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Department of Physiology (M.B., O-K.P-S.) University of
Kentucky Lexington, Kentucky 40536
Center for
Biotechnology (G.G.J.M.K.) and Department of Medical Nutrition
(J-Å.G.) Karolinska Institute S-14186 Huddinge, Sweden
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and ERß) genes in the rat ovary,
using Northern blotting, RT-PCR, and in situ hybridization
histochemistry. Northern blotting results show that the ovary expresses
both ER and ERß genes as single (
6.5-kb) and multiple (ranging
from 1.0-kb to 10.0-kb) transcripts, respectively. ER mRNA is
expressed at a level lower than ERß mRNA in immature rat ovaries.
This relationship appears unchanged between sexually mature adult rats
and immature rats. In sexually mature adult rats undergoing endogenous
hormonal changes, whole ovarian content of ERß mRNA, as determined by
RT-PCR, remained more or less constant with the exception of the
evening of proestrus when ERß mRNA levels were decreased. Examination
of ERß mRNA expression at the cellular level, by in situ
hybridization, showed that ERß mRNA is expressed preferentially in
granulosa cells of small, growing, and preovulatory follicles, although
weak expression of ERß mRNA was observed in a subset of corpora
lutea, and that the decrease in ERß mRNA during proestrous evening is
attributable, at least in part, to down-regulation of ERß mRNA in the
preovulatory follicles. This type of expression and regulation was not
typical for ER mRNA in the ovary. Although whole ovarian content of
ER mRNA was clearly detected by RT-PCR, no apparent modulation of
ER mRNA levels was observed during the estrous cycle. Examination of
ER mRNA expression at the cellular level, by in situ
hybridization, showed that ER mRNA is expressed at a low level
throughout the ovary with no particular cellular localization. To further examine the potential role of the preovulatory pituitary gonadotropins in regulating ERß mRNA expression in the ovary, we used immature rats treated with gonadotropins. In rats undergoing exogenous hormonal challenges, whole ovarian content of ERß mRNA, as determined by RT-PCR, remained more or less unchanged after an injection of PMSG. In contrast, a subsequent injection of human CG (hCG) resulted in a substantial decrease in whole ovarian content of ERß mRNA. In situ hybridization for ERß mRNA shows that small, growing, and preovulatory follicles express ERß mRNA in the granulosa cells. The preovulatory follicles contain ERß mRNA at a level lower than that observed for small and growing follicles. In addition, there is an abrupt decrease in ERß mRNA expression in the preovulatory follicles after hCG injection. The inhibitory effect of hCG on ERß mRNA expression was also observed in cultured granulosa cells. Moreover, agents stimulating LH/CG receptor-associated intracellular signaling pathways (forskolin and a phorbol ester) readily mimicked the effect of hCG in down-regulating ERß mRNA in cultured granulosa cells.
Taken together, our results demonstrate that 1) the ovary expresses
both ER and ERß genes, although ERß is the predominant form of
estrogen receptor in the ovary, 2) ERß mRNA is localized
predominantly to the granulosa cells of small, growing, and
preovulatory follicles, and 3) the preovulatory LH surge down-regulates
ERß mRNA. These results clearly implicate the physiological
importance of ERß in female reproductive functions.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(21). ER is believed to mediate many of estrogen’s actions in
a variety of reproductive tissues, including the uterus (22, 23). If
the same scenario is applied to the ovary, ER protein should be
present at a detectable level in the ovary and elimination of the ER
gene in vivo should disrupt all ovarian functions requiring
estrogen action, such as folliculogenesis. Although ER immunoreactivity
has been found in ovaries of a limited number of species, such as the
baboon and human (24, 25, 26), disruption of the ER-gene in
vivo did not eliminate the ability of small follicles to grow as
evident from the presence of secondary and antral follicles in
ER-knockout mice (27, 28), arguing for the possibility that
intraovarian action of estrogen may be mediated by molecular mechanisms
requiring estrogen-binding molecules other than ER. This possibility
is further supported by experimental results from our laboratory (29, 30) and others (31, 32) demonstrating that in the ovary, in contrast to
the uterus, estrogen does not stimulate one of the best known genes
targeted by ER, the progesterone receptor (PR) gene. In light of these
observations, the identification of a second subtype of ER (ERß) that
is expressed in the ovary (21) may provide a fundamental understanding
of the longstanding critical role of estrogen in ovarian function. This
receptor has been shown to bind to estrogen with high affinity and
steroid specificity very similar to that of the ER (Ref. 21 and
G.G.J.M. Kuiper, unpublished data). Thus, we sought to examine the
expression and hormonal regulation of these two ERs (ER and ERß)
in the rat ovary. We have used ovaries of immature rats treated with
gonadotropins to stimulate folliculogenesis, ovulation, and
luteinization, as well as of sexually mature rats during the estrous
cycle. Our results demonstrate that 1) the primary ER subtype in the
ovary is ERß, although the ovary expresses both ER and ERß
genes, 2) ovarian expression of ERß mRNA is decreased during
follicular development and differentiation, and 3) activation of LH/CG
receptors down-regulates ERß mRNA expression in cultured rat
granulosa cells. |
RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and ERß mRNA Species in the Rat
Ovary and ERß) are expressed
at a comparable level in the rat ovary, we performed Northern analysis
(Fig. 1A
) and RT-PCR (Fig. 1, B and C) to specifically
detect ER and ERß transcripts in rat ovarian RNA. An equal amount
of rat uterine RNA was used as a positive control since it expresses
ER mRNA at a high level. Forty micrograms of total RNA were
fractionated on a 1% denaturing gel that was transferred onto a nylon
membrane and probed for ER using the hormone-binding domain of the
rat ER (20) or for ERß using the EcoRI/PstI
fragment (750 bp most 5'-end) of the rat ERß (21). The results of
Northern analyses performed under stringent conditions show that the
ovary contains an ER transcript (6.5 kb), as the uterus does, but
at an extremely low level (Fig. 1A, left panel). When the
same blot was reprobed for ERß mRNA, the ovarian RNA was found to
contain multiple transcripts of the ERß gene (ranging from 1.0 kb
to 10.0 kb) at a detectable level whereas little ERß mRNA signal
was detected in uterine RNA (Fig. 1A, middle panel). These
multiple ERß transcripts were better shown in RNA of isolated
granulosa cells as compared with whole ovaries (Fig. 1A, right
panel), indicating that ERß mRNA is enriched in granulosa cells.
Similar quantification results were obtained using RT-PCR assays
performed under conditions generating a linear range of specific
amplification of ER and ERß mRNAs. A typical example of the
relationship between cycle numbers and RT-PCR amplification of ER
and ERß mRNAs from a fixed concentration of ovarian RNA (1/4 of cDNA
generated from 5 µg RNA) is shown in Fig. 1B. This concentration of
cDNA is within a linear range of amplification of ER and ERß mRNAs
(data not shown). Linear amplification of ER and ERß mRNAs in
ovarian samples was obtained using up to 30 cycles of PCR and thus,
subsequent experiments were performed using 25 cycles of amplification.
ER and ERß RT-PCR products from the same ovarian or uterine cDNA
samples show that the uterus predominantly expresses ER mRNA while
ERß is the predominant ER subtype expressed in the ovary (Fig. 1C).
These reactions amplified specific ER and ERß PCR products of the
expected size based upon our results using ER [the human ER
(33)] or ERß [the rat ERß (21)] cDNA plasmids as controls.
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and ERß mRNA Expression in Rat Ovaries in
Vivo or
ERß gene is temporally associated with particular stages of the
female reproductive cycle, we examined ER and ERß mRNA levels in
ovaries of 1) sexually mature adult rats throughout the reproductive
cycle and 2) immature rats treated with exogenous gonadotropin
combination (PMSG plus human CG), by performing RT-PCR and in
situ hybridization. After 25 cycles of amplification at 66 C
annealing temperature, ERß as well as ER RT-PCR products were
detectable in ovaries at all stages of the reproductive cycle (Fig. 2). Again, ERß mRNA levels were higher than ER mRNA
levels in these samples. This is consistent with our observations
showing a preferential amplification of ERß mRNA during
coamplification of ER and ERß mRNAs in the same samples (data not
shown). ER RT-PCR fragment was detected in all stages of the estrous
cycle with less variation than that observed for ERß. Quantification
of ERß mRNA levels shows little variation during the cycle with the
exception of the evening of proestrus when ERß mRNA levels were
decreased. This decrease in ERß mRNA levels occurred after the onset
of the preovulatory LH surge (our LH RIA results show that the
preovulatory LH surge was initiated at 1600 h and peaked at
1800 h of proestrus in these animals, data not shown). Our results
employing in situ hybridization followed by liquid emulsion
autoradiography show that ER mRNA is expressed at a low level with
no particular cellular localization within the ovary, and that ERß
mRNA in small and growing follicles remained more or less similar
throughout the estrous cycle (data not shown), whereas ERß and LH
receptor (LH-R) transcripts are coexpressed in preovulatory follicles
of cycling rats and are down-regulated during the transition from
proestrus to estrus (Fig. 3A). Before the peak of the
preovulatory LH surge (shown are 1400 h and 1800 h),
preovulatory follicles express both ERß and LH-R mRNA in the
granulosa cell layer. After the peak of the preovulatory LH surge,
levels of both ERß and LH-R mRNA are substantially decreased (shown
are 2200 h and 2400 h). An example of ER and ERß mRNA
expression in ovaries containing different follicular structures is
shown in Fig. 3B. A subset of corpora lutea which express LH-R mRNA
have been found to weakly express ERß mRNA (upper panel,
shown are corpora lutea from metestrus 1800 h). Interestingly,
small follicles expressing LH-R mRNA only in theca cells in the same
field clearly expressed ERß mRNA in granulosa cells. Throughout
folliculogenesis in adult rats during the estrous cycle as well as in
immature rats during PMSG treatment, all growing follicles, regardless
of their size (small, antral, or preovulatory), were found to express
ERß mRNA [shown are ovarian sections of PMSG-treated (10 IU, 48
h) immature rats, lower panel]; as previously shown (34),
preovulatory follicles express LH-R mRNA in granulosa cell layers (34).
The colocalization of LH-R mRNA and ERß mRNA in follicles at
different stages is shown at higher magnifications in Fig. 4A. Small follicles (indicated by an arrow, left
panel) with LH-R mRNA in the theca, but not granulosa, cell layer
(34) clearly express ERß mRNA in the granulosa, but not theca, cell
layer. Preovulatory follicles (also indicated by an arrow, right
panel) with LH-R mRNA in both granulosa and theca cell layers (34)
also express ERß mRNA, although at a decreased level, in the
granulosa cell layer. Figure 4B shows colocalization of LH-R or PR mRNA
with ERß mRNA in preovulatory follicles in immature rats challenged
with hCG. Three hours after hCG treatment, granulosa cell expression of
LH-R mRNA is persistent in preovulatory follicles. In contrast, ERß
mRNA expression in the same follicles is markedly decreased whereas
nonpreovulatory follicles (absence of LH-R mRNA in granulosa cells)
persistently express ERß mRNA. Six hours after hCG treatment,
granulosa cell expression of PR mRNA is evident in preovulatory
follicles (29), which express little ERß mRNA. Again, nonpreovulatory
follicles (absence of PR mRNA expression) persistently expressed ERß
mRNA.
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). PMSG treatment (up to 48 h) did not
significantly alter total ovarian content of ERß mRNA levels. In
contrast, a subsequent injection of hCG (up to 12 h) in rats
similarly treated with PMSG (48 h) substantially decreased ERß mRNA
levels. To examine the effects of PMSG and hCG on ERß mRNA expression
at a cellular level, we performed in situ hybridization on
ovaries of similarly treated rats. As shown in Fig. 6, the granulosa cells of preantral as well as antral follicles of
untreated immature rats clearly express ERß mRNA at a detectable
level. During folliculogenesis stimulated by PMSG treatment, ERß mRNA
expression in growing follicles appears to decrease. Forty-eight hours
after PMSG treatment, the granulosa cells of preovulatory follicles
clearly express LH-R mRNA as well as ERß mRNA. During short-term
treatment with hCG (3 h), down-regulation of ERß mRNA is evident in
the preovulatory follicles still expressing LH-receptors, suggesting
that ERß mRNA is rapidly down-regulated; six h after hCG injection,
both LH-R and ERß mRNA levels were reduced.
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(3-h
incubation), granulosa cells contain a fairly high level of ERß mRNA,
and incubation of these cells with hCG decreased ERß mRNA to 50% of
the original value. Activation of LH receptor-associated signaling
pathways by using activators for protein kinases A (forskolin,
10-5 M) and C [a phorbol 12-myristate
13-acetate (TPA), 10-7 M] results in ERß
mRNA down-regulation in cultured rat granulosa cells. Interestingly,
forskolin and TPA act together to further decrease ERß mRNA levels in
these cells. This inhibitory effect of forskolin or TPA on ERß mRNA
levels in granulosa cells lasts at least up to 12 h as shown in
Fig. 7B.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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in ER null mutant mice
(27, 28, 37) did not prevent the growth of follicles, which resulted in
high circulating estrogen levels (28, 37), suggesting the possibility
that the ovary expresses estrogen-binding molecule(s) other than ER.
Consistent with this possibility are our results demonstrating that
ERß is the primary ER subtype expressed in the ovary, in particular
granulosa cells of small, growing, and preovulatory follicles. Thus,
the ovarian responsiveness to estrogen in the ER knockout mice (27, 37) may result from the expression of ERß. Our results, however, do
not exclude the potential importance of ER in ovarian functions.
Indeed, experimental results of ours (this report) and others (38, 39)
show the evident, although low, expression of the ER gene in the
ovary. Coexpression of ER and ERß in the same follicles suggests
the interesting possibility that ER and ERß proteins may interact
with each other and discriminate between target sequences leading to
differential responsiveness to estrogen. Because both ER and ERß
proteins bind estrogen with an affinity of 10-9
M and display the same steroid specificity (20, 21), it is
highly unlikely that differential responsiveness to estrogen is
achieved at the level of interaction between ligand and receptor.
Because ERß, like ER, when expressed in transfected cells, has
been shown to be capable of transactivating a simple artificial
estrogen response element-containing promoter (21), it is possible that
differential responsiveness to estrogen may be achieved at the level of
interactions between ER and ERß or between ERs and DNA.
Interestingly, PRA, the N-terminally truncated naturally
occurring isoform of PRB, functions as a transactivator in
some cells as a homodimer whereas it serves as a repressor of
PRB when both isoforms are present (40, 41, 42). Perhaps
similar mechanisms for estrogen responsiveness could be operating in
cells expressing both ER and ERß. In this regard, it is
interesting to note that the N-terminal domain of the ERß protein is
not homologous to, and is much shorter than, the corresponding region
of the ER protein. In cells in vivo, the ratio of
ER/ERß mRNA varies significantly among estrogen target tissues
(Ref. 21 and our results). Our results show that the ratio of
ERß/ER in the ovary is much higher than in the uterus, a well
known estrogen target. In uterine cells expressing high levels of ER
but extremely low levels of ERß, estrogen clearly stimulates multiple
physiological processes including the synthesis of PRs (43). In
addition, ER knockout mice fail to initiate these physiological
processes in response to estrogen (37). In contrast, in the ovary,
which expresses relatively high levels of ERß but low levels of
ER, total elimination of ER failed to disrupt normal growth of
ovarian follicles (37). In addition, estrogen failed to directly
stimulate PR gene expression in normal preovulatory follicles (30, 31, 32).
Although one possible explanation is that these two cell types (uterus
and ovary) express different coactivators facilitating the interactions
between ERs and general transcription factors; another possibility is
that these two cell types respond to estrogen differently because of
the different ratio of ER/ERß expression. It will be important to
determine whether ER and ERß proteins are coexpressed within a
single cell, whether ER and ERß could potentially dimerize with
each other, and whether ER and ERß homodimers, as well as
ER/ERß heterodimers, would exhibit cell context-dependent and
promoter-dependent transactivation function.
Hormonal Regulation of ERß mRNA Expression by Gonadotropins
In many estrogen target tissues such as the uterus and pituitary,
estrogen and progesterone modulate levels of ER protein as well as
mRNA (22, 23, 36, 43). In the ovary, estrogen-binding sites have been
shown to be modulated by the pituitary gonadotropins (17). The
discrepancy between our RT-PCR results showing that ERß mRNA in whole
ovary remained more or less similar during folliculogenesis
(proliferation of granulosa cells) and our in situ
hybridization results showing an apparent decrease in ERß mRNA in
granulosa cells of large and preovulatory follicles may be explained by
the fact that whole ovary contains an increased number of granulosa
cells during folliculogenesis. This possibility is consistent with a
published observation that estrogen-binding sites per granulosa cells
decrease after PMSG treatment (48 h) (17). During PMSG treatment the
follicular synthesis of estrogen significantly increases and thus the
PMSG-induced decrease in granulosa cell expression of ERß mRNA may
result from PMSG-induced estrogen production and/or PMSG-induced
intracellular signaling pathways. Curiously, ERß mRNA levels in whole
ovary are substantially decreased after the onset of the gonadotropin
surge in cycling rats and after hCG injection in PMSG-primed immature
rats. Our in situ hybridization data showing colocalization
of ERß, LH-R, and PR mRNAs in immature rats treated with
gonadotropins argue that LH/CG-induced down-regulation of ERß mRNA in
whole ovaries is attributable, at least in part, to the loss of ERß
mRNA in preovulatory follicles responding to LH/CG. In these
experiments, only preovulatory follicles expressing LH-R mRNA lose
ERß mRNA in response to hCG challenge. Similarly, all preovulatory
follicles expressing PR mRNA in response to LH/CG (29, 30) express
little ERß mRNA while neighboring follicles without PR mRNA
(indicative of the lack of LH-R) express a normal level of ERß mRNA.
This observation is consistent with a previous report demonstrating
that hCG induces down-regulation of estrogen receptor-binding sites
(17). Our results demonstrating the ability of protein kinase A
(forskolin) and protein kinase C (TPA) activators to induce this type
of down-regulation of ERß mRNA levels in cultured granulosa cells
indicate that the intracellular signals generated by the preovulatory
LH surge in differentiated granulosa cells initiate the molecular
cascade of events leading to either rapid degradation of ERß mRNA
and/or transcriptional turn-off of the ERß gene. In MCF-7 breast
cancer cells in which the ER gene has been shown to be
down-regulated by estrogen (44), a phorbol ester TPA has also been
shown to down-regulate ER mRNA expression by facilitating rapid
degradation of ER messages (45). It remains to be determined whether
similar mechanisms perhaps mediate ER degradation in nontumor cells.
Although the importance of the LH-induced down-regulation of ERß mRNA
in preovulatory follicles is currently unknown, there must be another
hormonal cue that increases ERß mRNA expression by increasing either
mRNA stability or transcriptional rate, as evident from a subset of
corpora lutea of cycling rat ovaries expressing levels of ERß mRNA
that are slightly above background. Functional corpora lutea of
pregnant rats also express ERß mRNA (M. Byers and O-K. Park-Sarge,
unpublished observation). Thus, future studies will be required to
determine the molecular mechanisms by which ERß mRNA expression is
regulated in granulosa/luteal cells.
In summary, our results demonstrating that ERß is the primary ER subtype expressed in the ovary, that ERß is expressed in granulosa cells of virtually all healthy follicles, and that ERß is down-regulated by gonadotropins in granulosa cells suggests the possibility that in the ovary, the functional significance of estrogen action may be mediated primarily by ERß.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Immature Animals.
Sprague-Dawley female pups (21 days old) with a nursing mother were
purchased from Harlan Breeding Company (Indianapolis, IN) and housed in
a photoperiod of 14-h light, 10-h darkness, with lights on at 0500
h. Food and water were freely available. At 23 days of age, rats were
injected s.c. with 10 IU PMSG (Sigma, St Louis MO) in 0.1 ml PBS.
Forty-eight hours later, rats were injected s.c. with 10 IU hCG (Sigma)
in 0.1 ml PBS. Rats were killed by decapitation at various time points
throughout hormone treatments, and their ovaries were rapidly removed
from surrounding fat and oviduct, frozen on dry ice and stored until
use at -80 C.
Sexually Mature Adult Rats.
Adult female Sprague-Dawley rats (150–180 g body weight) were
purchased from Charles-Rivers Breeding Company (Wilmington, MA) and
were housed as above. Estrous cyclic stages were determined by daily
examination of vaginal cytology, and only those animals demonstrating
at least two consecutive 4-day cycles were used for the experiment.
Rats were euthanized at specific time intervals throughout the estrous
cycle. Trunk blood was collected for serum LH measurements, and ovaries
were rapidly removed from surrounding fat and oviduct, frozen on dry
ice, and stored until use at -80 C. Serum LH concentrations in these
cycling rats were determined by an RIA using the NIH NIDDK kit with the
exception of LH antibody CSU 120, which was generously provided by Dr.
Terry Nett.
Granulosa Cell Isolation and Culture
Immature rats at 22–23 days of age were primed with a single
s.c. injection of PMSG (10 IU) and 40–44 h later, granulosa cells were
isolated by the method of follicular puncture (30, 46, 47). Ovaries
were collected in cold serum-free medium (4F) consisting of 15
mM HEPES (pH 7.4), 50% Dulbecco’s MEM and 50% Ham’s F12
with bovine transferrin (5 µg/ml), human insulin (2 µg/ml),
hydrocortisone (40 ng/ml), and antibiotics. After incubation in warm
(37 C) 4F medium containing 0.5 M sucrose and 10
mM EGTA for 20–30 min to loosen cell junctions, ovaries
were washed in fresh 4F medium, and individual follicles were punctured
using 23-gauge needles. Extruded granulosa cells were collected, washed
twice, and plated in 4F medium supplemented with 5% FBS (GIBCO, Grand
Island, NY) at a density of approximately 2 x 106
cells per 100-mm dish, incubated overnight in the humidified atmosphere
of 5% CO2 at 37C, and treated with various hormones.
RNA Blot Analysis
Total RNA was prepared from ovaries at the indicated time points
by homogenization in guanidine isothiocynate and centrifugation through
cesium chloride (48, 49). Approximately 40 µg of each was separated
by electrophoresis on denaturing 1% agarose/formaldehyde gels. RNA was
transferred to a nylon membrane (Schleicher & Schuell, Keene, NH),
baked in a vacuum oven at 80 C for 2 h, and hybridized at 42 C
with 32P-dCTP-labeled ER [the 500-bp hormone-binding
domain (20)] or ERß [the 200-bp 5'-untranslated region (UTR) and
A/B region (21)]-specific probe in 50% formamide, 5 x SSPE (750
mM NaCl, 50 mM NaH2PO4,
pH 7.4, 1 mM EDTA), 2 x Denhardt’s reagent, 10%
dextran sulfate, 0.1% SDS, and 100 µg/ml salmon sperm DNA. The
membranes were subsequently washed in 0.1 x NaCl-sodium citrate
(SSC) at 65 C and exposed to Kodak XAR-5 film (Eastman Kodak,
Rochester, NY). After removal of probe in 50% formamide at 65 C, the
membranes were rehybridized to cDNA clone CHO-B (50), which detects the
LLRep3 gene family (51), to assess the amount of RNA present in each
lane.
In Situ Hybridization
Ovaries were removed from storage at -80 C and brought to -20
C, and 20-µm sections were cut using a Zeiss cryostat. Sections were
mounted onto positively charged glass slides, fixed in 5%
paraformaldehyde (pH 7.5) for 5 min, washed in 2 x SSC for 5 min,
rinsed in distilled deionized water, washed in 0.1 M
triethanolamine (pH 8.0), and incubated in 0.25% acetic anhydride in
0.1 M triethanolamine (pH 8.0) for 10 min. Sections were
dehydrated through an ethanol series and vacuum dried until
hybridization. Antisense [33P]UTP- or
[35S]UTP-labeled RNA probes were synthesized using SP6 or
T7 RNA polymerase (48, 49). Templates were an
EcoRI/PstI subclone of the rat ERß cDNA
encoding the 5'-UTR and N-terminal A/B region (21), a PCR clone
encoding the hormone-binding domain of the rat ER cDNA (20), the rat
LH-R PCR clone (34), and the rat PR PCR clone (29). The RNA probe
(2 x 107 cpm/ml in hybridization buffer: 50%
formamide, 5 x SSPE (750 mM NaCl, 50 mM
NaH2PO4, pH 7.4, 1 mM EDTA), 2
x Denhardt’s reagent, 10% dextran sulfate, 0.1% SDS, and 100 mg/ml
yeast tRNA) was applied to the tissue sections, and the sections were
overlaid with a coverslip. Slides were hybridized in a humidity chamber
at 47 C for 16–18 h. After hybridization, the coverslips were removed
and sections were treated with RNase A (20 µg/ml) at 37 C for 30 min,
washed in increasingly lower concentrations of SSC down to 0.1 x
SSC at 55 C, and dehydrated through an ethanol series. The slides were
exposed to Kodak XAR-5 film for 2–3 days at room temperature and were
then processed for liquid emulsion autoradiography using NTB-2 emulsion
(Kodak). Slides were developed using Kodak D-19 developer and fixer and
stained with hematoxylin.
RT-PCR Analysis
Oligonucleotide primer pairs of 20–22 nucleotides (40–60% GC
content) were designed based on the sequences of the rat ERß (21)
[amplifying nucleotides from 1018 to 1221 (21)], ER [amplifying
nucleotides from 1439 to 1847 or 1547 (20)], and rat ribosomal protein
S16 [amplifying nucleotides from 59 to 109 (52)]. The predicted sizes
of the amplified products are 203 bp (ERß), 115 or 400 bp depending
on the primer sets (ER), and 100 bp (S16). The conditions were such
that amplification of the product was linear with respect to the amount
of input RNA. Five micrograms of total RNA were reverse-transcribed at
37 C using random hexamer primers and MMLV reverse transcriptase (New
England Biolabs, Boston, MA) in a 20-µl reaction. Five microliters of
the cDNA samples were used for the subsequent PCR amplification of
ER, ERß, and S16 cDNAs. A 20 µl mix including the
oligonucleotide primers (50 ng each), -32P-dCTP (2 µCi
at 3000 Ci/mmol), and Taq DNA polymerase (2.5 U) in 1
x PCR buffer (10 mM Tris, pH 8.3, 50 mM KCl,
1.5 mM MgCl2, 0.01% gelatin) was added to each
cDNA sample and overlaid with light mineral oil. Amplification was
carried out for 25 cycles using an annealing temperature of 65 C on a
Perkin Elmer Cetus thermalcycler (Perkin Elmer Cetus, Norwalk, CT). The
samples were then electrophoresed on a 8% polyacrylamide gel. After
autoradiography, the gel was analyzed using a Molecular Dynamics
PhosphoImager and ImageQuant version 3 software (Molecular Dynamics,
Sunnyvale, CA). The intensity of the ER and ERß signal was
normalized to that of the ribosomal protein S16 internal control.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported, in part, by NIH Grant HD-30719 and NIH Research Career Development Award HD-01135 (to O-K.P-S.). M.B. was supported, in part, by the NIH Training Grant in Reproductive Sciences to the University of Kentucky (T32-HD07436). G.G.J.M.K. was supported, in part, by grants from the Netherlands Organization for Scientific Research (NWO) and by a visiting scientist fellowship from the Karolinska Institute. A.J.G. was supported in part, by Swedish Cancer Fund.
Received for publication September 10, 1996. Revision received November 4, 1996. Accepted for publication November 11, 1996.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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S. Ikeda, K. Nakamura, K. Kogure, Y. Omori, S. Yamashita, K. Kubota, T. Mizutani, K. Miyamoto, and T. Minegishi Effect of Estrogen on the Expression of Luteinizing Hormone-Human Chorionic Gonadotropin Receptor Messenger Ribonucleic Acid in Cultured Rat Granulosa Cells Endocrinology, April 1, 2008; 149(4): 1524 - 1533. [Abstract] [Full Text] [PDF]
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 C. Leigh Pearce, A. M. Near, J. L. Butler, D. Van Den Berg, P. Bretsky, D. V. Conti, D. O. Stram, M. C. Pike, J. N. Hirschhorn, and A. H. Wu Comprehensive Evaluation of ESR2 Variation and Ovarian Cancer Risk Cancer Epidemiol. Biomarkers Prev., February 1, 2008; 17(2): 393 - 396. [Abstract] [Full Text] [PDF]
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 K. S. Monkkonen, R. Aflatoonian, K.-F. Lee, W. S.B. Yeung, S.-W. Tsao, J. T. Laitinen, and A. Fazeli Hormonal regulation of G{alpha}i2 and mPR{alpha} in immortalized human oviductal cell line OE-E6/E7 Mol. Hum. Reprod., December 1, 2007; 13(12): 845 - 851. [Abstract] [Full Text] [PDF]
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 C. Stocco, C. Telleria, and G. Gibori The Molecular Control of Corpus Luteum Formation, Function, and Regression Endocr. Rev., February 1, 2007; 28(1): 117 - 149. [Abstract] [Full Text] [PDF]
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T. K. Woodruff and K. E. Mayo To {beta} or Not To {beta}: Estrogen Receptors and Ovarian Function Endocrinology, August 1, 2005; 146(8): 3244 - 3246. [Full Text] [PDF]
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J. F. Couse, M. M. Yates, B. J. Deroo, and K. S. Korach Estrogen Receptor-{beta} Is Critical to Granulosa Cell Differentiation and the Ovulatory Response to Gonadotropins Endocrinology, August 1, 2005; 146(8): 3247 - 3262. [Abstract] [Full Text] [PDF]
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 S. A. Milne, T. A. Henderson, R. W. Kelly, P. T. Saunders, D. T. Baird, and H. O. D. Critchley Leukocyte Populations and Steroid Receptor Expression in Human First-Trimester Decidua; Regulation by Antiprogestin and Prostaglandin E Analog J. Clin. Endocrinol. Metab., July 1, 2005; 90(7): 4315 - 4321. [Abstract] [Full Text] [PDF]
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 L. J. Spicer Effects of Estradiol on Bovine Thecal Cell Function In Vitro: Dependence on Insulin and Gonadotropins J Dairy Sci, July 1, 2005; 88(7): 2412 - 2421. [Abstract] [Full Text] [PDF]
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J. M. A. Emmen, J. F. Couse, S. A. Elmore, M. M. Yates, G. E. Kissling, and K. S. Korach In Vitro Growth and Ovulation of Follicles from Ovaries of Estrogen Receptor (ER){alpha} and ER{beta} Null Mice Indicate a Role for ER{beta} in Follicular Maturation Endocrinology, June 1, 2005; 146(6): 2817 - 2826. [Abstract] [Full Text] [PDF]
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