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-
I
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-
2951
2952
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.
Endo • 2000
Vol 141 • No 8
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.
ESTROGEN RECEPTOR ␣ AND ␤ IN THE SHEEP BRAIN
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.
2953
2954
ESTROGEN RECEPTOR ␣ AND ␤ IN THE SHEEP BRAIN
Endo • 2000
Vol 141 • No 8
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␤
ESTROGEN RECEPTOR ␣ AND ␤ IN THE SHEEP BRAIN
2955
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
2956
ESTROGEN RECEPTOR ␣ AND ␤ IN THE SHEEP BRAIN
Endo • 2000
Vol 141 • No 8
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.
ESTROGEN RECEPTOR ␣ AND ␤ IN THE SHEEP BRAIN
2957
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
2958
ESTROGEN RECEPTOR ␣ AND ␤ IN THE SHEEP BRAIN
Endo • 2000
Vol 141 • No 8
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-
ESTROGEN RECEPTOR ␣ AND ␤ IN THE SHEEP BRAIN
2959
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
2960
ESTROGEN RECEPTOR ␣ AND ␤ IN THE SHEEP BRAIN
Endo • 2000
Vol 141 • No 8
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
ESTROGEN RECEPTOR ␣ AND ␤ IN THE SHEEP BRAIN
2961
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,
2962
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
ESTROGEN RECEPTOR ␣ AND ␤ IN THE SHEEP BRAIN
Martin GB 1997 Gonadotrophin and prolactin secretion in castrated male
sheep following subcutaneous or intracranial treatment with testicular hormones. Endocrine 7:235–243
Lehman MN, Ebling FJ, Moenter SM, Karsch FJ 1993 Distribution of estrogen
receptor-immunoreactive cells in the sheep brain. Endocrinology 133:876 – 886
Hileman SM, Handa RJ, Jackson GL 1999 Distribution of estrogen receptor-␤
messenger ribonucleic acid in the male sheep hypothalamus. Biol Reprod
60:1279 –1284
Simmons DM, Arriza JL, Swanson LW 1989 A complete protocol for in situ
hybridization of messenger RNAs in brain and other tissues with radio-labeled
single-stranded RNA probes. J Histotechnol 12:169 –181
Ing NH, Spencer TE, Bazer FW 1996 Estrogen enhances endometrial estrogen
receptor gene expression by a posttranscriptional mechanism in the ovariectomized ewe. Biol Reprod 54:591–599
Mosselman S, Polman J, Dijkema R 1996 ER beta: identification and characterization of a novel human estrogen receptor. FEBS Lett 392:49 –53
Sambrook, J., Fritsch, E.F., and Maniatis, T 1989 Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Rosenfeld CS, Yuan X, Manikkam M, Calder MD, Garverick HA, Lubahn
DB 1999 Cloning, sequencing, and localization of bovine estrogen receptor-␤
within the ovarian follicle. Biol Reprod 60:691– 697
Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA 1996
Cloning of a novel receptor expressed in rat prostate and ovary. Proc Natl Acad
Sci USA 93:5925–5930
Tremblay GB, Tremblay A, Copeland NG, Gilbert DJ, Jenkins NA, Labrie
F, Giguere V 1997 Cloning, chromosomal localization, and functional analysis
of the murine estrogen receptor ␤. Mol Endocrinol 11:353–365
Madigou T, Tiffoche C, Lazennec G, Pelletier J, Thieulant ML 1996 The sheep
estrogen receptor: cloning and regulation of expression in the hypothalamopituitary axis. Mol Cell Endocrinol 121:153–163
Lauber AH, Romano GJ, Pfaff DW 1991 Gene expression for estrogen and
progesterone receptor mRNAs in rat brain and possible relations to sexually
dimorphic functions. J Steroid Biochem Mol Biol 40:53– 62
Shughrue PJ, Bushnell CD, Dorsa DM 1992 Estrogen receptor messenger
ribonucleic acid in female rat brain during the estrous cycle: a comparison with
ovariectomized females and intact males. Endocrinology 131:381–388
Thiery JC, Gayrard V, Le CS, Viguie C, Martin GB, Chemineau P, Malpaux
B 1995 Dopaminergic control of LH secretion by the A15 nucleus in anoestrous
ewes. J Reprod Fertil [Suppl] 49:285–296
Tilbrook AJ, Clarke IJ 1992 Evidence that dopaminergic neurons are not
involved in the negative feedback effect of testosterone on luteinizing hormone
in rams in the non-breeding season. J Neuroendocrinol 4:365–374
Lubbers LS, Hileman SM, Jansen HT, Lehman MN, Jackson GL 1995 Testosterone-induced activation of tyrosine hydroxylase-containing neurons of
the A14 and A15 hypothalamic nuclei in the male sheep. Soc Neurosci Abstr
754.2
Herbison AE, Skinner DC, Robinson JE, King IS 1996 Androgen receptorimmunoreactive cells in ram hypothalamus: distribution and co-localization
patterns with gonadotropin-releasing hormone, somatostatin and tyrosine
hydroxylase. Neuroendocrinology 63:120 –131
Endo • 2000
Vol 141 • No 8
29. Gallegos-Sanchez J, Delaleu B, Caraty A, Malpaux B, Thiery JC 1997 Estradiol acts locally within the retrochiasmatic area to inhibit pulsatile luteinizinghormone release in the female sheep during anestrus. Biol Reprod 56:
1544 –1549
30. Krege JH, Hodgin JB, Couse JF, Enmark E, Warner M, Mahler JF, Sar M,
Korach KS, Gustafsson JA, Smithies O 1998 Generation and reproductive
phenotypes of mice lacking estrogen receptor ␤. Proc Natl Acad Sci USA
95:15677–15682
31. Blache D, Batailler M, Fabre-Nys C 1994 Oestrogen receptors in the preopticohypothalamic continuum: immunohistochemical study of the distribution and
cell density during induced oestrous cycle in ovariectomized ewe. J Neuroendocrinol 6:329 –339
32. Shughrue PJ, Lane MV, Merchenthaler I 1997 Comparative distribution of
estrogen receptor-␣ and -␤ mRNA in the rat central nervous system. J Comp
Neurol 388:507–525
33. Shughrue PJ, Scrimo PJ, Merchenthaler I 1998 Evidence for the colocalization
of estrogen receptor-␤ mRNA and estrogen receptor-␣ immunoreactivity in
neurons of the rat forebrain. Endocrinology 139:5267–5270
34. Cowley SM, Hoare S, Mosselman S, Parker MG 1997 Estrogen receptors ␣
and ␤ form heterodimers on DNA. J Biol Chem 272:19858 –19862
35. Pettersson K, Grandien K, Kuiper GG, Gustafsson JA 1997 Mouse estrogen
receptor ␤ forms estrogen response element-binding heterodimers with estrogen receptor ␣. Mol Endocrinol 11:1486 –1496
36. Hall JM, McDonnell DP 1999 The estrogen receptor ␤-isoform (ER␤) of the
human estrogen receptor modulates ER␣ transcriptional activity and is a key
regulator of the cellular response to estrogens and antiestrogens. Endocrinology 140:5566 –5578
37. Legan SJ, Karsch FJ, Foster DL 1977 The endocrine control of seasonal reproductive function in the ewe: a marked change in response to the negative
feedback action of estradiol on luteinizing hormone secretion. Endocrinology
101:818 – 824
38. Skinner DC, Herbison AE 1997 Effects of photoperiod on estrogen receptor,
tyrosine hydroxylase, neuropeptide Y, and ␤-endorphin immunoreactivity in
the ewe hypothalamus. Endocrinology 138:2585–2595
39. Clarke IJ, Burman K, Funder JW, Findlay JK 1981 Estrogen receptors in the
neuroendocrine tissues of the ewe in relation to breed, season, and stage of the
estrous cycle. Biol Reprod 24:323–331
40. Glass JD, Amann RP, Nett TM 1984 Effects of season and sex on the distribution of cytosolic estrogen receptors within the brain and the anterior pituitary gland of sheep. Biol Reprod 30:894 –902
41. Bittman EL, Blaustein JD 1990 Effects of day length on sheep neuroendocrine
estrogen and progestin receptors. Am J Physiol 258:R135–R142
42. Lubbers LS, Jackson GL 1993 Neuroendocrine mechanisms that control seasonal changes of luteinizing hormone secretion in sheep are sexually differentiated. Biol Reprod 49:1369 –1376
43. Pelletier J, Caraty A 1981 Characterization of cytosolic 5␣-DHT and 17␤estradiol receptors in the ram hypothalamus. J Steroid Biochem 14:603– 611