared with WT subjects, and ERKO males have twice the testosterone concentrations as WT males (28). Down-regulation of TH expression in the adult AVPV by gonadal steroids has been previously described in rats (29). Thus, it is conceivable that the lower numbers of TH-ir cells in ERKO males described by Simerly et al. (9) was the product of higher levels of testosterone and/or estradiol in the brains of ERKO mice. An important caveat of this observation is that WT and ERKO mice undoubtedly have different concentrations of testosterone during development, and this difference may be additive with the loss of the ER in its effect on the differentiation of the AVPV.
Our data do confirm observations that whereas adult concentration of steroids can affect overall TH-ir cell numbers, the sex difference in the AVPV is determined by neonatal concentrations of steroids (29). In each of our three experiments, the mice were in different hormonal conditions (experiment 1: gonad intact, experiment 2: gonadectomized with E2 implants; and experiment 3: gonadectomized and treated with EB and P). In all cases WT males and females displayed the previously reported sex difference in the AVPV.
ER and ER have been shown to form heterodimers in vitro with retained DNA-binding ability and specific transcriptional activity (30, 31, 32). Moreover, it has been postulated that these heterodimers are present in vivo in cells coexpressing both types of ERs and play a role in estrogenic signal transduction in several areas of the rodent brain (10, 11, 33). Our results are consistent with this hypothesis because we did not find a difference in the phenotypes of the double-knockout males, compared with those in which only the ER or ER gene was disrupted, indicating that the presence of one of the receptors cannot compensate for the absence of the other. Alternatively, ER and ER may form homodimers acting sequentially during the process of differentiation, perhaps even on different areas of the hypothalamus. Although ER is abundantly expressed in the rat AVPV (34), its presence in the same area in adult mice is more controversial, with reports of low or no ER-ir cells (6, 35). In mouse embryos and neonates, little ER protein is present in the medial preoptic area (36). If ER is not present on the AVPV at any point during development, it may still mediate the effects of E2 indirectly through one or more of the many hypothalamic nuclei that function as afferents to the AVPV; possible candidate regions include the ventromedial nucleus and the bed nucleus of the stria terminalis (36).
Our data from normal C57BL/6J females that received neonatal treatments confirms that both ER and ER are playing a role in the sexual differentiation of this neural subpopulation and, furthermore, points to the neonatal testosterone surge taking place shortly after birth in males of most mammalian species (37) as the neuroendocrine event driving this differentiation. Remarkably, both specific agonists had an effect of their own in this case, suggesting that activation of either one of the ER isoforms is enough to reduce TH-ir cell numbers in the AVPV. However, it should be kept in mind that the experimental paradigm used to obtain this set of data has a number of caveats, which are different from the caveats relevant to the use of knockout models. The major drawback of knockout mice is that they provide no evidence as to when in development receptor activation is required. In our third experiment, we used normal females that express functional copies of both ER isoforms and attempted to selectively block one or both ERs for a short time during development. Of course, the caveats revolve around the use of pharmacological agents and timing of the drug injections. The selective agonists we used have been validated in vitro and in vivo (14, 15, 16, 17, 18, 19). However, different in vivo applications of these drugs may require different doses, and ours is the first use of these drugs in neonatal mice. In addition, more data on the timing of the testosterone peak postpartum in C57BL/6J mice are needed. In general, perinatal levels of estradiol in the female brain have been shown to be much lower than in males (38), but even the low levels of estradiol in female brain may be involved in important interactions between the ER isoforms, especially in presence of specific agonists.
For these reasons, we consider the evidence from the knockout models along with the evidence from the agonists to provide a more conclusive story than either data set alone. The same selective ER agonist approach has been used to examine differentiation of the AVPV using the rat as a model (39). Despite the obvious species difference, large differences in the timing of the treatment, and in the specific agonists used, the results reported from this study are remarkably similar to our findings in mice. This seems to imply that the role of ER in the sexual differentiation of the AVPV is a general phenomenon, not necessarily restricted to the model of choice for this study. Additional studies on other mammalian species commonly used in the field of neuroendocrinology are needed to confirm this assertion.
In summary, our data demonstrate that ER, along with ER, is required for the differentiation of the TH-ir cell subpopulation in the mouse AVPV and therefore may have an important role in the establishment of the sexual dimorphism characteristic of the HPG axis function. Moreover, ER seems to be acting in combination with ER, providing another example of interaction between the two ERs to transduce the estradiol signal into a concrete biological response, although the exact mechanism of this interaction awaits further studies.
Acknowledgments
The authors thank Aileen Wills, Jessi Gatewood, and Savera Shetty for excellent technical assistance.
Footnotes
This work was supported by National Institutes of Health Grants R01 MH57759 and K02 MH01349 (to E.F.R.) and F31 MH70092 (to A.E.K.).
First Published Online October 20, 2005
Abbreviations: AVPV, Anteroventral periventricular area; DMSO, dimethylsulfoxide; DPN, 2,3-bis(4-hydroxyphenyl)-propionitrile; E2, estradiol; EB, estradiol benzoate; ER, estrogen receptor; ERKO, ER knockout; ERKO, ER knockout; HPG, hypothalamic-pituitary-gonadal; -ir, immunoreactive; P, progesterone; PN, postnatal day; PPT, 4,4',4''-[4-propyl(1H)pyrazole-1,3,5-triyl] Tris-phenol; TH, tyrosine hydroxylase; WT, wild type.
Accepted for publication October 10, 2005.
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Abstract
Sexual dimorphisms in the hypothalamus are mediated in several cases by local aromatization of androgens to estrogens during the perinatal period. In this series of experiments, the contributions of the two estrogen receptors (ERs), ER and ER, to the differentiation of the sexually dimorphic subpopulation of dopaminergic neurons in the anteroventral periventricular area (AVPV) was examined. In the first experiment, numbers of tyrosine hydroxylase (TH) immunoreactive (-ir) AVPV neurons in ER knockout and wild-type (WT) mice of both sexes were measured. In the second experiment, the average number of TH-ir neurons in the medial portion of the AVPV in ER knockout, ER knockout, double-ER knockout, and WT mice of both sexes was calculated. In both experiments TH-ir cell numbers were sexually dimorphic as expected, with female individuals of all genotypes exhibiting more TH-ir neurons than WT males. Interestingly the average number of TH-ir neurons in all knockout males was significantly higher than in WT male littermates. In fact, TH-ir cell numbers in all knockout males were equivalent to females. In a final experiment, C57BL/6J female mice were treated during the first 3 postnatal days with either estradiol, or a specific agonist for one of the two ERs. Additional male and female pups received vehicle injections. Treatments with estradiol or either ER-specific agonist significantly reduced the number of TH-ir AVPV neurons in female brains. Our data demonstrate that both ER and ER are involved in the sexual differentiation of the AVPV in mice.
Introduction
REGULATION OF THE hypothalamic-pituitary-gonadal (HPG) axis is sexually dimorphic. Specifically, positive feedback of estradiol (E2) on LH secretion by the pituitary occurs only in female individuals and constitutes a requirement for the preovulatory gonadotropin surge (1). Estradiol has been postulated to exert this effect both at the level of pituitary gonadotrophs (2) and by modification of the GnRH secretion pattern from the hypothalamus (reviewed in Ref.1). In rodents, the anteroventral periventricular area (AVPV) of the hypothalamus contains a sexually dimorphic subpopulation of dopaminergic neurons, with a larger number present in females, compared with males (3). Furthermore, these cells have been shown to project to GnRH neurons, and both selective lesions (4) and local administration of estrogen antagonists (5) can prevent the normal display of the preovulatory LH surge in experimental animals. This suggests that this area is important for the positive feedback effect of estradiol on gonadotropin release.
The estrogen receptor (ER)- is widely distributed in the mouse brain (6, 7), including the AVPV. We recently described a role for ER in the defeminization of reproductive behavior in mice (8). It is not known whether this role is restricted to the differentiation of those neural circuits responsible for the display of female-like mating behavior or, alternatively, whether ER is involved in the differentiation of other sexually dimorphic functions known to differentiate under the influence of gonadal steroids during development.
The purpose of this study was to test the hypothesis that ER plays a role in the sexual differentiation of the subpopulation of dopaminergic neurons in the AVPV. Two strategies were used: mice with and without functional ER genes and administration of selective ER-specific agonists to neonatal mice. In adulthood we determined the average number of tyrosine hydroxylase (TH)-positive cells in the AVPV. Although Simerly et al. (9) previously described an incompletely masculinized phenotype in the TH-immunoreactive (ir) neurons in the AVPV of ER knockout (ERKO) male mice and postulated a role for ER in the process based on these results, cross-regulation between both types of ERs has been suggested to take place in other hypothalamic areas (10, 11), raising the possibility that ER may interact with ER during development to reduce the number of dopaminergic neurons in adult males.
Materials and Methods
Animals
For experiments 1 and 2, subjects were generated from breeding pairs in which either both the dams and sires were heterozygous for disruptions of only the ER gene (experiment 1) or both ER and ER genes (experiment 2) (12, 13). The breeders had undergone at least eight generations of backcrosses into the C57BL/6J inbred mouse strain. Genotypes were determined by PCR amplification of tail DNA (12, 13). In the third experiment, subjects were C57BL/6J mice born in our laboratory. The subjects were weaned at 18–20 d of age and group housed by sex on a 12-h light, 12-h dark cycle (lights off at 1900 h EDT) with mouse chow (Harlan Teklad 7912, Indianapolis, IN) and water ad libitum.
Experiment 1.
The subjects were generated from heterozygous breeding pairs carrying one functional and one disrupted copy of the ER gene. When the mice were between 50 and 80 d of age, they were killed and brains were collected. For this study, six females of each genotype and nine males of each genotype were used for a total of 30 mice.
Experiment 2.
The mice were generated from double-heterozygous breeding pairs, with each breeder carrying one functional and one disrupted copy of both the ER and ER genes. The subjects were gonadectomized after reaching puberty (at least 50 d of age). At the time of surgery, each mouse received a sc estradiol implant (50 μg 17-estradiol dissolved in 25 μl sesame oil placed in a SILASTIC brand capsule 3.18 mm outer diameter x 1.98 mm inner diameter; Dow Corning, Midland, MI). These implants yield a steady estradiol concentration of 85–120 pg/ml in plasma (Rissman, E., unpublished data). Brain tissue was collected 5 d after gonadectomy. Group sizes ranged from 4–7, and a total of 46 animals were used.
Experiment 3.
Pups were generated from C57BL/6J breeding pairs from our animal facility. On postnatal days (PN) 1–3 (the actual day of birth designated as PN1), neonates were injected sc with 0.01 ml of vehicle [dimethylsulfoxide (DMSO)] containing the ER-selective agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN; 0.001 mg per 0.01ml; Tocris Cookson Inc., Ellisville, MO), the ER-selective agonist, 4,4',4''-[4-propyl(1H)pyrazole-1,3,5-triyl] Tris-phenol (PPT; 0.003 mg per 0.01ml; Tocris Cookson), E2 (0.1 μg/0.01 ml; Sigma, St. Louis, MO), or no drug (vehicle control). DPN has a 70-fold greater relative binding and a 170-fold larger relative potency in transcriptional assays for ER than ER (14), whereas PPT is selective for ER with a 400-fold preference for this isoform and minimal binding to ER (15). Doses were selected based on previous studies and were within the range used effectively in rodents (16, 17, 18, 19). Because it is not possible to individually mark pups at these young ages, all members of the same litter received the same treatment; at least three litters were used for each treatment. Whereas this may not be the ideal design, given the individual variability between pups based both on genes and in uterine and maternal environments, we do not abide by the view that every litter should contribute only one individual to studies of this kind. Moreover, we do not believe that use of only one pup per litter is in keeping with modern animal husbandry and care practices. The subjects were weaned at 18–20 d of age, group housed by sex until gonadectomy (40–60 d of age), and then individually housed for the rest of the experiment. Each animal received an estradiol benzoate (EB) injection (0.5 μg per 0.05 ml sesame oil) 45 h before a progesterone (P) injection (400 μg per 0.03 ml sesame oil) and was killed 3–5 h after the progesterone injection was given. The subjects were between 75 and 95 d of age at the time of brain collection. Each group contained eight subjects for a total of 40 mice used.
Tissue collection and immunocytochemistry
All the experimental subjects were deeply anesthetized with Halothane inhalant and killed for tissue collection. All animal care was conducted in accordance with the National Institutes of Health Animal Care and Use Guidelines and with the approval of the University of Virginia Animal Care Committee. Brains were quickly removed from the skull and fixed in cold 4% acrolein for 4 h. After completion of the fixation step, the tissue was cryoprotected in 30% sucrose, and brains were frozen and stored at –20 C. Brains were sectioned coronally using a cryostat set to 30 μm, the sections were collected in antifreeze (containing Tris-buffered saline, sucrose, polyvinylpyrrolidone-40, and ethylene glycol) and stored again at –20 C until they were processed for immunocytochemistry. All sections (experiment 1), every fourth section (experiment 2), and every other section (experiment 3) were processed to identify TH-positive neurons in the AVPV. The sections were pretreated and then incubated in primary antisera (1:5000; mouse monoclonal from Incstar/Diasorin, Stillwater, MN) overnight at room temperature. The tissue was rinsed and incubated in biotinylated secondary antisera (1:500; horse antimouse IgG; Vector Laboratories, Burlingame, CA) for 90 min. The tissue was finally rinsed and treated with avidin-biotin complex (1:1000; Vector Laboratories). Immunoreactivity was visualized with nickel-intensified diaminobenzidine activated by 0.1% hydrogen peroxide. The primary antiserum had been previously used for detection of TH-positive cells in mouse brain tissue (20). We conducted control experiments with this antiserum, and no specific staining was noticed when either the primary or secondary antibodies were not included in the staining run.
Data analysis
The tissue sections were mounted on gel-coated glass slides and coded to make the operator blind to the sex/genotype of each subject. The sections were visualized using a BX60 microscope (Olympus, Tokyo, Japan) fitted with a charge-coupled device video camera (Photometrics, Tucson, AZ). In experiment 1, the total number of nucleated cells exhibiting positive staining in the area identified as anteroventral periventricular nucleus [0.50–0.02 mm anterior to Bregma, Figs. 27–31, Franklin and Paxinos Mouse Brain Atlas (21)] on both sides of the third ventricle was calculated. In experiments 2 and 3, only sections located approximately 0.26 mm anterior to Bregma (Fig. 29, Franklin and Paxinos Mouse Brain Atlas) were considered. In those cases in which no section matching the described area could be identified, the counting was performed in the immediately adjacent sections (Fig. 28 or 30, Franklin and Paxinos Mouse Brain Atlas), and if that was not possible, the subject was eliminated from the experiment. In all cases, sections were identified using the specific shape of the third ventricle, anterior commissure, and optic tract as landmarks, and counting was performed using image analysis software (MetaMorph, version 4.5; Molecular Devices, West Chester, PA).
Statistical analysis
Data from experiment 1 were analyzed using a two-way ANOVA with sex and genotype as the independent factors. Data from experiment 2 were analyzed using a three-way ANOVA with sex and presence or absence of functional copies of ER and/or ER as the independent factors. Data from male and female individuals were also analyzed separately by a two-way ANOVA with functional copy of ER and ER as the independent factors. The data from experiment 3 were analyzed using a one-way ANOVA with treatment group as the independent factor. In all cases, post hoc comparisons were performed using the Newman-Keuls multiple-comparison test.
Results
TH-ir sex difference in the AVPV depends on functional ER gene
In experiment 1, a significant interaction between genotype and sex was identified [F (1, 29) = 7.19; P < 0.02]. No effects of sex or genotype were found [F (1, 29) = 2.93 for sex and 2.22 for genotype]. The interaction was caused by the wild-type (WT) male group, which had fewer TH-ir neurons than any other group; WT females, ER knockout (ERKO) females and ERKO males (Figs. 1 and 2).
Deletion of either or both ERs affects TH-ir cell numbers in AVPV
In experiment 2, main effects of both ER [F (1, 38) = 16.99; P < 0.001] and ER [F (1, 38) = 4.63; P < 0.05] were identified, but no effect of sex [F (1, 38) = 1.63] or interaction between any of the factors was observed [F (1, 38) = 2.63, 3.14, 1.64]. When the data were analyzed for males only, absence of functional ER [F (1, 17) = 15.14; P < 0.005] and ER [F (1, 17) = 6.21; P < 0.05] had a significant effect on TH-ir neurons. The absence of neither of the receptors was significant in females [F (1, 21) = 3.18, 0.16]. Post hoc analysis showed that the average number of TH-ir cells in the WT males was significantly lower than in all the other male groups (P < 0.05; Fig. 3). The values for the knockout males were no different from females in the same genotypic group in any case. A tendency to exhibit higher TH-ir cell counts was observed in individuals from both sexes belonging to the double-knockout group, compared with the other knockout groups.
Neonatal treatment with E2 or ER-specific agonists reduced TH-ir cell number in females
In the final experiment, a main effect of the neonatal treatment was observed [F (4, 39) = 7.58; P < 0.01]. Post hoc analysis revealed a sex difference in the medial portion of the AVPV with female subjects treated with vehicle on PN1–3 having more TH-ir neurons than males subjected to the same treatment. Females treated on PN1–3 with either E2 or PPT had TH-ir cell numbers equivalent to control males and significantly lower than those of vehicle-treated females (P < 0.05). Females treated with the ER-specific agonist (DPN) had TH-ir cell numbers intermediate to and significantly different from both vehicle-treated males and females (P < 0.05; Fig. 4)
Discussion
The present results demonstrate that a functional ER is required for normal differentiation of the sexually dimorphic subpopulation of dopaminergic neurons in the AVPV. Furthermore, our data from knockouts for ER and double knockouts, as well as the perinatal treatment with PPT, the ER-specific agonist, confirms the previously characterized role for ER in the sexual differentiation of this area (9). Taken together the data show that permanent disruption of either ER isoform resulted in the female-like TH-ir cell phenotype in the AVPV. As predicted by the data from knockout mice, selective activation of ERs in neonatal females produced more male-like TH-ir phenotypes in the AVPV. Specifically, treatment with the ER selective agonist, PPT, matched the effect of estradiol. The ER-specific agonist, DPN, had only an intermediate effect; TH-ir cell counts in the AVPV were between those in control males and females. Given that these agonists have different specificities, that we only used a single dose, and that treatment lasted only 3 d, it is possible that a higher dose and/or longer administration of DPN would have resulted in male-like TH-ir neuron numbers in female brains. Based on the present results, we propose that during development estradiol in the male brain acts on both ERs to masculinize this important region. Thus, ER is involved in the differentiation of the HPG axis in mice, in addition to its previously characterized role in the defeminization of mating behavior (8).
After the original reports describing the expression of ER, but not ER, in GnRH-containing neurons in the rat (22, 23), evidence has accumulated that suggest a participation of ER in the regulation of their function, including phosphorylation of the cAMP response element binding protein (24) and the induction of galanin expression (25). The present set of data shows that, in addition to these acute actions on GnRH neurons in the adult, ER is also involved in the differentiation of one of their afferent inputs (1). Taken together, these results suggest that ER may regulate the function of the HPG axis in rodents by influencing GnRH neurons at multiple levels. If this were in fact true, ERKO individuals should exhibit specific deficits in the HPG axis function. ERKO female mice are known to exhibit subnormal fertility (12), and behavioral sexual maturity is delayed in ERKO males (26). These deficits may be explained by a disruption in endocrine physiology among other causes. However, confirmation of this hypothesis will require a more detailed characterization of neuroendocrine reflexes ERKO individuals.
Our results on TH-ir cell numbers in ERKO individuals confirm and extend previous work (9). In our study the phenotypic reversal of the ERKO males is more complete than reported earlier (9). One possible explanation for this divergence is a technical difference; the earlier study considered the full rostrocaudal extent of the AVPV (9), whereas our data from ERKO individuals is restricted to a single coronal section per subject in the middle portion of the nucleus. Increased size of the area in females relative to males has been described previously in several species of rodents (27), raising the possibility that the high number of TH-ir neurons in females previously reported may be explained by the inclusion of additional sections not present in males. However, this explanation seems unlikely because we failed to find any sex difference in the rostrocaudal extent of the AVPV in experiment 1, in which all coronal sections spanning the area were considered.
Another difference between the two studies is the hormonal status of the experimental subjects. Simerly et al. (9) used gonad-intact animals, whereas we standardized the levels of circulating steroids. In experiment 2, gonadectomized animals received equivalent E2 treatments. This reduced potential variation because it is well known that adult ERKO females have 10-fold higher E2 levels in plasma, compared with WT subjects, and ERKO males have twice the testosterone concentrations as WT males (28). Down-regulation of TH expression in the adult AVPV by gonadal steroids has been previously described in rats (29). Thus, it is conceivable that the lower numbers of TH-ir cells in ERKO males described by Simerly et al. (9) was the product of higher levels of testosterone and/or estradiol in the brains of ERKO mice. An important caveat of this observation is that WT and ERKO mice undoubtedly have different concentrations of testosterone during development, and this difference may be additive with the loss of the ER in its effect on the differentiation of the AVPV.
Our data do confirm observations that whereas adult concentration of steroids can affect overall TH-ir cell numbers, the sex difference in the AVPV is determined by neonatal concentrations of steroids (29). In each of our three experiments, the mice were in different hormonal conditions (experiment 1: gonad intact, experiment 2: gonadectomized with E2 implants; and experiment 3: gonadectomized and treated with EB and P). In all cases WT males and females displayed the previously reported sex difference in the AVPV.
ER and ER have been shown to form heterodimers in vitro with retained DNA-binding ability and specific transcriptional activity (30, 31, 32). Moreover, it has been postulated that these heterodimers are present in vivo in cells coexpressing both types of ERs and play a role in estrogenic signal transduction in several areas of the rodent brain (10, 11, 33). Our results are consistent with this hypothesis because we did not find a difference in the phenotypes of the double-knockout males, compared with those in which only the ER or ER gene was disrupted, indicating that the presence of one of the receptors cannot compensate for the absence of the other. Alternatively, ER and ER may form homodimers acting sequentially during the process of differentiation, perhaps even on different areas of the hypothalamus. Although ER is abundantly expressed in the rat AVPV (34), its presence in the same area in adult mice is more controversial, with reports of low or no ER-ir cells (6, 35). In mouse embryos and neonates, little ER protein is present in the medial preoptic area (36). If ER is not present on the AVPV at any point during development, it may still mediate the effects of E2 indirectly through one or more of the many hypothalamic nuclei that function as afferents to the AVPV; possible candidate regions include the ventromedial nucleus and the bed nucleus of the stria terminalis (36).
Our data from normal C57BL/6J females that received neonatal treatments confirms that both ER and ER are playing a role in the sexual differentiation of this neural subpopulation and, furthermore, points to the neonatal testosterone surge taking place shortly after birth in males of most mammalian species (37) as the neuroendocrine event driving this differentiation. Remarkably, both specific agonists had an effect of their own in this case, suggesting that activation of either one of the ER isoforms is enough to reduce TH-ir cell numbers in the AVPV. However, it should be kept in mind that the experimental paradigm used to obtain this set of data has a number of caveats, which are different from the caveats relevant to the use of knockout models. The major drawback of knockout mice is that they provide no evidence as to when in development receptor activation is required. In our third experiment, we used normal females that express functional copies of both ER isoforms and attempted to selectively block one or both ERs for a short time during development. Of course, the caveats revolve around the use of pharmacological agents and timing of the drug injections. The selective agonists we used have been validated in vitro and in vivo (14, 15, 16, 17, 18, 19). However, different in vivo applications of these drugs may require different doses, and ours is the first use of these drugs in neonatal mice. In addition, more data on the timing of the testosterone peak postpartum in C57BL/6J mice are needed. In general, perinatal levels of estradiol in the female brain have been shown to be much lower than in males (38), but even the low levels of estradiol in female brain may be involved in important interactions between the ER isoforms, especially in presence of specific agonists.
For these reasons, we consider the evidence from the knockout models along with the evidence from the agonists to provide a more conclusive story than either data set alone. The same selective ER agonist approach has been used to examine differentiation of the AVPV using the rat as a model (39). Despite the obvious species difference, large differences in the timing of the treatment, and in the specific agonists used, the results reported from this study are remarkably similar to our findings in mice. This seems to imply that the role of ER in the sexual differentiation of the AVPV is a general phenomenon, not necessarily restricted to the model of choice for this study. Additional studies on other mammalian species commonly used in the field of neuroendocrinology are needed to confirm this assertion.
In summary, our data demonstrate that ER, along with ER, is required for the differentiation of the TH-ir cell subpopulation in the mouse AVPV and therefore may have an important role in the establishment of the sexual dimorphism characteristic of the HPG axis function. Moreover, ER seems to be acting in combination with ER, providing another example of interaction between the two ERs to transduce the estradiol signal into a concrete biological response, although the exact mechanism of this interaction awaits further studies.
Acknowledgments
The authors thank Aileen Wills, Jessi Gatewood, and Savera Shetty for excellent technical assistance.
Footnotes
This work was supported by National Institutes of Health Grants R01 MH57759 and K02 MH01349 (to E.F.R.) and F31 MH70092 (to A.E.K.).
First Published Online October 20, 2005
Abbreviations: AVPV, Anteroventral periventricular area; DMSO, dimethylsulfoxide; DPN, 2,3-bis(4-hydroxyphenyl)-propionitrile; E2, estradiol; EB, estradiol benzoate; ER, estrogen receptor; ERKO, ER knockout; ERKO, ER knockout; HPG, hypothalamic-pituitary-gonadal; -ir, immunoreactive; P, progesterone; PN, postnatal day; PPT, 4,4',4''-[4-propyl(1H)pyrazole-1,3,5-triyl] Tris-phenol; TH, tyrosine hydroxylase; WT, wild type.
Accepted for publication October 10, 2005.
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