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Co-localization of Estrogen Receptor and Aromatase Enzyme Immunoreactivities in Adult Musk Shrew Brain
Hormones and Behavior 33, 151–162 (1998) Article No. HB981446
Co-localization of Estrogen Receptor and Aromatase Enzyme Immunoreactivities in Adult Musk Shrew Brain Sean L. Veney and Emilie F. Rissman1 Department of Biology, University of Virginia, Charlottesville, Virginia 22903 Received September 19, 1997; revised February 24, 1998; accepted March 30, 1998
Estrogens are produced by the aromatization of androgens. These steroids exert their actions after binding to their receptors. Past studies have shown that estrogen receptors (ER) and aromatase enzyme (AROM) reside in many of the same brain regions. Few studies, however, have examined the neural co-localization of these important components involved in estrogen-activated behaviors. In the present study we examined the co-localization of ER and AROM immunoreactive (ir) neurons in musk shrew (Suncus murinus) brains. Data were collected from a representative section from three neural regions, the bed nucleus of the stria terminalis (BNST), medial preoptic area (mPOA), and ventromedial nucleus of the hypothalamus (VMN). Here we report a sex difference in the number of ER-ir neurons from the analyzed section of the mPOA and BNST. Females have more ER-ir neurons in the mPOA and males have more in the BNST. In the sections we examined, males tended to have more aromatase containing neurons than females. Although there were no significant differences in the numbers of double-labeled cells, the VMN contains the greatest percentage of these cells in both males and females; followed by the mPOA and the BNST. In addition, in the mPOA of both sexes, a distinct nucleus of aromatase containing neurons which was devoid of ER immunoreactivity was noted. Area measurements of the AROM-ir nucleus showed that it was significantly larger in males than in females. Taken together, these data suggest that there is not extensive cellular co-localization of estrogen receptors and aromatase enzyme in the musk shrew brain. However, the presence of other genomic forms of ER (membrane and/or ERb) in AROM containing neurons has not been ruled out by this study. Thus, we hypothesize that estrogens produced in brain affect behavior by binding to ER in neurons other than those that contain aromatase enzyme. © 1998 Academic Press
1 To whom correspondence should be addressed. Fax: (804) 9825626. E-mail: efr2fVirginia.edu.
0018-506X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
Aromatase enzyme is a member of the cytochrome P450 family that irreversibly catalyzes the conversion of androstenedione to estrone and testosterone to estradiol. This enzyme is present in the brain, gonads, and other peripheral tissue (Naftolin, Ryan, Davies, Reddy, Flores, Petro, Kuhn, White, Takoka, and Wolin, 1975; Selmanoff, Brodkin, Weiner, and Siiteri, 1977). Neural aromatization of androgens to estrogens is necessary for the activation of sexual behavior in a number of vertebrate species. The majority of these data have been collected in male Japanese quail and rats (Adkins, Boop, Koutnik, Morris, and Pniewski, 1980; Balthazart, Foidart, and Hendrick, 1990; Christensen and Clemens, 1974). Similar to rats and quail, female musk shrew sexual behavior is also dependent upon neural aromatization of testosterone (T) to estradiol (E2); (Rissman, 1991). In all of these species, aromatase activity is highest in preoptic, hypothalamic, and limbic nuclei (Rissman, Harada, and Roselli, 1996; Roselli, Horton, and Resko, 1985; Roselli and Resko, 1989; Schumacher and Balthazart, 1987). Several of these regions, including the bed nucleus of the stria terminalis (BNST) and the medial preoptic area (mPOA), are important for the regulation of sexual behavior in rats. Implants containing either T or E2 placed in the mPOA can restore sexual behavior in castrated male quail and rats (Balthazart and Surlemont, 1990; Watson and Adkins-Regan, 1989; Christensen and Clemens, 1974). Also, T implants placed into the mPOA of female musk shrews reinstate sexual behavior after ovariectomy (Sharma and Rissman, 1994; Veney and Rissman, 1997a). Immunocytochemistry has been used to examine co-localization of estrogen receptors and aromatase containing neurons in male Japanese quail (Balthazart, Foidart, Surlemont, and Harada, 1991; Dellovade, Rissman, Thompson, Harada, and Ottinger, 1995), fe-
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tal and neonatal rat (Tsuruo, Ishimura, Hayashi, and Osawa, 1996), and mouse (Tsuruo, Ishimura, and Osawa, 1995) brain. In the musk shrew, the distribution of neurons that are immunoreactive for estrogen receptors (ER-ir; Dellovade, Blaustein, and Rissman, 1992) and aromatase enzyme (AROM-ir; Rissman et al., 1996) has been mapped. Neurons that express either ER or AROM immunoreactivity reside in many of the same brain regions. It is not known, however, whether the two are co-localized in the same cell. In the present study, we describe the co-expression of ER and AROM enzyme immunoreactivities in male and female musk shrew brains. At present, we do not know whether neural aromatization is involved in male musk shrew sexual behavior. Female musk shrews, however, require aromatization of T to E2 in order to show receptivity. We hypothesized that the neural co-expression of ER and AROM enzyme in females, and possibly male shrews, would be similar to that described previously in males of other species. The co-expression of ER and AROM enzyme immunoreactivities were examined in a representative section from three neural regions: the bed nucleus of the stria terminalis (BNST), medial preoptic area (mPOA), and ventromedial nucleus of the hypothalamus (VMN).
MATERIALS AND METHODS General Methods Musk shrews were born and raised in the colony at the University of Virginia. The colony was maintained on a light cycle of 14 h light/10 h dark (lights on at 0600 h, EST) at a temperature of 23 6 1°C. Food (Purina Cat Chow and Complete Mink Pellets from Milk Specialty Products, New Holstein, WI) and water were provided ad libitum. Animals were weaned at 18 –20 days of age and individually housed (cage dimensions 28 3 17 3 12 cm) with pine wood shavings and paper towels for bedding. Experimental Design and Treatment Males (60 days of age; n 5 7) and females (30 days of age; n 5 6) were gonadectomized and given a testosterone (T) implant (10 and 5 mm, respectively). Females are sexually mature by 26 days of age, and males are capable of siring young by 45 days. Thus, both groups were adult, mature individuals. The hormone was packed into Silastic tubing (1 mm ID 3 2.125 mm OD) and implanted subcutaneously (sc) in
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the back of the neck. Beginning 2 days after surgery, animals received daily (sc) injections of the anti-aromatase drug vorozole (5 mg/kg bw, generously provided by Janssen Research Foundation) for 7 days. Vorozole is a nonsteroidal anti-aromatase enzyme drug. In vivo it causes a drop in aromatase activity levels and estrogen concentrations, but it does not bind estrogen or other steroid receptors (Wouters, DeCoster, Beerens, Doolaege, Gruwez, van Camp, van der Pas, and Herendael, 1990). Previous studies have shown that injections of vorozole enhance specific staining of AROM-ir neurons in musk shrews (Rissman et al., 1996) and mice (Foidart, Harada, and Balthazart, 1995). Control animals (n 5 3 males and n 5 3 females) were gonadectomized, T-implanted, and given daily (sc) injections of the vehicle (PEG-400). Hormone Assay Prior to perfusion, 250 – 400 ml of blood was drawn from each animal by cardiac puncture. Blood samples were spun, and plasma was collected and stored at 270°C until assay. The plasma samples were assayed in singlet for T using the Diagnostic Products testosterone kit. This assay has been validated previously for use with musk shrew plasma (Fortman, Dellovade, and Rissman, 1992). The calculated dose of T at 90% binding was 0.18 ng/ml. The intra-assay variability averaged 6.94%. The antibody cross-reactivity was 100% with T, 7.8% with DHT, 2% with 11-oxy-testosterone, and 0.56% with androstenedione. Based on the results of the hormone assay, female plasma T concentrations ranged from 1.08 to 2.97 ng/ml; with a mean of 1.97 ng/ml. Male plasma T levels ranged from 3.95 to 5.76 ng/ml; with a mean of 5.18 ng/ml. These concentrations are higher than physiological hormone levels for gonad-intact male and female musk shrews (Rissman and Crews, 1988, 1989). Immunocytochemistry After 7 days of vorozole treatment, and within 2 h of the last injection, each animal was sacrificed by an overdose of sodium pentobarbital. The animals were perfused with heparinized saline (100 units/ml) followed by Zamboni’s fixative. Brains were cryoprotected overnight in 20% sucrose. The following day they were quick-frozen in cold methylbutane and stored at 270°C. Frozen brains were coronally cut to a thickness of 25 mm into a series of four wells and stored in antifreeze at 220°C until immunocytochemistry processing. Sections were collected from the ros-
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tral forebrain through the rostral portion of the midbrain central gray. Double-label immunocytochemistry was performed to visualize neurons immunoreactive for estrogen receptors and/or aromatase enzyme. One-quarter of the sections from each brain were treated with 1% NaBH4 in TBS (Tris-buffered saline, pH 7.8) to remove aldehydes, followed by avidin– biotin blocker for endogenous biotin. Tissue was incubated for 48 h in primary antibody diluted in 0.25% lambda carrageenan, 1% BSA, 0.3% Triton, and 0.1% sodium azide in TBS. Sections were simultaneously incubated with the rat monoclonal anti-estrogen receptor antiserum (H222, 1:500; generously provided by Abbott Laboratories) and a polyclonal aromatase antiserum (R-8-1, 1:500; kindly supplied by Dr. Y. Osawa; Kitawaki, Yoshida, and Osawa, 1989). Vector biotinylated donkey anti-rat IgG (double-bridged at 1:500) and Vector Elite avidin– biotin complex, followed by DAB, were used to visualize the ER-ir cells. To maximize the reaction product, the tissue was incubated in DAB for 20 min and then 15 ml of NiSO4(NH4)2SO4 was added for an additional 1–5 min. The addition of the NiSO4(NH4)2SO4 catalyzed the DAB reaction and increased the brown reaction product. Thus, total reaction time in DAB was 20 –25 min. Following development in DAB, sections were rinsed and stored overnight in TBS at 4°C. The following day, AROM immunoreactivity was visualized by a similar procedure. Vector biotinylated goat anti-rabbit IgG (double-bridged at 1:250) and SG (Vector Laboratories) were used to visualize the reaction product. The Vector SG substrate for peroxidase yields a blue-gray reaction product. Total incubation time in SG was 3–5 min. Following immunocytochemistry, sections were mounted on slides and allowed to airdry overnight. The following day, the sections were dehydrated and the slides were coverslipped. We have reported on the results of preadsorption controls with H222 in the past (Dellovade et al., 1992). To validate the specificity of AROM immunoreactivity, R-8-1 was preincubated with either 1 or 5 mg of immunoaffinity-purified aromatase P450 for 24 h before the tissue sections were added. Both concentrations of antigen eliminated specific staining. To examine the possible effect of vorozole treatment on ER immunoreactivity, brains from the control animals were examined. These animals (three males and three females) were gonadectomized, treated with a Silastic T-implant, and given vehicle injections in place of vorozole. Every fourth section was developed with H222 antibody (as described above) and the distribu-
tion of ER immunoreactivity was compared to that of animals treated with vorozole. Image Analysis Neurons that contained immunoreactive estrogen receptors or aromatase enzyme, as well as neurons that were double-labeled for both, were counted in one representative section from each of three brain regions: BNST, mPOA, and VMN. A comparable section from each brain region of each animal was matched and analyzed under the microscope at magnifications of 80 –1203. To select matched sections from each individual we used the following landmarks. For the mPOA, we selected a section comparable to the rat brain atlas, between plates 20 and 21 (Paxinos and Watson, 1986). The anterior commissure was caudal to its thickest extent and tapering on the lateral edges. The lateral ventricles were nearly joined at the midline. For the BNST, we selected a section comparable to the rat brain atlas, plate 22 (Paxinos and Watson, 1986). This was the next section in our series, 75 mm caudal to the mPOA. The lateral ventricles were joined at the midline and the anterior commissure was not present. The section examined for the VMN was similar to plate 28 in the rat atlas (Paxinos and Watson, 1986). On this section, all fields (CA1-3) of the hippocampus were present, as was the dentate gyrus. The median eminence was just forming along the midline. Images were captured on an Olympus U-CMAD-2 color video camera and cell counts were conducted using Mocha statistical image analysis software (Jandel Scientific). AROM-ir cells were defined as such based on the appearance of a clear nucleus and the presence of at least one cell process. A large, sexually dimorphic nucleus containing AROM-ir neurons is present in the musk shrew mPOA (Rissman et al., 1996). This nucleus is present in Nissl-stained material and these were used to help define the AROM-ir region as well as the areas that we selected for the counts (Veney and Rissman, unpublished data). In the present study, the nucleus was so densely stained with AROM-ir reaction product that counts of individual cells were not possible. Therefore, we quantified the area of the nucleus. Using Mocha image analysis software, a calibration was made for the magnification used to measure the nucleus and the number of pixels within a 1 mm by 1 mm square was determined. An outline was drawn free-handed around the outermost edges of the AROM-ir nucleus. The resulting number of pixels was converted to an area measurement (mm2) based on the calibration.
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TABLE 1 Estrogen Receptor and Aromatase Enzyme Immunoreactive Cell Counts
? ? ? / / /
BNST mPOA VMN BNST mPOA VMN
ER-ir
AROM-ir
# Double Labeled
% Double Labeled
71.0 6 6.0* 179.3 6 20.7* 179.6 6 22.0 42.5 6 9.0 431.0 6 36.5 217.8 6 13.9
1037.6 6 50.7a 196.7 6 7.9b 98.1 6 8.4c 973.2 6 58.9a 133.2 6 10.8b 47.8 6 3.9c
13.7 6 3.1 12.0 6 1.7 9.3 6 1.5 9.2 6 1.5 9.8 6 2.1 6.5 6 1.5
1.32 6 1.2a 6.1 6 1.2b 9.5 6 1.2c 0.95 6 1.3a 7.4 6 1.3b 13.6 6 1.3c
Note. Cell counts were taken from three neural regions in male (n 5 7) and female (n 5 6) brains. Data are presented as means 6 SEM. * Significant sex difference in that brain region; P , 0.05 at least. Values with different letters are significantly different from each other; P , 0.05 at least.
Data Analysis Cell count data were analyzed by analysis of variance (two-way ANOVA). The two factors were sex and brain area. When interactions were noted, twotailed Student–Newman–Keuls tests were used. Student’s t tests, corrected for multiple comparisons, were used to analyze the size of the aromatase nucleus in the mPOA. Effects were considered significant at an a of 0.05 or less.
RESULTS Counts of ER-ir and AROM-ir, as well as the numbers and percentage of dual-labeled cells, were recorded from a representative section of the BNST, mPOA, and VMN in males and females (Table 1). In the mPOA, the sexually dimorphic nucleus of densely packed aromatase containing neurons did not contain immunoreactive estrogen receptors. Brains from control animals, treated only with the vehicle and incubated with the anti-ER antiserum, confirmed that the nucleus was devoid of ER immunoreactivity (Figs. 1 and 2).Therefore, in the mPOA, only ER-ir and/or AROM-ir cells outside of the large aromatase nucleus were counted. Estrogen Receptor Immunoreactivity There was a significant difference in the number of neurons that contained estrogen receptors based on sex (F(1, 33) 5 27.7, P , 0.0001) and brain area (F(2, 33) 5 75.4, P , 0.0001), and an interaction between sex and brain area was revealed (F(2, 33) 5 26.0, P , 0.0001; Table 1). A Newman–Keuls follow-up of the interaction to determine the effect of sex on the number of ER-ir cells in each brain region was conducted.
Females had significantly more ER-ir neurons in the mPOA than males did (P , 0.05). However, males had more ER-ir cells in the BNST than females (P , 0.05). There was no sex difference in ER-ir cells in the VMN. Aromatase Enzyme Immunoreactivity There was a significant difference in the number of AROM-ir neurons based on sex (F(1, 33) 5 5.09, P 5 0.031) and brain area (F(2, 33) 5 507.92, P , 0.0001; Table 1). There was no interaction between the two variables (F(2, 33) 5 0.030). Pair tests revealed that the number of AROM-ir neurons in the BNST was significantly greater than in the VMN or mPOA, and more AROM-ir neurons were present in the mPOA than in the VMN (P , 0.05). In addition, males had more AROM-ir neurons in each region than females (P , 0.05; Table 1). It is important to keep in mind that the cell counts of AROM-ir neurons in the mPOA did not include the large AROM-ir nucleus. Based on our previous work (Rissman et al., 1996), we are certain that if we had been able to include neurons in that area, the total number of AROM-ir cells in the mPOA would have exceeded that in the BNST. Student’s t tests revealed that the area of the aromatase-rich nucleus in the mPOA was significantly larger in males (mean 6 SEM 5 0.020 6 0.00037 mm2) than in females (mean 6 SEM 5 0.0047 6 0.00013 mm2) (t(14) 5 21, P , 0.002). Co-expression of ER and AROM Immunoreactivity The overall number of cells that were double-labeled for both ER and AROM immunoreactivity did not differ significantly based on sex (F(1, 33) 5 3.56 or brain area (F(2, 33) 5 1.74), nor was an interaction
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FIG. 1. Photomicrograph of the mPOA in control male (A) and female (B) musk shrews. Sections were incubated with antiserum for estrogen receptors only (Abbott H222). Note that in both sexes there is a region within the mPOA that is devoid of ER immunoreactivity (arrows). This region contains AROM immunoreactivity. 3V, third ventricle.
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FIG. 2. Double-immunocytochemical labeling of estrogen receptor containing neurons (brown nuclear stain) and aromatase enzyme containing neurons (bluish gray cytoplasmic stain) in the brain of adult male (A, C, E, and F) and female (B and D) musk shrews. In the mPOA, under low magnification, the densely packed, sexually dimorphic cluster of AROM-ir neurons that is devoid of ER immunoreactivity is shown. In A and B, the boxed areas outline the regions that are shown with higher magnification (1203) in C (male) and D (female). Higher magnifications (1203) of the VMN (E) and BNST (F) in a male musk shrew are also presented. Arrows in C–F point out double-labeled ER-ir and AROM-ir neurons. Bar in C represents 10 mm. 3V, third ventricle.
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found (F(2, 33) 5 0.18; Table 1). However, the proportion of double-labeled cells did differ significantly in different brain areas (F(2, 33) 5 32.35, P , 0.0001; Table 1). We did not detect a sex difference (F(1, 33) 5 1.83) nor was there an interaction between sex and brain area (F(2, 33) 5 1.13). Post hoc pair tests revealed that the VMN contained the largest percentage of double-labeled cells, followed by the mPOA and BNST (P , 0.05). In each of these regions, the neurons that co-localized ER and AROM enzyme were randomly distributed. The data for the mPOA did not include counts of cells in the AROM-ir nucleus. Clearly, if cells in this nucleus had been counted, the percentage of aromatase containing neurons co-expressing ER immunoreactivity would have been much lower in this area. An interesting trend was noted: the number of single-labeled AROM-ir cells was inversely related to the percentage of double-labeled cells in all three areas (Fig. 3).
DISCUSSION Based on the representative section of each neural region examined, our results demonstrate that only a minority of aromatase enzyme containing neurons in male and female musk shrew brain also contain estrogen receptors. Therefore, estrogen may exert its effects in cells other than those that contain aromatase enzyme. Co-localization of estrogen receptor and aromatase neurons has been quantified in male quail and rodents. In the Japanese quail, Balthazart et al. (1991) reported that the majority of aromatase and estrogen receptor double-immunoreactive neurons occurred in the hypothalamic region. About 60% of the AROM-ir neurons co-localized ER immunoreactivity in the VMN, 5% in the septum, and less than 20% in the POA. Tsuruo et al. (1996) reported similar findings in fetal and neonatal rat brain in the same regions. In addition, in the BNST and medial amygdala (MeA), about 75% of AROM-ir neurons co-localized ER immunoreactivity. In a similar study in mice, Tsuruo et al. (1995) reported that about 80% of the AROM-ir neurons co-localized ER immunoreactivity in the BNST and MeA, but few dual-labeled neurons were found in the mPOA and VMN. Dellovade et al. (1995) examined changes in the numbers of neurons that co-localized AROM and ER immunoreactivities in the mPOA during aging in Japanese quail. They found no significant difference in the numbers of co-labeled neurons in young sexually active males, old active males, and inactive males. However, the relative per-
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centage of dual-labeled cells increased in old males compared to young males. In contrast to these studies, we report that the musk shrew does not show a high degree of co-localization in limbic or hypothalamic brain regions. The VMN contains the highest percentage of double-labeled cells (9.5 and 13.6% in males and females, respectively); followed by the mPOA and BNST. Although we did not quantify the numbers of immunoreactive cells, we also noted a large population of AROM-ir neurons present in the MeA. Few of the AROM-ir cells in the MeA contained ER-ir. However, many other neurons in this area are ER-ir positive. Our ability to visualize AROM-ir neurons in the mPOA and hypothalamus of musk shrews depends upon high testosterone concentrations in plasma and the use of the aromatase inhibitor vorozole (Rissman et al., 1996, Veney and Rissman, unpublished results). This procedure is not necessary for visualizing AROM immunoreactivity in neonatal rodents or quail. One explanation for this difference is that aromatase enzyme levels may be relatively lower in musk shrew brains than in quail and young rodent brains. It is not clear why vorozole and fadrazole, another nonsteroidal aromatase inhibitor, increases immunoreactivity for aromatase enzyme. However, this effect has been noted in quail, mice, and musk shrews (Foidart, Tlemcani, Harada, Abe-Dohmae, and Balthazart, 1995; Foidart et al., 1995a; Rissman et al., 1996). In quail, the distribution of AROM-ir neurons is not altered by drug treatment; the density of stain is merely increased in individual cells (Foidart, Harada, and Balthazart, 1994). In mice, treatment with vorozole reveals AROM-ir neurons in several hypothalamic nuclei including the mPOA and nucleus accumbens (Foidart et al., 1995a). These same regions, although they contain high levels of AROM enzyme activity, show very little or no AROM immunoreactivity in brains of untreated animals. Thus, we speculate that vorozole simply reveals cells that contain AROM immunoreactivity, but are too faint to be seen without its enhancing effects. The mechanism for this enhancement is unknown, but Foidart et al. (1995b) speculate that vorozole increases the half-life of aromatase enzyme and/or increases translation of its message. Because we needed to employ vorozole to visualize neurons that are immunoreactive for aromatase enzyme, we assessed the possibility that this drug interferes with the ER immunoreactivity. We know that the presence of E2 in shrew brains could affect the detection of ER-ir cells since the antibody we employed (H222) preferentially recognizes unbound estrogen re-
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FIG. 3. The number of AROM-ir neurons and the percentage of colocalized ER-ir and AROM-ir neurons in each of three brain regions in males (n 5 7) and females (n 5 6). In each region, the number of single-labeled AROM-ir cells was inversely related to the number of double-labeled cells.
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ceptors (Blaustein, 1993; Clancy and Michael, 1994). Vorozole treatment in theory should reduce E2 concentrations and should, if anything, increase ER-ir. In fact, Foidart et al. (1994) and Rissman et al. (1996) have reported that aromatase activity in brain drops soon after a single injection of vorozole. Up to 4 h after the injection, aromatase enzyme activity is significantly decreased from baseline. However, in quail brain, 16 h after treatment aromatase enzyme activity rebounds and exceeds baseline (Foidart et al., 1994). In the present experiment, each animal received a single injection of vorozole, every 24 h for 7 days. It is possible that these repeated, spaced injections produced dramatic fluctuations in E2 concentrations in brain. Control animals that did not receive vorozole had denser appearing ER immunoreactivity in the mPOA and VMN, yet the AROM-ir nucleus in the mPOA did not contain ER immunoreactivity in these brains or in brains of shrews that were treated with vorozole. The available data suggest that vorozole did not interfere with the pattern of ER-ir. In the mPOA, there is a sexually dimorphic, densely packed cluster of AROM-ir neurons that lack ER immunoreactivity. These neurons are also present in Nissl-stained musk shrew material (Veney and Rissman, unpublished data). Based on the rat brain atlas (Paxinos and Watson, 1986) we tentatively identify this nucleus as the homologue to the rodent central medial preoptic area. Although many studies have shown that aromatase enzyme activity is regulated by testosterone, we believe that different levels of testosterone in males and females do not cause the sex difference in immunoreactivity in this nucleus. This dimorphism is present in gonad-intact animals (Rissman et al., 1996) as well as in males and females receiving equivalent testosterone treatments (Freeman, Arora, and Rissman, 1997). We hypothesize that the large cluster of AROM-ir neurons in the mPOA produces estrogens that bind to receptors just outside of this region. Many ER-ir neurons reside just lateral and ventral to this nucleus. Based on previous work, we know that the mPOA is essential for female sexual behavior. Unilateral testosterone implants in this region reinstate sexual behavior in ovariectomized animals (Sharma and Rissman, 1994; Veney and Rissman, 1997a) and unilateral chemical lesions with an excitatory neurotoxin block behavior (Veney and Rissman, 1997b). Thus, data from females suggest that the subpopulation of AROM-ir and ER-ir neurons in the POA is essential for sexual behavior. Paradoxically, we have yet to determine the function of AROM in the male shrew. In future studies we will examine the role
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of aromatase enzyme acting in the mPOA of the male musk shrew. In musk shrews, what is the significance of having such limited co-localization of estrogen receptors and aromatase enzyme? Does this near separation of AROM-ir and ER-ir neurons suggest anything about possible steroid regulation of aromatase enzyme? Androgens can increase hypothalamic aromatase activity (Roselli and Resko, 1993; Schumacher and Balthazart, 1986) and aromatase mRNA (Abdelgadir, Resko, Ojeda, Lephart, McPhaul, and Roselli, 1994) through androgen receptor mediated processes in adult rats and quail. In birds, estradiol can enhance levels of preoptic aromatase (Hutchison and Steimer, 1986; Foidart, Houbart, Harada, and Balthazart, 1996). Finally, synergistic actions of both androgens and estrogens on aromatase enzyme activity (Roselli and Resko, 1993) and aromatase mRNA (Harada, Abe-Dohmae, Loeffen, Foidart, and Balthazart, 1993) have been found in adult rats and quail. In the musk shrew, because so few cells contain both AROM-ir and ER-ir neurons, we predict that if estrogen affects aromatase enzyme, it does so indirectly. For example, estrogen may be part of a biochemical cascade involving many neuromodulatory hormones that eventually leads to changes in aromatase enzyme. Estrogen receptors are located in dopaminergic neurons (Sar, 1984), and there have been suggestions in the literature that estrogens can interact with dopamine to regulate aromatase enzyme (Balthazart and Ball, 1992). Alternatively, androgens could regulate aromatase enzyme in musk shrews. Unfortunately, to our knowledge, there have not been any immunohistochemical studies which have addressed the possibility of androgen receptors co-localizing with AROM-ir neurons in mammalian brain. Currently, we are examining this possibility in the musk shrew. It is also possible that estrogen may be affecting aromatase enzyme by acting through a membrane or other form of genomic ER, such as ERb. Estrogen bound to these alternative forms of ER may not have been detected by the H222 antibody. In male rats and quail, aromatase is involved in copulation (Adkins et al., 1980; Christensen and Clemens, 1975). In addition, in male quail, implants of T or estradiol benzoate (EB), but not the nonaromatizable androgen dihydrotestosterone (DHT), are able to reinstate aggressive behaviors after castration (Schlinger and Callard, 1989, 1990). Likewise, estrogen has been shown to mimic the effects of T in stimulating aggressive behaviors in castrated mice (Edwards and Burge, 1971), rats (Christie and Barfield, 1979),
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and hamsters (Vandenbergh, 1971). Estrogens may also be critical for the expression of chemoinvestigatory behaviors. In male Syrian hamsters for example, integration of chemosensory and hormonal cues in the MeA, mPOA, and BNST are critical for male mating behavior (Wood and Newman, 1995a). Neural implants of T or E2, but not DHT, in the MeA or BNST/ mPOA can increase the numbers of interactions between males and females and the number of anogenital investigations (Wood and Newman, 1995b; Wood, 1996). In male musk shrews, initial attempts to block sexual behavior with vorozole have not met with success (Veney and Rissman, unpublished data). However, we have yet to look at any other social behaviors in males. In female musk shrews, aromatase enzyme is necessary for receptivity (Rissman, 1991), but it may also play a role in other aspects of social behavior. For example, female shrews are highly aggressive toward males when they are first introduced in a mating test. When a male approaches a female, she lunges and displays open mouth threats accompanied by short, high-pitched vocalizations. Females also approach males and sniff and investigate male-soiled regions in the test cages. These behaviors continue until the female becomes receptive and receives mounts and intromissions from the male. We have not yet specifically examined the effects of aromatase inhibition on the expression of these other social behaviors in female musk shrews. One intriguing observation may shed some light on this issue. Recently we have examined c-fos immunoreactivity after mating in male and female musk shrews (Gill, Cholbi, and Rissman, 1996). In mated females, fos-ir neurons are present in the same region that contains the dense cluster of AROM-ir neurons. In mated males, however, this same area is devoid of fos-ir cells. This observation may reflect the different roles that aromatase enzyme plays in the expression of sexual behavior in males versus females. Sexual dimorphisms in the mPOA have been reported in several other mammals (Cherry, Basham, Weaver, Krohmer, and Baum, 1990; Ulibarri and Yahr, 1993; Gorski, Harlan, Jacobson, Shryne, and Southam, 1980). The direction of the sex differences are typically, as we report here, that males have more cells in the region than do females, but in the cases where a function has been found, the nuclei appear to regulate some aspect of masculine social behavior (Yahr and Stephens, 1987; Arendash and Gorski, 1983; Paredes and Baum, 1995). In musk shrews our data suggest the opposite—that
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the nucleus may have a function in female social behavior.
ACKNOWLEDGMENTS The authors thank Drs. Y. Osawa and C. P. Yarborough for generously supplying the R-8-1 aromatase antibody. We thank the reviewers of the manuscript and Michael Baum for their excellent comments and suggestions. We also acknowledge assistance from the Ligand Preparation and Assay Core (NIH Grant P30-HD28934, Center for Cellular and Molecular Studies in Reproduction). This work was supported by NSF Grant IBN-9412605 (E.F.R.) and NIH Grants MH01349, NS35429 (E.F.R.), and HD04945 (Y. Osawa). Sean Veney was supported, in part, by NIH Training Grant HD072323.
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Paxinos, G., and Watson, C. (1986). The Rat Brain in Stereotaxic Coordinates. Academic Press, San Diego. Rissman, E. F., and Crews, D. (1988). Hormonal correlates of sexual behavior in the female musk shrew: The role of estradiol. Physiol. Behav. 44, 1–7. Rissman, E. F., and Crews, D. (1989). Effect of castration on epididymal sperm storage in male musk shrews (Suncus murinus) and mice (Mus musculus). J. Reprod. Fertil. 86, 219 –222. Rissman, E. F. (1991). Evidence that neural aromatization of androgen regulates the expression of sexual behavior in female musk shrews. J. Neuroendocrinol. 3, 441– 448. Rissman, E. F., Harada, N., and Roselli, C. E. (1996). Effect of vorozole, an aromatase enzyme inhibitor, on sexual behavior, aromatase activity and neural immunoreactivity. J. Neuroendocrinol. 8, 199 –210. Roselli, C. E., Horton, L. E., and Resko, J. A. (1985). Distribution and regulation of aromatase activity in the rat hypothalamus and limbic system. Endocrinology 117, 2471–2477. Roselli, C. E., and Resko, J. A. (1989). Testosterone regulates aromatase activity in discrete brain areas of the male rhesus macaques. Biol. Reprod. 40, 1– 6. Roselli, C. E., and Resko, J. A. (1993). Aromatase activity in the rat brain: Hormonal regulation and sex differences. J. Steroid Biochem. Mol. Biol. 44, 499 –508. Sar, M. (1984). Estradiol is concentrated in tyrosine hydroxylasecontaining neurons of the hypothalamus. Science 223, 938 –940. Selmanoff, M. K., Brodkin, L. D., Weiner, R. I., and Siiteri, P. K. (1977). Aromatization and 5a-reduction of androgens in discrete hypothalamic and limbic regions of the male and female rat. Endocrinology 101, 841– 848. Schlinger, B. A., and Callard, G. V. (1989). Aromatase activity in quail brain: Correlation with aggressiveness. Endocrinology 124, 437– 443. Schlinger, B. A., and Callard, G. V. (1990). Aromatization mediates aggressive behavior in quail. Gen. Comp. Endocrinol. 79, 39 –53. Schumacher, M., and Balthazart, J. (1986). Testosterone-induced brain aromatase is sexually dimorphic. Brain Res. 370, 285–293. Schumacher, M., and Balthazart, J. (1987). Neuroanatomical distribution of testosterone metabolizing enzymes in the Japanese quail. Brain Res. 422, 137–148. Sharma, U. R., and Rissman, E. F. (1994). Testosterone implants in specific neural sites activate female sexual behavior. J. Neuroendocrinol. 6, 423– 432. Tsuruo, Y., Ishimura, K., and Osawa, Y. (1995). Presence of estrogen receptors in aromatase-immunoreactive neurons in the mouse brain. Neurosci. Lett. 195, 49 –52. Tsuruo, Y., Ishimura, K., Hayashi, S., and Osawa, Y. (1996). Immunohistochemical localization of estrogen receptors within aromatase-immunoreactive neurons in the fetal and neonatal rat brain. Anat. Embryol. 193, 115–121. Ulibarri, C. M., and Yahr, P. (1993). Ontogeny of the sexually dimorphic area of the gerbil hypothalamus. Brain Res. Dev. Brain Res. 74, 14 –24. Vandenbergh, J. A., (1971). The effects of gonadal hormones on the aggressive behavior of adult golden hamsters. Anim. Behav. 19, 589 –594. Veney, S. L., and Rissman, E. F. (1997a). Neural testosterone implants activate female sexual behavior. Neurosci. Abstr. 23(2), 1853. Veney, S. L., and Rissman, E. F. (1997b). Axon sparing lesions of the medial preoptic area block female sexual behavior. Brain Res. 756, 273–277. Watson, J. T., and Adkins-Regan, E. (1989). Activation of sexual
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sexual behavior in the male Syrian hamster. Horm. Behav. 29, 338 –353. Wouters, W., De Coster, R., Beerens, D., Doolaege, R., Gruwez, J. A., van Camp, K., van der Pas, H., and van Herendael, B. (1990). Potency and selectivity of the aromatase inhibitor R 76713. A study in human ovarian, adipose stromal, testicular and adrenal cells. J. Steroid Biochem. 36(1/2), 57– 65. Yahr, P., and Stephens, D. R. (1987). Hormonal control of sexual and scent marking behaviors of male gerbils in relation to the sexually dimorphic area of the hypothalamus. Horm. Behav. 21, 331–346.
Co-localization of Estrogen Receptor and Aromatase Enzyme Immunoreactivities in Adult Musk Shrew Brain Sean L. Veney and Emilie F. Rissman1 Department of Biology, University of Virginia, Charlottesville, Virginia 22903 Received September 19, 1997; revised February 24, 1998; accepted March 30, 1998
Estrogens are produced by the aromatization of androgens. These steroids exert their actions after binding to their receptors. Past studies have shown that estrogen receptors (ER) and aromatase enzyme (AROM) reside in many of the same brain regions. Few studies, however, have examined the neural co-localization of these important components involved in estrogen-activated behaviors. In the present study we examined the co-localization of ER and AROM immunoreactive (ir) neurons in musk shrew (Suncus murinus) brains. Data were collected from a representative section from three neural regions, the bed nucleus of the stria terminalis (BNST), medial preoptic area (mPOA), and ventromedial nucleus of the hypothalamus (VMN). Here we report a sex difference in the number of ER-ir neurons from the analyzed section of the mPOA and BNST. Females have more ER-ir neurons in the mPOA and males have more in the BNST. In the sections we examined, males tended to have more aromatase containing neurons than females. Although there were no significant differences in the numbers of double-labeled cells, the VMN contains the greatest percentage of these cells in both males and females; followed by the mPOA and the BNST. In addition, in the mPOA of both sexes, a distinct nucleus of aromatase containing neurons which was devoid of ER immunoreactivity was noted. Area measurements of the AROM-ir nucleus showed that it was significantly larger in males than in females. Taken together, these data suggest that there is not extensive cellular co-localization of estrogen receptors and aromatase enzyme in the musk shrew brain. However, the presence of other genomic forms of ER (membrane and/or ERb) in AROM containing neurons has not been ruled out by this study. Thus, we hypothesize that estrogens produced in brain affect behavior by binding to ER in neurons other than those that contain aromatase enzyme. © 1998 Academic Press
1 To whom correspondence should be addressed. Fax: (804) 9825626. E-mail: efr2fVirginia.edu.
0018-506X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
Aromatase enzyme is a member of the cytochrome P450 family that irreversibly catalyzes the conversion of androstenedione to estrone and testosterone to estradiol. This enzyme is present in the brain, gonads, and other peripheral tissue (Naftolin, Ryan, Davies, Reddy, Flores, Petro, Kuhn, White, Takoka, and Wolin, 1975; Selmanoff, Brodkin, Weiner, and Siiteri, 1977). Neural aromatization of androgens to estrogens is necessary for the activation of sexual behavior in a number of vertebrate species. The majority of these data have been collected in male Japanese quail and rats (Adkins, Boop, Koutnik, Morris, and Pniewski, 1980; Balthazart, Foidart, and Hendrick, 1990; Christensen and Clemens, 1974). Similar to rats and quail, female musk shrew sexual behavior is also dependent upon neural aromatization of testosterone (T) to estradiol (E2); (Rissman, 1991). In all of these species, aromatase activity is highest in preoptic, hypothalamic, and limbic nuclei (Rissman, Harada, and Roselli, 1996; Roselli, Horton, and Resko, 1985; Roselli and Resko, 1989; Schumacher and Balthazart, 1987). Several of these regions, including the bed nucleus of the stria terminalis (BNST) and the medial preoptic area (mPOA), are important for the regulation of sexual behavior in rats. Implants containing either T or E2 placed in the mPOA can restore sexual behavior in castrated male quail and rats (Balthazart and Surlemont, 1990; Watson and Adkins-Regan, 1989; Christensen and Clemens, 1974). Also, T implants placed into the mPOA of female musk shrews reinstate sexual behavior after ovariectomy (Sharma and Rissman, 1994; Veney and Rissman, 1997a). Immunocytochemistry has been used to examine co-localization of estrogen receptors and aromatase containing neurons in male Japanese quail (Balthazart, Foidart, Surlemont, and Harada, 1991; Dellovade, Rissman, Thompson, Harada, and Ottinger, 1995), fe-
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tal and neonatal rat (Tsuruo, Ishimura, Hayashi, and Osawa, 1996), and mouse (Tsuruo, Ishimura, and Osawa, 1995) brain. In the musk shrew, the distribution of neurons that are immunoreactive for estrogen receptors (ER-ir; Dellovade, Blaustein, and Rissman, 1992) and aromatase enzyme (AROM-ir; Rissman et al., 1996) has been mapped. Neurons that express either ER or AROM immunoreactivity reside in many of the same brain regions. It is not known, however, whether the two are co-localized in the same cell. In the present study, we describe the co-expression of ER and AROM enzyme immunoreactivities in male and female musk shrew brains. At present, we do not know whether neural aromatization is involved in male musk shrew sexual behavior. Female musk shrews, however, require aromatization of T to E2 in order to show receptivity. We hypothesized that the neural co-expression of ER and AROM enzyme in females, and possibly male shrews, would be similar to that described previously in males of other species. The co-expression of ER and AROM enzyme immunoreactivities were examined in a representative section from three neural regions: the bed nucleus of the stria terminalis (BNST), medial preoptic area (mPOA), and ventromedial nucleus of the hypothalamus (VMN).
MATERIALS AND METHODS General Methods Musk shrews were born and raised in the colony at the University of Virginia. The colony was maintained on a light cycle of 14 h light/10 h dark (lights on at 0600 h, EST) at a temperature of 23 6 1°C. Food (Purina Cat Chow and Complete Mink Pellets from Milk Specialty Products, New Holstein, WI) and water were provided ad libitum. Animals were weaned at 18 –20 days of age and individually housed (cage dimensions 28 3 17 3 12 cm) with pine wood shavings and paper towels for bedding. Experimental Design and Treatment Males (60 days of age; n 5 7) and females (30 days of age; n 5 6) were gonadectomized and given a testosterone (T) implant (10 and 5 mm, respectively). Females are sexually mature by 26 days of age, and males are capable of siring young by 45 days. Thus, both groups were adult, mature individuals. The hormone was packed into Silastic tubing (1 mm ID 3 2.125 mm OD) and implanted subcutaneously (sc) in
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the back of the neck. Beginning 2 days after surgery, animals received daily (sc) injections of the anti-aromatase drug vorozole (5 mg/kg bw, generously provided by Janssen Research Foundation) for 7 days. Vorozole is a nonsteroidal anti-aromatase enzyme drug. In vivo it causes a drop in aromatase activity levels and estrogen concentrations, but it does not bind estrogen or other steroid receptors (Wouters, DeCoster, Beerens, Doolaege, Gruwez, van Camp, van der Pas, and Herendael, 1990). Previous studies have shown that injections of vorozole enhance specific staining of AROM-ir neurons in musk shrews (Rissman et al., 1996) and mice (Foidart, Harada, and Balthazart, 1995). Control animals (n 5 3 males and n 5 3 females) were gonadectomized, T-implanted, and given daily (sc) injections of the vehicle (PEG-400). Hormone Assay Prior to perfusion, 250 – 400 ml of blood was drawn from each animal by cardiac puncture. Blood samples were spun, and plasma was collected and stored at 270°C until assay. The plasma samples were assayed in singlet for T using the Diagnostic Products testosterone kit. This assay has been validated previously for use with musk shrew plasma (Fortman, Dellovade, and Rissman, 1992). The calculated dose of T at 90% binding was 0.18 ng/ml. The intra-assay variability averaged 6.94%. The antibody cross-reactivity was 100% with T, 7.8% with DHT, 2% with 11-oxy-testosterone, and 0.56% with androstenedione. Based on the results of the hormone assay, female plasma T concentrations ranged from 1.08 to 2.97 ng/ml; with a mean of 1.97 ng/ml. Male plasma T levels ranged from 3.95 to 5.76 ng/ml; with a mean of 5.18 ng/ml. These concentrations are higher than physiological hormone levels for gonad-intact male and female musk shrews (Rissman and Crews, 1988, 1989). Immunocytochemistry After 7 days of vorozole treatment, and within 2 h of the last injection, each animal was sacrificed by an overdose of sodium pentobarbital. The animals were perfused with heparinized saline (100 units/ml) followed by Zamboni’s fixative. Brains were cryoprotected overnight in 20% sucrose. The following day they were quick-frozen in cold methylbutane and stored at 270°C. Frozen brains were coronally cut to a thickness of 25 mm into a series of four wells and stored in antifreeze at 220°C until immunocytochemistry processing. Sections were collected from the ros-
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tral forebrain through the rostral portion of the midbrain central gray. Double-label immunocytochemistry was performed to visualize neurons immunoreactive for estrogen receptors and/or aromatase enzyme. One-quarter of the sections from each brain were treated with 1% NaBH4 in TBS (Tris-buffered saline, pH 7.8) to remove aldehydes, followed by avidin– biotin blocker for endogenous biotin. Tissue was incubated for 48 h in primary antibody diluted in 0.25% lambda carrageenan, 1% BSA, 0.3% Triton, and 0.1% sodium azide in TBS. Sections were simultaneously incubated with the rat monoclonal anti-estrogen receptor antiserum (H222, 1:500; generously provided by Abbott Laboratories) and a polyclonal aromatase antiserum (R-8-1, 1:500; kindly supplied by Dr. Y. Osawa; Kitawaki, Yoshida, and Osawa, 1989). Vector biotinylated donkey anti-rat IgG (double-bridged at 1:500) and Vector Elite avidin– biotin complex, followed by DAB, were used to visualize the ER-ir cells. To maximize the reaction product, the tissue was incubated in DAB for 20 min and then 15 ml of NiSO4(NH4)2SO4 was added for an additional 1–5 min. The addition of the NiSO4(NH4)2SO4 catalyzed the DAB reaction and increased the brown reaction product. Thus, total reaction time in DAB was 20 –25 min. Following development in DAB, sections were rinsed and stored overnight in TBS at 4°C. The following day, AROM immunoreactivity was visualized by a similar procedure. Vector biotinylated goat anti-rabbit IgG (double-bridged at 1:250) and SG (Vector Laboratories) were used to visualize the reaction product. The Vector SG substrate for peroxidase yields a blue-gray reaction product. Total incubation time in SG was 3–5 min. Following immunocytochemistry, sections were mounted on slides and allowed to airdry overnight. The following day, the sections were dehydrated and the slides were coverslipped. We have reported on the results of preadsorption controls with H222 in the past (Dellovade et al., 1992). To validate the specificity of AROM immunoreactivity, R-8-1 was preincubated with either 1 or 5 mg of immunoaffinity-purified aromatase P450 for 24 h before the tissue sections were added. Both concentrations of antigen eliminated specific staining. To examine the possible effect of vorozole treatment on ER immunoreactivity, brains from the control animals were examined. These animals (three males and three females) were gonadectomized, treated with a Silastic T-implant, and given vehicle injections in place of vorozole. Every fourth section was developed with H222 antibody (as described above) and the distribu-
tion of ER immunoreactivity was compared to that of animals treated with vorozole. Image Analysis Neurons that contained immunoreactive estrogen receptors or aromatase enzyme, as well as neurons that were double-labeled for both, were counted in one representative section from each of three brain regions: BNST, mPOA, and VMN. A comparable section from each brain region of each animal was matched and analyzed under the microscope at magnifications of 80 –1203. To select matched sections from each individual we used the following landmarks. For the mPOA, we selected a section comparable to the rat brain atlas, between plates 20 and 21 (Paxinos and Watson, 1986). The anterior commissure was caudal to its thickest extent and tapering on the lateral edges. The lateral ventricles were nearly joined at the midline. For the BNST, we selected a section comparable to the rat brain atlas, plate 22 (Paxinos and Watson, 1986). This was the next section in our series, 75 mm caudal to the mPOA. The lateral ventricles were joined at the midline and the anterior commissure was not present. The section examined for the VMN was similar to plate 28 in the rat atlas (Paxinos and Watson, 1986). On this section, all fields (CA1-3) of the hippocampus were present, as was the dentate gyrus. The median eminence was just forming along the midline. Images were captured on an Olympus U-CMAD-2 color video camera and cell counts were conducted using Mocha statistical image analysis software (Jandel Scientific). AROM-ir cells were defined as such based on the appearance of a clear nucleus and the presence of at least one cell process. A large, sexually dimorphic nucleus containing AROM-ir neurons is present in the musk shrew mPOA (Rissman et al., 1996). This nucleus is present in Nissl-stained material and these were used to help define the AROM-ir region as well as the areas that we selected for the counts (Veney and Rissman, unpublished data). In the present study, the nucleus was so densely stained with AROM-ir reaction product that counts of individual cells were not possible. Therefore, we quantified the area of the nucleus. Using Mocha image analysis software, a calibration was made for the magnification used to measure the nucleus and the number of pixels within a 1 mm by 1 mm square was determined. An outline was drawn free-handed around the outermost edges of the AROM-ir nucleus. The resulting number of pixels was converted to an area measurement (mm2) based on the calibration.
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TABLE 1 Estrogen Receptor and Aromatase Enzyme Immunoreactive Cell Counts
? ? ? / / /
BNST mPOA VMN BNST mPOA VMN
ER-ir
AROM-ir
# Double Labeled
% Double Labeled
71.0 6 6.0* 179.3 6 20.7* 179.6 6 22.0 42.5 6 9.0 431.0 6 36.5 217.8 6 13.9
1037.6 6 50.7a 196.7 6 7.9b 98.1 6 8.4c 973.2 6 58.9a 133.2 6 10.8b 47.8 6 3.9c
13.7 6 3.1 12.0 6 1.7 9.3 6 1.5 9.2 6 1.5 9.8 6 2.1 6.5 6 1.5
1.32 6 1.2a 6.1 6 1.2b 9.5 6 1.2c 0.95 6 1.3a 7.4 6 1.3b 13.6 6 1.3c
Note. Cell counts were taken from three neural regions in male (n 5 7) and female (n 5 6) brains. Data are presented as means 6 SEM. * Significant sex difference in that brain region; P , 0.05 at least. Values with different letters are significantly different from each other; P , 0.05 at least.
Data Analysis Cell count data were analyzed by analysis of variance (two-way ANOVA). The two factors were sex and brain area. When interactions were noted, twotailed Student–Newman–Keuls tests were used. Student’s t tests, corrected for multiple comparisons, were used to analyze the size of the aromatase nucleus in the mPOA. Effects were considered significant at an a of 0.05 or less.
RESULTS Counts of ER-ir and AROM-ir, as well as the numbers and percentage of dual-labeled cells, were recorded from a representative section of the BNST, mPOA, and VMN in males and females (Table 1). In the mPOA, the sexually dimorphic nucleus of densely packed aromatase containing neurons did not contain immunoreactive estrogen receptors. Brains from control animals, treated only with the vehicle and incubated with the anti-ER antiserum, confirmed that the nucleus was devoid of ER immunoreactivity (Figs. 1 and 2).Therefore, in the mPOA, only ER-ir and/or AROM-ir cells outside of the large aromatase nucleus were counted. Estrogen Receptor Immunoreactivity There was a significant difference in the number of neurons that contained estrogen receptors based on sex (F(1, 33) 5 27.7, P , 0.0001) and brain area (F(2, 33) 5 75.4, P , 0.0001), and an interaction between sex and brain area was revealed (F(2, 33) 5 26.0, P , 0.0001; Table 1). A Newman–Keuls follow-up of the interaction to determine the effect of sex on the number of ER-ir cells in each brain region was conducted.
Females had significantly more ER-ir neurons in the mPOA than males did (P , 0.05). However, males had more ER-ir cells in the BNST than females (P , 0.05). There was no sex difference in ER-ir cells in the VMN. Aromatase Enzyme Immunoreactivity There was a significant difference in the number of AROM-ir neurons based on sex (F(1, 33) 5 5.09, P 5 0.031) and brain area (F(2, 33) 5 507.92, P , 0.0001; Table 1). There was no interaction between the two variables (F(2, 33) 5 0.030). Pair tests revealed that the number of AROM-ir neurons in the BNST was significantly greater than in the VMN or mPOA, and more AROM-ir neurons were present in the mPOA than in the VMN (P , 0.05). In addition, males had more AROM-ir neurons in each region than females (P , 0.05; Table 1). It is important to keep in mind that the cell counts of AROM-ir neurons in the mPOA did not include the large AROM-ir nucleus. Based on our previous work (Rissman et al., 1996), we are certain that if we had been able to include neurons in that area, the total number of AROM-ir cells in the mPOA would have exceeded that in the BNST. Student’s t tests revealed that the area of the aromatase-rich nucleus in the mPOA was significantly larger in males (mean 6 SEM 5 0.020 6 0.00037 mm2) than in females (mean 6 SEM 5 0.0047 6 0.00013 mm2) (t(14) 5 21, P , 0.002). Co-expression of ER and AROM Immunoreactivity The overall number of cells that were double-labeled for both ER and AROM immunoreactivity did not differ significantly based on sex (F(1, 33) 5 3.56 or brain area (F(2, 33) 5 1.74), nor was an interaction
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FIG. 1. Photomicrograph of the mPOA in control male (A) and female (B) musk shrews. Sections were incubated with antiserum for estrogen receptors only (Abbott H222). Note that in both sexes there is a region within the mPOA that is devoid of ER immunoreactivity (arrows). This region contains AROM immunoreactivity. 3V, third ventricle.
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FIG. 2. Double-immunocytochemical labeling of estrogen receptor containing neurons (brown nuclear stain) and aromatase enzyme containing neurons (bluish gray cytoplasmic stain) in the brain of adult male (A, C, E, and F) and female (B and D) musk shrews. In the mPOA, under low magnification, the densely packed, sexually dimorphic cluster of AROM-ir neurons that is devoid of ER immunoreactivity is shown. In A and B, the boxed areas outline the regions that are shown with higher magnification (1203) in C (male) and D (female). Higher magnifications (1203) of the VMN (E) and BNST (F) in a male musk shrew are also presented. Arrows in C–F point out double-labeled ER-ir and AROM-ir neurons. Bar in C represents 10 mm. 3V, third ventricle.
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found (F(2, 33) 5 0.18; Table 1). However, the proportion of double-labeled cells did differ significantly in different brain areas (F(2, 33) 5 32.35, P , 0.0001; Table 1). We did not detect a sex difference (F(1, 33) 5 1.83) nor was there an interaction between sex and brain area (F(2, 33) 5 1.13). Post hoc pair tests revealed that the VMN contained the largest percentage of double-labeled cells, followed by the mPOA and BNST (P , 0.05). In each of these regions, the neurons that co-localized ER and AROM enzyme were randomly distributed. The data for the mPOA did not include counts of cells in the AROM-ir nucleus. Clearly, if cells in this nucleus had been counted, the percentage of aromatase containing neurons co-expressing ER immunoreactivity would have been much lower in this area. An interesting trend was noted: the number of single-labeled AROM-ir cells was inversely related to the percentage of double-labeled cells in all three areas (Fig. 3).
DISCUSSION Based on the representative section of each neural region examined, our results demonstrate that only a minority of aromatase enzyme containing neurons in male and female musk shrew brain also contain estrogen receptors. Therefore, estrogen may exert its effects in cells other than those that contain aromatase enzyme. Co-localization of estrogen receptor and aromatase neurons has been quantified in male quail and rodents. In the Japanese quail, Balthazart et al. (1991) reported that the majority of aromatase and estrogen receptor double-immunoreactive neurons occurred in the hypothalamic region. About 60% of the AROM-ir neurons co-localized ER immunoreactivity in the VMN, 5% in the septum, and less than 20% in the POA. Tsuruo et al. (1996) reported similar findings in fetal and neonatal rat brain in the same regions. In addition, in the BNST and medial amygdala (MeA), about 75% of AROM-ir neurons co-localized ER immunoreactivity. In a similar study in mice, Tsuruo et al. (1995) reported that about 80% of the AROM-ir neurons co-localized ER immunoreactivity in the BNST and MeA, but few dual-labeled neurons were found in the mPOA and VMN. Dellovade et al. (1995) examined changes in the numbers of neurons that co-localized AROM and ER immunoreactivities in the mPOA during aging in Japanese quail. They found no significant difference in the numbers of co-labeled neurons in young sexually active males, old active males, and inactive males. However, the relative per-
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centage of dual-labeled cells increased in old males compared to young males. In contrast to these studies, we report that the musk shrew does not show a high degree of co-localization in limbic or hypothalamic brain regions. The VMN contains the highest percentage of double-labeled cells (9.5 and 13.6% in males and females, respectively); followed by the mPOA and BNST. Although we did not quantify the numbers of immunoreactive cells, we also noted a large population of AROM-ir neurons present in the MeA. Few of the AROM-ir cells in the MeA contained ER-ir. However, many other neurons in this area are ER-ir positive. Our ability to visualize AROM-ir neurons in the mPOA and hypothalamus of musk shrews depends upon high testosterone concentrations in plasma and the use of the aromatase inhibitor vorozole (Rissman et al., 1996, Veney and Rissman, unpublished results). This procedure is not necessary for visualizing AROM immunoreactivity in neonatal rodents or quail. One explanation for this difference is that aromatase enzyme levels may be relatively lower in musk shrew brains than in quail and young rodent brains. It is not clear why vorozole and fadrazole, another nonsteroidal aromatase inhibitor, increases immunoreactivity for aromatase enzyme. However, this effect has been noted in quail, mice, and musk shrews (Foidart, Tlemcani, Harada, Abe-Dohmae, and Balthazart, 1995; Foidart et al., 1995a; Rissman et al., 1996). In quail, the distribution of AROM-ir neurons is not altered by drug treatment; the density of stain is merely increased in individual cells (Foidart, Harada, and Balthazart, 1994). In mice, treatment with vorozole reveals AROM-ir neurons in several hypothalamic nuclei including the mPOA and nucleus accumbens (Foidart et al., 1995a). These same regions, although they contain high levels of AROM enzyme activity, show very little or no AROM immunoreactivity in brains of untreated animals. Thus, we speculate that vorozole simply reveals cells that contain AROM immunoreactivity, but are too faint to be seen without its enhancing effects. The mechanism for this enhancement is unknown, but Foidart et al. (1995b) speculate that vorozole increases the half-life of aromatase enzyme and/or increases translation of its message. Because we needed to employ vorozole to visualize neurons that are immunoreactive for aromatase enzyme, we assessed the possibility that this drug interferes with the ER immunoreactivity. We know that the presence of E2 in shrew brains could affect the detection of ER-ir cells since the antibody we employed (H222) preferentially recognizes unbound estrogen re-
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FIG. 3. The number of AROM-ir neurons and the percentage of colocalized ER-ir and AROM-ir neurons in each of three brain regions in males (n 5 7) and females (n 5 6). In each region, the number of single-labeled AROM-ir cells was inversely related to the number of double-labeled cells.
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ceptors (Blaustein, 1993; Clancy and Michael, 1994). Vorozole treatment in theory should reduce E2 concentrations and should, if anything, increase ER-ir. In fact, Foidart et al. (1994) and Rissman et al. (1996) have reported that aromatase activity in brain drops soon after a single injection of vorozole. Up to 4 h after the injection, aromatase enzyme activity is significantly decreased from baseline. However, in quail brain, 16 h after treatment aromatase enzyme activity rebounds and exceeds baseline (Foidart et al., 1994). In the present experiment, each animal received a single injection of vorozole, every 24 h for 7 days. It is possible that these repeated, spaced injections produced dramatic fluctuations in E2 concentrations in brain. Control animals that did not receive vorozole had denser appearing ER immunoreactivity in the mPOA and VMN, yet the AROM-ir nucleus in the mPOA did not contain ER immunoreactivity in these brains or in brains of shrews that were treated with vorozole. The available data suggest that vorozole did not interfere with the pattern of ER-ir. In the mPOA, there is a sexually dimorphic, densely packed cluster of AROM-ir neurons that lack ER immunoreactivity. These neurons are also present in Nissl-stained musk shrew material (Veney and Rissman, unpublished data). Based on the rat brain atlas (Paxinos and Watson, 1986) we tentatively identify this nucleus as the homologue to the rodent central medial preoptic area. Although many studies have shown that aromatase enzyme activity is regulated by testosterone, we believe that different levels of testosterone in males and females do not cause the sex difference in immunoreactivity in this nucleus. This dimorphism is present in gonad-intact animals (Rissman et al., 1996) as well as in males and females receiving equivalent testosterone treatments (Freeman, Arora, and Rissman, 1997). We hypothesize that the large cluster of AROM-ir neurons in the mPOA produces estrogens that bind to receptors just outside of this region. Many ER-ir neurons reside just lateral and ventral to this nucleus. Based on previous work, we know that the mPOA is essential for female sexual behavior. Unilateral testosterone implants in this region reinstate sexual behavior in ovariectomized animals (Sharma and Rissman, 1994; Veney and Rissman, 1997a) and unilateral chemical lesions with an excitatory neurotoxin block behavior (Veney and Rissman, 1997b). Thus, data from females suggest that the subpopulation of AROM-ir and ER-ir neurons in the POA is essential for sexual behavior. Paradoxically, we have yet to determine the function of AROM in the male shrew. In future studies we will examine the role
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of aromatase enzyme acting in the mPOA of the male musk shrew. In musk shrews, what is the significance of having such limited co-localization of estrogen receptors and aromatase enzyme? Does this near separation of AROM-ir and ER-ir neurons suggest anything about possible steroid regulation of aromatase enzyme? Androgens can increase hypothalamic aromatase activity (Roselli and Resko, 1993; Schumacher and Balthazart, 1986) and aromatase mRNA (Abdelgadir, Resko, Ojeda, Lephart, McPhaul, and Roselli, 1994) through androgen receptor mediated processes in adult rats and quail. In birds, estradiol can enhance levels of preoptic aromatase (Hutchison and Steimer, 1986; Foidart, Houbart, Harada, and Balthazart, 1996). Finally, synergistic actions of both androgens and estrogens on aromatase enzyme activity (Roselli and Resko, 1993) and aromatase mRNA (Harada, Abe-Dohmae, Loeffen, Foidart, and Balthazart, 1993) have been found in adult rats and quail. In the musk shrew, because so few cells contain both AROM-ir and ER-ir neurons, we predict that if estrogen affects aromatase enzyme, it does so indirectly. For example, estrogen may be part of a biochemical cascade involving many neuromodulatory hormones that eventually leads to changes in aromatase enzyme. Estrogen receptors are located in dopaminergic neurons (Sar, 1984), and there have been suggestions in the literature that estrogens can interact with dopamine to regulate aromatase enzyme (Balthazart and Ball, 1992). Alternatively, androgens could regulate aromatase enzyme in musk shrews. Unfortunately, to our knowledge, there have not been any immunohistochemical studies which have addressed the possibility of androgen receptors co-localizing with AROM-ir neurons in mammalian brain. Currently, we are examining this possibility in the musk shrew. It is also possible that estrogen may be affecting aromatase enzyme by acting through a membrane or other form of genomic ER, such as ERb. Estrogen bound to these alternative forms of ER may not have been detected by the H222 antibody. In male rats and quail, aromatase is involved in copulation (Adkins et al., 1980; Christensen and Clemens, 1975). In addition, in male quail, implants of T or estradiol benzoate (EB), but not the nonaromatizable androgen dihydrotestosterone (DHT), are able to reinstate aggressive behaviors after castration (Schlinger and Callard, 1989, 1990). Likewise, estrogen has been shown to mimic the effects of T in stimulating aggressive behaviors in castrated mice (Edwards and Burge, 1971), rats (Christie and Barfield, 1979),
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and hamsters (Vandenbergh, 1971). Estrogens may also be critical for the expression of chemoinvestigatory behaviors. In male Syrian hamsters for example, integration of chemosensory and hormonal cues in the MeA, mPOA, and BNST are critical for male mating behavior (Wood and Newman, 1995a). Neural implants of T or E2, but not DHT, in the MeA or BNST/ mPOA can increase the numbers of interactions between males and females and the number of anogenital investigations (Wood and Newman, 1995b; Wood, 1996). In male musk shrews, initial attempts to block sexual behavior with vorozole have not met with success (Veney and Rissman, unpublished data). However, we have yet to look at any other social behaviors in males. In female musk shrews, aromatase enzyme is necessary for receptivity (Rissman, 1991), but it may also play a role in other aspects of social behavior. For example, female shrews are highly aggressive toward males when they are first introduced in a mating test. When a male approaches a female, she lunges and displays open mouth threats accompanied by short, high-pitched vocalizations. Females also approach males and sniff and investigate male-soiled regions in the test cages. These behaviors continue until the female becomes receptive and receives mounts and intromissions from the male. We have not yet specifically examined the effects of aromatase inhibition on the expression of these other social behaviors in female musk shrews. One intriguing observation may shed some light on this issue. Recently we have examined c-fos immunoreactivity after mating in male and female musk shrews (Gill, Cholbi, and Rissman, 1996). In mated females, fos-ir neurons are present in the same region that contains the dense cluster of AROM-ir neurons. In mated males, however, this same area is devoid of fos-ir cells. This observation may reflect the different roles that aromatase enzyme plays in the expression of sexual behavior in males versus females. Sexual dimorphisms in the mPOA have been reported in several other mammals (Cherry, Basham, Weaver, Krohmer, and Baum, 1990; Ulibarri and Yahr, 1993; Gorski, Harlan, Jacobson, Shryne, and Southam, 1980). The direction of the sex differences are typically, as we report here, that males have more cells in the region than do females, but in the cases where a function has been found, the nuclei appear to regulate some aspect of masculine social behavior (Yahr and Stephens, 1987; Arendash and Gorski, 1983; Paredes and Baum, 1995). In musk shrews our data suggest the opposite—that
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the nucleus may have a function in female social behavior.
ACKNOWLEDGMENTS The authors thank Drs. Y. Osawa and C. P. Yarborough for generously supplying the R-8-1 aromatase antibody. We thank the reviewers of the manuscript and Michael Baum for their excellent comments and suggestions. We also acknowledge assistance from the Ligand Preparation and Assay Core (NIH Grant P30-HD28934, Center for Cellular and Molecular Studies in Reproduction). This work was supported by NSF Grant IBN-9412605 (E.F.R.) and NIH Grants MH01349, NS35429 (E.F.R.), and HD04945 (Y. Osawa). Sean Veney was supported, in part, by NIH Training Grant HD072323.
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