The Distribution of Cells Containing Estrogen Receptor- (ER ) and ER ...

0013-7227/00/$03.00/0 Endocrinology Copyright © 2000 by The Endocrine Society

Vol. 141, No. 8 Printed in U.S.A.

The Distribution of Cells Containing Estrogen Receptor␣ (ER␣) and ER␤ Messenger Ribonucleic Acid in the Preoptic Area and Hypothalamus of the Sheep: Comparison of Males and Females CHRISTOPHER J. SCOTT, ALAN J. TILBROOK, DONNA M. SIMMONS, JOHN A. RAWSON, SIMON CHU, PETER J. FULLER, NANCY H. ING, AND IAIN J. CLARKE Department of Physiology, Monash University (C.J.S., A.J.T., J.A.R.,), and Prince Henry’s Institute of Medical Research (C.J.S., S.C., P.J.F., I.J.C.), Clayton, Victoria 3168, Australia; Department of Biological Sciences, University of Southern California (D.M.S.), Los Angeles, California 90089; and Department of Animal Science, Texas A&M University (N.H.I.), College Station, Texas 77843 ABSTRACT We have used in situ hybridization to compare the distributions of estrogen receptor ␣ (ER␣) and ER␤ messenger RNA (mRNA)-containing cells in the preoptic area and hypothalamus of ewes and rams. Perfusionfixed brain tissue was collected from luteal phase ewes and intact rams (n ⫽ 4) during the breeding season. Matched pairs of sections were hybridized with sheep-specific, 35S-labeled riboprobes, and semiquantitative image analysis was performed on emulsion-dipped slides. A number of sex differences were observed, with females having a greater density of labeled cells than males (P ⬍ 0.001) and a greater number of silver grains per cell (P ⬍ 0.01) in the ventromedial nucleus for both ER subtypes. In addition, in the retrochiasmatic area, males had a greater (P ⬍ 0.05) cell density for ER␣ mRNA-containing cells than females, whereas in the paraventricular nucleus, females had a greater density (P ⬍ 0.05) of ER␣ mRNA-containing cells than males. There was a trend (P ⫽ 0.068) in the arcuate nucleus for males to have a greater number of silver grains per cell labeled for ER␣ mRNA. In both sexes, there was considerable overlap in the distributions of ER␣ and ER␤ mRNA-containing cells, but the density of labeled cells within each nucleus differed in a number of instances. Nuclei that con-

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T IS WELL established that estrogen has a critical role in the regulation of GnRH secretion in female sheep (1). Evidence is also accumulating that this is the case in male sheep (2– 4). The nature of this estrogenic regulation of GnRH secretion is sexually dimorphic, however, as estrogen can induce GnRH and LH surges in females but not males (5), although it does have an inhibitory action on LH secretion in males (6). The mechanism(s) responsible for this sex difference is unknown, but may relate to the location of estrogen receptors (ER) in the brain and their relationship to GnRH neurons. Using conventional immunocytochemistry, and in situ hybridization, the majority of studies indicate that GnRH neurons do not contain ER (7, 8). A recent study by Butler et al. (9) reported, however, the presence of ER␣ immunoreactivity in 17% of GnRH neurons in acrolein-fixed rat brain tissue. Most recently, Skynner et al. (10) used multiplex RT-PCR on Received February 7, 2000. Address all correspondence and requests for reprints to: Dr. Christopher J. Scott, Department of Physiology, Monash University, P.O. Box 13F, Victoria 3800, Australia. E-mail: [email protected]. edu.au.

tained a higher (P ⬍ 0.001) density of ER␣ than ER␤ mRNA-containing cells included the preoptic area, bed nucleus of the stria terminalis, and ventromedial nucleus, whereas the subfornical organ (P ⬍ 0.001), paraventricular nucleus (males only, P ⬍ 0.05), and retrochiasmatic nucleus (females only, P ⬍ 0.05) had a greater density of ER␣ than ER␤ mRNAcontaining cells. The anterior hypothalamic area and supraoptic nucleus had similar densities of cells containing both ER subtypes. The lateral septum and arcuate nucleus contained only ER␣, whereas only ER␤ mRNA-containing cells were seen in the zona incerta. The sex differences in the populations of ER mRNA-containing cells in the ventromedial and arcuate nuclei may explain in part the sex differences in the neuroendocrine and behavioral responses to localized estrogen treatment in these nuclei. Within sexes, the differences between the distributions of ER␣ and ER␤ mRNA-containing cells may reflect differential regulation of the actions of estrogen in the sheep hypothalamus. Low levels of ER␤ mRNA in the preoptic area and ventromedial and arcuate nuclei, regions known to be important for the regulation of reproduction, suggest that ER␤ may not be involved in these functions. (Endocrinology 141: 2951–2962, 2000)

messenger RNA (mRNA) from the contents of GnRH cells aspirated from tissue slices and reported that more than 50% of GnRH neurons contained ER␣ mRNA and 10% contained ER␤. As most laboratories working in this area report that ER can be seen in various cell types with conventional immunocytochemistry and are not seen in GnRH cells, we continue to work on the assumption that GnRH cells either do not possess ER or have very low levels of ER. Thus, we have adopted the hypothesis that the actions of estrogen to regulate GnRH secretion are mediated via neurons that contain ER and also synapse onto or relay to GnRH neurons. The sites of action of estrogen to regulate GnRH are unknown. Microimplants of estrogen in the ventromedial nucleus (but not the preoptic area) induced a LH surge in ovariectomized ewes (11), and implants into the mediobasal hypothalamus and preoptic area of castrated rams suppressed LH secretion (4, 12), suggesting that these sites are important for estrogen feedback. The possibility remains, however, that estrogen may act in other brain sites to regulate GnRH secretion. The distribution of neurons containing ER␣ in the hypo-

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ESTROGEN RECEPTOR ␣ AND ␤ IN THE SHEEP BRAIN

thalamus of the ewe has been well described using immunocytochemistry (13), and the distribution of the ER␤ mRNA in the hypothalamus of the ram has recently been determined using in situ hybridization (14). Conversely, the distribution of ER␣-containing neurons in the ram hypothalamus and that of ER␤-containing neurons in the ewe hypothalamus are unknown. In the present study we used in situ hybridization with species-specific riboprobes to compare the distributions of ER␣ mRNA and ER␤ mRNA in the hypothalamus of male and female sheep. Materials and Methods This work was conducted in accordance with the Prevention of Cruelty to Animals Act, Victorian Government, 1986, and the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. The work was approved by the ethics committees of Monash University and the Victorian Institute of Animal Science.

Experimental animals and tissue collection Tissue was obtained from adult Corriedale and Romney Marsh ewes and rams during the breeding season (n ⫽ 4/sex). The ewes were killed during the luteal phase, 12 days after a timed estrus. The stage of the cycle was verified by visual inspection of the ovaries and by RIA for progesterone from a jugular blood sample taken at necropsy (data not shown). The sheep were killed by an overdose of sodium pentobarbital (Lethabarb, Virbac, Peakhurst, Australia). The head was removed and perfused through both carotid arteries with 2 liters normal saline containing 25,000 U heparin, followed by 3 liters 4% paraformaldehyde in 0.1 m phosphate buffer, pH 7.4; the final liter contained 20% sucrose. The brain was then removed, the hypothalamus was dissected out, and the tissue was postfixed at 4 C in fixative containing 30% sucrose for 7 days. Cryostat sections were cut in the coronal plane at a thickness of 20 ␮m and collected into tissue culture plate wells containing cryoprotectant (15) with 2% paraformaldehyde; these were stored at ⫺20 C.

Ovine ER␣ and ER␤ complementary DNAs (cDNAs) ER␣. A partial cDNA for ovine ER␣ was cloned by PCR by Ing et al. (16) from the endometrium of a cyclic ewe. The resultant 336-base ovine ER␣ mRNA sequence, which encodes for the N-terminal region and part of the DNA-binding region of the protein, has 96%, 91%, and 83% identity with the nucleotide sequences of the pig, human, and mouse ER␣ cDNAs, respectively (16). This sequence shares 60.7% identity with the corresponding portion of the ovine ER␤ cDNA based on an unpublished sequence recently deposited in GenBank (accession no. AF177936). ER␤. A partial cDNA for ovine ER␤ was cloned from the ovary of a cycling ewe. One microgram of total RNA was reverse transcribed at 42 C using 11 pmol oligo(deoxythymidine)15 (Roche Molecular Biochemicals, Mannheim, Germany) and AMV reverse transcriptase (Roche Molecular Biochemicals) in a total volume of 20 ␮l. Oligonucleotide primers were designed from the published human ER␤ sequence (17) with OLIGO Primer Analysis Software, version 5.0 (Natural Biosciences, North Plymouth, MN). The oligonucleotide primers used were as follows, with the number of the 5⬘-nucleotide in the ER␤ sequence given in parentheses: 5⬘-CCTGGCAACTACTTCAAGGTTTC-3⬘ (999) and 5⬘ACACACTGGAGTTCACGCTTCAG-3⬘ (1515). Two microliters of the RT reaction were amplified in a single stage PCR for 40 cycles with 10 pmol of each primer together with 2.5 U Taq polymerase (Roche Molecular Biochemicals) in a total volume of 50 ␮l. The thermal cycling profile for the receptor consisted of a denaturing step at 95 C, initially for 5 min and subsequently for 30 sec, annealing at 42 C for 30 sec, and extension at 72 C for 40 sec, with a final 72 C incubation for 5 min. Products were analyzed on a 2% agarose gel stained with ethidium bromide. A 516-bp PCR product was isolated and subcloned into pCR2.1 (Invitrogen, San Diego, CA) and sequenced by the dideoxy chain termination method using an ABI Prism 377 DNA sequencer.

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In situ hybridization In situ hybridization was conducted according to the protocol of Simmons et al. (15). cDNAs for ovine ER␣ and ER␤ were linearized from their recombinant plasmids (pGEM-4Z and pCR 2.1, respectively) with BamHI and HindIII, respectively, using standard techniques (18). Complementry RNA probes were synthesized using a Gemini System II kit (Promega Corp., Hawthorn, Victoria, Australia). The reaction included 5 ⫻ transcription buffer; 100 mm dithiothreitol (DTT); 40 U RNasin; 1.25 mm ATP, CTP, and GTP; 1 ␮g linearized DNA template; 30 U RNA polymerase (T7 for antisense, SP6 for sense); and approximately 100 ␮Ci [35S]UTP (NEN Life Science Products, Boston, MA). The transcription mixture was incubated for 60 min at 37 C with an additional 30 U RNA polymerase added after 30 min. This reaction was terminated by the addition of 30 ␮l 1% SDS in 10 mm Tris/1 mm EDTA/10 mm DTT, and the unincorporated nucleotides were removed by centrifugation through a Sephadex G-25 spin column. The probe was heated for 5 min at 65 C in a solution containing 50 mm DTT, 2.5 mg/ml transfer RNA, and 2.5 mg fish sperm DNA (Roche Molecular Biochemicals). It was then diluted in a hybridization buffer to produce a final concentration of 50% formamide, 10% dextran sulfate, 1 ⫻ Denhardt’s solution, 1 mm EDTA, 10 mm Tris, and 12 mm sodium chloride, with a final specific activity for the probe of 107 cpm/ml. Matched pairs of ewe and ram sections were mounted on polyl-lysine-coated slides and air-dried overnight. The sections were taken at 360-␮m intervals through the preoptic area and hypothalamus, with a parallel set of sections for each probe. All sections from each pair of ewes and rams were hybridized together. Before hybridization the sections were treated with proteinase K (0.001%; for 30 min at 37 C), 0.1 m triethanolamine (3 min), and 0.25% acetic anhydride (10 min); rinsed twice in 2 ⫻ SSC (1 ⫻ SSC is 0.15 m NaCl and 15 mm trisodium citrate, pH 7.0), dehydrated in increasing concentrations of ethanol, delipidated in chloroform, and rinsed in 100% ethanol. Hybridization solution was applied to the section (⬃2 ⫻ 106 cpm/slide) and covered with a glass coverslip, and the slides were placed in a humidified plastic container and incubated at 53 C for 16 h. After soaking the coverslips off in 4 ⫻ SSC, the sections were treated with 20 ␮g/ml ribonuclease A, rinsed in decreasing concentrations of SSC to 0.5 ⫻ SSC, and then washed in 0.1 ⫻ SSC at 65 C for 30 min. The sections were dehydrated in increasing concentrations of ethanol, air-dried, and exposed to Kodak BMR film (Eastman Kodak Co., Rochester, NY) for 7 days, then dipped in Ilford K5 (Ilford, Australia, Mt. Waverly, Victoria, Australia) photographic emulsion and exposed for 2 weeks. The dipped slides were then developed using Ilford phenisol x-ray developer, fixed, and lightly counterstained with 1% cresyl violet.

Image analysis The distribution of labeled cells in the hypothalamus was mapped with an X-Y plotting system (M.D. plot, MN Datametrics, Shoreview, MN). Semiquantitative image analysis was conducted on dipped autoradiograms. All image analysis was conducted by a single operator using coded slides. Grain counting was conducted under brightfield conditions at a magnification of ⫻400 using a Fuji Photo Film Co. Ltd. HC-2000 high resolution digital camera and Analytical Imaging Station 4.0 software (Imaging Research, Inc., St. Catherine’s, Canada). Cells were regarded as labeled if grain counts were more than 5 times background (which was typically 5–15 grains per equivalent cellular area). From each treatment group, 1 section was selected from the middle region of each nucleus. These selected sections were carefully matched between groups. From each section, 10 labeled cells were selected at random from throughout the whole nucleus. For the calculation of cell density, cells were counted manually under darkfield conditions within an eyepiece grid placed in the center of the nucleus. When used at ⫻100 magnification, this grid covers 0.81 mm2. All densities were converted to cell number per mm2. For each nucleus, univariate ANOVA was used to compare the density of labeled cells between ER receptor subtype and sex as well as for sex comparison of the number of silver grains per labeled cell. Homogeneity of variance was tested using Leverne’s test of equality of error variances, and when necessary, square root transformations were performed.


FIG. 1. Comparison of the nucleic acid sequence of the ovine ER␤ cDNA clone with the corresponding sequences in bovine (19), human (17), rat (20), and mouse (21) ER␤ cDNAs. Numbers on the left and right indicate nucleotide positions.

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Results Cloning and sequencing of partial cDNA for ovine ER␤

A partial cDNA for ovine ER␤ was cloned and sequenced (Fig. 1). The 516-base cDNA sequence shared 97%, 87%, 80%, and 83% identity with the bovine (19), human (17), rat (20), and mouse (21) sequences, respectively. The sequence shows 55.9% identity with the corresponding portion of the ovine ER␣ cDNA (22). The deduced amino acids were 96%, 88%, 83%, and 85% identical to those of the bovine, human, rat, and mouse, respectively. ER mRNA-producing cells

ER mRNA-producing cells were identified as clusters of silver grains over single neurons (e.g. Fig. 2A, showing ER␣). The specificity of this labeling was demonstrated by the fact that no accumulation of silver grains at levels above background occurred when sections were hybridized with the sense strand (Fig. 2B; ER␣), or when sections hybridized with the antisense probe were pretreated with ribonuclease A (not shown). Distribution of ER mRNA-producing cells in the hypothalamus

ER␣ mRNA-producing cells. The distribution of cells in the preoptic area/hypothalamus that contain ER␣ mRNA is shown in a series of film autoradiograms from a representative ewe and ram (Fig. 3). ER␣ mRNA-producing cells were detected in a number of hypothalamic nuclei in both female and male sheep. High levels of expression were found in the medial preoptic area (Fig. 4), bed nucleus of the stria terminalis, supraoptic nucleus, ventromedial nucleus (Fig. 5), and arcuate nucleus (Fig. 6). Strong expression was also found in the lateral septum, subfornical organ, anterior hypothalamic area, retrochiasmatic area (Fig. 7), and paraventricular nucleus. Scattered cells were found in the ventral limb of the diagonal band of Broca and posterior hypothalamus. The density of ER␣ mRNA-containing cells in the 11 regions studied is shown graphically in Fig. 8. There was a significantly lower density of ER␣ mRNA-containing cells in the ventromedial nucleus (P ⬍ 0.001) and paraventricular nucleus (P ⬍ 0.05) and a significantly higher density of ER␣ mRNA-containing cells in the retrochiasmatic area (P ⬍ 0.05) of rams compared with ewes. In all other nuclei the density of ER␣ mRNA-producing cells was the same in rams and ewes. The number of silver grains per labeled cell in the 11 regions studied is shown in Fig. 9 and was significantly (P ⬍ 0.01) lower in the ventromedial nucleus of rams compared with ewes. There were no sex differences in the number of silver grains per ER␣ mRNA-containing cell in any other nucleus examined, although there was a trend (P ⫽ 0.068) toward a greater number of silver grains per cell in the arcuate nucleus of rams compared with ewes. ER␤ mRNA-producing cells. The distribution in the preoptic area/hypothalamus of cells that contain ER␤ mRNA is shown in a series of film autoradiograms from a representative ewe and ram (Fig. 3). The distribution of ER␤ mRNA-containing cells in the ewe was broadly similar to the distribution of ER␣ mRNA-containing cells, but with several specific differences.

FIG. 2. High power photomicrographs of MBH hybridized with either antisense (A) or sense (B) mRNA for ovine ER␣. The arrowhead in A indicates a Nissl-stained cell. Scale bar, 10 ␮m.

Labeling for ER␤ mRNA in the rostral preoptic area was limited to a thin strip close to the midline, whereas labeling for ER␣ was throughout the preoptic area (Fig. 3). In the ventromedial nucleus, labeling for ER␤ mRNA more confined to the ventral part of the nucleus than with ER␣ mRNA (Fig. 3). There was no labeling for ER␤ mRNA in the lateral septum and arcuate nucleus, but strong labeling was observed in the zona incerta, a region that did not show any labeling for ER␣ (Fig. 3). The density of cells labeled for ER␤ mRNA was significantly (P ⬍ 0.001) lower than that for ER␣ mRNA in the preoptic area, bed nucleus of the stria terminalis, and ventromedial nucleus but was significantly higher (P ⬍ 0.001) in the subfornical organ (Fig. 8). There was a greater density of cells labeled for ER␤


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FIG. 3. Film autoradiograms showing the distributions of ER␣ and ER␤ mRNA-containing cells in the hypothalamus of a representative ewe and ram. Scale bar, 5 mm. Diagrams A–H show 20-␮m sections at 720-␮m intervals (rostral to caudal). 3V, Third ventricle; AC, anterior commissure; AHA, anterior hypothalamic area; ARC, arcuate nucleus; BNST, bed nucleus of the stria terminalis; LS, lateral septum; ME, median eminence; MR, mammillary recess of the third ventricle; MPOA, medial preoptic area; OC, optic chiasm; OVLT, organum vasculosum of the lamina terminalis; PVN, paraventricular nucleus; RCh, retrochiasmatic area; SFO, subfornical organ; SON, supraoptic nucleus; VMN, ventromedial nucleus; ZI, zona incerta.

mRNA than ER␣ mRNA in the paraventricular nucleus of rams only (probe ⫻ sex interaction, P ⫽ 0.05), whereas the density of cells labeled for ER␤ mRNA tended to be greater than that of cells labeled for ER␣ in the retrochiasmatic area of ewes only (probe ⫻ sex interaction, P ⫽ 0.055; Fig. 8). The density of ER␤ mRNA-containing cells in the ventromedial nucleus was significantly (P ⬍ 0.001) lower in rams compared with ewes (Fig. 8). There were no sex differences in the density of ER␤ mRNA-containing cells in any other region studied, nor was there a sex difference in the number

of silver grains per ER␤ mRNA-containing cell, although there was a trend (P ⫽ 0.07) toward fewer silver grains per cell in the ventromedial nucleus of rams compared with ewes (Fig. 10). Discussion

In this study we have mapped the distribution of ER␣ and ER␤ mRNA-containing cells in the preoptic area and hypothalamus of both ewes and rams. The design of this study has

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FIG. 4. Low power darkfield photomicrographs of the preoptic area in a ewe (A and C) and a ram (B and D) hybridized with antisense mRNA for ovine ER␣ (A and B) and ER␤ (C and D). Scale bar, 100 ␮m. 3V, Third ventricle.

allowed us to compare, for the first time in any species, the hypothalamic distribution of both receptors in both sexes. Several sex differences were observed that have important implications for the physiological regulation of neuroendo-

crine function and reproductive behavior. In both sexes, there was considerable overlap in the distribution of mRNA for the two receptor subtypes, although a number of receptor subtype differences were observed.


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FIG. 5. Low power darkfield photomicrographs of the ventromedial nucleus in a ewe (A and C) and a ram (B and D) hybridized with antisense mRNA for ovine ER␣ (A and B) and ER␤ (C and D). The midline is to the left of the figure. Scale bar, 100 ␮m.

This study is the first to describe the distribution of ER␣ in the preoptic area/hypothalamus of the ram and has identified a major sex difference with regard to the ventromedial

nucleus. In this nucleus, rams showed substantially fewer ER␣-containing cells and less ER␣ mRNA per cell than ewes. There was a trend toward a similar result in the arcuate

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FIG. 6. Low power darkfield photomicrographs of the arcuate nucleus in a ewe (A and C) and a ram (B and D) hybridized with antisense mRNA for ovine ER␣ (A and B) and ER␤ (C and D). Scale bar, 100 ␮m. 3V, Third ventricle.

nucleus. Similar sex differences in these nuclei have been noted in rats (23, 24). Estrogen has a sexually dimorphic action on GnRH secretion, with a positive feedback action in females to induce a GnRH/LH surge and a negative feedback action in males. Furthermore, exogenous estrogen treatment at a dose that can induce a LH surge in ewes does not do so in rams (5). Thus, this is not simply a dose effect, but a major sex difference in the response to estrogen. The ventromedial nucleus is an important site of estrogen action to stimulate the GnRH/LH surge in ewes (11) and estrogen negative feedback in rams (4, 12). The ventromedial nucleus is also an important site of action for estrogen in the regulation of reproductive behavior (11), which is also sexually dimorphic. The sex difference in the number of ER mRNAcontaining cells as well as the amount of ER mRNA per cell

may explain in part these major sexual dimorphisms in the action of estrogen in this nucleus. Notably, the ventromedial and arcuate nuclei contained little or no ER␤ mRNA despite high levels of ER␣ mRNA, and our results suggest that the actions of estrogen at this level are most likely through ER␣ and not ER␤. The sexually dimorphic distribution of ER␣ mRNA in the retrochiasmatic area is more difficult to interpret. Although there is good evidence that dopaminergic cells in this nucleus are important in mediation of the action of estrogen to inhibit LH secretion in the ewe during anestrus (25), this region has few ER␣-containing cells. Paradoxically, although the retrochiasmatic area in the ram contains greater levels of ER␣ than that in ewes, this dopaminergic system may not regulate GnRH secretion in rams (26). Despite this, preliminary evi-


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FIG. 7. Low power darkfield photomicrographs of the retrochiasmatic area in a ewe (A and C) and ram (B and D) hybridized with antisense mRNA for ovine ER␣ (A and B) and ER␤ (C and D). The midline is to the left of the figure. Scale bar, 100 ␮m.

dence (27) indicates that dopaminergic cells in the retrochiasmatic area of rams express the Fos-related antigens after testosterone treatment during the nonbreeding season, indicating that these cells are undergoing long term activation. As this region does not contain androgen receptors (28), the actions of testosterone must be through ER after aromatization to estrogen. Thus, the role of the retrochiasmatic area in the regulation of GnRH secretion in rams requires further clarification. Implantation of estrogen directly into the retrochiasmatic area suppressed plasma LH levels in ovariectomized ewes during an inhibitory photoperiod (29), yet no ER␣-immunoreactive cells (13) and few ER␣ mRNA-containing cells (present study) have been detected in this nucleus. The large population of ER␤ mRNA-containing cells observed within

the retrochiasmatic nucleus raises the possibility that estrogen acts via ER␤ within this nucleus to inhibit LH secretion during the nonbreeding season in the sheep. This would represent a novel action for ER␤, as there is currently no evidence for ER␤ involvement in the regulation of GnRH secretion in any species. Male mice that lack ER␤ are fully fertile, and female mice produce litters, albeit with reduced frequency (30), thus indicating the generation of effective preovulatory LH surges. The distribution of ER␣ mRNA-containing cells in the ewe hypothalamus is nearly identical to that reported for ER␣immunoreactive (-ir) cells (13, 31), with the notable exception of the supraoptic and paraventricular nuclei, which contain few ER␣-ir cells but abundant ER␣ mRNA-containing cells. It remains to be determined whether these differences reflect

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FIG. 8. Histogram showing the mean (⫾SEM) number of labeled cells per mm2 for ER␣ and ER␤ in various hypothalamic regions of ewes and rams. *, P ⬍ 0.05; ***, P ⬍ 0.001.

FIG. 9. Histogram showing the mean (⫾SEM) number of silver grains per cell for ER␣ mRNA in various hypothalamic regions of ewes and rams. **, P ⬍ 0.01; a, P ⫽ 0.068. For abbreviations, see Fig. 3.

methodology and model or whether some of these ER␣ mRNA-containing cells do not produce the ER␣ protein. Our study confirms and extends the work of Hileman et al. (14) concerning the distribution of ER␤ mRNA-containing cells in the hypothalamus of the ram, although, in addition, we note a small population of strongly labeled cells within the subfornical organ. ER␤ mRNA-containing cells have not previously been described in the subfornical organ in any species, although this structure does contain a large population of ER␣ mRNA-containing cells in the rat (8, 32), whereas the sheep subfornical organ contains little ER␣. The overlap in the distribution of cells containing ER␣ and ER␤ mRNA raises the possibility that the two receptor subtypes may be colocalized and may interact in the regulation of the actions of estrogen. Evidence in the rat (33) indicates that ER␣ and ER␤ are colocalized in the preoptic area and bed nucleus of the stria terminalis as well as sites outside the

diencephalon. Further work is required to determine the colocalization of ER␣ and ER␤ in the brain of the sheep and whether there are any sex differences in the degree of colocalization. ER␣ and ER␤ can form heterodimers in vitro (34, 35), although the ability of the ER subtypes to form heterodimers in vivo and the activity are unknown. It is possible, however, that the actions of estrogen may differ depending on whether a cell expresses ER␣, ER␤, or both. Recent evidence (36) suggests that one role of ER␤ is to modulate ER␣ transcriptional activity. This has implications for the hypothalamus, especially in females, where cyclical variation in estrogen concentration is critical for its function (e.g. gonadotropin secretion and reproductive behavior), whereas other functions of estrogen may benefit more from a more constant level of estrogen. It is unknown to what degree, if any, the levels of the two ER subtypes may vary throughout the estrous cycle, but one might speculate that the relative levels


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FIG. 10. Histogram showing the mean (⫾SEM) number of silver grains per cell for ER␣ mRNA in various hypothalamic regions of ewes and rams. a, P ⫽ 0.07. For abbreviations, see Fig. 3.

of ER␣ and ER␤ may vary throughout the cycle in some hypothalamic regions to modulate the response to estrogen. If this is the case, then some of the sex differences in the levels of the two ER subtypes in some hypothalamic nuclei may be due to the fact that although estrogen levels in the ewe fluctuate greatly throughout the estrous cycle, they are relatively constant in the ram. An obvious concern when comparing the distribution of two closely related mRNAs is that there is no cross-hybridization of the probe with other mRNA. Thus, although there is a degree of sequence similarity between the probes and the corresponding mRNA from the other ER, this is relatively low compared with that in most other portions of the ER genes. Furthermore, it is notable that both probes labeled at least one region of the hypothalamus that did not contain any specific labeling for the other mRNA species. This would indicate that the probes did not cross-hybridize with the mRNA of the other ER subtype. This study was conducted during the breeding season, and it is possible that the number of cells expressing detectable quantities of ER mRNA may change with season. There is an acute change in the sensitivity of GnRH secretion to estrogen negative feedback in the ewe (37), and this may reflect changes in ER number in the brain. Indeed, studies by Skinner and Herbison (38) indicate that the number of ER␣-ir cells in the preoptic area of the ewe (but not in other hypothalamic sites) increases by about 20% during the nonbreeding season compared with the breeding season. Studies measuring ER binding (which would cover both ER␣ and ER␤) are not as clear, with results suggesting an increase (39), decrease (40), or no change (41) in the number of estrogen-binding sites in the hypothalamus during the nonbreeding season compared with the breeding season. These differences may reflect the techniques used and the animal model, but the studies lack anatomical resolution. The ram does not show the same seasonal alteration in sensitivity to estrogen feedback on LH secretion (42), so one may expect less seasonal change in hypothalamic ER numbers. Binding studies, however, suggest that hypothalamic ER numbers are higher in the breeding than the nonbreeding season in the ram (40, 43). This

needs to be confirmed with quantitative histochemical studies. In summary, we have compared the distributions of ER␣ and ER␤ mRNA-containing cells in the preoptic area and hypothalamus of rams and ewes. We have identified major sex differences in the distribution of ER-containing cells that may explain in part the sex differences in the gonadotropic and behavioral responses to estrogen. Within sexes, the region-specific distribution of cells containing ER␣ and ER␤ mRNA or, in some regions, possibly both allows for differential regulation of the actions of estrogen in the sheep hypothalamus. References 1. Goodman RL 1994 Neuroendocrine control of the ovine estrous cycle. In: Knobil ENJ (ed) The Physiology of Reproduction. Raven Press, New York, pp 659 –709 2. Schanbacher BD 1984 Regulation of luteinizing hormone secretion in male sheep by endogenous estrogen. Endocrinology 115:944 –950 3. Tilbrook AJ, De Kretser DM, Cummins JT, Clarke IJ 1991 The negative feedback effects of testicular steroids are predominantly at the hypothalamus in the ram. Endocrinology 129:3080 –3092 4. Scott CJ, Kuehl DE, Ferreira SA, Jackson GL 1997 Hypothalamic sites of action for testosterone, dihydrotestosterone, and estrogen in the regulation of luteinizing hormone secretion in male sheep. Endocrinology 138:3686 –3694 5. Herbosa CG, Dahl GE, Evans NP, Pelt J, Wood RI, Foster DL 1996 Sexual differentiation of the surge mode of gonadotropin secretion: prenatal androgens abolish the gonadotropin-releasing hormone surge in the sheep. J Neuroendocrinol 8:627– 633 6. D’Occhio MJ, Schanbacher BD, Kinder JE 1983 Androgenic and oestrogenic steroid participation in feedback control of luteinizing hormone secretion in male sheep. Acta Endocrinol (Copenh) 102:499 –504 7. Herbison AE 1995 Neurochemical identity of neurones expressing oestrogen and androgen receptors in sheep hypothalamus. J Reprod Fertil [Suppl] 49:271–283 8. Laflamme N, Nappi RE, Drolet G, Labrie C, Rivest S 1998 Expression and neuropeptidergic characterization of estrogen receptors (ER␣ and ER␤) throughout the rat brain: anatomical evidence of distinct roles of each subtype. J Neurobiol 36:357–378 9. Butler JA, Sjoberg M, Coen CW 1999 Evidence for oestrogen receptor ␣immunoreactivity in gonadotrophin-releasing hormone-expressing neurones. J Neuroendocrinol 11:331–335 10. Skynner MJ, Sim JA, Herbison AE 1999 Detection of estrogen receptor ␣ and ␤ messenger ribonucleic acids in adult gonadotropin-releasing hormone neurons. Endocrinology 140:5195–5201 11. Blache D, Fabre-Nys CJ, Venier G 1991 Ventromedial hypothalamus as a target for oestradiol action on proceptivity, receptivity and luteinizing hormone surge of the ewe. Brain Res 546:241–249 12. Blache D, Tjondronegoro S, Blackberry MA, Anderson ST, Curlewis JD,

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