| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
and ß in the Rat Corpus Luteum of Pregnancy: Regulation by Prolactin and Placental Lactogens1
Department of Physiology and Biophysics (C.M.T., L.Z., S.D., R.K.S., N.S., G.G.), College of Medicine, University of Illinois, Chicago, Illinois 60612; and Department of Physiology (K.S.P., O.-K.P.-S.), University of Kentucky, Lexington, Kentucky 40536
Address all correspondence and requests for reprints to: Dr. Geula Gibori, Department of Physiology and Biophysics (M/C 901), University of Illinois, 835 South Wolcott Avenue, Chicago, Illinois 60612-7342. E-mail: ggibori{at}uic.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and ERß) (by RT-PCR and in situ
hybridization) in the rat corpus luteum, studied their developmental
changes throughout pregnancy, and investigated the regulation of ER
and ERß messenger RNA (mRNA) expression by PRL and placental
lactogens. The RT-PCR studies showed that both ER mRNA species (ER
and ERß) are coexpressed in the rat corpus luteum during pregnancy.
Whereas ER mRNA increased from early pregnancy, reached a maximum at
midpregnancy, and had a remarkable decline before parturition; ERß
mRNA remained constant throughout pregnancy, with a significant decline
at parturition. Examination of ER and ERß mRNA expression at the
cellular level, by in situ hybridization, showed ER
expressed in both follicles and corpus luteum, with maximal expression
at midpregnancy. In parallel with the RT-PCR studies, ERß mRNA was
similarly expressed throughout pregnancy in the corpus luteum, but it
was less abundant when compared with small and growing follicles.
Western blot analysis revealed two ER immunoreactive proteins in the
nuclear fraction obtained from pregnant rat corpus luteum: a 67-kDa
moiety, highly expressed at midpregnancy but barely detectable in early
and late gestation; and a 61-kDa form that remained developmentally
unchanged. Hypo- physectomy, performed early in pregnancy, induced a
sharp decline in ER mRNA expression but a less-marked reduction in
ERß mRNA levels. PRL treatment reverted the inhibition induced by
hypophysectomy in both receptor subtypes. When primary luteinized cells
were used to test the effect of PRL, rat placental lactogen I, and rat
placental lactogen II on the expression of ER and ERß mRNA, all
these lactogenic hormones stimulated both ER mRNA species in a
dose-dependent manner. The regulation of ER mRNA expression was further
evaluated in a luteal cell line, termed GG-CL, which apparently
expresses only the ERß mRNA species. Culture of the GG-CL cells, in
the presence of PRL, resulted in a dose-related up-regulation of ERß
mRNA expression. In addition, PRL treatment enhanced the binding
activity of GG-CL cell nuclear proteins to a classical estrogen
response element. Furthermore, in these cells, estradiol treatment
induced a dose-dependent up-regulation of the mRNA encoding protein
kinase C delta isoform, a well-known estrogen target gene in the corpus
luteum of the pregnant rat. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
) (15), may provide an explanation for the
selective actions of estradiol in the rat corpus luteum.
In the rat, PRL (secreted by the anterior pituitary) and PRL-like
hormones from the placenta [placental lactogens I and II (rPL-I and
rPL-II)] seem to be required for the luteotropic effect of estradiol
in corpora lutea of pregnant rats (11, 16). Experimental evidences
indicate that such a synergistic action of PRL with estradiol in the
luteotropic process may involve, at least in part, PRL stimulation of
luteal receptors for estradiol (17, 18). Despite the fact that binding
experiments demonstrated the presence of estrogen-binding sites in the
rat corpus luteum of pregnancy (19), no evidence exists as to whether
the rat corpus luteum expresses the ER genes. Thus, the aims of the
present investigation were to examine: 1) the expression and
developmental changes of the two ER (ER and ERß) genes in the rat
corpus luteum during pregnancy; and 2) the regulation of ER and
ERß messenger RNA (mRNA) expression by PRL and placental
lactogens.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Animal model and tissue preparation
Pregnant (day 1 = sperm positive) and immature female (day
26 of age) Sprague-Dawley rats were obtained from Sasco Animal Labs.
(Madison, WI). They were kept under controlled conditions of light
(lights on 0500–1900 h) and temperature (22–24 C) with free access to
standard rat chow and water. All experiments were conducted in
accordance with the principles and procedures of the NIH Guide for the
Care and Use of Laboratory Animals and were approved by the
Institutional Animal Care and Use Committee.
For the developmental studies, rats were obtained at various stages of pregnancy, from days 4–22 (day of parturition) and at the day after parturition. Corpora lutea were dissected from the ovaries under a stereoscopic microscope. All tissues were frozen in liquid nitrogen and stored at -80 C until processed for RNA or protein preparation. In particular, for the in situ hybridization experiments, whole ovaries obtained from animals at different stages of pregnancy were frozen in dry ice and stored at -80 C until processed.
To determine the effect of PRL on ER mRNA expression in corpora lutea, pregnant rats were hypophysectomized, using a transauricular approach, on day 3 of pregnancy. Surgery was performed under ether anesthesia, with minimal stress to the animals. Completeness of hypophysectomy was evaluated by examination of the pituitary, removed at the time of operation, and visualization of the pituitary fossa at autopsy. Hypophysectomized rats were injected sc with 125 µg PRL (NIDDK oPRL-18, 30 IU/mg) twice daily in 50% polyvinylpyrrolidone, pH 9.0, for 4 days. Control rats were treated with vehicle. An additional group of intact pregnant rats was included in the study.
Granulosa cell culture
Maturation of preovulatory follicles was stimulated by treatment
of immature rats at day 28 of age, with 0.15 IU hCG sc, twice daily for
2 days (20). Luteinization of these preovulatory follicles was
subsequently achieved by an ovulatory dose (10 IU) of hCG on the third
day, via the tail vein. Luteinized granulosa cells were harvested from
preovulatory follicles 7 h after the iv injection of hCG. Briefly,
follicles were incubated sequentially in DMEM/F12 (1:1), containing 6
mM EGTA and 0.5 M sucrose, respectively; and
granulosa cells were harvested by needle-pricking the follicles. The
cells were plated in 60-mm culture dishes at 8 x 105
cells/ml and were incubated at 37 C, under 95% air-5% CO2
atmosphere, in DMEM/F12 containing 15 mM HEPES, 1% FBS,
100 IU/ml penicillin G, 100 µg/ml streptomycin, and 0.25 mg/ml
amphotericin B. After 72 h of incubation, the medium was changed,
and the cells were treated, during 12 h, with different doses of
oPRL (0.01–10 µg/ml), rPL-I (0.01–1 µg/ml), or rPL-II (0.1–1
µg/ml). Cells were washed with PBS several times after treatment, and
they were stored at -80 C until RNA isolation.
Generation of the GG-CL cell culture
The luteal cell line, termed GG-CL, was developed from large
luteal cells that were purified to homogeneity by flow cytometry from
corpora lutea of day-14 pregnant rats, as reported previously (21).
Cells were cultured in McCoy’s 5A: Ham’s F12 (1:1) containing 25
mM HEPES and 2% antibiotic-antimycotic solution, and 5%
FBS for 24 h, washed several times, and then infected with the
SV-40tsA209 mutant virus, as reported (22). The transformed
cells were maintained at the permissive temperature (33 C) until
colonies were identified. Several colonies of the transformed cells
were isolated and passed, and one clone (designated GG-CL cells) was
used in this study.
Transfection of GG-CL cells with the long form of the PRL
receptor
For stable transfection of GG-CL cells with the long form of PRL
receptor (PRL-RL), we adopted the procedure of Felgner
et al. (23), with slight modification. The complementary DNA
(cDNA) of the PRL-RL was subcloned into pMT2poly and
transformed to prepare large quantities of plasmid DNA. We used another
vector pSV2neo to confer the neomycin resistance to the cells. Plasmid
DNA was purified after transformation by equilibrium centrifugation in
cesium chloride-ethidium bromide gradient. GG-CL cells were plated in
6-well dishes and grown to 33% confluency in RPMI-1640 medium
containing 5% FBS. Cells were washed with serum-free medium without
antibiotics, and 1.8 ml of this medium was added to each well.
Meanwhile, for each well, 10 µl lipofectin reagent was mixed with 10
µg plasmid DNA (isolated from pMT2poly and pSV2neo) and 180 µl of
RPMI-1640 (without serum and antibiotics) and incubated for 15 min at
room temperature. This mixture of lipofectin and DNA complex was laid
on the cells in 1.8 ml of medium and was incubated for 24 h at 37
C, under an atmosphere consisting of 95% air-5% CO2.
Medium was replaced with the growth medium containing 5% FBS and
antibiotics and was incubated for 48 h. After 48 h, medium
was again replaced with fresh growth medium and treated with 100
µg/ml G418 sulfate. G418 sulfate addition was continued every
alternate day until G418 sulfate resistant colonies were identified.
These colonies were picked and cultured in the growth medium containing
5% FBS until cells were confluent. For identifying the successful
stable transfection with the PRL-RL, cells were grown and
passed at several times, and the presence of PRL-RL mRNA
was identified in the extracted RNA by RT-PCR using specific primers,
as previously described (24).
Untransfected GG-CL cells, as well as GG-CL cells expressing the PRL-RL (GG-CL PRL-RL), were cultured at 33 C under 95% air-5% CO2 in RPMI-1640 medium supplemented with 1 x glutamine, 2 x antibiotic-antimycotic solution, 1 x nonessential amino acids, 1 x sodium pyruvate, 0.45% D-glucose, and 1% FBS. The cells were treated with different hormones, and either RNA or proteins were extracted.
To study the effect of PRL on ER mRNA expression, GG-CL PRL-RL cells were treated for 8 h with different doses of oPRL (0.01–1 µg/ml). Cells were washed with PBS several times, after treatment, and were stored at -80 C until RNA isolation. When the effect of PRL on ER immunoreactive proteins was investigated, GG-CL PRL-RL cells were cultured for 8 h in the presence of either 0.1 µg/ml oPRL or vehicle, washed with PBS, and immediately harvested for protein isolation. For the studies involving the interaction of protein extracted from GG-CL cells and DNA, GG-CL PRL-RL cells were cultured for 48 h in the presence of 17ß-estradiol (10 ng/ml) and/or oPRL (0.1 µg/ml). Cells were then washed, and purified nuclear extracts were obtained (see below).
To test the effect of estradiol on protein kinase C delta isoform
(PKC-
) mRNA expression, GG-CL cells were cultured with different
doses of 17ß-estradiol (10-11-10-8
M) for 6 h, then washed with PBS, and stored at -80 C
until RNA extraction.
RNA isolation and RT-PCR analysis
Total RNA from frozen corpora lutea was purified by
homogenization in guanidinium thiocyanate and centrifugation through a
cesium chloride cushion (25), whereas total RNA from cultured cells was
isolated by a one-step guanidinium-thiocyanate-phenol-chloroform
extraction procedure (26).
Oligonucleotide primer pairs were based on the sequence of the rat
ER gene (27) (5'-AATTCTGACAATCGACGCCAG-3' and
5'-GTGCTTCAACATTCTCCCTCCTC-3') and rat ERß gene (15)
(5'-AAAGCCAAGAGAAACGGTGGGCAT-3' and 5'-GCCAATCATGTGCACCAGTTCCT T-3').
For the PKC- isoform, the following primers were used:
5'-CACCATCTTCCAGAAAGAACG-3' and 5'-CTTGCCATAGGTCCCGTTGTTG-3', as
previously reported (28). In each reaction, an additional pair of
oligonucleotides specific to the rat ribosomal protein S16 (29) was
included for use as an internal control (5'-TCCAAGGGTCCGCTGCAGTC-3' and
5'-CGTTCACCTTGATGAGC CCATT-3'). The predicted sizes of the
PCR-amplified products were 344 bp for ER, 204 bp for ERß, 352 bp
for PKC-, and 100 bp for S16. One to three micrograms of total RNA
were reverse transcribed at 42 C using random hexamer primers
(Pharmacia, Piscataway, NJ) and Maloney murine leukemia virus RT (Life
Technologies, Gaithersburg, MD) in a 20-µl reaction mixture. The
reaction mixture was added to tubes containing specific oligonucleotide
primers (50 pmol each) for amplification of either form of the ER or
PKC- cDNAs. A mix containing the oligonucleotide primers for S16
mRNA (50 pmol each), Taq DNA polymerase (2.5 U), and
[32P]-deoxycytidine triphosphate (2 µCi of 3000
Ci/mmol) was added to each tube; and the final vol was increased to 90
µl with 1 x PCR buffer (20 mM Tris-HCl (pH 8.4), 50
mM KCl, and 2.5 mM MgCl2). The
samples were overlaid with light mineral oil, and PCR was carried out
for 25 cycles using 95 C for denaturing, 65 C for annealing, and 72 C
for extension, in a Perkin-Elmer/Cetus Thermal Cycler (Perkin-Elmer,
Norwalk, CT). In particular, when RNA from GG-CL cells was used, 30 PCR
cycles were performed. The conditions were such that the amplification
of the products was in the exponential phase, and the assay was linear,
with respect to the amount of input RNA. Reaction products were
electrophoresed on a 8% polyacrylamide nondenaturing gel. After
autoradiography, data were analyzed using a Molecular Dynamics
PhosphorImager and ImageQuant version 3 sofware (Molecular Dynamic,
Sunnyvale, CA). The intensity of the ER, ERß, and PKC- signals
was normalized to that of the ribosomal protein S16 internal
control.
Immunoblot analysis
Isolated corpora lutea were homogenized in 2-ml ice-cold
homogenization buffer containing 25 mM Tris-HCl (pH 7.4), 2
mM MgCl2, 1 mM EDTA, 1
mM PMSF, 1 mM DTT, 1 µM
leupeptin, 1 µM pepstatin-A, and 1 µg/ml aprotinin in a
Polytron (Brinkmann Instruments, Rexdale, Ontario, Canada) homogenizer.
To obtain the protein extracts from cultured cells after appropriate
treatments, cells were washed twice with cold PBS (pH 7.4), scrapped
off the dishes using a rubber policeman, and disrupted with a 25-gauge
needle/1-cc syringe in the same homogenization buffer. To obtain the
nuclear and cytosolic fractions, the total homogenates were centrifuged
at 1,000 x g for 10 min. The pellets containing the
crude nuclear fraction were resuspended in homogenization buffer. The
supernatants were centrifuged at 105,000 x g during 30
min, and the new supernatants were considered the cytosolic fraction.
Homogenates were assayed for protein content (30), appropriately
diluted in 6 x concentrated electrophoresis sample buffer, boiled
for 10 min, and stored at -80 C until electrophoresed. Equivalent
amounts of protein (50 µg) were separated through 10% SDS-PAGE gels
and electrotransferred to nitrocellulose membranes (Amersham).
Immunoblotting was performed by blocking nonspecific binding with 5%
nonfat milk in TBS buffer containing 0.1% Tween 20. Blots were then
incubated overnight with a 1:750 dilution of the polyclonal antibody
ER-715 (kindly provided by the NIDDK). This polyclonal rabbit antiserum
was raised against a synthetic peptide corresponding to the hinge
region of the rat ER molecule (31). The membranes were washed and
incubated with a secondary antibody linked to horseradish-peroxidase
labeled antirabbit IgG (Sigma) for 2 h. After extensive washing,
blots were developed using an enhanced chemiluminescence Western
blotting detection system (Amersham Corp., Arlington Heights, IL) and
exposed for 15–120 sec to x-ray films. Data were analyzed using a
Molecular Dynamics Densitometer and ImageQuant version 3 software
(Molecular Dynamics).
Specificity of the stained protein band was ascertained by exposing the antiserum to excess antigen peptide (1.25 µg/ml) overnight before exposure to a blotted membrane. The molecular size of immunoreactive bands was estimated by the comigration of a prestained SDS-PAGE molecular mass standard of proteins ranging from 10–200 kDa, approximately every 10 kDa (Benchmark, Gibco/BRL, Gaithersburg, MD).
Nuclear extract preparation and electrophoretic mobility shift
assay (EMSA)
Crude nuclear extracts, obtained as reported above, were
subjected to a further purification and concentration. Briefly, crude
nuclear extracts were resuspended in high-salt buffer (20
mM HEPES (pH 7.9), 350 mM KCl, 0.2
mM EDTA, 20% glycerol, 1.5 mM
MgCl2, 0.1 mM EGTA, 1 mM DTT, and
0.5 mM PMSF) and incubated for 1 h on ice. Insoluble
proteins and particles were removed by centrifugation at 10,000 x
g for 15 min at 4 C. The nuclear extracts were dialyzed
overnight against dialysis buffer (20 mM HEPES (pH 7.9),
100 mM KCl, 0.1 mM EDTA, 0.5 mM
DTT, 0.1 mM PMSF, and 20% glycerol) at 4 C. Typically, 10
µg of purified nuclear protein extract were used in each binding
reaction. DNA-binding reactions were carried out in binding buffer (5
mM Tris (pH 8.0), 40 mM KCl, 6% glycerol, 1
mM DTT, and 0.05% Nonidet P-40), 2 µg of
poly(deoxyinosinic-deoxycytidylic)acid, 0.1 µg of denatured salmon
sperm DNA, and 10 µg BSA with 10,000 cpm of oligonucleotide probe
that was labeled by end-filling with Klenow (Life Technologies,
Gaithersburg, MD) in the presence of [32P]-deoxyguanidine
triphosphate. Preincubation with unlabeled probe was conducted on ice
for 15 min, then the labeled probe was added and allowed to bind at
room temperature for 30 min. The entire reaction was loaded onto a 4%
polyacrylamide gel and electrophoresed at 160 V at room temperature.
Gels were dried and autoradiographed with intensifying screens. The
following oligonucleotide and its complement were used as probe and
competitor:
5'-GATCCGTCAGGTCACAGTGACCTGATG-3'(32). The
underlined portions represent the palindromic sequence of
the estrogen response element (ERE) reported in the vitellogenin
A2 gene (33).
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 saline sodium
citrate (SSC) for 5 min, rinsed in distilled deionized water, washed in
0.1 M thiethanolamine (pH 8.0), and incubated in 0.25%
acetic anhydride in 0.1 M thiethanolamine (pH 8.0) for 10
min. Sections were dehydrated through an ethanol series and vacuum
dried until hybridization. Sense and antisense
[33P]-uridine triphosphate RNA probes were synthesized
using SP6 or T7 RNA polymerase. Templates were an EcoRI/Pstl
(750 bp) subclone of the rat ERß cDNA encoding the 5'-UTR and
N-terminal A/B region (34), and a PCR clone encoding the
hormone-binding domain of the rat ER cDNA (34). The RNA probe
[1 x 107 cpm/ml in hybridization buffer: 50%
formamide, 5 x SSPE (750 mM NaCl, 50 mM
NaH2PO4 (pH 7.4), and 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 coverslips. Slides were hybridized in a humidity
chamber at 47 C for 12 h. After hybridization, the coverslips were
removed and sections were treated with ribonuclease A (20 µg/ml) at
37 C for 30 min, washed in increasingly lower concentrations of SSC
down to 0.1 x SSC at 60 C, and dehydrated through an ethanol
series. The slides were exposed to Kodak XAR-5 film (Eastman Kodak) for
2–3 days at room temperature and were then processed for liquid
emulsion autoradiography using NTB-2 emulsion (Eastman Kodak). Slides
were developed three weeks later using Kodak D-19 developer and fixer
(Eastman Kodak) and stained with hematoxylin.
Statistics
Data were examined by one-way ANOVA followed by Duncan’s
multiple-range test. When appropriate, the Student’s t
-test was used. A level of P < 0.05 was accepted as
statistically significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and ERß mRNA species in the rat
corpus luteum during pregnancy and ERß) are
expressed in the pregnant rat corpus luteum, and to determine whether
such expression is temporally associated with particular stages of
pregnancy, we examined the ER and ERß mRNA levels by in
situ hybridization and RT-PCR analysis.
Our results, employing in situ hybridization followed by
emulsion autoradiography performed in whole ovaries taken from rats at
different days of pregnancy, show that ER mRNA is expressed within
the ovary in small and growing follicles, as well as in corpora lutea,
with maximal expression observed at midpregnancy (Fig. 1
, upper panel). Despite the
fact that ERß mRNA was detected in both follicles and corpora lutea,
the signal was much stronger in the follicles than in the luteal
structures. On the other hand, the ovarian expression of ERß mRNA
remained more or less similar throughout pregnancy in both the
follicles and the corpora lutea (Fig. 1, lower panel).
|
revealed that both forms of the ER mRNA are expressed in the corpus
luteum throughout pregnancy, with, however, a different pattern of
expression. ER mRNA was expressed at low levels early in pregnancy;
it increased thereafter and declined just at the day of parturition,
remaining at low levels 1 day after parturition. In contrast, ERß
mRNA remained constant throughout pregnancy, with a significant decline
at parturition (Fig. 2B).
|
. Also shown in Fig. 3A is an extract
from uteri taken from cycling rats. Two ER isoforms were found in
luteal tissue: a high-molecular-mass moiety of 67 kDa and a smaller ER
isoform of 61 kDa, both forms being located mainly in the nuclear cell
fraction (Fig. 3A, left panel). When antiserum ER-715 was
preabsorbed with excess antigen peptide before incubation on the blot,
both bands observed in the corpus luteum extracts did not show
immunoreaction ascertaining the specificity of the immunoreactive
proteins (Fig. 3A, right panel). Fig. 3B depicts a
representative autoradiograph of an immunoblot of ER protein prepared
from corpora lutea obtained at different stages of pregnancy. The
high-molecular-mass ER immunoreactive protein (67 kDa) was highly
expressed at midpregnancy (days 12, 17, and 20), but it was barely
detectable in early (day 7) and late (day 22) gestation. In contrast to
the high-molecular-mass isoform, the smaller ER moiety of 61 kDa was
abundantly expressed throughout pregnancy.
|
and ERß mRNA expression in
vivo, hypophysectomy caused a remarkable
decrease (P < 0.01) in the expression of ER mRNA,
an effect that was totally reversed by treatment with PRL. In contrast
to the marked effect on ER, hypophysectomy had a less marked, but
significant (P < 0.05), inhibitory effect on ERß
mRNA expression, which also was reversed by PRL treatment (Fig. 4B).
|
and ERß mRNA species in cultured luteinized granulosa
cells: regulation by PRL and placental lactogens
(Fig. 5, A and C) and ERß (Fig. 5, B,
and C) mRNA expression in a dose-dependent manner.
|
and ERß. As shown in Fig. 6, whereas both ER and ERß mRNA were
detected in the corpora lutea, only the mRNA encoding for ERß could
be detected in the GG-CL cells. When we examined for ER immunoreactive
isoforms in the corpus luteum and in the GG-CL cells (Fig. 7A), we detected both the 67-kDa and the
61-kDa proteins in the corpus luteum, but only the 61-kDa form in the
GG-CL cells, which was localized in the nuclear cell fraction. No
signal could be detected when the antiserum was absorbed with the
antigen peptide used to develop the antibody (Fig. 7B).
|
|
mRNA, the luteal cell line (GG-CL) that apparently
expresses ERß mRNA (but not ER mRNA) was used. However, because
this cell line does not express the PRL receptor, we stably transfected
these cells with the long form of the PRL receptor (36). Culture of the
transfected GG-CL PRL-RL cells in the presence of PRL
resulted in a dose-related up-regulation of the ERß mRNA expression
within 8 h of incubation (Fig. 8).
The increase in the ERß mRNA levels induced by PRL treatment was
accompanied by a significant increase (P < 0.05) in
the expression of the 61-kDa immunoreactive protein detected by
immunoblot (Fig. 9).
|
|
indicated the formation of several
shifted complexes that are specific, given that the interaction
disappeared when an excess of the unlabeled probe was added to the
reaction mixture. Interestingly, the nuclear extract, obtained from
cells treated with estradiol plus PRL, showed an increased interaction
with the DNA, when compared with that exhibited by the cells treated
with estradiol alone.
|
mRNA levels in the GG-CL cell
line
isoenzyme as a target gene, because it was previously shown to be
highly up-regulated by estradiol in the rat corpus luteum of pregnancy
(37, 38) and because the GG-CL cells express this PKC isoform. Results
depicted in Fig. 11 indicate that
estradiol induced a dose-dependent up-regulation of PKC- mRNA
expression in the luteal-derived cells within 6 h of culture.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and ERß, are also
expressed in the luteal tissue throughout pregnancy, whereas PRL and
placental lactogens up-regulate their expression. Taken together, these
data indicate that multiple signaling steps, triggered through
different types of PRL receptor and ER, could be involved in the
synergism between PRL and estradiol, to regulate luteal function, and
that the up-regulation of ER by PRL is one such mechanism.
Ovarian estradiol regulates reproductive and nonreproductive tissue
function, generally by modulating gene transcription through the ER
(39). The ER-mediated gene transcription is initiated by binding of the
DNA binding domain of the dimerized receptor to a consensus palindromic
DNA sequence, the ERE (32). Until 1995, only one ER type was cloned
(27), but the fact that disruption of the ER gene did not completely
eliminate the ability of small ovarian follicles to grow, as evidenced
by the presence of secondary and antral follicles in the knock-out
mouse ovary (40, 41), suggested the possibility that the ovary
expresses an estrogen-binding molecule(s) other than the classical ER.
An important approach to answering such a question came from the
cloning of a novel ER cDNA from rat prostate (15). This ER subtype was
named ERß to distinguish it from the previously cloned ER, now called
ER. A recent study revealed that ER and ERß are both expressed
in the rat ovarian follicles and that ERß is the more abundant
subtype in this ovarian tissue (34). In the present study, we report
that both ER subtypes are expressed also in the corpus luteum of
pregnancy, but displaying a different pattern of expression depending
on the day of pregnancy studied. The pattern of ER expression in the
corpora lutea of pregnancy follows that of the serum progesterone
levels and corpora lutea growth (11, 16), i.e. low at the
beginning of pregnancy, with a sharp increase at midpregnancy and a
decline at the time of parturition. In contrast, the ERß expression
does not seem to change substantially throughout pregnancy, displaying
only a slight decline at the end of the gestational period.
It is difficult to determine how estradiol signals in a tissue
coexpressing both receptor subtypes. No significant differences in the
affinity for the ligand have been measured between the two ER subtypes
(42). Two recent and independent studies demonstrated that homodimers
ER/ER and ERß/ERß, or heterodimers ER/ERß, can be formed
in vitro, can bind to the EREs in the DNA, and can stimulate
the transcription of a reporter gene (43, 44). The DNA binding affinity
of the heterodimer is similar to that of the ER homodimer but
greater than that of the ERß homodimer (44). Therefore, the relative
expression of ER and ERß in the corpus luteum and the difference
in DNA binding activity between heterodimers and homodimers could be a
mechanism for differential responsiveness of the luteal tissue to
estrogens at different stages of pregnancy.
Two ER immunoreactive proteins, of 67-kDa and 61-kDa, are detected in
the corpus luteum of pregnancy by using the polyclonal antibody ER-715,
which was raised against a peptide corresponding to a 15-amino-acid
sequence lying in the hinge region of the rat ER (27, 31). The
high-molecular-mass 67-kDa isoform is the expected full-length ER
protein demonstrated in several reports by using polyclonal and
monoclonal antibodies (31, 45, 46, 47). Accordingly, the pattern of the
67-kDa protein expression in the corpora lutea during pregnancy mirrors
the ER mRNA expression. In contrast, it is difficult to explain the
identity of the 61-kDa ER immunoreactive protein recognized by the
ER-715 antibody. Our observation that under the RT-PCR condition of the
linear range of amplification, only ERß mRNA could be detected in the
GG-CL cells, together with the fact that these cells express only the
61-kDa ER immunoreactive isoform, suggest that this protein could be
ERß. Also, the size of this protein (61-kDa) corresponds to that
reported for the ERß synthesized in an in vitro
translation system (15). However, it is possible also that the 61-kDa
isoform detected in the rat corpus luteum and in the luteal cell line
GG-CL represents either an ER degradation product, an ER isoform
caused by alternative splicing, or a protein that is not related to
ER but that cross-reacts with the ER-715 antibody. Therefore,
because the expression of this 61-kDa ER immunoreactive isoform is
up-regulated by PRL in the GG-CL cells, it will be of interest to
reveal its identity.
Estrogen binding activity has been observed in the nucleus of the rat
corpus luteum throughout pregnancy, with maximum levels at midpregnancy
(19), and a PRL stimulation of such activity has been proposed (17, 18). PRL and GH also were shown to stimulate estradiol binding activity
in the rat liver (48). In the present investigation, we demonstrated
clearly that PRL and placental lactogens up-regulate the luteal
estradiol receptors by stimulating both ER and ERß mRNA
expression. In the GG-CL cells that seem to express only ERß, we have
shown that not only the specific ERß mRNA, but also the 61-kDa
immunoreactive protein, were increased by PRL treatment. This increase
in the levels of the protein could explain the increased interaction
between nuclear extracts obtained from GG-CL cells and the labeled DNA
probe containing the palindromic ERE. However, we cannot rule out the
possibility that other factors, such as coactivators and corepressors,
could be affected by PRL treatment and therefore modulate the
interaction with the response element.
The luteotropic action of PRL and PRL-related hormones is mediated through two subtypes of the PRL receptor that bind PRL and placental lactogens (49, 50) and are classified as long or short, depending upon the length of the cytoplasmic domain (51, 52, 53). The expression of the two PRL receptor forms remains elevated in the corpora lutea during most of the period of pregnancy, with, however, a dramatic decline occurring before parturition (24). Interestingly, despite the existence of high levels of placental lactogen in the circulation before parturition (16), a drop in the luteal ER expression was observed. Most probably, as a consequence of the dramatic decline in the luteal content of PRL receptor mRNA and protein observed at the end of pregnancy (24), the corpora lutea become unresponsive to the circulating lactogen, which can no longer stimulate the expression of the ER genes. Results from this investigation indicate that PRL and placental lactogen stimulation of ER mRNA expression occurs through the long form of the PRL receptor, because the GG-CL cells used express only the PRL receptor long-form mRNA (36). In the rat corpus luteum, PRL can signal through the Janus tyrosine kinase JAK2 and members of the Stat5 family of transcription factors, Stat5a and Stat5b (54, 55, 56). Whether the Stat5 family of proteins is essential for the PRL-mediated stimulation of ER expression in the corpus luteum remains to be investigated.
The fact that 17ß-estradiol is able to stimulate the mRNA levels of
an estrogen target gene in the GG-CL cells expressing ERß mRNA, and
apparently lacking ER mRNA, suggests a mechanism of action involving
ERß in this luteal cell line. One classical estrogenic effect
reported in several estrogen target tissues is the increase in the
expression of the progesterone receptor-gene (57). However, the rat
corpus luteum does not seem to express the progesterone receptor (58).
Therefore, as an estrogen-target gene, we used the isoform of the
PKC, because it was shown to be strongly up-regulated by estradiol in
rabbit and rat corpus luteum (38, 59, 60). Although the role of PKC-
in luteal function is still unknown, the increase in its expression
before parturition suggests a possible participation of this kinase in
the luteolytic process (38).
In conclusion, the results of this investigation have established
that: 1) ER and ERß mRNAs are differentially coexpressed in the
rat corpus luteum during pregnancy; 2) PRL and placental lactogens
up-regulate the expression of both ER mRNA species in luteal cells; and
3) the GG-CL luteal-derived cells, expressing ERß mRNA and apparently
lacking ER mRNA, respond to estrogen in functional studies. This
luteal cell line could prove to be an important tool with which to
study the regulation of ERß gene expression.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Present address: Faulkner Centre for Reproductive Medicine, 1153
Centre Street, Boston, Massachusetts 02130.
3 NIH Merit Awardee (HD-11119).
Received November 10, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-hydroxy- steroid dehydrogenase expression in the rat
corpus luteum through the glucocorticoid receptor. Endocrinology 138:4497–4500 protein kinase C during
rat ovarian differentiation. Biochem Biophys Acta 1179:260–270[Medline]
protein kinase-C in the rat ovary: estrogen regulation and
localization. Endocrinology 135:1669–1678[Abstract]
and ß. Endocrinology 138:863–870. Mol Endocrinol 11:1486–1496 and ß form heterodimers on DNA. J Biol
Chem 272:19858–198622-macroglobulin
gene in rat ovarian granulosa cells: stat 5 activation and binding to
the interleukin-6 response element. Mol Endocrinol 10:171–184-hydroxysteroid dehydrogenase
gene expression and the tyrosine kinase system. Biochem
Biophys Res Commun 235:587–592[CrossRef][Medline]
. J Biol Chem 267:17061–17068 protein and mRNA levels in the
rabbit corpus luteum. Mol Cell Endocrinol 122:213–221[CrossRef][Medline]
This article has been cited by other articles:
 |
|
 |
|
  Y. S. Devi, A. Shehu, C. Stocco, J. Halperin, J. Le, A. M. Seibold, M. Lahav, N. Binart, and G. Gibori Regulation of Transcription Factors and Repression of Sp1 by Prolactin Signaling Through the Short Isoform of Its Cognate Receptor Endocrinology, July 1, 2009; 150(7): 3327 - 3335. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. van den Driesche, V. M Smith, M. Myers, and W C. Duncan Expression and regulation of oestrogen receptors in the human corpus luteum Reproduction, April 1, 2008; 135(4): 509 - 517. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
|
|
A. A Goyeneche, J. M Harmon, and C. M Telleria Cell death induced by serum deprivation in luteal cells involves the intrinsic pathway of apoptosis Reproduction, January 1, 2006; 131(1): 103 - 111. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Kindblom, K. Dillner, L. Sahlin, F. Robertson, C. Ormandy, J. Tornell, and H. Wennbo Prostate Hyperplasia in a Transgenic Mouse with Prostate-Specific Expression of Prolactin Endocrinology, June 1, 2003; 144(6): 2269 - 2278. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. A. Goyeneche, I. L. Martinez, R. P. Deis, G. Gibori, and C. M. Telleria In Vivo Hormonal Environment Leads to Differential Susceptibility of the Corpus Luteum to Apoptosis In Vitro Biol Reprod, June 1, 2003; 68(6): 2322 - 2330. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Lopez, M. D. Sanchez, W. Shea-Eaton, and M. P. McLean Estrogen Activates the High-Density Lipoprotein Receptor Gene via Binding to Estrogen Response Elements and Interaction with Sterol Regulatory Element Binding Protein-1A Endocrinology, June 1, 2002; 143(6): 2155 - 2168. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. A. Goyeneche, V. Calvo, G. Gibori, and C. M. Telleria Androstenedione Interferes in Luteal Regression by Inhibiting Apoptosis and Stimulating Progesterone Production Biol Reprod, May 1, 2002; 66(5): 1540 - 1547. [Abstract] [Full Text]
|
|
|
|
|
|
N. Baran, P. A. Kelly, and N. Binart Characterization of a Prolactin-Regulated Gene in Reproductive Tissues Usingthe Prolactin Receptor Knockout Mouse Model Biol Reprod, April 1, 2002; 66(4): 1210 - 1218. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. E. Thompson, I. G. Sipes, B. D. Greenstein, and P. B. Hoyer 17{beta}-Estradiol Affords Protection against 4-Vinylcyclohexene Diepoxide-Induced Ovarian Follicle Loss in Fischer-344 Rats Endocrinology, March 1, 2002; 143(3): 1058 - 1065. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. A. LaVoie, D. C. DeSimone, C. Gillio-Meina, and Y. Y. Hui Cloning and Characterization of Porcine Ovarian Estrogen Receptor {beta} Isoforms Biol Reprod, March 1, 2002; 66(3): 616 - 623. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Frasor, K. Park, M. Byers, C. Telleria, T. Kitamura, L.-y. Yu-Lee, J. Djiane, O.-K. Park-Sarge, and G. Gibori Differential Roles for Signal Transducers and Activators of Transcription 5a and 5b in PRL Stimulation of ER{alpha} and ER{beta} Transcription Mol. Endocrinol., December 1, 2001; 15(12): 2172 - 2181. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Frasor, U. Barkai, L. Zhong, A. T. Fazleabas, and G. Gibori PRL-Induced ER{alpha} Gene Expression Is Mediated by Janus Kinase 2 (Jak2) While Signal Transducer and Activator of Transcription 5b (Stat5b) Phosphorylation Involves Jak2 and a Second Tyrosine Kinase Mol. Endocrinol., November 1, 2001; 15(11): 1941 - 1952. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Gray, F. F. Bartol, B. J. Tarleton, A. A. Wiley, G. A. Johnson, F. W. Bazer, and T. E. Spencer Developmental Biology of Uterine Glands Biol Reprod, November 1, 2001; 65(5): 1311 - 1323. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Cardenas, K.A. Burke, R.M. Bigsby, W.F. Pope, and K.P. Nephew Estrogen Receptor {beta} in the Sheep Ovary During the Estrous Cycle and Early Pregnancy Biol Reprod, July 1, 2001; 65(1): 128 - 134. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. F. Palter, A. B. Tavares, A. Hourvitz, J. D. Veldhuis, and E. Y. Adashi Are Estrogens of Import to Primate/Human Ovarian Folliculogenesis? Endocr. Rev., June 1, 2001; 22(3): 389 - 424. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Guo, L. Savage, K. D. Sarge, and O.-K. Park-Sarge Gonadotropins Decrease Estrogen Receptor-{beta} Messenger Ribonucleic Acid Stability in Rat Granulosa Cells Endocrinology, June 1, 2001; 142(6): 2230 - 2237. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. K. Kang, K.-C. Choi, C.-J. Tai, N. Auersperg, and P. C. K. Leung Estradiol Regulates Gonadotropin-Releasing Hormone (GnRH) and its Receptor Gene Expression and Antagonizes the Growth Inhibitory Effects of GnRH in Human Ovarian Surface Epithelial and Ovarian Cancer Cells Endocrinology, February 1, 2001; 142(2): 580 - 588. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Iwata, G. Kawahara, K. Yamamoto, C. J. Zhou, S. Nakajo, S. Shioda, and S. Kikuyama Effect of Prolactin and Androgen on the Expression of the Female-Attracting Pheromone Silefrin in the Abdominal Gland of the Newt, Cynops ensicauda Biol Reprod, December 1, 2000; 63(6): 1867 - 1872. [Abstract] [Full Text]
|
|
|
|
|
|
H. Wang, H. Eriksson, and L. Sahlin Estrogen Receptors {alpha} and {beta} in the Female Reproductive Tract of the Rat During the Estrous Cycle Biol Reprod, November 1, 2000; 63(5): 1331 - 1340. [Abstract] [Full Text]
|
|
|
|
 |
|
 C.-H. Chiang, K. W. Cheng, S. Igarashi, P. S. Nathwani, and P. C. K. Leung Hormonal Regulation of Estrogen Receptor {alpha} and {beta} Gene Expression in Human Granulosa-Luteal Cells in Vitro J. Clin. Endocrinol. Metab., October 1, 2000; 85(10): 3828 - 3839. [Abstract] [Full Text]
|
|
|
|
|
|
C. Tessier, S. Deb, A. Prigent-Tessier, S. Ferguson-Gottschall, G. B. Gibori, R. P. C. Shiu, and G. Gibori Estrogen Receptors {alpha} and {beta} in Rat Decidua Cells: Cell-Specific Expression and Differential Regulation by Steroid Hormones and Prolactin Endocrinology, October 1, 2000; 141(10): 3842 - 3851. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. K. Olson and D. H. Townson Prolactin-Induced Expression of Intercellular Adhesion Molecule-1 and the Accumulation of Monocytes/Macrophages During Regression of the Rat Corpus Luteum Biol Reprod, June 1, 2000; 62(6): 1571 - 1578. [Abstract] [Full Text]
|
|
|
|
|
|
D. M. Duffy, C. L. Chaffin, and R. L. Stouffer Expression of Estrogen Receptor {alpha} and {beta} in the Rhesus Monkey Corpus Luteum during the Menstrual Cycle: Regulation by Luteinizing Hormone and Progesterone Endocrinology, May 1, 2000; 141(5): 1711 - 1717. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. M. Taylor, C. A. Gray, M. M. Joyce, M. D. Stewart, F. W. Bazer, and T. E. Spencer Neonatal Ovine Uterine Development Involves Alterations in Expression of Receptors for Estrogen, Progesterone, and Prolactin Biol Reprod, April 1, 2000; 63(4): 1192 - 1204. [Abstract] [Full Text]
|
|
|
|
|
|
S. Takiguchi, N. Sugino, S. Kashida, Y. Yamagata, Y. Nakamura, and H. Kato Rescue of the Corpus Luteum and an Increase in Luteal Superoxide Dismutase Expression Induced by Placental Luteotropins in the Rat: Action of Testosterone Without Conversion to Estrogen Biol Reprod, February 1, 2000; 62(2): 398 - 403. [Abstract] [Full Text]
|
|
|
|
|
|
D. L. Russell and J. S. Richards Differentiation-Dependent Prolactin Responsiveness and Stat (Signal Transducers and Activators of Transcription) Signaling in Rat Ovarian Cells Mol. Endocrinol., December 1, 1999; 13(12): 2049 - 2064. [Abstract] [Full Text]
|
|
|
|
|
|
G. Pelletier, V. Luu-The, A. Charbonneau, and F. Labrie Cellular Localization of Estrogen Receptor Beta Messenger Ribonucleic Acid in Cynomolgus Monkey Reproductive Organs Biol Reprod, November 1, 1999; 61(5): 1249 - 1255. [Abstract] [Full Text]
|
|
|
|
|
|
H. Wang, B. Masironi, H. Eriksson, and L. Sahlin A Comparative Study of Estrogen Receptors {alpha} and {beta} in the Rat Uterus Biol Reprod, October 1, 1999; 61(4): 955 - 964. [Abstract] [Full Text]
|
|
|
|
|
|
S. C. Sharma, J. W. Clemens, M. D. Pisarska, and J. S. Richards Expression and Function of Estrogen Receptor Subtypes in Granulosa Cells: Regulation by Estradiol and Forskolin Endocrinology, September 1, 1999; 140(9): 4320 - 4334. [Abstract] [Full Text]
|
|
|
|
|
|
S. L. Fitzpatrick, J. M. Funkhouser, D. M. Sindoni, P. E. Stevis, D. C. Deecher, A. R. Bapat, I. Merchenthaler, and D. E. Frail Expression of Estrogen Receptor-{beta} Protein in Rodent Ovary Endocrinology, June 1, 1999; 140(6): 2581 - 2591. [Abstract] [Full Text]
|
|
|
|
 |
|
 R. Misao, Y. Nakanishi, W.S. Sun, J. Fujimoto, S. Iwagaki, R. Hirose, and T. Tamaya Expression of oestrogen receptor {alpha} and ß mRNA in corpus luteum of human subjects Mol. Hum. Reprod., January 1, 1999; 5(1): 17 - 21. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. M. Telleria, J. Ou, N. Sugino, S. Ferguson, and G. Gibori The Expression of Interleukin-6 in the Pregnant Rat Corpus Luteum and Its Regulation by Progesterone and Glucocorticoid Endocrinology, August 1, 1998; 139(8): 3597 - 3605. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
