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Molecular Biology and Functional Morphology Divisions, Women’s Health Research Institute, Wyeth-Ayerst Research, Radnor, Pennsylvania 19087
Address all correspondence and requests for reprints to: Dr. Susan L Fitzpatrick, Women’s Health Research Institute, Wyeth-Ayerst Research, 145 King of Prussia Road, Radnor, Pennsylvania 19087. E-mail: fitzpas2{at}war.wyeth.com
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-iodovinyl-11ß-methoxyestradiol
([125I]-VME2), bound to cytosolic granulosa cell
preparations with high affinity (estimated KD value of
401 ± 83 pM, and Bmax value of 102
± 9 fmol/mg protein). ER-ß protein levels rapidly declined following
hCG treatment consistent with the reported decrease in binding activity
and ER-ß mRNA levels by high levels of gonadotropins. Overall, we
have demonstrated that 1) ER-ß protein is the dominant estrogen
receptor subtype present in rodent granulosa cells, 2) this receptor is
functional, and 3) it is regulated by ovulatory doses of gonadotropins.
Thus, ER-ß is likely to be a mediator of estrogen action in rodent
granulosa cells during follicular development. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Presumably, estrogen exerts its biological effects in granulosa cells
through classical nuclear receptors. Earlier reports, using
radiolabeled hormone, demonstrated that estradiol was taken up by the
ovary (5), and binding sites were observed in rat granulosa cell nuclei
(6, 7). Later reports further characterized these estrogen binding
sites in the ovary (7, 8, 9, 10). These binding sites were believed to be
estrogen receptors, which are now referred to as estrogen receptor-
(ER-). ER- messenger RNA (mRNA) has been detected in rodent
granulosa cells using in situ hybridization and RNase
protection assays but is also present in residual ovarian tissue
(thecal and interstitial) (11, 12). ER- protein has been identified
in granulosa cell nuclear extracts by electrophoretic mobility shift
assays (13, 14). In contrast, other studies detected ER- protein in
interstitial and theca cells rather than in granulosa cells (15). The
importance of ER- for reproduction can be observed in female mice
lacking ER- (ERKO). These mice are usually infertile, and the
ovaries are blocked in follicular development at the small antral stage
[ (16, 17, 18), reviewed in (19)]. However, it is not known if this
block in follicular development is due to a primary deficiency in
ovarian follicles or if it is secondary to a deficiency in the
hypothalamus or pituitary. Nevertheless, it suggests that ER- is not
required for early follicular development in mice.
With the discovery of a second estrogen receptor, ER-ß (10, 20), it
is possible that some of the estrogen effects in the ovary are mediated
through this receptor. ER-ß was initially identified in the rat
prostate but found to be highly expressed in the rodent ovary
(10). The ligand binding domain (LBD) of ER-ß is only 55% homologous
to the LBD of ER- (10), suggesting that specific receptor subtype
ligands might be identified. Many estrogen-like compounds have similar
affinities for ER- and ER-ß, although genistein, a phytoestrogen,
has a higher affinity for ER-ß than for ER- (21). Additionally,
ER-ß and ER- differ in their responses to agonists and antagonist
in cell type and promoter-specific manners (22, 23, 24).
In the ovary, ER-ß mRNA expression can be detected by RT-PCR (21, 25, 26, 27, 28), RNase protection assays (11, 26, 29), Northern blot analysis
(20, 25, 26, 28, 30) and in situ hybridization (10, 26, 27, 28).
In addition, splice variants of ER-ß are observed in ovary RNA
(31, 32, 33, 34). ER-ß mRNA is specifically localized to granulosa cells
using in situ hybridization and absent (12) or weakly
present in interstitial cells (10, 26, 27, 28). Using this method, ER-ß
mRNA was not detected in primordial follicles or oocytes (10), and
expression was very low in corpora lutea (11, 27). ER-ß mRNA was
demonstrated in granulosa cells from ER- deficient ERKO mice (29).
Recent evidence suggests that ER-ß mRNA expression in granulosa cells
is regulated by the hypothalamic-pituitary axis. Rat ER-ß mRNA levels
in granulosa cells are relatively constant during follicular
development (11, 28) but dramatically decline after high doses of
gonadotropins, e.g. the LH surge (28) or after treatment
with high levels of hCG (11, 28).
Despite the abundance of information on ER-ß mRNA, there is little characterization of the ER-ß protein in any cell type. Recent reports demonstrate ER-ß protein in rat granulosa cells by electrophoretic mobility shift assay (35) and by immunocytochemistry (15). In this paper, we demonstrate the presence of ER-ß protein in rodent granulosa cells and its hormonal regulation by ovulatory doses of gonadotropins. A detailed description of ER-ß protein was completed using several molecular and biochemical approaches to enhance our understanding of the nature of this protein. The results suggest that ER-ß is likely to be a mediator of estrogen action in rodent granulosa cells during follicular development.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Antisera
Antisera were obtained commercially or prepared in-house. The
following commercial rabbit antipeptide antisera and the corresponding
rat (10) or human (20) ER-ß peptide sequences used to make the
antisera are as follows: PC168: rat ER-ß 467–485 (Oncogene Research Products division of Calbiochem,
Cambridge, MA also known as PA1–310 (Affinity BioReagents, Inc., Golden, CO); 6–629: rat ER-ß 54–71 (Upstate Biotechnology, Inc., Lake Placid, NY); no. 91: human ER-ß
171–185 (human specific, prepared in-house). Each of these antisera
recognizes ER-ß and does not recognize ER-. For the detection of
ER-, the antisera SRA-1000 (StressGen, Victoria, BC, Canada) and
MC-20 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and
FMS-ER7 (in house, raised against last 21 aa of rat ER-) (37) were
used.
An additional antiserum against ER-ß, RB2.3, was raised in rabbits
(polyclonal) using the AF-2 region of rat ER-ß (R182-
Q485). The protein was expressed from a DNA sequence
subcloned into bacterial expression vector pET23b (Novagen, Madison,
WI), which contains a histidine tag, and was purified using a His-Bind
Resin (Novagen) and preparative SDS-PAGE. Rabbits were immunized with
RB2.3 and the antiserum collected, affinity purified using a SulfoLink
kit (Pierce Chemical Co., Rockford, IL), dialyzed against
PBS containing 15 mM sodium azide and 0.2% BSA, and stored
at -70 C in aliquots for future use. The titer of the purified
antiserum was determined by Western blot analysis using the recombinant
protein. This antiserum did not cross-react with a recombinant protein
containing the AF-1 region of ER-ß (unpublished observation) or
full-length ER-. RB2.3 recognizes both rat and human ER-ß
protein.
Antiserum to HA1 (Roche Molecular Biochemicals, Indianapolis, IN) was obtained commercially.
Immunocytochemistry
Rat ovaries were fixed by either perfusion or immersion using
2% paraformadehyde and 2% acrolein buffered with sodium phosphate to
pH 7.2. The tissue was held in fixative for 1–2 days, cryoprotected
overnight with 20% sucrose, frozen, and stored at -80 C. Sections
were cryosectioned at 10 µm, air-dried, and stored at -80 C.
Immunocytochemistry was done using the buffers of Slayden and
Brenner’s (38, 39). The sections were pretreated by incubating with a
solution containing PBS, 1% sodium borohybride, 0.1%
polyvinylpyrrolidone (PVP) 360 (Sigma Chemical Co.),
washed, incubated with a solution containing PBS, 1%
H2O2, 0.3% Triton X-100, PVP 360; then
incubated with a solution containing PBS, 10% normal donkey serum
(Chemicon), and 0.1% gelatin. The primary antisera for ER-ß (PC 168,
Oncogene Research Products or PA1 310, Affinity BioReagents, Inc.) were used at 1:2000 or 5 µg/ml dilution,
respectively. The secondary antiserum, donkey antirabbit
F(ab)2 fragment conjugated to biotin SP (Jackson
Immunoresearch Laboratory, West Grove, PA) was used at a 1:1,000
dilution. The Elite standard ABC kit (Vector Laboratories, Inc., Burlingame, CA) and diaminobenzidine (40 mg/ml in Tris, pH
7.6 and 0.005% H2O2) were used for avidin
peroxidase detection.
Immunoprecipitation
Granulosa cells from PMSG-primed female mice or estradiol-primed
female rats were plated in dishes in the presence of DMEM:Ham’s F-12
(1:1, Life Technologies, Inc., Gaithersburg, MD),
supplemented with 1% FBS (HyClone Laboratories, Inc.,
Logan, UT), 100 U/ml penicillin, 100 U/ml streptomycin, and 1
mM GlutaMAX-1 (Life Technologies, Inc.). The
next day, the cells were rinsed with PBS then incubated with DMEM
without L-methionine and L-cystine (Life Technologies, Inc.) supplemented with
[35S]-EXPRESS Methionine/Cysteine Protein Labeling Mix
(NEN Life Science Products, Boston, MA) (122 µCi/35 mm
dish or 300 µCi/100 mm dish), 10 mM HEPES buffer solution
(Life Technologies, Inc.), 2 mM
L-glutamine (Life Technologies, Inc.), and
1 x penicillin/streptomycin solution (Life Technologies, Inc.) for 6 h at 37 C. Cells were washed three times with
cold PBS and lysed in cold RIPA buffer [50 mM Tris-HCl, pH
7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate,
0.1% SDS containing protease inhibitors (Roche Molecular Biochemicals). Cell extracts were incubated with normal rabbit
serum for 30 min at 4 C with rotation and incubated with protein G
Sepharose [50% slurry in buffer B (2 mM Tris, pH 7.5,
0.1% SDS, 0.1% NP-40)] (Amersham Pharmacia Biotech,
Piscataway, NJ) for 30 min at 4 C with rotation. Extracts were
centrifuged for 2 min at 10,000 rpm (4 C) and the supernatant was
removed. Precleared extracts were incubated with antiserum for 12
h at 4 C with rotation and incubated with protein G Sepharose for
1 h at 4 C with rotation. Samples were centrifuged for 2 min at
10,000 rpm (4 C) and the precipitates were washed five times in cold
buffer C (2 mM Tris-HCl, pH 7.5, 0.1% SDS, 0.1% NP-40,
150 mM NaCl). Proteins were solubilized in 2 x
Laemmli sample buffer (Bio-Rad Laboratories, Inc.,
Hercules, CA) and separated under reducing conditions on 10%
Tris-Glycine gels (Novex) by electrophoresis. Gels were
fixed in 30% methanol, 10% acetic acid for 30 min at room
temperature, followed by immersion in Amplify (Amersham Pharmacia Biotech) for 30 min at room temperature. Gels were dried under
vacuum at 80 C for 30 min and exposed for 12 h to X-Omat AR film
(Eastman Kodak Co., Rochester, NY) at -80 C.
Western blot
Granulosa cell nuclear extracts were prepared from immature
estradiol-treated rats as described previously (40, 41) and homogenized
in RIPA buffer. Whole ovary tissue extracts from immature rats
(Charles River) and liver extracts from adult rats
(Harlan Sprague Dawley, Inc.) were prepared by
homogenization in RIPA buffer. Rat ER- and rat ER-ß (485 aa, short
form) (10) protein were prepared by in vitro translation
using the TNT kit (Promega Corp., Madison, WI). Rat ER-ß
protein, long form, was prepared using the full-length rER-ß
complementary DNA (cDNA) and the TNT kit (Promega Corp.).
Protein (40–50 µg) from tissue extracts were electrophoresed under reducing conditions in a 4–20% or 10% Tris-Glycine gel (Novex, San Diego, CA) and transferred onto Immobilon-P membrane (Millipore Corp., Bedford, MA). The membrane was incubated for 1 h at room temperature in blocking buffer [Dulbecco’s PBS (D-PBS), 5% Carnation nonfat dry milk, 0.3% Tween-20]. RB2.3 antiserum was applied at a 1:1000 dilution in PMT buffer (PBS, 3% milk, 0.3% Tween-20) and incubated overnight at 4 C. The membrane was washed (4x for 5 min) in wash buffer (PBS, 0.3% Tween-20) and incubated with horseradish peroxidase (HRP)-linked antirabbit IgGs (from donkey) (Amersham Pharmacia Biotech) at a 1:3000 dilution in PMT for 1 h at room temperature. The membrane was washed again (4 times for 5 min each) in wash buffer and two more times for 10 min each in PBS. The membrane was immersed into SuperSignal Ultra Substrate working solution (Pierce Chemical Co.) for 5 min and exposed to X-Omat AR film (Eastman Kodak Co.).
For the hCG time course experiment, the proteins were detected by chemifluorescence according to the directions of the manufacturer (Amersham Pharmacia Biotech). The gel was transferred to membrane, incubated with 5% blocking agent (provided by Amersham Pharmacia Biotech) (1 h, room temperature), washed, then incubated with RB2.3 antiserum (1 h, room temperature). Next, the membrane was washed, incubated with fluorescein-linked antirabbit antibody (1 h, room temperature), washed, and incubated with antifluorescein alkaline phosphatase conjugate (1 h, room temperature). To detect protein, ECF substrate was added to the membrane (20 min, room temperature). The membrane was dried and then scanned using a 570 nm filter with a Storm Imager (Molecular Dynamics, Inc.). The data from two experiments was quantified using ANOVA and LSD tests. Data are presented as mean ± SEM with significance indicated (*, P < 0.05).
Electrophoretic mobility shift assays (EMSA)
All reactions were performed in EMSA buffer (20 mM
HEPES, pH 7.8, 80 mM KCl, 10% glycerol, 2 mM
MgCl2, 0.2 mM EDTA, 2 mM DTT, 200
µg/ml poly d[I-C], 25 µg/ml denatured salmon sperm DNA) in a
final reaction volume of 25 µl. Recombinant human (h) ER- (HA1
tagged) and hER-ß [long = 530 aa, (42)], were derived from a
baculovirus (bv) expression system (BAC-TO-BAC, Life Technologies) and prepared in extraction buffer (20
mM HEPES, pH 7.6, 500 mM NaCl, 1.5
mM MgCl2, 20% glycerol, 2 mM DTT,
1 mM phenylmethylsulfonyl fluoride, 10 mg/ml leupeptin, 20
mg/ml aprotinin). Granulosa cell nuclear extracts were prepared from
estrogen-primed immature rats as described (40, 41). EMSA buffer was
supplemented with the indicated protein source (approximately 0.5 µg
bv hER-, 0.5 µg bv hER-ß, or 10 µg granulosa nuclear extract)
and preincubated for 15–30 min at room temperature. For standard EMSA
assays, the reaction was supplemented with 30,000 cpm of
[32P]-dCTP-labeled vitellogenin estrogen response element
(vitERE) (43) and allowed to proceed for 1 h. Unlabeled vitERE or
progesterone response element (PRE) oligos (100-fold molar excess) were
added for the competition experiments.
vitERE: 5'-CCAAAGTCAGGTCACAGTGACCTGATCAAAGTTAATGTAACCTCA-3' 3'-TTCAGTCCAGTGTCACTGGACTAGTTTCAATTACATTGGAGTACG-5'
PRE: 5'-AGCAAAGTCAGAACACAGTGTTCTGATCAA TTTCAGTCTTGTGTCACAAGACTAGTTCGA-5'
In the ligand-based EMSA assays, the reaction
was supplemented with 1 pmol of unlabeled vitERE and 0.75 µl of
[125I]-17-iodovinyl-11ß-methoxyestradiol (E isomer)
([125I]-VME2) (1.67 mCi/ml; 2200Ci/mmol, NEN Life Science Products) and allowed to proceed for 1 h.
Supershift analysis of the DNA/protein and DNA/protein/ligand
interactions were accomplished by adding approximately 1 µg of the
indicated antiserum during the preincubation step. Samples were
resolved by PAGE for 1.5–2 h at 4 C in 0.5 x TBE. Gels were
dried on Whatman paper and exposed to film with an
intensifying screen for 16–48 h at -70 C. For the radiolabeled ligand
EMSA, after an initial exposure of the gel, the wells were cut off and
the gel reexposed to more easily detect the binding complexes in
granulosa cells extracts.
Radioligand binding assays
Harvested granulosa cells from immature, untreated rats were
washed in PBS, then pelleted by centrifugation (2,000 x
g, 5 min). Cells were homogenized (30 sec, setting 3, PT1200
polytron, Kimematica; Switzerland) in binding buffer (10 mM
Tris HCl, 1 mM EDTA, 1 mM dithiothreitol, pH
7.4 at 37 C) and centrifuged at 50,000 x g for 1
h. The supernatant was collected and used for radioligand binding
assays. Protein concentration was determined on cytosolic preparations
using the Pierce Chemical Co. BCA protein assay with
BSA as the standard (44). The cytosol routinely contained approximately
42 µg protein/reaction.
Binding reactions were prepared in triplicate in 96-well microtiter
plates. Cytosol preparation (50 µl) was added to each well followed
by 25 µl binding buffer for determining total bound or 1
µM 17-ß-estradiol (Sigma Chemical Co.) for
determining nonspecific bound. Reactions were initiated by the addition
of 25 µl of increasing concentrations of 50–2000 pM
[125 I]-VME2 (2200 Ci/mmol specific activity; NEN Life Science Products) in estrogen binding buffer for a final
reaction volume of 100 µl. The reactions were incubated on an orbital
shaker for 2 h (25 C). Ice-cold buffer (100 µl) containing 1%
(wt/vol) Norit A-activated carbon (Fisher Scientific, Fair
Lawn, NJ) and 0.01% (wt/vol) dextran T500 (Amersham Pharmacia Biotech, Uppsala, Sweden) was added to trap unbound estradiol.
The microtiter plates were centrifuged at 2,500 x g
for 10 min and 150 µl of supernatant was removed from each well. The
amount of radioactivity present in each sample was measured using an
ICN Micromedic 10-channel 
counter (ICN Micromedic Systems, Huntsville, AL). The amount of specifically bound
radioactivity was calculated by subtracting nonspecific counts from
total counts after correcting the data for a total sample volume (200
µl).
A three-parameter logistic model with parameters KD, Bmax, and slope was fitted to evaluate the 2-site saturation models. If the slope estimate indicated a 1-site model (slope not significantly different than 1), the slope was locked to 1, and the analysis was rerun to provide a linear Rosenthal plot. In contrast, if the slope differed significantly from 1, a curvilinear plot was generated, and the 2-site saturation model was run to determine the binding parameters of each binding site. Using this procedure, we were able to demonstrate that a one-site model was suited for our data. The customized JMP applications were developed by Biometrics Research (Wyeth-Ayerst Laboratories, Inc., Princeton, NJ).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and hER-ß protein. As shown
in Fig. 1
, antisera SRA-1000, and MC-20
specifically recognized ER- but not ER-ß protein. In contrast,
Ab91, 6–629, PC168, and RB2.3 specifically recognized ER-ß but not
ER- protein.
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A and B). No staining was observed in
the interstitium or theca cells. In contrast, ER- immunoreactivity
was detected in theca and some interstitial cells using the ER-
specific antiserum FMS-ER7 (Fig. 2, C and D). The same ER- antiserum
strongly stained cells in uterine tissue. As a negative control, normal
donkey serum did not detect any specific staining in the ovary.
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, left panel). No band
was seen with NRS. Using rat granulosa cells, a protein of
approximately 60 kDa was observed using RB2.3 but not NRS or rabbit IgG
(Fig. 3, right panel). This experiment was performed in
triplicate and a representative result is shown in Fig. 3.
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were included. Using peptide-purified antiserum RB2.3, a
band of approximately 60 kDa was observed in the granulosa cell lane
(Fig. 4). The data shown is typical of
the results from three experiments.
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, lane 1, arrows). The binding was competed for by an excess
of an unlabeled ERE but not a PRE oligomer (lanes 6 and 7). The
complexes were supershifted by the presence of ER-ß antiserums PC
168, 6–629, and RB2.3 but not by SRA-1000, an ER- specific
antiserum (lanes 2–5). The slower mobility of the RB2.3 complex is
likely due to multiple antibodies binding to the protein because RB2.3
is an antiserum directed against the AF-2 domain of rat ER-ß, whereas
PC168 and 6–629 are antisera directed against peptide sequences. As
additional controls, bv hER-ß (long form) was supershifted by
antiserum 91, which is specifically directed against human ER-ß
(lanes 9, 10), and by antiserum 6–629 (data not shown and Fig. 4B
, lane 6). Baculovirus-expressed hER- was supershifted by
antiserum SRA-1000, which is specific for ER- (lanes 11 and 12).
Interestingly, the ERE oligomer complex formed with granulosa cell
proteins was larger than that formed with bv hER-ß (long) protein and
the ERE oligo (compare lanes 1 to 8). Although ER- protein was not
detected in granulosa cells in this autoradiograph, it was detected in
granulosa cells using another ER- antiserum (MC-20) and a longer
exposure (data not shown).
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and ER-ß (unpublished observation). The labeled ligand, in the
presence or absence of ERE oligomer, migrated near the top of the gel
in the absence of protein (data not shown). The labeled ligand bound to
bv hER- or bv hER-ß complexed with ERE oligomer, thus detecting
the shifted complex (Fig. 5B, lanes 1 and 5). The bv hER- complex
was supershifted with SRA-1000, an ER--specific antiserum (lane 3),
but not with 6–629, an ER-ß-specific antiserum (lane 2). Because bv
hER- contains the HA1 epitope, antiserum to HA1 also supershifted
the bv ER--DNA binding complex. Alternatively, the ER-ß complex
was supershifted with 6–629 and 91, both hER-ß specific antisera
(lanes 6 and 8), but not with SRA-1000, an ER--specific antiserum
(lane 7). Using granulosa cell nuclear extracts, the labeled ligand
detected a protein-ERE complex (lane 9), which was supershifted by
6–629 but not SRA-1000.
Specific estradiol binding sites in rat granulosa cells
To characterize the I-VME binding sites in granulosa cells,
cytosol extracts from immature, unprimed rat granulosa cells
were incubated with increasing concentrations of
[125I]-VME2 in the presence or absence of
17ß-estradiol. Specific binding increased as the concentration of
[125I]-VME2 increased (Fig. 6). A Scatchard analysis revealed a
linear relationship between Bound/Free and Bound indicating a single
class of receptors with a weighted estimated KD value of
401 ± 83 pM and a Bmax value of 102
± 9 fmol/mg protein (Fig. 6, inset).
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. ER-ß protein was demonstrated in
granulosa cells of preantral, small antral, and large antral follicles
from vehicle-treated and PMSG-treated rats. There was little change in
staining 3 h after hCG. In contrast, 9–12 h after hCG, there was
a dramatic decrease in ER-ß immunoreactivity in large ovulatory
follicles but not small antral or preantral follicles. After 24 h,
ER-ß staining was still observed in small follicles but not in newly
formed corpora lutea.
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. There
was no statistical difference in ER-ß protein levels in granulosa
cells from PMSG- compared with vehicle-treated animals. Statistical
analysis revealed that ER-ß protein was reduced 9–24 h following hCG
treatment compared with PMSG, vehicle, or control values (*,
P < 0.05).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mRNA and protein are preferentially localized
in theca and interstitial cells with little or no detection in
granulosa cells, the response of granulosa cells to estrogens is likely
to be primarily through ER-ß rather than ER-.
The size of the rat ER-ß protein produced in vivo has not
been previously demonstrated and the predicted size has been an issue.
The original description of ER-ß predicted a protein of 485 aa
(
54.2 kDa) (10) although more recent data (42, 48) suggests that the
rat ER-ß sequence was incomplete. The predicted rat protein, could be
either 530 aa [59.2 kDa, similar to the human long form (42)] or
549 aa (61 kDa, longest open reading frame with a stop codon before
the first methionine). Immunoprecipitations and Western blot analyses
with an antiserum directed against the rat ER-ß AF-2 domain, RB2.3,
revealed a single protein of approximately 60 kDa for ER-ß in
PMSG-primed mouse and ± E2-primed rat granulosa cells.
The presence of ER-ß protein in rodent granulosa cells was further
confirmed using electrophoretic mobility shift assays. Several
different ER-ß antisera supershifted a rat granulosa nuclear
extract-ERE binding complex. Detection of a supershifted complex with
ER- antisera depended on the antiserum used and required a much
longer exposure, suggesting that the amount of ER- protein in
granulosa cells is quite low. These results are consistent with those
recently reported by Clemens et al. (35). An additional
level of specificity was examined by a ligand based EMSA with the
synthetic estrogen [125I]-VME2. [125I]-VME2
has been shown to accumulate in the ovary when injected into rats (45),
bind cells containing ER- and ER-ß in vivo (49), and,
in data presented in this paper, bind both ER- and ER-ß in a
ligand-based EMSA. In comparison, 16-[125I]iodo-E2 has
a higher binding affinity for ER- than for ER-ß (21). Nuclear
extracts from rat granulosa cells specifically bound to an ERE, forming
a complex that bound [125I]-VME2, and the complex was
entirely supershifted by an antiserum specific to ER-ß. Furthermore,
[125I]-VME2 recognizes a single class of high affinity
binding sites in granulosa cells (Kd = 0.4 nM
and Bmax = 102 fmol/mg protein). The binding data were
similar to that previously seen with [3H]-estrogen for
rat ovary or granulosa cell extracts (7, 8, 9, 10). Thus, the present
radioligand studies are consistent with those from over 20 yr ago using
rat granulosa cells (6). Data presented here suggests that this binding
activity is ER-ß and identifies ER-ß as the dominant estrogen
binding species in rat granulosa cells.
ER-ß mRNA in rat granulosa cells has been shown to be hormonally regulated by gonadotropins. Low levels of gonadotropins appear to slightly decrease ER-ß mRNA levels but high doses, e.g. the LH surge or hCG after PMSG treatment, dramatically reduce ER-ß mRNA expression (11, 28). We demonstrate here that ER-ß protein is not regulated by low levels of gonadotropins but is down-regulated within 9–24 h by high levels of gonadotropins. Specifically, the expression of ER-ß protein is greatly reduced in granulosa cells from large ovulatory follicles but not in granulosa cells from small antral or preantral follicles. Likewise, binding of granulosa cell extracts to an ERE was reduced when the extracts were obtained from rats treated with a high dose of hCG compared with control animals (35) and binding of [3H]-estradiol to granulosa cells was reduced in estrogen- and FSH-primed rats treated with an ovulatory doses of LH (6). These results indicate that ER-ß mRNA and protein are coordinately down regulated in granulosa cells.
The change in ER-ß levels following the LH surge is similar to that
seen for a number of other genes expressed in granulosa cells. For
example, within 6 h after a surge of LH or hCG, P450 aromatase and
LH receptor mRNAs dramatically decline [reviewed in (50)] and ER-
mRNA levels partially decline (11). In contrast, the expression of
progesterone receptor (PR) and prostaglandin synthetase-2 (also known
as cyclooxygenase-2) mRNAs are induced following the gonadotropin surge
(reviewed in Ref. 50). The effects of high doses of gonadotropins on
ER-ß expression could be a direct stimulation of transcription or an
indirect effect, perhaps through the induction of PR. Regardless, there
is a complex change in gene and protein expression patterns as
granulosa cells start to differentiate and undergo luteinization. The
gene expression pattern would suggest that estrogen, ER-, and ER-ß
play a role in maintaining and facilitating follicular development but
are not required for luteinization.
The precise role and mechanism of action of estrogen in the rodent
ovary is not well understood. There are two well documented effects of
estrogen in the rat ovary; concomitant increase in granulosa cell
number and ovary wet weight (reviewed in Refs. 2, 4, 51) and
enhancement of FSH-induced gene expression (reviewed in Refs. 2, 3, 4).
Studies of female mice lacking a functional aromatase gene would
suggest that estrogen is not required in mice for follicular
recruitment but contributes to growth to the antral stage and is
required for follicular development beyond the preovulatory stage and
for ovulation (52). The relative contributions of ER- and ER-ß to
estrogen responses in the ovary are being elucidated. Female mice that
lack ER- are infertile in the absence of exogenous gonadotropins and
follicular development is arrested at the preovulatory stage (16, 17, 53). In female adult mice, ovaries appear hyperemic, and follicles
contain few granulosa cells (16, 17, 53). However, because the
regulation of gonadotropin synthesis and secretion in the hypothalamus
and pituitary is impaired in ER- -/- female mice, the arrest of
follicular development and cyst formation that occurs may result from
deficiencies directly within the ovary or indirectly within the
hypothalamic-pituitary axis. In contrast, the fertility of female mice
that lack ER-ß is compromised as demonstrated by a reduced number and
size of litters. In the ER-ß -/- ovaries, follicles of all stages
of development are present, although the presence of more atretic
follicles and fewer corpora lutea compared with wild-type ovaries
suggest a partial arrest of follicular development and less frequent
follicular maturation (54). Furthermore, superovulation of these mice
results in an increase in mature follicles, but the number of ovulated
oocytes is reduced compared with wild-type animals (54). The reduction
in ovulated oocytes could be due to fewer ovulatory follicles or a
reduced ability of those follicles to respond to gonadotropins.
Therefore, ER-ß is necessary for modulating and maintaining
follicular health and may also contribute to the responsiveness of
granulosa cells to high levels of gonadotropins.
The hypothesis that emerges from the combined localization, regulation,
and knockout animal studies is that the ovary requires at least two
estrogen receptors, ER- primarily in theca and interstitial cells,
and ER-ß primarily in granulosa cells, each of which binds estradiol
and differentially activates gene transcription through independent
cellular and molecular mechanisms. ER- is likely involved directly
or indirectly in earlier events in follicular development and ER-ß
likely provides a facilitatory role in later events of follicular
development before luteinization.
In this paper, we have demonstrated that ER-ß protein expression in the ovary is primarily within granulosa cells and that ER-ß is the predominant estrogen receptor in rat granulosa cells, consistent with the data of Sar and Welsch (15). ER-ß protein is present in granulosa cells of preantral and antral follicles but the level dramatically declines following the ovulatory surge of gonadotropins. Therefore, ER-ß likely mediates the estrogen responsiveness of granulosa cells during the follicular phase. The confirmation of ER-ß as the mediator of estrogen responses in rodent granulosa cells awaits further analysis of functional studies of granulosa cells from normal and ER-ß -/- animals.
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Acknowledgments |
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Received November 10, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(ER-) and ß
(ER-ß) mRNA in the rat pituitary, gonad, and reproductive tract.
Steroids 63:498–504[CrossRef][Medline]
in the rat ovary.
Endocrinology 140:963–971 and ß. Endocrinology 138:863–870 and ß.
Biochem Biophys Res Commun 236:140–145[CrossRef][Medline]
and ERß at AP1 sites. Science 277:1508–1510 and ß by
mutations of a conserved tyrosine can be abolished by antiestrogens.
Cancer Res 58:877–881 (ER-) and ß (ER-ß) mRNAs in normal ovary, ovarian
serous cystadenocarcinoma and ovarian cancer cell lines:
down-regulation of ER-ß in neoplastic tissues. J Clin Endocrinol
Metab 83:1025–1028 and ß in the rat corpus luteum of
pregnancy—regulation by prolactin and placental lactogens.
Endocrinology 139:2432–2442 (ER) and estrogen receptor-ß (ERß) messenger
ribonucleic acid in the wild-type and ER-knockout mouse.
Endocrinology 138:4613–4621 immunoreactivity in neurons of the rat forebrain.
Endocrinology 139:5267–5270
in vivo and in vitro. Biochem Biophys Res Commun 243:122–126[CrossRef][Medline]
-iodovinyl-11 ß-methoxyestradiol in the immature female rat.
Steroids 62:244–252[CrossRef][Medline]
-iodovinyl-11
ß-methoxyestradiol and 2-iodo-1,1-bis(4-hydroxyphenyl)-phenylethylene
estrogens in the immature female rat. J Nucl Med 34:272–280-knockout mice.
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