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NEUROENDOCRINOLOGY |
and Progestin Receptor Proteins in the Female Rat Forebrain: Effects of Estradiol Treatment
Center for Neuroendocrine Studies, University of Massachusetts (B.G., M.J.T., J.D.B.), Amherst, Massachusetts 01003; and DuPont Pharmaceuticals Co. (E.A.A.), Wilmington, Delaware 19880
Address all correspondence and requests for reprints to: Dr. Béatrice Gréco, Department of Neurology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, Massachusetts 01655. E-mail: beatrice.greco{at}umassmed.edu
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Abstract |
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and PgR. To determine whether ERß is found within
ER- or PgR-containing neurons in female rat, we used dual label
immunocytochemistry. ERß-immunoreactivity (ERß-ir) was
primarily detected in the nuclei of cells in the periventricular
preoptic area (PvPO), the bed nucleus of the stria terminalis (BNSTpr),
the paraventricular nucleus, the supraoptic nucleus, and the medial
amygdala (MEApd). Coexpression of ERß-ir with ER-ir or PgR-ir was
observed in the PvPO, BNSTpr, and MEApd in ovariectomized rats. E2
treatment decreased the number of ERß-ir cells in the PvPO and BNSTpr
and the number of ER-ir cells in the MEApd and paraventricular
nucleus, and therefore decreased the number of cells coexpressing
ERß-ir and ER-ir in the PvPO, BNSTpr, and MEApd. E2 treatment
increased the amount of PgR-ir in cells of the PvPO, BNSTpr, and MEApd,
a portion of which also contained ERß. These results demonstrate that
ERß is expressed in ER- or PgR-containing cells, and they suggest
that E can modulate the ratios of these steroid receptors in a brain
region-specific manner. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ERß shares a high level of sequence identity with ER in some
receptor domains, and both receptors bind to estradiol (E2) with
similar affinity (7). However, ERß and ER can exhibit
different transcriptional activities in cotransfection experiments
depending on the ligands and promoters tested (8, 9).
Furthermore, ERß and ER have the ability to homodimerize and
heterodimerize and display different transcriptional activities
dependent on the particular dimer that is formed (10, 11).
Thus, cellular responses to estrogenic ligands may depend in part on
the presence and ratio of the ER subtypes. Therefore, to study E
signaling in the brain, it is important to determine the extent of
cellular coexpression of ERß and ER in specific neuroanatomical
areas.
Recent studies have demonstrated that progesterone can alter the
expression of ERß mRNA in human breast tumors (12) and
monkey corpus luteum (13). Additionally, one study showed
that E2 acts through ERß to induce PgR expression in a colon cancer
cell line (14). In the brain virtually all cells in which
E2 induces PgR expression contain ER (15). However, E2
also induces some PgR expression in ER knockout mice (ERKO). This
response could be due to the presence of residual ER variants
(16, 17). Alternatively, ERß may mediate PgR induction
upon E2 treatment in the ERKO (17). To examine whether
the presence of ERß alters the expression of PgR in female rat brain,
it is essential to first determine whether ERß and PgR are present
within the same cells.
ERß mRNA has been detected in the brain by both RT-PCR and in
situ hybridization (5, 7). Substantial quantities of
ERß mRNA exist in hypothalamic and limbic regions of the brain, which
are known to contain high levels of ER and PgR. ERß mRNA has also
been detected in other regions of the brain [supraoptic nucleus and
paraventricular nucleus (PVN)] known to express little or no ER or
PgR (5). Recently, Shughrue et al.
(18) also observed the presence of ER-immunoreactivity
(ER-ir) in ERß mRNA-containing neurons of limbic and hypothalamic
regions of the female rat brain, suggesting that in vivo,
ER and ERß proteins may be expressed within the same cells. ERß
protein has also been detected in rat brain by use of
immunocytochemistry (19). In this study, which used an
affinity-purified polyclonal antibody raised against the C-terminus of
rat ERß, ERß protein was seen in a more limited number of cells and
in fewer regions than ERß mRNA by in situ
hybridization.
In the present study we determined the extent of cellular coexpression
of ERß-ir with ER-ir as well as ERß-ir with PgR-ir in different
brain regions, using a newly developed monoclonal antibody raised
against a peptide from the N-terminal region of ERß. The resulting
distribution of ERß-ir cells closely matched the distribution of
cells containing moderate to high levels of ERß mRNA in female rat
brain (5). A double label immunofluorescence technique was
used to determine whether ERß-ir was present in ER-ir- or
PgR-ir-containing neurons in forebrain regions of ovariectomized female
rats. Moreover, we tested the hypothesis that E2 treatment influenced
the distribution and the ratio of coexpression of these receptors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immunoblotting
Protein extracts were from Sf21 insect cells infected without or
with human (h) ERß (1–485) or wild-type hER recombinant virus.
Cells were harvested, washed, and lysed in ice cold buffer containing
0.4 M KCl, 10 mM Tris (pH 7.5), 0.5
mM phenylmethylsulfonylfluoride, 1 µg/ml aprotinin, and 1
mM dithiothreitol. Soluble protein extracts were obtained
by centrifugation at 100,000 x g for 45 min at 4 C.
Protein concentration was determined, and extracts were added to
SDS-PAGE buffer, subjected to SDS-PAGE, and transferred to
nitrocellulose membranes (Bio-Rad Laboratories, Inc.,
Richmond, CA). Membranes were blocked in 5% Carnation instant nonfat
dry milk in Tris-buffered saline (TBS; Bio-Rad Laboratories, Inc.) for 15 min. Primary hERß antibody (1 µg/ml) was
incubated with membranes overnight at room temperature in 0.5% milk in
TBS/0.1% Tween-20. Goat antimouse secondary antibodies conjugated to
alkaline phosphatase (Bio-Rad Laboratories, Inc.) were
incubated with membranes for 2 h at room temperature in 0.5%
milk/TBS/0.1% Tween-20. Washes and development of color were performed
according to the Bio-Rad Laboratories, Inc., protocol.
Characterization of ERß and ER antibodies: ligand-bound vs.
unbound receptors
To determine whether variations in ERß or ER immunostaining
after E2 treatment may be attributable to differential recognition of
ligand-bound vs. unbound ERs by the respective antibodies,
OVX female rats were injected sc with either 50 µg 17ß-E2 dissolved
in 0.1 ml vehicle (n = 5) or the 50% ethanol-water vehicle
(n = 5), and they were perfused 20 min later. This treatment with
a large amount of free E2 for a short period of time is believed to
induce maximal ligand occupancy of ER without resulting in
down-regulation of the receptor protein (20).
Control for ER down-regulation
To determine whether recognition of the antibody for the
receptor is influenced by binding of EB at the same concentration as
that used in the 48-h experiment and to determine whether any
variations in ER or ERß immunostaining at 48 h could be due
to ER down-regulation, a second group of female rats was injected sc
with the same dose of EB as the experimental group (10 µg; n =
4) or oil vehicle (0.1 ml; n = 4), but they were perfused 2 h
later, instead of 48 h later. This treatment is expected to result
in higher levels of circulating E2 (21) and occupation of
ERs (22) than in the 48 h experimental group, but
considerably less than in the group receiving 50 µg free E2 20 min
before perfusion.
Perfusions
All animals received a lethal dose of sodium pentobarbital (89
mg/kg), and they were perfused with 0.9% physiological saline (25 ml)
for 1 min followed by 4% paraformaldehyde (25 ml/min) for 10 min.
After the brains were removed from the cranium, they were placed into
0.1 M sodium phosphate buffer (pH 7.2) containing 20%
sucrose for 48 h. Thirty-five-micron sections from the preoptic
area to the midbrain region were cut on a freezing microtome, and the
sections were placed into a cryoprotectant solution.
Double label immunofluorescence for ERß-ir with either ER-ir
or PgR-ir
For all animals, two sets of sections were removed from
cryoprotectant and rinsed three times for 5 min each time in 0.05
M TBS, pH 7.6. Sections were placed into a solution
containing 1% H2O2, 20%
normal goat serum, and 1% BSA for 20 min. Sections were then incubated
for 2 d at 4 C in a solution containing the mouse monoclonal
hERß antibody (hERßNT-221.3, Ligand Pharmaceuticals, Inc., San Diego, CA; 1 µg/ml) raised against a synthetic
peptide corresponding to the 14-amino acid N-terminal sequence of
hERß (1–485 form) and either the rabbit polyclonal rat ER
antibody (C1355; gift from M. Shupnik, University of Virginia,
Charlottesville, CA; 1:30,000) raised against the last 14-amino acid
C-terminal sequence of rat ER or the rabbit polyclonal human PgR
antibody (DAKO Corp., Carpinteria, CA; 1:500) raised
against a synthetic peptide corresponding to the DNA-binding domain of
human PgR (PgR-A and PgR-B forms; 533–547), in a buffer containing
TBS, 0.1% gelatin, 0.02% sodium azide, 0.5% Triton X-100, and 1%
normal goat serum (TBS-gel). After three washes in TBS-gel buffer, the
sections were incubated in a solution containing the cyanine goat
antimouse (7 µg/ml, to reveal ERß-ir) and the fluorescein goat
antirabbit (10 µg/ml, to reveal ER-ir or PgR-ir) secondary
antisera for 90 min at room temperature. The sections were then rinsed
three times for 5 min each time in TBS, mounted onto slides, air-dried,
and coverslipped with Vectashield mounting medium (Vector Laboratories, Inc., Burlingame, CA).
Immunocytochemistry controls
ERß-ir preadsorption. To determine the specificity of the
anti-ERß antibody immunostaining for ERß, the anti-ERß antibody
was preadsorbed at 4 C overnight with a 100-fold molar excess of its
corresponding synthetic peptide before use in the immunostaining assays
and was used to immunostain brain sections.
PgR-ir preadsorption. To determine the specificity of the PgR immunostaining, the rabbit anti-PgR antibody was preadsorbed at 4 C overnight with a 100-fold molar excess of a mix of human PgR-A and PgR-B recombinant proteins (Tissue Culture Core Facility of the University of the Colorado Cancer Center) before use in immunostaining.
Double label immunofluorescent controls. To control for nonspecific immunofluorescent staining, cross-immunoreactivity, and "bleed-through" of the fluorochromes, sections were also incubated either in solutions in which the primary antibodies were omitted and the secondary antibodies were present or in solutions in which only one primary antibody was present and both secondary antibodies were present. No nonspecific immunofluorescent staining, cross-immunostaining, or bleed-through was observed (data not shown).
Data analysis
Quantification of double labeled immunofluorescent cells.
Quantifications were performed on one matched section per brain region
for each rat. Using the NIH Image computer analysis system (developed
at the NIH and available at
http://rsb.info.nih.gov/nih-image/), digitized pictures of the
same microscopic field were captured with two different bandpass
filters, specific for cyanine and fluorescein. The images were
superimposed, and the numbers of single or double labeled cells were
counted by eye in the brain region chosen. Because the main point of
the present study was to report the level of colocalization of ERß
with either ER or PgR, the regions examined were selected based upon
the presence of ERß-ir cells, that is, in the principal nucleus of
the bed nucleus of the stria terminalis (BNSTpr), the periventricular
zone of the preoptic area (PvPO), the posterodorsal nucleus of the
medial amygdala (MEApd), and PVN. The quantification for all groups was
performed with a x10 objective for each brain region (BNSTpr, 340
x 300 µm; PvPO, 580 x 120 µm; MEApd, 870 x 240 µm;
PVN, 390 x 480 µm), except for the 2 h control groups, in
which the quantification was performed with a x20 objective (BNSTpr,
180 x 160 µm; PvPO, 420 x 90 µm; MEApd, 370 x 160
µm). In all microscopic fields observed, only cells that were clearly
distinguishable were counted.
Variation in PgR immunofluorescent intensity in cells of oil- and EB-treated females. Many studies have shown that variations in immunostaining intensity can be measured by variations in OD (mean pixel densities) (23, 24, 25). Using inverted digitized pictures for PgR-ir, the mean pixel density of PgR-ir was measured in the PvPO, BNSTpr, MEApd, and PVN in oil- and EB-treated females, as described in detail previously (25). In brief, the gain and black levels of the camera were set so that the gray level ranged from 0 to 255 pixel density for PgR immunoreactivity (0 being white and 255 being black). The density threshold was set to determine the contribution of the background immunoreactivity and to exclude it from the contribution of the foreground immunoreactivity. The OD of PgR-ir (i.e. the intensity of PgR immunofluorescence) was determined for each area and was represented by the average and SEM of the pixel densities of the sections analyzed.
Variation in PgR immunofluorescent intensity in cells
coexpressing ERß-ir. PgR-ir was apparent in many cells with or
without EB treatment. However, the OD of PgR-ir in EB-treated females
was considerably brighter (see above, Table 3
). Therefore, to determine
whether cells in which EB treatment increased PgR expression contained
ERß-ir, the darkly immunostained PgR neurons were counted in
EB-treated females using a method similar to that described above. The
density threshold was set to eliminate E2-independent PgR
immunoreactivity in oil-treated females, and was used to measure
foreground PgR immunoreactivity in EB-treated females. Processed images
were superimposed on corresponding digitized pictures containing
ERß-ir, and the darkly immunostained PgR neurons coexpressing
ERß-ir were counted by eye in the PvPO, BNSTpr, and MEApd.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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55 kDa) was observed on blots
probed with the ERß antibody in lanes containing protein extract from
insect cells infected with hERß recombinant virus, but not in lanes
containing extracts from mock- or hER-infected cells (Fig. 1A). Therefore, this antipeptide ERß
monoclonal antibody reacts with ERß, but not with ER, on a Western
blot. Additionally, this antibody reacted with ligand-occupied ERß,
but not with holo-ER, as assessed by ligand binding
immunoprecipitation of the recombinant proteins (data not shown).
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antibodies: ligand-bound vs.
unbound receptors-ir and ERß-ir cells after a 20-min injection of 50 µg
17ß-E2. The number of ER-ir cells did not vary in any brain region
20 min after the injection of 50 µg free 17ß-E2 (vehicle
vs. E2: in PvPO, 116 ± 10.5
vs. 116.2 ± 5.2; in BNSTpr, 178 ± 15.1
vs. 178.2 ± 16.8; in MEApd, 177.6 ± 8.1
vs. 178.6 ± 18.6), demonstrating that the polyclonal
ER antibody used in the study recognizes both ligand-bound and
unbound ER. In contrast, immunoreactivity for ERß was not
detectable 20 min after treatment with 50 µg free 17ß-E2 (vehicle
vs. E2: in PvPO, 43.2 ± 6
vs. 0 ± 0; in BNSTpr, 54 ± 11.5 vs.
0 ± 0; in MEApd, 77.8 ± 4.6 vs. 0 ± 0),
suggesting that ERß immunostaining is greatly affected by occupation
of the receptor with a saturating dose of free E2.
Neuronal distribution of ER-ir, ERß-ir, and PgR-ir, and their
regulation by EB treatment
The pattern of distribution of ER-ir in oil- or EB-treated
female brains was consistent with previous reports
(27, 28, 29). ER-ir was present in nuclei of neurons of the
preoptic area; the PvPO; the principal and medial nuclei of the BNST;
the anterior, lateral and ventromedial hypothalamus; the parvocellular
part of the PVN; the central, medial, and cortical amygdala; the
arcuate nucleus; the piriform cortex; and, to a lesser extent, the
hippocampus. The number of ER-ir neurons in the MEApd and PVN
significantly decreased 48 h after EB treatment (P
< 0.05; Table 1). The number of ER-ir
cells did not vary in any brain region 2 h after treatment with 10
µg EB vs. treatment with vehicle (oil vs. EB:
in PvPO, 106.6 ± 10.8 vs. 128.3 ± 5.2; in
BNSTpr, 208 ± 7.9 vs. 181.4 ± 40; in MEApd,
185.6 ± 23 vs. 164 ± 24.6), consistent with the
finding that occupancy of ER by E2 does not change the interaction
of the ER antibody, C1355, with ER. Therefore, the decrease in
ER-ir cells observed at 48 h is probably due to down-regulation
of ER by E2.
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), and
the immunostaining with this antibody was completely blocked by
preadsorption of the antibody with the peptide that it was generated
against (Fig. 1B). In the forebrain, ERß-ir was found in nuclei of
cells in the PvPO, the dorsal and ventral BNSTpr, the dorsal and
magnocellular part of the PVN, the anterior and caudal supraoptic
nucleus, and the anterior and posterodorsal MEA (Fig. 2). The number of ERß-ir cells
significantly decreased in the PvPO and BNSTpr 48 h after EB
treatment vs. treatment (P < 0.05; Tables 1 and 2). Even though the level of
E2-occupied ERß is probably higher at 2 h than at 48 h, the
number of ERß-ir cells did not change in any brain region 2 h
after treatment with 10 µg EB vs. treatment with vehicle
(oil vs. EB: in PvPO, 49.3 ± 13 vs.
59.3 ± 15; in BNSTpr, 69 ± 4.9 vs. 63.3 ±
9.5; in MEApd, 91.3 ± 10 vs. 77.6 ± 20). This
suggests that the dose of EB (10 µg) used in the experiment did not
interfere with antibody binding to the receptor, even though a much
larger dose of free E2 (50 µg) interfered with immunostaining.
Therefore, the decrease in ERß-ir seen at 48 h represents
down-regulation of ERß-ir by E2.
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). However, E2 treatment resulted in a
significant increase (P < 0.05) in staining intensity
(i.e. pixel density) of PgR-ir in the PvPO, BNSTpr, and
MEApd of EB-treated females (Table 3),
consistent with previous findings that there is E2-independent PgR
expression throughout the brain, and that E2 can also increase PgR
levels.
Coexpression of ER-ir and ERß-ir
Colocalization of ER-ir and ERß-ir was observed in cells of
the PvPO, the BNSTpr, and the MEApd (Table 1 and Fig. 3). The number of neurons expressing both
ER-ir and ERß-ir decreased significantly after EB treatment in the
PvPO, BNSTpr, and MEApd (Table 1). The percentage of ER-ir neurons
containing ERß-ir in the PvPO and MEApd decreased significantly in
the EB-treated group compared with the oil-treated group
(P < 0.05; Fig. 4A).
Conversely, the percentage of ERß-ir neurons containing ER-ir in
the MEApd decreased significantly in the EB-treated group compared with
the oil-treated group (P < 0.05; Figs. 4B). In the
PVN, because ER-ir and ERß-ir were in anatomically distant sites,
very little colocalization of ER-ir and ERß-ir was observed (Table 1 and Fig. 4, A and B).
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). The number of
neurons coexpressing both PgR-ir and ERß-ir in all regions
observed did not differ between groups (Table 2). The percentage of
PgR-ir neurons containing ERß-ir in the PvPO decreased significantly
in the EB-treated group compared with the oil-treated group
(P < 0.05, Fig. 5A). In all
regions observed, there was no difference in the percentage of
ERß-ir neurons containing PgR-ir between groups (Fig. 5B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-ir- and PgR-ir-containing neurons. Furthermore, E2 treatment
modulated the expression of ERß, ER, and PgR in a brain
region-specific manner. This caused a change in the ratio of cells
coexpressing both ER subtypes or ERß and PgR in specific brain areas.
Also as expected, E2 treatment induced the expression of PgR in
hypothalamic and limbic regions, which occurred in part in
ERß-ir-containing neurons.
The hERß antibody used in the present study appears to be very
specific for ERß as assessed by immunoblotting and
immunoprecipitation assays. The antibody reacted with ERß, but not
ER, PgR, or AR, by Western blot (Fig. 1A and data not shown) and by
the elimination of immunostaining after antibody preadsorption with its
specific peptide (Fig. 1B). Although the antibody recognized the
ligand-bound ERß by in vitro immunoprecipitation assays
using recombinant proteins (data not shown), we found that sc treatment
of ovariectomized female rats with a large amount of free E2 (50 µg)
eliminated ERß immunostaining in all brain regions. This suggests
that in brain tissue, this antibody does not react with ligand-bound
ERß. Discrepancies between in vitro and in vivo
characteristics of antibodies have previously been reported, such as
for the specific ER antibody, H222 (20). Although
antibodies raised against the N-terminus of ER were not reported to
show differential interactions with ER (20), this may
not be the case for the ERß antibody used in these studies. Blocking
of the ERß antibody epitope by ligand binding may occur in
vivo through conformational changes in ERß upon binding of
ligand, by dimerization of the receptor with ER or ERß, or by
interaction of ERß with other accessory proteins, such as
cofactors.
The distribution of ERß-ir (Fig. 2) as assessed with the N-terminus
hERß monoclonal antibody was different from that in previous studies
in which a polyclonal C-terminal rat ERß antibody was used (19, 33). Using the C-terminal ERß antibody, fiber and cytoplasmic
stainings were observed in the lateral septum and the hippocampus
(19), which were not observed using the N-terminus ERß
antibody. In contrast to the C-terminal antibody, which labeled cells
in the lateral septum, the medial BNST, the PVN, the supraoptic
nucleus, the anterior MEA, and the hippocampus (19), the
N-terminal antibody labeled numerous cells within the PvPO, the BNSTpr,
the PVN, the supraoptic nucleus, and the MEApd, where the highest
density of ERß mRNA-containing cells and the highest level of ERß
mRNA have been reported (5). This suggests that the human
N-terminus ERß antibody used in the present study may specifically
recognize cells containing moderate to high levels of ERß. Thus, the
extent of colocalization of ERß with ER or PgR and the changes in
the ratio of steroid-containing cells after hormonal treatment reported
here may be underestimated. These should, of course, be taken as
relative indexes of colocalization and not as absolute numbers. It is
also possible that ERß mRNA is expressed at a higher level than ERß
protein. Differential protein expression of ERß variants that have
been reported in the brain (34) could also be an
explanation for the differences in ERß distribution observed between
in situ hybridization and immunocytochemical approaches.
Little work has been reported on the hormonal regulation of ERß in the brain. Previous studies reported that E2 treatment caused down-regulation of ERß mRNA expression in the PVN (35) and the MEA (36). Although we did not observe a decrease in the number of ERß-ir cells in the PVN or the MEApd 48 h after EB injection, the number of ERß-ir cells decreased in the PvPO and the BNSTpr. This decrease is probably due to down-regulation of ERß in specific brain regions. Although an injection of 50 µg free E2 20 min before perfusion interfered with ERß antibody binding to ERß, an injection of 10 µg E2 benzoate 2 h before perfusion resulted in apparent levels of ERß-ir not different from those observed after vehicle treatment. This suggests that even though ERß immunostaining can be influenced by occupation of the receptor with a saturating dose of free E2 (50 µg), exposure to a low level of circulating E2 (10 µg EB) does not interfere with the ERß immunostaining. Thus, decreases in ERß immunostaining observed after EB injection are probably due to down-regulation of ERß. Although this issue needs to be further examined with molecular approaches, the present results are consistent with reports of down-regulation of ERß mRNA in the brain (35, 36) and other tissues (37) after E2 treatment. Therefore, these findings suggest that ERß protein expression may be differentially regulated by E2 in various regions of the brain.
Our observations on the presence of ER-ir in hypothalamic and
limbic regions, including the PvPO, the BNSTpr, the parvocellular part
of the PVN, and the MEApd, agree with previous reports using various
ER antibodies (27, 28, 29). E2 treatment significantly
decreased the number of ER-ir neurons in the MEApd and the PVN. This
decrease is also probably due to a brain region-specific
down-regulation of ER, as ligand binding did not influence ER-ir
staining (with the C1355 antibody used in this study) in any brain
region. Moreover, these findings agree with previous reports of
down-regulation of ER or ER mRNA in the brain after E2
administration (38, 39).
In vitro studies suggest that ERß and ER may
heterodimerize (10). For this heterodimerization to occur
in the brain, both ER and ERß must be expressed in the same
neurons. Colocalization of ER-ir and ERß-ir was observed in
neurons of the PvPO, the BNSTpr, the MEApd, as well as the PVN.
Depending on the region and the hormonal treatment, this represented
less than 40% of the ER-containing cell population, but this
represented over 60% of the ERß-ir neurons, except in the PVN where
very little colocalization was observed. Shughrue et al.
(18) previously reported a comparable, high level of ERß
cells coexpressing ER in the PvPO, the BNST, and the MEApd when
examining the colocalization of ERß mRNA and ER-ir. In contrast in
the present study because the number of ERß-ir cells is lower than
the number of ERß mRNA cells in the brain regions observed, the
levels of ER-containing cell coexpressing ERß are lower than those
reported in the earlier study (18). Nevertheless, these
findings provide evidence that ER and ERß have the opportunity to
interact in vivo intracellularly in a small population of
limbic and hypothalamic neurons. Furthermore, it suggests that in these
brain regions, transcriptional regulation by E2 could occur via at
least three classes of cells: cells containing ER, ERß, or
both.
Because ERß expression decreased in the PvPO after E2 treatment, the
number of ER neurons containing ERß decreased significantly. This
decrease in neurons coexpressing both ERs also results in an increase
in the number of cells expressing only ER-ir. In contrast to the
E2-induced decrease in ERß in the PvPO, in the MEApd ER expression
significantly decreased after E2 treatment, which induced an increase
in the number of cells expressing ERß only. As previously mentioned,
ER and ERß isoforms have the ability to form heterodimers
(10, 34), which have different transcriptional activities
than ER homodimers (11). Together with the present data,
this further suggests that differential regulation of ERs by E2 may
lead to cells with different profiles of E responsiveness in a
brain region-specific manner.
The neural distribution of PgRs in females and the increase in its expression by E2 in some brain regions have been described extensively using a variety of techniques (30, 31, 40). In other immunocytochemical studies, a limited distribution of PgR-ir has been observed in the brain due to the sensitivity of the immunocytochemical procedure adjusted so that either only cells in which E2-induced PgR-ir were labeled (30) or only dark nuclear staining was observed (32). Using a higher concentration of antibody than had been used previously, we detected PgR-ir in neurons of regions previously described as containing E2-induced PgR, such as in the PvPO, the preoptic area, the BNSTpr, the lateral ventromedial hypothalamus, the arcuate nucleus and the MEApd, as well as E2-independent PgR such as in the PVN and the cerebral cortex (31, 41, 42, 43). The requirement of a higher concentration of antibody necessary to visualize PgR in the PVN and the cortex suggests a lower abundance of the receptor in these regions. Both isoforms of PgR (PgR-A and PgR-B) have been detected in the brain (41). Although it has been suggested that the expression of one of the two PgR isoforms (PgR-B) has less dependence on E2 (41), the PgR antibody used here recognizes both PgR isoforms and does not allow us to discriminate the two. In accordance with previous reports (30), E2 injection increased the expression of PgR-ir, as seen in the present study by an increase in optical intensity in the PvPO, BNSTpr, and MEApd, but no effect of E2 on PgR expression was detected in the PVN. This finding agrees with earlier ligand binding studies in which progesterone binding was seen throughout the brain, but E2 treatments increased PgR expression only in limited areas (44).
Although the coexpression of PgR and ERß has been reported in cell
lines and human tumor cells (12, 45), we report here the
in vivo coexpression of PgR-ir and ERß-ir in neurons of
the PvPO, the BNSTpr, the MEApd, and the PVN of female rat brains.
Depending on the region and the hormonal status of the females,
20–50% of the PgR-ir cells coexpressed ERß-ir. Conversely in the
PvPO and PVN, over 70% of the ERß-ir cells coexpressed PgR-ir,
whereas only about 45% did in the BNSTpr and the MEApd. It is well
known that E2 induces the expression of PgR in ER-containing cells
of the female brain (15, 46). In the present study
E2-induced PgR-ir was measured by an increase in the intensity of
PgR-ir. It is important to note that ERß-ir was found in cells
containing E2-induced PgR-ir (EB-treated) as well as cells containing
E2-independent PgR-ir (i.e. in oil-treated rats) in all
regions tested. More specifically, about 30% of cells that showed an
increased expression of PgR-ir coexpressed ERß-ir. We did not test
for the presence of ER in these cells. However, as mentioned
earlier, E2-induced PgR-ir is almost exclusively in ER-
containing cells (15), strongly supporting the idea
that cells with E2-induced PgR and ERß-ir may also contain ER.
In ER-gene disrupted (ERKO) mice, E2 treatment increased the
expression of PgR mRNA in the PvPO (16) and of PgR-ir in
the MEApd as well as in the arcuate nucleus and the lateral
ventromedial hypothalamus (17). Although our present data
suggest the involvement of both ER and ERß in the regulation of
PgR expression, future studies using various steroid receptor
gene-disrupted models will be needed to further examine whether the
induction of PgR expression by E2 is mediated by ER variants, ERß,
or both.
It has recently been shown that ERß plays a modest role, if any, in the regulation of female sexual behavior. Indeed, although sexual behavior is not impaired in ERß gene- disrupted mice, the duration of sexual receptivity in females is extended compared with that in wild-type animals (47). On the other hand, the brain regions in which ERß-ir is present are known to be involved in gonadal hormone regulation of a variety of neuroendocrine systems. For example, the posterior medial BNST (level equivalent to the BNSTpr) is involved in the regulation of the CRH expression in the PVN (48), the expression of which is regulated by gonadal hormones. Likewise, in the supraoptic nucleus and PVN, ERß has been located in oxytocin- and vasopressin-containing cells (33), which are involved in reproductive functions.
Our results demonstrated that ERß is present in cells containing
other steroid receptors in brain regions that are of importance in
females, and they suggest that ERß alone or in combination with ER
and/or PgR may participate in the hormonal regulation of a variety of
neuroendocrine functions. Furthermore, our results suggest that E2 may
play a crucial role in regulating amounts of ERs ( and ß) and PgRs
in cells in a brain-region specific manner, which may, in turn, induce
differential expression of relevant genes.
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Acknowledgments |
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Footnotes |
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Abbreviations: BNSTpr, Bed nucleus of the stria terminalis; E,
estrogen; E2, estradiol; EB, E2 benzoate; ERß-ir,
ERß-immunoreactivity; ERKO, ER knockout mice; hER, human ER;
MEApd, medial amygdala; OVX, ovariectomized; PgR, progestin receptor;
PVN, paraventricular nucleus; PvPO, periventricular preoptic area; TBS,
Tris-buffered saline.
Received March 21, 2001.
Accepted for publication September 5, 2001.
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) and EB-treated (
)
females. *, Significantly different from the oil group (by
t test, P < 0.05).
