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(ER) and ERß Messenger Ribonucleic Acid in the Preoptic Area and Hypothalamus of the Sheep: Comparison of Males and Females
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
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: chris.scott{at}med.monash edu.au.
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Abstract |
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(ER) and ERß messenger RNA
(mRNA)-containing cells in the preoptic area and hypothalamus of ewes
and rams. Perfusion-fixed 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 ERß mRNA-containing cells, but the density
of labeled cells within each nucleus differed in a number of instances.
Nuclei that contained a higher (P < 0.001) density
of ER than ERß 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 ERß mRNA-containing 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 ERß 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 ERß mRNA-containing cells may
reflect differential regulation of the actions of estrogen in the sheep
hypothalamus. Low levels of ERß mRNA in the preoptic area and
ventromedial and arcuate nuclei, regions known to be important for the
regulation of reproduction, suggest that ERß may not be involved in
these functions.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 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 ERß. 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 hypothalamus of the
ewe has been well described using immunocytochemistry (13), and the
distribution of the ERß 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 ERß-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 ERß mRNA in the hypothalamus of male
and female sheep.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 µm and collected into
tissue culture plate wells containing cryoprotectant (15) with 2%
paraformaldehyde; these were stored at -20 C.
Ovine ER and ERß 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
ERß cDNA based on an unpublished sequence recently deposited in
GenBank (accession no. AF177936).
ERß. A partial cDNA for ovine ERß 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 µl. Oligonucleotide primers were designed from the published human ERß 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 ERß 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 µl. 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.
In situ hybridization
In situ hybridization was conducted according to the
protocol of Simmons et al. (15). cDNAs for ovine ER and
ERß 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 x transcription buffer; 100 mM
dithiothreitol (DTT); 40 U RNasin; 1.25 mM ATP,
CTP, and GTP; 1 µg linearized DNA template; 30 U RNA polymerase (T7
for antisense, SP6 for sense); and approximately 100 µCi
[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 µl 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 x 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
poly-L-lysine-coated slides and air-dried overnight. The
sections were taken at 360-µm 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 x
SSC (1 x 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 x
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 x SSC, the sections were treated with 20 µg/ml
ribonuclease A, rinsed in decreasing concentrations of SSC to
0.5 x SSC, and then washed in 0.1 x 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 x400 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 x100 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). 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.
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, 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).
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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.
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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.
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). The distribution of ERß mRNA-containing cells in the ewe was
broadly similar to the distribution of ER mRNA-containing cells, but
with several specific differences. Labeling for ERß 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 ERß mRNA more confined to
the ventral part of the nucleus than with ER mRNA (Fig. 3). There
was no labeling for ERß 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 ERß 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 ERß mRNA than ER mRNA
in the paraventricular nucleus of rams only (probe x sex
interaction, P = 0.05), whereas the density of cells
labeled for ERß mRNA tended to be greater than that of cells labeled
for ER in the retrochiasmatic area of ewes only (probe x sex
interaction, P = 0.055; Fig. 8).
The density of ERß 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
ERß mRNA-containing cells in any other region studied, nor was there
a sex difference in the number of silver grains per ERß
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).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ERß
mRNA-containing cells in the preoptic area and hypothalamus of both
ewes and rams. The design of this study has 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
neuroendocrine 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.
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 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 mRNA-containing 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 ERß 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 ERß.
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
evidence (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 ERß mRNA-containing cells observed
within the retrochiasmatic nucleus raises the possibility that estrogen
acts via ERß within this nucleus to inhibit LH secretion during the
nonbreeding season in the sheep. This would represent a novel action
for ERß, as there is currently no evidence for ERß involvement in
the regulation of GnRH secretion in any species. Male mice that lack
ERß 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 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 ERß 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.
ERß 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 ERß
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 ERß 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 ERß in the
brain of the sheep and whether there are any sex differences in the
degree of colocalization. ER and ERß 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, ERß, or both. Recent
evidence (36) suggests that one role of ERß 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 of ER and ERß 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 ERß) 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 ERß
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 ERß mRNA
or, in some regions, possibly both allows for differential regulation
of the actions of estrogen in the sheep hypothalamus.
Received February 7, 2000.
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References |
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hormone-expressing neurones. J Neuroendocrinol 11:331–335[CrossRef][Medline]
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gonadotropin-releasing hormone neurons. Endocrinology 140:5195–5201 and -ß mRNA in the
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C. J. Scott, I. J. Clarke, and A. J. Tilbrook Neuronal Inputs from the Hypothalamus and Brain Stem to the Medial Preoptic Area of the Ram: Neurochemical Correlates and Comparison to the Ewe Biol Reprod, April 1, 2003; 68(4): 1119 - 1133. [Abstract] [Full Text] [PDF]
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S. L. Hardy, G. M. Anderson, M. Valent, J. M. Connors, and R. L. Goodman Evidence That Estrogen Receptor Alpha, but Not Beta, Mediates Seasonal Changes in the Response of the Ovine Retrochiasmatic Area to Estradiol Biol Reprod, March 1, 2003; 68(3): 846 - 852. [Abstract] [Full Text] [PDF]
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L. Dufourny and D. C. Skinner Progesterone Receptor, Estrogen Receptor {alpha}, and the Type II Glucocorticoid Receptor Are Coexpressed in the Same Neurons of the Ovine Preoptic Area and Arcuate Nucleus: A Triple Immunolabeling Study Biol Reprod, November 1, 2002; 67(5): 1605 - 1612. [Abstract] [Full Text] [PDF]
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N. Scanlan and D. C. Skinner Estradiol Modulation of Growth Hormone Secretion in the Ewe: No Growth Hormone-Releasing Hormone Neurons and Few Somatotropes Express Estradiol Receptor {alpha} Biol Reprod, May 1, 2002; 66(5): 1267 - 1273. [Abstract] [Full Text]
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 C. Orikasa, Y. Kondo, S. Hayashi, B. S. McEwen, and Y. Sakuma Sexually dimorphic expression of estrogen receptor beta in the anteroventral periventricular nucleus of the rat preoptic area: Implication in luteinizing hormone surge PNAS, February 14, 2002; (2002) 52707299. [Abstract] [Full Text] [PDF]
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S. Pompolo, C. J. Scott, and I. J. Clarke Selective Regulation of Glutamic Decarboxylase Isoform 65, But Not Isoform 67, in the Bed Nucleus of the Stria Terminalis and the Preoptic Area of the Ewe Brain Across the Estrous Cycle Endocrinology, February 1, 2002; 143(2): 544 - 550. [Abstract] [Full Text] [PDF]
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 D. A. Jacob, J. L. Temple, H. B. Patisaul, L. J. Young, and E. F. Rissman Coumestrol Antagonizes Neuroendocrine Actions of Estrogen via the Estrogen Receptor {{alpha}} Experimental Biology and Medicine, April 1, 2001; 226(4): 301 - 306. [Abstract] [Full Text]
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C. Orikasa, Y. Kondo, S. Hayashi, B. S. McEwen, and Y. Sakuma Sexually dimorphic expression of estrogen receptor beta in the anteroventral periventricular nucleus of the rat preoptic area: Implication in luteinizing hormone surge PNAS, March 5, 2002; 99(5): 3306 - 3311. [Abstract] [Full Text] [PDF]
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