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12/5/2014 Stress Levels of Glucocorticoids Inhibit LHβ-Subunit Gene Expression in Gonadotrope Cells
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Mol Endocrinol. Oct 2012; 26(10): 1716–1731.
Published online Jul 31, 2012. doi: 10.1210/me.2011-1327
PMCID: PMC3458227
Stress Levels of Glucocorticoids Inhibit LHβ-Subunit Gene Expression in
Gonadotrope Cells
Kellie M. Breen, Varykina G. Thackray, Tracy Hsu, Rachel A. Mak-McCully, Djurdjica Coss, and Pamela L. Mellon
Department of Reproductive Medicine and Center for Reproductive Science and Medicine, University of California, San Diego, La Jolla,
California 92093-0674
Corresponding author.
Address all correspondence and requests for reprints to: Pamela L. Mellon Ph.D., Department of Reproductive Medicine/Neuroscience,
University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0674., E-mail: [email protected].
Received November 18, 2011; Accepted July 2, 2012.
Copyright © 2012 by The Endocrine Society
Abstract
Increased glucocorticoid secretion is a common response to stress and has been implicated as a
mediator of reproductive suppression upon the pituitary gland. We utilized complementary in vitro and
in vivo approaches in the mouse to investigate the role of glucocorticoids as a stress-induced
intermediate capable of gonadotrope suppression. Repeated daily restraint stress lengthened the
ovulatory cycle of female mice and acutely reduced GnRH-induced LH secretion and synthesis of LH β-
subunit (LHβ) mRNA, coincident with increased circulating glucocorticoids. Administration of a stress
level of glucocorticoid, in the absence of stress, blunted LH secretion in ovariectomized female mice,
demonstrating direct impairment of reproductive function by glucocorticoids. Supporting a pituitary
action, glucocorticoid receptor (GR) is expressed in mouse gonadotropes and treatment with
glucocorticoids reduces GnRH-induced LHβ expression in immortalized mouse gonadotrope cells.
Analyses revealed that glucocorticoid repression localizes to a region of the LHβ proximal promoter,
which contains early growth response factor 1 (Egr1) and steroidogenic factor 1 sites critical for GnRH
induction. GR is recruited to this promoter region in the presence of GnRH, but not by dexamethasone
alone, confirming the necessity of the GnRH response for GR repression. In lieu of GnRH, Egr1
induction is sufficient for glucocorticoid repression of LHβ expression, which occurs via GR acting in a
DNA- and dimerization-independent manner. Collectively, these results expose the gonadotrope as an
important neuroendocrine site impaired during stress, by revealing a molecular mechanism involving
Egr1 as a critical integrator of complex formation on the LHβ promoter during GnRH induction and
GR repression.
Stress profoundly disrupts reproductive function. Whether the nature of the stressor is physical (e.g.
foot-shock, exercise), immunological (e.g. infection, administration of cytokines or endotoxins), or
psychological (e.g. isolation, mental performance tasks), each has been shown to decrease circulating
levels of gonadotropins in mammals (1–5). Associated with this reproductive disturbance is an
activation of the hypothalamic-pituitary-adrenal axis and an elevation in circulating glucocorticoids
from the adrenal cortex, the final hormonal effectors of the hypothalamic-pituitary-adrenal axis.
Evidence that the glucocorticoid receptor (GR) antagonist, RU486, attenuates the inhibitory effect of
immobilization stress on LH secretion in male rats or psychosocial stress on pituitary responsiveness to
GnRH in ovariectomized ewes implies a physiological role for glucocorticoids in mediating the
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inhibitory effects of stress on LH secretion, although RU486 can also block the effects of progesterone
(6–8).
Although there is little doubt that glucocorticoids suppress gonadotropin secretion, the neuroendocrine
mechanism underlying this effect is not well understood. Inhibition at the hypothalamic level is
supported by evidence that glucocorticoids reduce the frequency of LH pulses in ovary-intact female
sheep, ovariectomized female rats, and women during the follicular phase of the ovulatory cycle (9–11).
Because LH pulse frequency is generally modulated by the GnRH neurosecretory system, these findings
suggest an action of glucocorticoids to suppress the frequency of GnRH pulses. A recent study in
follicular phase sheep provides the first definitive evidence that glucocorticoids inhibit GnRH pulses in
pituitary portal blood (12). GR is expressed within hypothalamic neurons implicated in GnRH
regulation (13), and such neurons provide a potential indirect target by which glucocorticoids may
inhibit GnRH secretion or GnRH synthesis (14); however, a direct action within the GnRH neuron
itself is supported by evidence that glucocorticoids blunt GnRH synthesis and release from immortalized
GnRH neurons, GT1–7 cells (15, 16). Thus, the mechanism whereby glucocorticoids suppress GnRH
and LH remains unclear and may involve direct actions upon the GnRH neuron itself, indirect actions
via another neuronal cell type, or actions upon an extrahypothalamic site.
With regard to a site outside of the central nervous system, the most obvious possibility is that
glucocorticoids act via GR located within gonadotrope cells of the anterior pituitary gland. Evidence
that glucocorticoids reduce the amplitude of the LH response to a GnRH challenge in rodents, pigs,
cows, and women is consistent with this possibility (17–20). Further, suppression of responsiveness to
GnRH in vitro has been observed in rodent, porcine, and bovine pituitary cell cultures, indicating that
glucocorticoids can act directly upon the gonadotrope cell to inhibit responsiveness to GnRH (19–21).
Consistent with an action upon the gonadotrope cell, GR has been identified in rat gonadotropes (22),
and studies in rat and pig primary cells suggest that glucocorticoids inhibit signaling mechanisms
downstream of the GnRH receptor, including protein kinase C and cAMP (20, 23). It is not known,
however, whether these nongenomic actions of glucocorticoids that inhibit intracellular signaling
pathways ultimately lead to a reduction in LH release. Alternatively, evidence suggests that
glucocorticoids can act genomically to suppress gonadotrope responsiveness by regulating transcription
and translation of the GnRH receptor gene (24, 25).
Another potential mechanism whereby glucocorticoids could diminish GnRH responsiveness of the
gonadotrope is via regulation of gonadotropin synthesis. At the molecular level, LH and FSH are
glycoprotein hormones that exist as heterodimers, consisting of a common and abundantly expressed
glycoprotein hormone alpha-subunit (αGSU) complexed with a unique β-subunit that confers biological
specificity (26). Synthesis of the β-subunit gene of each hormone is the rate-limiting step in the overall
production of LH and FSH (26, 27). Because expression of each β-subunit is tightly controlled by
endocrine, paracrine, and autocrine actions, including hypothalamic GnRH, the activin-inhibin-
follistatin system, and steroid hormones of gonadal origin (27, 28), it is possible that GR regulation of
transcriptional activity underlies the inhibitory effects of stress on the gonadotrope.
Transcriptional effects of steroid hormones within the gonadotrope have been shown for the
gonadotropin genes, including Cga, Fshb, and Lhb (29, 30). With regard to Lhb, androgen repression of
Lhb involves protein-protein interactions between the androgen receptor and steroidogenic factor 1
(SF1) and is localized to a bipartite SF1 element within the LHβ proximal promoter, critical for
mediating GnRH responsiveness (31, 32). Progesterone repression also involves indirect receptor binding
but differs from androgen repression of LHβ gene expression in that, rather than SF1 elements,
progesterone repression involves two novel promoter regions located upstream of the SF1 sites. Similar
to progestins and androgens, glucocorticoids have been shown to inhibit LHβ gene expression (29, 33),
although the mechanism is unclear, raising the possibility that stress impairs fertility by way of
disruption of gene expression within the gonadotrope cell.
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We initiated two lines of investigation in the mouse to tease apart the mechanisms whereby elevated
glucocorticoids inhibit gonadotrope responsiveness during episodes of stress. First, we tested the
hypothesis that restraint stress, and/or an elevation in glucocorticoids mimicking the level induced by
restraint stress, can disrupt reproductive function and inhibit gonadotrope production of LH in female
mice. Second, we conducted a series of studies to examine the molecular mechanisms underlying
glucocorticoid regulation of the LHβ promoter utilizing the immortalized LβT2 gonadotrope cell line.
Materials and Methods
Animals
Female C57Bl/6 mice (6 wk of age) were purchased from The Jackson Laboratory (Bar Harbor, ME),
and housed in a UCSD vivarium animal facility under standard conditions. All animals were housed
under a 12-h light, 12-h dark cycle (lights on at 0700 h) and provided with food and water ad libitum.
Mice were group housed (four females per cage) for 2 wk of acclimatization. All experimentation was
performed between 0900 and 1300 h in a room within the vivarium. Care was taken to minimize pain
and discomfort for the animals. Mouse colonies were maintained in agreement with protocols approved
by the Institutional Animal Care and Use Committee at the University of California, San Diego. All
procedures were approved by the University of California, San Diego IACUC.
Determination of phase of estrous cycle
At 8 wk of age, vaginal lavage was performed daily (at 0900 h) by flushing the vagina with distilled
H O. Collected smears were mounted on glass slides and examined microscopically for cell type (34).
The smears were classified into one of three phases of estrus: diestrus, proestrus, or estrus. Female mice
exhibiting two consecutive 4- to 6-d estrous cycles, including positive classification of diestrus, proestrus,
and estrus, were used in animal experiments. Estrous cycle length was calculated as the length of time
between two successive occurrences of estrus. The time spent in each cycle stage was calculated as the
proportion of time classified in each cycle phase during the observation period, and values were
analyzed by two-way repeated measures ANOVA, group (no stress vs. stress) × time (d 1–d 18 vs. d 19–
d 36). All statistics were performed using JMP 7.0 (SAS Institute, Cary, NC), and significance was
established as P < 0.05.
Restraint stress protocol
After vaginal lavage and estrus classification, mice were either returned to their group home cage (no
stress) or placed individually into clear plastic restraint tubes (stress). The ventilated tubes (Harvard
Apparatus, Holliston, MA) are designed to be small enough to restrain a mouse so that it is able to
breathe but unable to move freely. The restraint devices were cleaned between uses with soap, water,
and ethanol (70%). Mice are continually observed by experienced personnel during the 180-min
restraint period. After the restraint period, stress mice are returned to individual home cages or killed by
decapitation, and trunk blood or pituitary tissue was collected from individual animals. Hormone values
were analyzed by one-way ANOVA followed by Tukey's post hoc test or two-way ANOVA, group (no
stress vs. stress) × time (vehicle vs. GnRH).
Corticosterone response protocol
Female mice (8 wk of age) were ovariectomized and allowed to recover for 2 wk before
experimentation. To test the LH response to a stress level of corticosterone, animals received a bolus
injection of corticosterone (200 ng/kg, sc) or vehicle. After 90 min, animals were killed by decapitation,
and trunk blood was collected from individual animals. Hormone values were analyzed by one-way
ANOVA followed by Tukey's post hoc test.
Hormone analysis
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Trunk blood was collected and serum separated by centrifugation and stored frozen at −20 C before
analysis at the Center for Research in Reproduction Ligand Assay and Analysis Core at the University of
Virginia (Charlottesville, VA). Corticosterone concentrations were determined by RIA in single 25- to
50-μl aliquots of serum. Assay sensitivity averaged 20.0 ng/ml. LH concentrations were determined by
two-site sandwich immunoassay in duplicate 50-μl aliquots of serum. Assay sensitivity averaged 0.07
ng/ml.
Plasmids
The −1800 rat LHβ-luc in pGL3 was kindly provided by Dr. Mark Lawson. The 5′-truncations of the
−1800-bp LHβ-luc reporter plasmid were created by inserting fragments between KpnI and HindIII in
pGL3 have been partially described by Thackray et al. (35): −400, −300, −200, −150, −87. The 5′- and
3′-mutations of either Egr1 or SF1 binding sites in the −200 rat LHβ-luc have been reported previously
(35). The reporter plasmids with the multimerized consensus SF1 site (TGACCTTGA) or consensus Egr1
site (CGCCCCCGC) were created by ligating an oligonucleotide, containing four copies of the indicated
site, between KpnI and NheI in pGL3, upstream of the 81-bp thymidine kinase (TK) promoter. The
sequences of all promoter fragments were confirmed by dideoxynucleotide sequencing.
The mouse Ptx1 pcDNA3 expression vector has been previously described (36). The rat Egr1 and mouse
SF1 cDNA were kindly provided by Dr. Jacques Drouin and Dr. Bon-chu Chung, respectively. The cDNA
were cloned into the pCMV expression vector using the ClaI/XbaI restriction sites of both plasmids. The
human Egr1 cDNA was provided by Dr. Hermann Pavenstadt and was cut using XhoI/EcoRI and
inserted into pGEX-5X expression vector using the SmaI restriction site by blunt end cloning. Dr.
Douglass Forbes provided the green fluorescent protein (GFP) expression plasmid. The wild-type rat GR
pSG5 plasmid was provided by Dr. Keith Yamamoto. Both GR mutants are in pSG5 and have been
previously described; the GRdim4 mutant contains four point mutations that prevent dimerization and
DNA binding of the receptor (29), and the GR DNA-binding domain (DBD) mutant contains a
mutation in the DNA-binding domain (33).
Cell culture and transient transfection
LβT2 cells, cultured as previously described (29), were seeded into 12-well plates at 3 × 10 cells per well
and incubated overnight at 37 C. Each well was transfected with 400 ng of the luciferase-reporter
plasmid or control pGL3 vector and 100 ng of a β-galactosidase reporter gene regulated by the TK
promoter (TK-βgal) as a control for transfection efficiency using FuGENE 6 transfection reagent
(Roche Applied Science, Indianapolis, IN). In experiments utilizing Egr1, SF1, or Ptx1 to induce
promoter activity, cells were also transfected with 100 ng Egr1 (unless indicated otherwise) or empty
pCMV vector, 100 ng SF1 or empty pCMV vector, 50 ng Ptx1 or empty pcDNA3 vector. Eighteen hours
after transfection, cells were transferred to serum-free DMEM (supplemented with 0.1% BSA, 5 mg/liter
transferrin, and 50 nM sodium selenite) containing either the natural glucocorticoid, corticosterone (100
nM, Sigma Aldrich, St. Louis, MO), synthetic glucocorticoid, dexamethasone (100 nM, Sigma Aldrich), or
vehicle (0.1% ethanol). When cells were treated with GnRH (10 nM; Sigma Aldrich), treatment with
GnRH or vehicle (0.1% BSA) began 6 h before harvest. Cells were harvested and extracts were prepared
for assay of luciferase and β-galactosidase activity as previously described (33).
CV-1 cells, a monkey kidney cell line that does not express detectable endogenous GR (37), were seeded
into 12-well plates at 1.5 × 10 cells per well as previously described (38) and treated as indicated above
with the following addition. Each well was transfected with 50 ng of the GR expression vector (wild
type or mutant) or empty pGS5 vector.
Luciferase reporter experiments were performed in triplicate and were repeated at least three times. To
normalize for transfection efficiency, all luciferase values were divided by β-galactosidase, and the
triplicate values were averaged. To control for interexperimental variation, the control pGL3 reporter
5
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plasmid was transfected with TK-βgal and any relevant expression vectors, and the average pGL3/βgal
value was calculated. Average luc/βgal values were divided by the corresponding pGL3/βgal value.
Individual values obtained from each independent experiment were averaged and analyzed by Student's
t test or one-way ANOVA followed by Tukey's post hoc test.
Quantitative real-time PCR
Preparation of cDNA from mouse pituitary or LβT2 cells was performed as previously described (39).
Briefly, RNA was extracted with Trizol reagent (Invitrogen/GIBCO, Carlsbad, CA) according to the
manufacturer's instructions, treated to remove contaminating DNA (DNA-free; Ambion, Austin, TX),
and reverse transcribed using Superscript III First-Strand Synthesis System (Invitrogen). Quantitative
real-time PCR was performed in an iQ5 Real-Time PCR instrument (Bio-Rad Laboratories, Inc.,
Hercules, CA) and used iQ SYBR Green Supermix (Bio-Rad Laboratories) with specific primers for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), LHβ, or Egr1 cDNA.
LHβ forward: CTGTCAACGCAACTCTGG
LHβ reverse: ACAGGAGGCAAAGCAGC
Egr1 forward: ATTTTTCCTGAGCCCCAAAGC
Egr1 reverse: ATGGGAACCTGGAAACCACC
GAPDH forward: TGCACCACCAACTGCTTAG
GAPDH reverse: GGATGCAGGGATGATGGTTC
The iQ5 real-time PCR program was as follows: 95 C for 15 min, followed by 40 cycles at 95 C for 15
sec, 55 C for 30 sec, and 72 C for 30 sec. Within each experiment, the amount of LHβ or Egr1 and
GAPDH mRNA was calculated by comparing a threshold cycle obtained for each sample with the
standard curve generated from serial dilutions of a plasmid containing GAPDH, ranging from 1 ng to 1
fg. All samples were assayed (in triplicate) within the same run, and the experiment was conducted
three times. Values were analyzed by one-way ANOVA followed by Tukey's post hoc test or two-way
ANOVA, group (no stress vs. stress) × time (vehicle vs. GnRH).
Dual immunofluorescence
Adult mouse pituitary paraffin tissue sections (Zyagen, San Diego, CA) were dewaxed in xylene,
rehydrated through a series of graded ethanol baths, and washed in H 0. Sections were immersed in 10
mM sodium citrate buffer (pH 6.0), heated in a standard microwave twice for 5 min, and allowed to
stand for 20 min at room temperature. After a brief wash in PBS, nonspecific binding was blocked with
5% goat serum/0.3% Triton X-100 for 60 min at room temperature. Dual fluorescence labeling was
tested on the same section with a guinea pig antirat LHβ primary antibody (anti-rβ LH-IC-2, NIDDK
NHPP; 1:200 dilution in 5% goat serum/0.3% Triton X-100) plus a rabbit antimouse GR primary
antibody (sc-1004, Santa Cruz Biotechnology, Inc., Santa Cruz, CA; 1:500 dilution) for 48 h at 4 C.
LHβ- and GR-containing cells were revealed using a goat rhodamine antiguinea pig IgG secondary
antibody (ab6905, 1:300 dilution; Abcam, Cambridge, MA) plus a goat fluorescein antirabbit IgG
secondary antibody (FI-1000; Vector Laboratories, Burlingame, CA; 1:300 dilution) for 60 min at room
temperature. After rinsing with PBS, sections were coverslipped with Vectashield HardSet mounting
medium with 4′,6-diamidino-2-phenylindole (Vector Laboratories). Exclusion of each primary antibody
was run as a negative control, and each antibody was run separately to confirm that each
immunostaining pattern was similar to published reports (40, 41). Specificity of the GR antibody was
confirmed by immunoblot analysis of LβT2 and CV-1 cell lysates, cell lines previously shown to contain
and lack GR (29, 37), respectively, which detected a single band of the expected size.
Sections were viewed using an inverted fluorescence microscope (Nikon Eclipse TE 2000-U; Nikon,
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Tokyo, Japan), and digital images were collected using a Sony CoolSNAP EZ cooled charge-coupled
device camera (Roper Scientific, Trenton, NJ) and analyzed with Nikon Imaging System—Elements
image analysis system (version 2.3; Nikon).
Western blot analysis
Nuclear extracts were prepared as previously described (36) from LβT2 cells treated with
dexamethasone (100 nM, 18 h), GnRH (10 nM, 45 min), GnRH + dexamethasone or vehicle (0.1%
BSA/0.1% ethanol). Nuclear extract (20 μg) was boiled for 5 min in 5× Western-loading buffer,
fractionated on a 10% SDS-PAGE gel, and electroblotted for 90 min at 300 mA onto polyvinylidene
difluoride (Millipore Corp., Billerica, MA) in 1× Tris-glycine-sodium dodecyl sulfate/20% methanol.
Blots were blocked overnight at 4 C in 3% BSA and then probed for 1 h at room temperature with rabbit
antihuman Egr1 antibody (sc-110, Santa Cruz Biotechnology) diluted 1:750 in blocking buffer. Blots
were then incubated with a horseradish peroxidase-linked secondary antibody (Santa Cruz
Biotechnology), and bands were visualized using the SuperSignal West Pico chemiluminescent substrate
(Pierce Biotechnology, Inc., Rockford, IL). Bio-Rad Pre-stained Protein Ladder Plus serves as a size
marker.
Chromatin immunoprecipitation (ChIP)
ChIP assays were performed as previously described (29, 35). Briefly, confluent LβT2 cells in 10-cm
plates were treated with dexamethasone (100 nM, 1 h), GnRH (10 nM, 1 h), GnRH + dexamethasone
(cotreatment 1 h), or vehicle (0.1% BSA/0.1% ethanol) and cross-linked with 1% formaldehyde. The
nuclear fraction was obtained, and chromatin was sonicated to an average length of 300–500 bp using
a Branson Sonifier 250 (Branson Ultrasonics Corp., Danbury, CT). Protein-DNA complexes were
incubated overnight with nonspecific rabbit IgG (sc-2027, Santa Cruz Biotechnology) or rabbit
antihuman GR (ab3579, Abcam) and precipitated with protein A/G beads. Immunoprecipitated DNA
and DNA from input chromatin were analyzed by quantitative PCR using primers specific for a 220-bp
sequence of the mouse LHβ proximal promoter (−180 LHβ/+40 LHβ). Primers specific to the mouse
FSHβ promoter (−223 FSHβ/+57 FSHβ) and FSHβ coding region were used as positive and negative
controls, respectively. DNA from immunoprecipitated samples was quantified relative to a standard
curve representing percent of input chromatin. For ChIP assays comparing chromatin from hormone-
treated LβT2 cells, the fold enrichment of antibody signal over IgG was calculated for each primer set,
and data from each independent experiment were normalized to the indicated negative control gene.
ChIP and input samples were quantified using a standard curve made from ChIP input DNA. ChIP
samples were normalized to their appropriate input samples and are expressed as fold enhancement
over nonspecific IgG.
−180 LHβ-forward: CGAGTGTGAGGCCAATTCACTGG
+40 LHβ-reverse: GGCCCTACCCATCTTACCTGGAGC
−223 FSHβ-forward: GGTGTGCTGCCATATCAGATTCGG
+57 FSHβ-reverse: GCATCAAGTGCTGCTACTCACCTGTG
FSHβ-coding forward: GCCGTTTCTGCATAAGC
FSHβ-coding reverse: CAATCTTACGGTCTCGTATACC
The iQ5 real-time PCR program was as follows: 95 C for 15 min, followed by 40 cycles at 95 C for 15
sec, 55 C for 30 sec, and 72 C for 30 sec. All samples were assayed within the same run, and the
experiment was conducted three times. Individual values obtained from each independent experiment
were averaged and analyzed by one-way ANOVA followed by Tukey's post hoc test.
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Glutathione-S-transferase (GST) interaction assay
S-labeled proteins were produced using the TnT Coupled Reticulolysate System (Promega Corp.,
Madison, WI). Bacteria transformed with the GST plasmids were grown to an OD of 0.6 and then
induced with isopropyl-β-d-thiogalactoside overnight at 30 C. The bacterial pellets were sonicated in
0.1% Triton X-100 and 5 mm EDTA in 1× PBS and centrifuged, and the supernatant was bound to
glutathione Sepharose 4B resin (Amersham Pharmacia Biotech, Piscataway, NJ). The beads were
washed four times in PBS and then in HEPES/Nonidet P-40/dithiothreitol (HND) buffer [10 mg/ml
BSA, 20 mm HEPES (pH 7.8), 50 mm NaCl, 5 mm dithiothreitol, and 0.1% Nonidet P-40]. For the
interaction assay, S-labeled in vitro-transcribed and -translated GR (20 μl), SF1 (5 μl), or GFP (5 μl)
was added to the beads with 400 μl HND buffer. The beads were incubated for 1 h at 4 C and then
washed twice with HND buffer and twice with 0.1% Nonidet P-40 in PBS. Thirty microliters of 2×
Laemmli load buffer were added, and the samples were boiled and then electrophoresed on a 10%
sodium dodecyl sulfate-polyacrylamide gel. One tenth of the S-labeled in vitro-transcribed and -
translated product was loaded onto the gel as input. The gel was dried, and the proteins were visualized
by autoradiography.
Results
Chronic restraint stress compromises estrous cyclicity
To evaluate the mechanism whereby elevated glucocorticoids impair reproduction, we developed a
model to assess whether daily restraint stress disrupts estrous cyclicity. Vaginal cytology was examined
daily by vaginal lavage in a cohort of female mice during an 18-d control period (d 1–d 18). After this
period of observation, mice were randomly assigned to either the stress or no stress group (n =
9/group), and estrous cyclicity was monitored for an additional 18 d (d 19–d 36). Figure 1A illustrates
profiles of vaginal histological classification during the prestress and stress period of three female mice
exposed to 180 min of daily restraint stress. In mice not subjected to stress, cycle length was not
significantly different between the two periods of assessment (P > 0.05; d 1–d 18 vs. d 19–d 36, 5.33 ±
0.31 vs. 4.81 ± 0.45 d, Fig. 1B). In contrast, stressed mice exhibited a significant increase in average
cycle length in the stress period compared with the prestress period (P < 0.05; d 1–d 18 vs. d 19–d 36,
5.25 ± 0.35 vs. 6.75 ± 0.55 d). Specifically, exposure to daily restraint stress significantly increased the
time spent in diestrus during the stress period, d 19–d 36, without altering the time spent in estrus or
proestrus as compared with the prestress period, d 1–d 18 (P > 0.05; Fig. 1C).
Acute restraint stress disrupts gonadotrope function
Having found that repeated exposure to stress compromises reproductive neuroendocrine activity as
evidenced by a disruption in estrous cyclicity in female mice, the next step was to focus on the role of
the pituitary gland and test whether gonadotrope responsiveness, as assessed by GnRH-induced LH
synthesis or secretion, is diminished by acute restraint stress. Separate cohorts of female mice, in which
estrus cyclicity was confirmed and diestrus females selected, were used to assess gonadotrope
responsiveness to GnRH in the absence or presence of stress. In the first study, GnRH-induced LH
secretion was monitored in groups of control mice (no stress) or mice exposed to 180 min of restraint
stress (Fig. 2A). Blood was collected before stress, as well as 30 and 180 min after the initiation of the
control or stress period for measurement of corticosterone (n = 7 per time point per group). To assess
LH release in response to GnRH, female diestrus mice were administered GnRH (200 ng/kg, sc; n = 7
per group per treatment) or saline vehicle and killed 10 min after injection. Blood was collected and
processed for measurement of LH at 180 min after the initiation of stress. This GnRH dose was selected
because it has been shown to produce a significant, yet submaximal, LH response, that peaks 10 min
after administration in a mouse model of diestrus in which female mice are ovariectomized and
estrogen primed (42, 43). Serum levels of corticosterone remained low in no stress control animals (
Fig. 2B, open circles); corticosterone levels were significantly increased in stressed mice at 30 min (P <
35
35
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0.05; stress vs. no stress, 588.6 ± 26.4 vs. 108.8 ± 35.5 ng/ml) and remained significantly elevated 180
min after initiation of restraint (Fig. 2B, gray circles). Stress did not significantly induce serum levels of
progesterone at either 30 min or 180 min after the initiation of restraint (data not shown). Mean LH in
the no stress diestrus females receiving vehicle was not significantly different from values in the vehicle-
treated stress animals, indicating that stress does not significantly impair responsiveness to endogenous
GnRH in this animal model (P > 0.05; stress Veh vs. no stress Veh, Fig. 2C). In the no stress group,
exogenous GnRH caused a robust increase in circulating LH levels as compared with vehicle-treated
animals (P < 0.05; 1.83 ± 0.23 vs. 0.56 ± 0.11 ng/ml, Fig. 2C). GnRH also significantly increased LH in
stressed animals (P < 0.05, Fig. 2C). However, the LH response to exogenous GnRH was significantly
blunted in restraint-stressed animals compared with the response in no stress controls (P < 0.05; stress
GnRH vs. no stress GnRH, 1.19 ± 0.18 vs. 1.83 ± 0.23 ng/ml; Fig. 2C), suggesting that stress diminishes
the ability of the pituitary to respond to GnRH. Taken together, these experiments reveal an interplay
between GnRH and stress and implicate responsiveness of the pituitary gonadotrope as a potential
neuroendocrine site of LH suppression.
We further investigated the response of the gonadotrope by analyzing GnRH-induced LHβ mRNA
transcript level in mice exposed to restraint stress. Initially, diestrus female mice received saline vehicle
or GnRH (200 ng/kg, sc; n = 7 per group per treatment) and were subsequently sorted into no stress or
stress treatment groups (Fig. 2D). Animals were killed and the pituitary glands were collected for mRNA
analysis by quantitative RT-PCR. Stress did not alter basal LHβ transcript levels compared with
expression in no stress vehicle controls (P < 0.05; stress Veh vs. no stress Veh; Fig. 2E). In the absence
of stress, exogenous GnRH resulted in a 4-fold increase in LHβ mRNA compared with values in vehicle-
treated animals (P < 0.05; Fig. 2E). In stressed mice, the LHβ transcript level in response to GnRH was
significantly reduced by approximately 45% (P < 0.05; no stress GnRH vs. stress GnRH, 16.5 ± 2.3 vs.
8.9 ± 1.1; Fig. 2E). Collectively, these observations raise the possibility that stress can interfere with
ovarian cyclicity by disrupting the synthesis and secretion of LH at the level of the anterior pituitary
gonadotrope cell.
Glucocorticoids impair LH secretion in vivo potentially via receptors expressed in mousegonadotrope cells
Our studies thus far demonstrate that circulating glucocorticoids are increased within 30 min and
remain significantly elevated for the 180-min stress paradigm (Fig. 2). Because stress likely induces a
host of inhibitory intermediates, any of which could alter reproductive activity, we directly assessed the
role of glucocorticoids by testing the hypothesis that a stress-like level of glucocorticoid in female mice
reduces LH secretion. Pilot studies were conducted to identify a dose of glucocorticoid that approximated
a stress level (∼750 ng/ml) and an animal model that eliminated confounding effects of ovarian
steroids. Blood was collected 90 min after a bolus administration of vehicle or corticosterone (200
ng/kg, sc; n = 6 per treatment) to ovariectomized female mice (Fig. 3A). Corticosterone remained low
in mice treated with vehicle, yet values were markedly elevated after administration of corticosterone (P
< 0.05; Fig. 3B). Treatment with corticosterone significantly reduced mean LH as compared with the
value in mice treated with vehicle (P < 0.05; Cort vs. Veh, 3.4 ± 0.3 vs. 1.9 ± 0.6 ng/ml, Fig. 3C),
demonstrating sufficiency of glucocorticoids to disrupt reproductive neuroendocrine function and
relevancy as an inhibitory factor induced during stress.
Evidence that GR is expressed in gonadotrope cells in the rat (22) supports our hypothesis of a direct
action of glucocorticoids via GR within mouse gonadotrope cells. To confirm the presence of this
receptor in mouse gonadotrope, we used dual-label immunofluorescence of adult mouse anterior
pituitary sections for LHβ and GR (rhodamine- and fluorescein isothiocyanate-conjugated secondary
antibodies, respectively; Fig. 3, D–F). LHβ immunostaining identified gonadotropes that accounted for a
small proportion of labeled anterior pituitary cells (Fig. 3D), whereas GR immunostaining occurred in
an extensive population of pituitary cells (Fig. 3E). Of interest, numerous LHβ-containing gonadotropes
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showed GR staining, confirming the presence of GR in this anterior pituitary cell type in the mouse
(merge, white stars, Fig. 3F).
Glucocorticoids regulate LHβ gene expression in gonadotrope cells
Having confirmed the presence of GR in adult mouse pituitary gland, we next tested the hypothesis that
the stress-induced decrease in LHβ mRNA expression could be recapitulated in cultured gonadotrope
cells. As expected, immortalized pituitary gonadotrope cells, LβT2, responded to GnRH with a 2-fold
increase in endogenous LHβ mRNA as measured by quantitative RT-PCR (Fig. 4A). Treatment of LβT2
cells with a physiological, stress level of corticosterone decreased GnRH-induced LHβ mRNA expression
(P < 0.05; GnRH vs. GnRH + Cort, 2.1 ± 0.3 vs. 1.6 ± 0.1; Fig. 4A); however, corticosterone alone did
not reduce LHβ expression. These findings indicate that repression by glucocorticoids occurs in the
presence of GnRH and confirm the LβT2 gonadotrope cell is an appropriate model for investigating the
role of glucocorticoid-induced suppression of LHβ gene expression.
We began to dissect out the mechanism whereby glucocorticoids reduce LHβ-subunit induction in LβT2
cells, by investigating the regulation of an LHβ-luciferase reporter after incubation with either natural
or synthetic glucocorticoids. For this purpose, LβT2 cells were transiently transfected with −1800 bp of
the rat LHβ regulatory region fused upstream of a luciferase reporter gene (LHβ-luc) or pGL3 control
plasmid and treated with corticosterone (100 nM), dexamethasone (100 nM), or vehicle (0.1% ethanol)
for 24 h before harvest. Neither the natural glucocorticoid, corticosterone, nor the synthetic
glucocorticoid, dexamethasone, significantly altered basal expression of the −1800-bp LHβ promoter (P
> 0.05; Veh vs. Cort or Dex; Fig. 4B). In contrast, both glucocorticoids significantly blunted the robust
increase in promoter activity induced by GnRH (10 nM, final 6 h of glucocorticoid treatment; P < 0.05;
Fig. 4B). Specifically, GnRH resulted in a 4.6-fold induction of LHβ-luc, which was reduced 35% by
corticosterone, or 45% by dexamethasone. Although both glucocorticoids are capable of transcriptional
repression of GnRH induction of the LHβ promoter, we focused on the synthetic glucocorticoid,
dexamethasone, based on the intensity of its effect and evidence for its potent interaction with the
endogenous steroid receptor, GR, expressed in LβT2 cells (29) and identified in mouse gonadotrope cells
(Fig. 3).
Using a promoter truncation approach, we identified regions of the LHβ gene that are functionally
involved in glucocorticoid regulation. LβT2 cells were transiently transfected with a series of truncated
LHβ reporter plasmids, ranging in length from −1800 to −87 bp of the 5′-regulatory sequence. Figure 4
C illustrates the effect of treatment with GnRH in the presence or absence of dexamethasone on
progressive 5′-LHβ promoter truncations. As observed previously, GnRH induction of the LHβ gene
declined incrementally as the promoter was progressively truncated from −1800 to −87 (Fig. 4C and
Ref. 44). Dexamethasone repressed LHβ promoter activity by approximately 40% when the region
contained at least −150 bp of the proximal promoter. Interestingly, further truncation of the region
from −150 to −87, which removed the proximal GnRH responsive elements [such as early growth
response factor 1 (Egr1) and SF1], resulted in a loss of GnRH induction and elimination of
dexamethasone repression, indicating that GnRH responsiveness of the LHβ gene is required for
glucocorticoid repression. Collectively, these data suggest that GR exerts its inhibitory effects upon the
LHβ proximal promoter, likely via interactions with GnRH-responsive factors, such as Egr1 and SF1.
Proximal binding elements are important for glucocorticoid repression
The proximal 150 bp of the rat LHβ promoter contain multiple binding elements that are critically
important for GnRH regulation of LHβ transcription (Fig. 5A), including tandem elements for both
Egr1 and SF1 that are arranged on either side of a homeobox element, previously shown to bind
pituitary homeobox factor 1 (Ptx1). Ptx1 is expressed throughout pituitary development and plays a
critical role in activation of a number pituitary genes, including Lhb (45). SF1 is specifically expressed in
the gonadotrope of the anterior pituitary gland, and its importance for reproduction is underscored by a
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lack of gonads in SF1-null mice (46). Although highly important for LHβ transcription, SF1 and Ptx1
are not regarded as factors induced or regulated by GnRH (47). On the other hand, Egr1 is rapidly
induced by GnRH and considered a critical regulator of LHβ gene expression (47), because Egr1 null
mice lack LHβ expression in the pituitary gonadotropes (48). Therefore, we focused on the role of Egr1
in glucocorticoid repression of LHβ gene expression and tested the hypothesis that glucocorticoids
interfere with GnRH induction of LHβ by blunting the GnRH-stimulated increase in Egr1 mRNA and
protein. To investigate hormone regulation of Egr1 mRNA, LβT2 cells were treated with hormone, and
after 45 min, RNA was harvested for Egr1 mRNA analysis by quantitative RT-PCR. Egr1 mRNA was
low in vehicle-treated control cells, and expression was unchanged by treatment with dexamethasone
alone (Fig. 5B). As expected, GnRH caused a dramatic 27.2-fold induction in Egr1 mRNA (P < 0.05 vs.
Veh). This increase in GnRH-induced Egr1 transcript, however, was not significantly decreased by
dexamethasone (P > 0.05; GnRH vs. GnRH + Dex, 27.2± 4.6 vs. 23.1 ± 4.5; Fig. 5B). Because we
found that treatment with glucocorticoids does not suppress levels of GnRH-induced Egr1 transcript, we
next tested the hypothesis that dexamethasone regulates translation or degradation of Egr1 in LβT2 cells
using Western blot analysis. Protein expression of Egr1 was undetectable in nuclear extracts of LβT2
cells after treatment with vehicle or dexamethasone (Fig. 5C). After treatment with GnRH, Egr1 protein
was readily detected, yet protein expression did not significantly change after cotreatment with GnRH
and dexamethasone, indicating that GR does not interfere with GnRH induction of Egr1 protein.
We further examined how glucocorticoids might influence GnRH-induced LHβ expression by
performing ChIP assays on the endogenous mouse LHβ promoter in LβT2 cells. Cells were treated with
dexamethasone or GnRH alone or in combination for 60 min before cross-linking. Sonicated chromatin
was immunoprecipitated using either anti-GR or nonspecific IgG. Cross-linking was reversed and
precipitates analyzed by quantitative PCR for 220 bp of the LHβ promoter (−180 to +40).
Dexamethasone treatment alone did not increase GR binding to the proximal promoter over vehicle
treatment (P < 0.05; Veh vs. Dex; Fig. 5D). However, we can only rule out a change in GR occupancy
of the LHβ promoter at 60 min, and that later (or earlier) changes in the response to glucocorticoids are
still possible. In contrast, GnRH treatment increased GR binding 3.8-fold compared with vehicle (P <
0.05; Veh vs. GnRH), indicating that recruitment of GR to the LHβ promoter is not dependent on
dexamethasone binding, but rather is dependent on a GnRH-responsive factor. No difference in GR
binding was observed when the cells were concomitantly stimulated with dexamethasone and GnRH as
compared with GnRH alone, suggesting that a change in GR conformation or recruitment due to
ligand does not underlie the mechanism of glucocorticoid repression of LHβ transcription.
Glucocorticoids interfere with Egr1 actions at the level of the promoter
Having determined that GR recruitment is dependent on GnRH (i.e. a responsive factor such as Egr1),
yet the inhibitory effect of glucocorticoids is downstream of GnRH-induced Egr1 mRNA or protein, we
investigated the role of Egr1 at the level of the LHβ promoter. We began by testing the hypothesis that
glucocorticoids reduce activity of the LHβ promoter when induced by Egr1 itself, in the absence of
GnRH. Egr1 is a potent activator of LHβ transcription, and transfection of an Egr1 expression plasmid
in LβT2 cells treated with vehicle caused a robust 35-fold increase in LHβ activity (P < 0.05; Veh:
Empty Vec vs. Egr1; Fig. 6A). Treatment with dexamethasone significantly blunted the increase in LHβ
activity induced by Egr1 compared with the response in cells treated with vehicle (P < 0.05; Egr1 (black
bars): Dex vs. Veh; Fig. 6A), indicating that glucocorticoids can interfere with activation by Egr1 at the
level of the LHβ promoter in gonadotrope cells and that GR suppression does not require factors
involved in GnRH signaling upstream of Egr1.
We next attempted to rescue the glucocorticoid repression of GnRH-induced LHβ activity in LβT2 cells.
We hypothesized that if Egr1 were the sole factor affected by glucocorticoids, then titrating increasing
amounts of Egr1 into LβT2 cells would restore full induction and prevent diminishment by
glucocorticoids. LHβ-luc/βgal values are represented as fold induction of GnRH or GnRH + Dex relative
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to the vehicle control condition containing the same amount of Egr1. In the absence of exogenous Egr1,
dexamethasone significantly blunted GnRH-induced LHβ activity (P < 0.05; empty vector: GnRH vs.
GnRH + Dex; Fig. 6B). In the presence of increasing amounts of overexpressed Egr1 (50–200 ng), the
percent repression significantly decreased (P < 0.05; empty vector vs. 100 or 200 ng Egr1; Fig. 6C),
indicating that repression occurs, in part, via interfering with Egr1 function on the promoter.
We focused our attention on the action of glucocorticoids at the level of the LHβ promoter and analyzed
the necessity of known GnRH-responsive elements within the proximal 150 bp of the LHβ gene for
glucocorticoid repression by creating cis mutations in the 5′-SF1, 3′-SF1, 5′-Egr1, and 3′-Egr1 binding
elements in the context of a minimal −200 bp LHβ promoter (WT). We used this minimal LHβ
promoter because it is sufficient for responsiveness to GnRH and glucocorticoids. cis mutation of either
the 5′- or 3′-SF1 or the 5′-Egr1 site preserved significant GnRH induction and was sufficient for
glucocorticoid repression, suggesting that these elements are not required for either GnRH induction or
GR repression (P < 0.05; *, significant induction by GnRH; #, significant repression of GnRH
induction; Fig. 6D). In contrast, the 3′-Egr1 cis mutation was the only mutation to abrogate
glucocorticoid repression (Fig. 6D, 3′-Egr1 mutation). Of interest to our study, this Egr1 binding site has
been shown to be critical for GnRH induction as well (49), and cis mutation of this element eliminated
significant induction by GnRH in our hands, implying that GnRH responsiveness is necessary for
repression by glucocorticoids.
Interaction and involvement of Lhb promoter proximal binding factors
Egr1 conveys GnRH induction of the LHβ promoter by interaction with other regulators of gene
expression in the gonadotrope (47, 49–51). To assess the complex and cooperative roles of Egr1, SF1,
and Ptx1, we used heterologous CV-1 cells. Unlike gonadotrope cells, CV-1 cells lack GnRH receptors, an
Egr1 response to GnRH, and are devoid of endogenous SF1, Ptx1, and GR, which allowed us to
reconstitute these factors and determine the necessity and sufficiency of proteins involved in suppression
by dexamethasone. In addition to GR, CV-1 cells were cotransfected with 1800-bp LHβ-luc reporter
plasmid or pGL3 control plasmid and Egr1, SF1, or Ptx1 alone, or in combination, and tested for
activation of LHβ transcription in the presence of dexamethasone or vehicle. As shown in Fig. 7A (white
bars), overexpression of Egr1, SF1, or Ptx1 alone induced small increases in LHβ transcription, with
induction by Egr1 reaching significance. Dexamethasone significantly diminished induction by Egr1 (P
< 0.05; Egr1: Dex vs. Veh). Interestingly, dexamethasone also significantly repressed LHβ induction
when Egr1 was cotransfected with SF1 alone or SF1 plus Ptx1 (P < 0.05; Egr1: Dex vs. Veh, Egr1/SF1:
Dex vs. Veh; Fig. 7A).
To address whether the Egr1-binding site is sufficient for glucocorticoid repression of LHβ transcription,
reporter constructs containing four copies of the Egr1 consensus site ligated into pGL3 upstream of a
minimal TK promoter were created (Egr1 multimer, Fig. 7B). CV-1 cells were cotransfected with the
Egr1 multimer or control TKluc pGL3 plasmid, GR, and either Egr1 alone, or Egr1 in combination with
SF1 and Ptx1, to test for sufficiency to activate transcription in the presence or absence of
dexamethasone. Egr1 was sufficient to induce transcriptional activation of the Egr1 multimer, and this
effect was blunted by glucocorticoids (P < 0.05; #, significant repression by Dex; Fig. 7B). Of interest,
this site was sufficient to allow for enhanced activation by the combination of Egr1, SF1, and Ptx1, an
affect that was also diminished by glucocorticoids. In contrast to the sufficiency of the Egr1 site, a
consensus SF1-binding site multimer does not convey responsiveness to dexamethasone when induced
by SF1 alone, but activation of the SF1 site by the combination of Egr1, SF1, and Ptx1 is disrupted by
dexamethasone. Taken together, these findings indicate that the Egr1 site activated by Egr1 alone is
sufficient for mediating repression. In contrast, the SF1 site activated by SF1 alone is not sufficient
although a complex with SF1, Egr1, and Ptx1 on the SF1 site can be repressed.
Role of the GR at the level of the Lhb promoter
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Because CV-1 cells lack GR, this cell model allowed us to determine the necessity of DNA binding or
dimerization by GR for repression in gonadotrope cells. In the absence of transfected GR in CV-1 cells,
dexamethasone does not inhibit LHβ promoter activity (P > 0.05; no GR: Veh vs. Dex; Fig. 8A),
confirming that GR is necessary for repression of LHβ by glucocorticoids. To determine whether direct
DNA binding by GR plays a critical role in the repression of LHβ by glucocorticoids, we transfected CV-1
cells with two different GR mutants that are incapable of binding DNA. The first mutant, GR Dim mut,
contains four point mutations in the dimerization domain of GR. These mutations prevent
homodimerization of GR, but do not prevent indirect DNA binding through interactions with other
transcription factors (52, 53). The second GR mutant, GR DBD mut, prevents direct DNA binding by
the mutant. Dexamethasone elicited suppression in the presence of either transfected GR mutant,
indicating that GR dimerization and DNA binding are not necessary for repression of GnRH-induced
LHβ promoter activity (P < 0.05; Dim mut or DBD mut: Veh vs. Dex; Fig. 8A).
After demonstrating that GR does not require DNA binding to elicit repression upon the LHβ promoter,
we investigated the ability of GR to be tethered to DNA by a GnRH-induced factor, by testing the
hypothesis that GR is capable of binding Egr1. We asked whether in vitro-transcribed and -translated
GR could bind bacterially expressed GST-Egr1 in pull-down experiments. Figure 8B demonstrates
precipitation of S-labeled GR with GST-Egr1, as compared with GST alone, illustrating a physical
interaction between GR and Egr1. As a positive control, we show that S-labeled SF1 binds GST-Egr1,
a strong interaction that has been previously reported (54). In contrast, the interaction between S-
labeled GFP with either GST construct was undetectable. Together with the ChIP results in Fig. 5D,
these findings indicate that GR does not directly bind the LHβ promoter; rather it physically interacts
with Egr1 and thus is recruited to the LHβ promoter as a complex with Egr1, identifying Egr1 as a
critical factor mediating GnRH induction and GR repression of LHβ gene expression.
Discussion
Utilizing a restraint stress paradigm that robustly stimulates corticosterone to investigate stress-induced
suppression of gonadotrope function in female mice, we demonstrate that chronic exposure to stress
impairs estrous cyclicity and reduces GnRH-induced LH synthesis and secretion in diestrus female mice.
Whether the increase in glucocorticoids is induced by stress or exogenously administered, both
conditions lead to repression of gonadotrope function, confirming the inhibitory role of glucocorticoids
within the reproductive axis and demonstrating the capacity for suppression during stress. Although we
acknowledge that stress likely increases a variety of mediators, including other hormones of the adrenal
stress axis that have been shown to alter reproductive function during stress (2, 55, 56), we conclude,
based on our current investigation, that glucocorticoids are sufficient to disrupt reproductive
neuroendocrine function.
Seminal work by Dr. Neena Schwartz and colleagues (57) clearly identified inhibitory effects of
glucocorticoids within the reproductive neuroendocrine axis and postulated that glucocorticoids may
alter hypothalamic and pituitary function. Indeed, in vivo analyses demonstrated that glucocorticoids
could prevent the postcastration rise in LH in rats but also blunt the response to GnRH in anterior
pituitary fragments in culture (20, 57). Our present investigation expands upon these early studies by
demonstrating that glucocorticoids can act directly upon the anterior pituitary gonadotrope cell to
suppress GnRH induction of LHβ gene expression. Intriguingly, we observe a similar suppression in
LHβ gene expression after stress, but it remains to be determined whether this is a direct effect of
glucocorticoids within the pituitary gland. With regard to direct actions within the gonadotrope, we
demonstrate a novel mechanism whereby GR is recruited to the 5′-region of the mouse LHβ gene in
live LβT2 cells and blunts GnRH induction of LHβ by interfering with the genomic effects of Egr1 on the
proximal LHβ promoter.
Glucocorticoid repression via GR maps to a highly active region of the LHβ promoter, which contains
35
35
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elements critical for GnRH induction. Promoter analyses revealed that glucocorticoid-mediated
repression is lost upon 5′-truncation of the bipartite SF1 and Egr1 elements in the LHβ proximal
promoter. Truncation of this region, however, also eliminated GnRH induction of the promoter (Fig. 4
C, −87 LHβ-luc), suggesting that the ability of glucocorticoids to repress is closely tied to the highly
coordinated and complex mechanism of GnRH action on Lhb. Not surprisingly, the only cis mutation
that relieved suppression by glucocorticoids (Fig. 6D, the 3′-Egr site) also prevented significant
induction of the LHβ promoter by GnRH, supporting the conclusion that glucocorticoid-induced
repression is dependent on the response to GnRH and highlights the importance of Egr1 for repression
by glucocorticoids.
Glucocorticoids have the potential to interfere with GnRH induction of LHβ via interference of GnRH
receptor signaling upstream and downstream of Egr1 induction. For example, studies performed using
rat and pig pituitary cell cultures suggest that chronic exposure to glucocorticoids can disrupt GnRH
receptor-signaling mechanisms involved in gonadotropin release, including activation of protein kinase
C and cAMP (20, 23). Further, GR has been shown to interact rapidly with c-Jun N-terminal kinase, a
kinase implicated in mediating effects of GnRH in gonadotrope cells (58). In contrast to these actions of
glucocorticoids on GnRH signaling, our findings support an action of glucocorticoids downstream of
Egr1 induction by GnRH via GR acting within the LHβ chromatin. Glucocorticoids do not alter GnRH-
induced Egr1 mRNA or protein in LβT2 cells, demonstrating that the pathway between GnRH binding
its receptor and induction of the immediate early gene, Egr1, is intact. Furthermore, glucocorticoids
blunt the ability of overexpressed Egr1 to induce LHβ activity, providing evidence that GR inhibition
does not require elements upstream of Egr1 and focus our attention on a mechanism involving Egr1 at
the promoter level. We show that titrating in increasing amounts of Egr1 partially overcomes
glucocorticoid repression of Lhβ in LβT2 cells, solidifying a mediatory role of Egr1.
With regard to the mechanism of repression, similar to other steroid hormone receptors, GR mediates
transcriptional regulation of target genes via a host of direct and indirect mechanisms. For example,
within the gonadotrope cell, induction of either the murine FSHβ or human αGSU gene occurs via
direct GR binding to DNA at conserved glucocorticoid response elements (29, 30). In contrast, using cell
models either devoid of or expressing GR (CV-1 vs. LβT2 cells, respectively), we find that neither an
intact DBD nor dimerization domain is necessary for GR repression of the rat LHβ gene, implicating a
genomic action that occurs via indirect GR binding. In addition, GR is recruited to the LHβ promoter
chromatin by GnRH induction in the presence or absence of glucocorticoids. Coupled with our findings
that GR physically interacts with Egr1 in GST-pulldown experiments, this provides evidence in support
of our hypothesis that GR is tethered to the LHβ promoter via a GnRH-induced factor, and in particular
by Egr1.
The requirement for a GnRH-induced factor may also contribute to the differential effect of
glucocorticoids on transcription within the gonadotrope cell. On the one hand, genes encoding FSHβ,
αGSU, and GnRH receptor are each induced by glucocorticoids alone (30, 33, 59). On the other hand,
our data reveal that GR repression requires GnRH, likely due to the deficiency of GR recruitment to the
proximal promoter in the absence of GnRH. On the GnRH receptor gene in gonadotrope cells, GnRH-
induced GR recruitment has been shown to be involved in glucocorticoid induction of transcription
(59). In that case, however, the recruitment of GR to the GnRH receptor promoter is dependent on
rapid phosphorylation of GR by GnRH-signaling pathways, which differs from our finding that GR
repression of LHβ transcription does not require GnRH signaling upstream of Egr1 activation. Rather,
we conclude that GR is recruited by a factor induced by GnRH and hypothesize that Egr1 itself
mediates the balance between GnRH induction and GR repression of the LHβ promoter.
In summary, the present study shows that restraint stress potently activates the adrenal stress axis in
mice and interferes with gonadotropin synthesis and secretion. We expand upon this finding by
demonstrating that administration of a stress level of glucocorticoids inhibits LH secretion in female
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mice and detailing a mechanism whereby glucocorticoids repress activity of the anterior pituitary gland
via regulation of LHβ gene expression in gonadotrope cells. We identify GR in native gonadotrope cells
in the mouse and demonstrate that the recruitment of GR to the mouse LHβ promoter via Egr1
interferes with the tripartite transcriptional complex necessary for mediating responsiveness of this
gonadotropin gene to GnRH. Utilizing dual in vivo and in vitro approaches, collectively, this work
reveals new insights regarding the interaction between the adrenal stress and reproductive
neuroendocrine axes by identifying a mechanism whereby LHβ expression is dampened after a stress
elevation in glucocorticoid.
Acknowledgments
We thank Dr. Alexander (Sasha) Kauffman (University of California, San Diego, La Jolla, CA) and Dr.
Catherine Rivier (Salk Institute, La Jolla, CA) for insightful discussions regarding the in vivo
experiments. We are grateful to Dr. Jacques Drouin (University of Montreal, Quebec, Canada) for
generously providing the mouse Ptx1 and rat Egr1 cDNAs; to Dr. Hermann Pavenstadt (University of
Munster, Munster, Germany) for providing the human Egr1 cDNA; and to Dr. Douglass Forbes
(University of California, San Diego, La Jolla, CA) for providing the GFP expression plasmid. The
mouse SF1 pCMV expression plasmid was a kind gift of Dr. Bon-chu Chung (Academia Sinica, Taipei,
Taiwan), and the rat LHβ-luciferase plasmid was kindly provided by Dr. Mark Lawson (University of
California, San Diego, La Jolla, CA). We thank Dr. Keith Yamamoto (University of California, San
Francisco, San Francisco, CA) for providing the rat GR plasmid and Dr. Al Parlow of the National
Hormone and Peptide Program for providing the NIDDK-anti-rβ LH-IC-2 antibody. We also thank
Jason Meadows, Emily Witham, Chuqing (Carol) Yao, Courtney Benson, and Dr. Suzanne Rosenberg
(University of California, San Diego, La Jolla, CA) for technical assistance and helpful discussions
throughout this work.
This work was supported by National Institutes of Health (NIH) grants R01 HD020377, R01
HD072754, and R01 DK044838 (to P.L.M.) and by the Eunice Kennedy Shriver National Institute of
Child Health and Human Development/NIH through cooperative agreement (U54 HD012303) as part
of the Specialized Cooperative Centers Program in Reproduction and Infertility Research (to P.L.M.).
P.L.M. was also partially supported by P30 CA023100, P30 DK063491, and P42 ES010337. K.M.B. was
partially supported by NIH Grant K99 HD060947. V.G.T. was partially supported by NIH Grants K01
DK080467, and R01 HD067448. D.C. was partially supported by NIH Grants R01 HD057549, R21
HD058752, and R03 HD054595. The LHβ antibody was provided by Dr. A. F. Parlow from the National
Hormone and Pituitary Program, Harbor-UCLA Medical Center. DNA sequencing was performed by
the DNA-sequencing shared resource, University of California, San Diego, Cancer Center, which is
funded in part by National Cancer Institute Cancer Support Grant P30 CA023100. Serum hormone
assays were performed by The University of Virginia Ligand Assay Core Laboratory, which is supported
through National Institute of Child Health and Human Development Grant U54 HD028934.
Disclosure Summary: The authors have nothing to disclose.
Notes
NURSA Molecule Pages :
Ligands: Corticosterone.
Annotations provided by Nuclear Receptor Signaling Atlas (NURSA) Bioinformatics Resource. Molecule Pages can be
accessed on the NURSA website at www.nursa.org.
Abbreviations:
†
†
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ChIP Chromatin immunoprecipitation
DBD DNA-binding domain
Egr1 early growth response factor 1
GAPDH glyceraldehyde-3-phosphate dehydrogenase
GFP green fluorescent protein
GR glucocorticoid receptor
GST glutathione-S-transferase
αGSU glycoprotien hormone alpha-subunit
SF1 steroidogenic factor 1
TK thymidine kinase.
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Figures and Tables
Fig. 1.
Daily stress disrupts estrous cy clicity in the mouse. A, Representative profiles depicting estrous cy clicity , as
measured by vaginal cy tology , during the prestress period (d 1–d 18) and subsequent daily restraint stress
period (d 19–d 36) in three female mice subjected to 180 min of daily restraint stress. E, Estrus; P, proestrus; D,
diestrus. Shading indicates period of exposure to daily restraint stress. B, Average estrous cy cle length in the
no stress group and stress group (n = 9/group) during the two periods of assessment, d 1–d 18 and d 19–d 36. C,
Time spent in each stage of the cy cle in stress mice during the prestress period, d 1–d 18, and stress period, d
19–d 36. *, Significant (P < 0.05) group (no stress vs. stress) × time (d 1–d 18 vs. d 19–d 36) interaction.
Fig. 2.
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Acute stress disrupts pituitary responsiveness to GnRH. A, Schematic depicting events during the 180-min
observation period in which animals were maintained in no stress conditions (white bar) or subjected to
restraint stress (gray bar) for measurement of circulating corticosterone and GnRH-induced LH. Time of
euthanasia and blood collection (Blood coll'n) are indicated: 0, 30, and 180 min. At 17 0 min, no stress and
stressed animals (group) are div ided into two treatments (n = 7 /group per treatment) receiv ing either GnRH
(200 ng/kg, sc) or vehicle (Veh). B, Serum corticosterone (ng/ml) was measured in no stress (white circles) and
stress animals (gray circles). *, Significant (P < 0.05) effect of stress. C, Serum LH (ng/ml) was measured in no
stress (white bars) and stressed (gray bars) animals that received vehicle or GnRH, respectively , 10 min before
euthanasia. *, Significant (P < 0.05) effect of GnRH; #, difference between no stress and stress. D, Schematic
depicting events during 180-min observation period in which animals are maintained in no stress conditions
(white bar) or subjected to restraint stress (gray bar) for measurement of GnRH-induced LHβ mRNA. No stress
and stressed animals are div ided into two groups (n = 7 /group) receiv ing either GnRH (200 ng/kg, sc) or
vehicle (Veh) at 0 min of observation. Time of euthanasia and blood collection (Blood coll'n) occurred at 180
min. E, Quantitative RT-PCR analy sis of LHβ mRNA was performed on indiv idual mouse pituitary glands, and
the amount of LHβ mRNA was compared with the amount of GAPDH mRNA and expressed as relative transcript
level. *, Significant (P < 0.05) effect of GnRH; #, difference between no stress and stress. grp, Group; trt,
treatment.
Fig. 3.
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Glucocorticoids inhibit LH and GR are expressed in mouse gonadotrope cells. A, Schematic depicting
experimental events in which animals were administered corticosterone (200 ng/kg, sc; gray bar) or vehicle
(white bar), 90 min before euthanasia and blood collection (Blood coll'n). B and C, Serum corticosterone
(ng/ml, panel B) or LH (ng/ml, panel C) measured in animals administered vehicle (white bars) or
corticosterone (gray bars). *, Significant (P < 0.05) effect of treatment. D–F, Photomicrographs of
representative mouse pituitary sections subjected to two-color immunofluorescence staining using a
fluorescein isothiocy anate-conjugated anti-GR (green fluorescent signal), followed by rhodamine-conjugated
anti-LHβ (red fluorescent signal). Red (panel D), green (panel E), and merge (panel F) immunofluorescence
images were taken of the same microscopic field using appropriate filters. White stars, GR-positive/LHβ-
positive cells. Scale bar, 20 μm. Cort, Corticosterone; trt, treatment; Veh, vehicle.
Fig. 4.
Glucocorticoid repression localizes to the LHβ proximal promoter. A, Quantitative RT-PCR analy sis of LHβ
mRNA extracted from LβT2 cells cultured in the presence of GnRH (10 nM, 6 h), corticosterone (Cort; 100 nM,
24 h), GnRH + corticosterone [Cort (100 nM, entire 24 h); GnRH (10 nM, final 6 h)], or vehicle (Veh; 0.1%
BSA/0.1% ethanol). Results are expressed as LHβ mRNA levels normalized to GAPDH mRNA levels and are the
mean of three separate experiments performed in triplicate. Results shown are average ± SEM relative to the
vehicle treatment. *, Significant (P < 0.05) effect of GnRH; #, significant repression by corticosterone. B, The
−1800-bp rat LHβ-luc reporter gene was transfected into LβT2 cells, and cells were subsequently treated with
corticosterone (Cort; 100 nM, 24 h), dexamethasone (Dex; 100 nM, 24 h), or vehicle alone (Veh; 0.1% BSA/0.1%
ethanol) or either glucocorticoid (100 nM, entire 24 h) in combination with GnRH (10 nM, final 6 h), and
harvested for luciferase as a measure of LHβ promoter activ ity . Results are depicted as fold induction by
hormone treatment relative to vehicle (dashed line) as indicated. *, Significant induction by GnRH vs. vehicle
control; #, significant repression by glucocorticoid treatment on GnRH-induced LHβ expression. C, LβT2 cells
were transfected with a series of 5′-truncated LHβ-luc reporter plasmids and treated with GnRH (10 nM, final 6
h) or GnRH + dexamethasone [Dex (100 nM, entire 24 h); GnRH (10 nM, final 6 h)] to determine regions of the
LHβ promoter that are responsive to glucocorticoids. Results for each truncation are depicted as LHβ fold
induction relative to vehicle of that truncation (dashed line). #, Significant repression by glucocorticoid
treatment on GnRH-induced LHβ expression.
Fig. 5.
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GnRH-responsive factor necessary for GR recruitment. A, Schematic of the proximal 150 bp of the rat LHβ 5′-
regulatory region illustrating the known promoter elements involved in expression of the LHβ gene. Proteins
binding each site are indicated. B, Quantitative RT-PCR analy sis of Egr1 mRNA extracted from LβT2 cells
cultured in the presence of dexamethasone (Dex; 100 nM, 18 h), GnRH (10 nM, 45 min), GnRH + dexamethasone
[Dex (100 nM, entire 18 h); GnRH (10 nM, final 45 min)], or vehicle (Veh; 0.1% BSA/0.1% ethanol). Results are
expressed as Egr1 mRNA levels normalized to GAPDH mRNA levels and are the mean of three separate
experiments performed in triplicate. Results shown are average ± SEM. *, Significant induction by GnRH vs.
vehicle control. C, Western blotting analy sis of nuclear extracts from LβT2 cells treated with dexamethasone
(Dex; 100 nM, 18 h), GnRH (10 nM, 2 h), GnRH + dexamethasone [Dex (100 nM, entire 18 h); GnRH (10 nM, final 2
h)], or vehicle (Veh; 0.1% BSA/0.1% ethanol) was performed using an antibody for Egr1 . A protein band was
detected at the expected size of 82 kDa for Egr1 . The experiment was repeated three times with similar results,
and a representative gel is shown. D, ChIP was performed using cross-linked chromatin from LβT2 cells treated
with dexamethasone (Dex; 100 nM, 1 h), GnRH (10 nM, 1 h), GnRH + dexamethasone [Dex (100 nM, 1 h); GnRH
(10 nM, 1 h)], or vehicle (Veh; 0.1% BSA/0.1% ethanol) and antibodies directed against GR or nonspecific IgG as
a negative control. ChIP and input DNA were analy zed by quantitative PCR using primers encompassing the
proximal promoter of Lhb to determine the amount of immunoprecipitated DNA. ChIP samples were
normalized to the respective input samples and then expressed as fold enrichment relative to the nonspecific
IgG ChIP samples.
Fig. 6.
Glucocorticoids interfere with Egr1 actions at the level of the proximal promoter. A, To test whether
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glucocorticoids can interfere with Egr1-induced LHβ expression, Egr1 (black bars) or empty vector (Empty
Vec, open bars) was transfected with the −1800-bp LHβ-luc reporter plasmid into LβT2 cells and subsequently
treated with dexamethasone (Dex; 100 nM, 24 h) or vehicle (Veh; 0.1% BSA/0.1% ethanol). Data are shown
relative to vehicle-treated in the absence of Egr1 . *, Significant induction by Egr1 vs. vector control; #,
significant repression by glucocorticoid treatment on Egr1-induced LHβ expression. B, To determine whether
titrating in increasing levels of Egr1 can rescue glucocorticoid repression of −1800-bp LHβ-luc activ ity , LβT2
cells were transfected with empty vector (EV, 200 ng) or increasing amounts of Egr1 [50 ng (plus 150 ng empty
vector), 100 ng (plus 100 ng empty vector), 200 ng] and treated with GnRH (white hatched bars, 10 nM, final 6
h) or GnRH + dexamethasone [gray hatched bars, Dex (100 nM, entire 24 h); GnRH (10 nM, final 6 h)]. Results
are depicted as LHβ-fold induction relative to the vehicle-treated condition in the presence of the same amount
of Egr1 (dashed line) as indicated. *, Significant induction by GnRH; #, significant repression by glucocorticoid
treatment on GnRH-induced LHβ expression. C, The effect of increasing Egr1 on the ratio of LHβ-fold induction
in cells treated with GnRH alone vs. GnRH with dexamethasone was calculated and expressed as percent
repression. *, Significant difference in the ratio as compared with EV determined by Student's t test. D, LβT2
cells were transfected with either the −200-bp LHβ-luc reporter plasmid (WT) or reporter plasmids containing
the −200-bp LHβ region with mutations in SF1 or Egr1 binding elements as indicated and cultured in the
presence of GnRH (white hatched bars) or GnRH + Dex (gray hatched bars). Data are shown for each mutant
promoter, relative to its own vehicle treatment. See Fig. 6B legend for more details. n.s., Not significant P =
0.08; WT, wild ty pe.
Fig. 7.
Interruption of LHβ transcriptional complex formation by glucocorticoids. A, To investigate glucocorticoid-
mediated interference of Egr1 , SF1, and/or Ptx1 induction of LHβ promoter activ ity , CV-1 cells were transfected
with a GR expression vector and the −1800-bp LHβ-luc reporter plasmid, along with Egr1 , SF1, or Ptx1 alone or
in combination, as indicated, and treated with dexamethasone (gray bars, Dex; 100 nM, 24 h) or vehicle (white
bars, Veh; 0.1% BSA/0.1% ethanol). *, Significant induction of LHβ promoter activ ity vs. vector control
(dashed line); #, significant repression by glucocorticoid treatment. B and C, Induction of the Egr1 multimer (B)
or SF1 multimer (C) by the indicated Egr1 or SF1 alone, respectively , or in combination with Ptx1 , was assessed
after treatment with dexamethasone (gray bars, Dex; 100 nM, 24 h) or vehicle (white bars, Veh; 0.1% BSA/0.1%
ethanol) and depicted as fold induction relative to induction of the control luciferase reporter driven by the TK
promoter. *, Significant induction of multimer activ ity vs. vector control (dashed line); #, significant repression
by glucocorticoid treatment.
Fig. 8.
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GR does not require dimerization or DNA binding, but is capable of phy sically interacting with Egr1 . A, The
necessity of dimerization or DNA binding by GR, or by GR itself, was assessed in CV-1 cells. The −1800-bp LHβ-
luc reporter plasmid was maximally induced with a combination of Egr1 , SF1, and Ptx1 in CV-1 cells
cotransfected with empty vector (No GR), wild-ty pe GR (WT GR), GR dimerization mutant (Dim mut), or GR DBD
mutant (DBD mut) and subsequently treated with dexamethasone (gray bars, Dex; 100 nM, 24 h) or vehicle
(open bars, Veh; 0.1% BSA/0.1% ethanol). Data are presented relative to the −1800-bp LHβ-luc reporter
cotransfected with the empty vectors for Egr1 , SF1, and Ptx1 for each GR expression vector. #, Significant
repression by glucocorticoid treatment. B, GST interaction assay s were performed using bacterially expressed
Egr1-GST fusion protein and S-labeled in vitro translated GR, SF1, or GFP as a control. One tenth of the
protein input (10% Input) and the GST tag-alone (GST, negative control) are shown (note that the SF1 complex
with GST-Egr1 has spilled over into the intermediate empty lane between the GST and the GST-Egr1 lanes).
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