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Animal Reproduction and Biotechnology Laboratory, Department of Physiology, Colorado State University College of Veterinary Medicine and Biomedical Sciences, Fort Collins, Colorado 80523
Address all correspondence and requests for reprints to: Dr. Colin M. Clay, Animal Reproduction and Biotechnology Laboratory, Foothills Campus, Colorado State University, Fort Collins, Colorado 80523. E-mail: cclay{at}vines.colostate.edu
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Abstract |
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Introduction |
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Although a number of groups have demonstrated changes in the number of GnRH receptors associated with different physiological states or hormonal treatments (1), particular attention has been paid to endocrine mechanisms underlying the increase in GnRH receptor number during the preovulatory period (4, 8). There appears to be general agreement that both 17ß-estradiol and GnRH itself are particularly important in mediating this increase (1, 2, 7, 9), although a role for inhibin has been suggested (10). Since the initial isolation of the complementary DNA encoding the murine GnRH receptor (11), hormonal regulation of GnRH receptor numbers has been correlated with concomitant fluxes in steady state concentrations of messenger RNA (mRNA) for the GnRH receptor (5, 6, 8, 12, 13, 14). Thus, it seems likely that regulation of expression of the GnRH receptor gene by both hypothalamic and gonadal inputs is an important determinant of the concentration of GnRH receptors in the anterior pituitary gland.
To study the molecular mechanisms underlying the regulation of GnRH
receptor gene expression, we isolated the promoter for the gene
encoding the murine GnRH receptor and, consistent with others (15),
found that 1900 bp of the proximal 5'-flanking region are sufficient to
direct expression of luciferase to the gonadotrope tumor-derived
T3–1 (16), but not to nongonadotrope cell lines (17). Since then,
we have made progress toward identifying cis-acting elements
necessary for the activity of the GnRH receptor gene promoter in the
T3–1 cell line (18). For several reasons, however, we believed that
it was important to expand our in vitro analyses to
transgenic mice. First, there are clear examples of fundamental
discrepancies between conclusions derived from in vitro vs.
in vivo analyses of promoter function (19, 20). Second,
although T3–1 cells are useful as a model for cell-specific
expression, they do not recapitulate either the native phenotype of
gonadotropes (16) or the normal patterns of endocrine responsiveness of
gonadotropes (15, 21, 22). Thus, analyses of hormonal regulation of
GnRH receptor gene expression in this cell line are problematic.
In light of these considerations, we sought to test the physiological
relevance of GnRH receptor promoter activity in T3–1 cells through
the construction and analysis of transgenic mice. Herein, we report
that approximately 1900 bp of proximal 5'-flanking region are
sufficient to direct expression of a heterologous reporter gene to the
pituitary gland of transgenic mice, thus providing the first in
vivo validation of T3–1 cells as a model for
pituitary-specific expression of the GnRH receptor gene. Furthermore,
diminution of transgene expression after the administration of an
anti-GnRH serum and abrogation of this effect by replacement with GnRH
agonist suggest that 1900 bp of the proximal 5'-flanking region contain
one or more enhancer elements that mediate transcriptional
responsiveness of the GnRH receptor gene to GnRH.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Enzymes and plasmids
Restriction and modifying enzymes were purchased from New
England Biolabs (Beverly, MA) and were used according to the
supplier’s specifications. The vector pGL3-Basic was purchased from
Promega (Madison, WI). Isolation of the murine GnRH receptor gene
promoter and construction of the plasmid pMGR-1900Luc have been
described (17).
Animals
All mice used in these experiments were DBA/C57 hybrid
intercrosses propagated in our laboratory under 12 h of light/day.
The positive transgenic offspring used in these studies were no more
than two generations from the founder animal and harbored the transgene
at a single integration site. All experiments were performed under
veterinary supervision with approval from the Colorado State University
Animal Care and Use Committee and in accordance with NIH Animal Care
and Use guidelines.
Generation and screening of transgenic mice
A fusion gene consisting of approximately 1900 bp of the
proximal 5'-flanking region from the murine GnRH receptor gene
subcloned upstream of the complementary DNA (cDNA) encoding firefly
luciferase (Fig. 1
) was released from pMGR-1900Luc by
digestion with SacI and BamHI and purified by
electroelution. After extraction with phenol-chloroform-isoamyl alcohol
(24:24:1), the DNA was precipitated by the addition of 0.1 vol 3
M sodium acetate and 2 vol ice-cold ethanol. The
precipitated DNA was resuspended in 10 mM Tris-HCl (pH 7.5)
and 0.2 mM EDTA to a final concentration of 5 ng/µl. The
linearized fusion gene was microinjected with continuous positive flow
into pronuclei of fertilized mouse oocytes. Injected embryos were
reimplanted into oviducts of recipient females and allowed to develop
to term (27). Genomic DNA was extracted from tail biopsies and analyzed
for the presence of the transgene by slot blot hybridization (28).
Briefly, approximately 10 µg tail DNA were denatured in 0.3
N NaOH at 95 C for 5 min, blotted by vacuum aspiration onto
nylon membranes in 1.0 M ammonium acetate (pH 7.0), and
hybridized with random primed, radiolabeled luciferase cDNA.
Nonspecific binding was removed by sequential 10-min washes in 2
x SSC (1 x SSC = 150 mM NaCl and 15
mM sodium citrate)-0.1% SDS at 42 C, 1 x SSC-0.1%
SDS at 65 C, and 0.1 x SSC-0.1% SDS at 65 C. Autoradiography was
performed with Kodak X-Omat film (Eastman Kodak, Rochester, NY) and an
intensifying screen at -80 C for 24–72 h.
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Luciferase assays
Tissue extracts were prepared by homogenization in 200 µl cold
lysis buffer [25 mM glycylglycine (pH 7.8), 1.0% Triton
X-100, 10 mM MgSO4, 1.0 mM
dithiothreitol, and 0.5 mM phenylmethylsulfonylfluoride]
(17). Cellular debris was pelleted by microcentrifugation at
16,000 x g for 10 min at 4 C. Cellular lysates were
immediately assayed for luciferase activity by the addition of 20 µl
lysate to 100 µl luciferin substrate (Promega, Madison, WI).
Luminescence was measured using a Turner model TD-20E luminometer
(Turner Designs, Sunnyvale, CA) (17). Total protein was precipitated
from 50 µl lysate with 10% trichloroacetic acid and then dissolved
in 0.1 N NaOH. Protein concentrations were determined using
bicinchoninic acid (Pierce Chemical Co., Rockford, IL). Luciferase
activity was adjusted for protein content by dividing the arbitrary
light units by the protein concentration.
Animal treatments
Exp 1: determination of GnRH antiserum (AS) dose.
To
determine an appropriate dose of AS, sexually mature, nontransgenic
mice were ovariectomized (ovx) under general anesthesia using a
combination of pentobarbital and metofane. Seven days after
ovariectomy, animals were divided into four groups for treatment with
increasing doses of antiserum (24). Doses included 0 µl (n = 4),
50 µl (n = 3), 200 µl (n = 4), and 500 µl (n = 3),
and each was administered as a single ip injection. All injections were
adjusted to a final volume of 500 µl with the appropriate quantity of
normal sheep serum (NSS). Tail blood was collected 24 h after
injection, and 7 days later, animals were killed for collection of
trunk blood. The concentrations of LH in serum were determined by RIA
(19).
Exp 2: GnRH regulation of transgene expression.
Female
transgenic mice were ovx at 2–3 months of age and divided into 4
groups of 8 animals each. One week postovariectomy, a single ip
injection of 300 µl AS or NSS was administered to 16 animals, whereas
8 animals were not treated and served as ovx controls. The remaining 8
animals were administered 200-µl sc injections of the GnRH
antagonist, antide (300 ng/ml in 20% propylene glycol and normal
saline) (19), starting 1 week postovariectomy and continuing every
other day thereafter. Two weeks postovariectomy, animals were killed,
and pituitary, brain, and liver were harvested for assay of luciferase
activity. Trunk blood samples were collected for assay of serum LH
concentrations (19).
Exp 3: estradiol regulation of transgene expression.
Female
transgenic mice were ovx at 2–3 months of age and divided into 3
groups of 8 animals each. One week postovariectomy, 16 animals received
a single ip injection of 300 µl AS, whereas 8 animals remained
untreated to serve as ovx controls. Thirty minutes after antiserum
injection, 8 of the AS-treated animals received sc implants of 2.5-mg
17ß-estradiol pellets (23). Two weeks after ovariectomy, animals were
killed, and pituitary, brain, and liver were harvested for assay of
luciferase activity. Trunk blood samples were collected for assay of
serum LH concentrations (19).
Exp 4: GnRH and testosterone regulation of transgene
expression.
To determine whether the effects of AS on transgene
expression were sex dependent and to investigate the potential for
androgen regulation of transgene expression in the testes, adult
transgenic males were treated with a single 300-µl ip injection of AS
alone (n = 7) or in combination with sc implanted 5-mg
testosterone pellets (23) (n = 7). Nontreated adult transgenic
male mice served as controls (n = 8). One week after treatment,
animals were killed, and pituitary, brain, testes, and liver were
harvested for assay of luciferase activity. Trunk blood samples were
collected for assay of serum LH concentrations (19).
Exp 5: GnRH replacement in immunoneutralized animals.
To
determine whether diminution of pituitary transgene expression detected
in AS-treated animals could be prevented by simultaneous treatment with
a noncross-reacting GnRH agonist, 19 male transgenic mice were treated
with a single 300-µl ip injection of AS. Nine of these animals served
as controls; the remaining 10 mice also received a 100-µl ip
injection containing 1 ng of the GnRH agonist, D-Ala, in
0.15 M NaCl every 2 h for 48–52 h (19). Two days
after AS treatment and 15 min after the final D-Ala
injection, animals were killed, and pituitary, brain, testes, and liver
were harvested for assay of luciferase activity. Trunk blood samples
were collected for assay of serum LH concentrations (19).
Data analysis
Any tissue that expressed luciferase above the mean plus 2
SDs of the mean of luciferase values in nontransgenic
tissues was considered positive for expression of the transgene. Only
animals that were positive for luciferase expression in the pituitary
and brain were used for further analysis in the hormone regulation
studies (19). The serum LH concentration and luciferase activity were
log transformed to normalize the data, then analyzed by one-way ANOVA.
Differences among treatments were determined by using the least
significant difference test (SAS statistical package, SAS Institute,
Cary, NC).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
Specifically, XbaI digestion resulted in a 2.3-kilobase (kb)
fragment, ScaI digestion yielded two fragments of 0.8 and
3.1 kb in length, and HindIII digestion excised a 2.4-kb
fragment from transgene concatamers. These results indicated a single
integration site with no gross structural rearrangements of the
transgene.
Tissue specificity of transgene expression
To investigate tissue expression of the transgene, cellular
lysates were prepared from brain, heart, kidney, liver, lung, ovary,
pancreas, pituitary, spleen, and testis of six transgenic and three
nontransgenic mice at 30–35 days of age and assayed for luciferase
activity. Luciferase activity was not detected in ovary, liver, spleen,
pancreas, kidney, heart, or lung of either transgenic or nontransgenic
mice (Table 1). In contrast, luciferase activity was
detected in pituitary gland, brain, and testes of transgenic mice, but
was absent in nontransgenic tissues. Pituitary glands exhibited the
highest mean concentrations, which ranged from 42–2179 arbitrary light
units/mg protein. Thus, three tissues tested positive for expression of
the transgene; however, luciferase activity in the pituitary gland was
approximately 10- and 100-fold greater than that in the brain or
testes, respectively.
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). Higher doses of AS did not further suppress serum LH
concentrations at 24 h. In contrast, at least 200 µl AS were
required to maintain suppressed serum LH levels for 8 days after
injection (1.3 ± 0.1 ng/ml) compared with ovx controls (12.9
± 2.3 ng/ml; P < 0.01). In subsequent studies with
transgenic mice we chose to administer a 300-µl dose of AS to ensure
immunoneutralization for at least 7 days.
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). Although not significant at the 0.05 level, there
was a trend (P = 0.13) for suppression of pituitary
luciferase activity in animals receiving antide. As expected, serum LH
concentrations were lowest (P < 0.01) in AS- and
antide-treated mice. The expression of luciferase in brain was not
affected by any of the treatments (P = 0.7), and
luciferase activity was not detected in the liver samples of any
mice.
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). Consistent with
immunoneutralization of GnRH, serum LH concentrations were lower
(P < 0.01) in mice receiving AS than in ovx controls.
No effect of 17ß-estradiol treatment on pituitary luciferase
expression was detected (P = 0.8). Consistent with our
initial tissue surveys, luciferase activity was not detected in liver
samples of any of the mice. There was no effect (P =
0.25) of any treatment on brain expression of luciferase, which, as in
the original tissue survey, was approximately 10-fold lower than
luciferase activity in the pituitary gland (data not shown).
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). Neither brain nor testicular expression of
luciferase was affected (P = 0.65) by treatment with AS
or T. Luciferase activity was not detected in any liver sample.
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). Neither
brain nor testicular expression of luciferase was affected by AS or
treatment with D-Ala. Luciferase activity above background
was not detected in any liver sample.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T3–1 cell line (17). Furthermore, we have
suggested that T3–1 specific activity of the murine GnRH receptor
gene promoter is partially due to two elements located within 500 bp of
the proximal 5' flanking region. One of these elements is capable of
binding the nuclear orphan receptor steroidogenic factor-1, whereas the
protein(s) that binds the second element has not been identified. Thus,
we have suggested that a complex enhancer that includes a binding site
for steroidogenic factor-1 mediates gonadotrope-specific expression of
the GnRH receptor gene (18). Formulation of this model, however, is
ultimately dependent on the validity of the T3–1 cell line as a
model for tissue/cell-specific expression. At issue, then, is whether
the proximal promoter of the murine GnRH receptor gene is sufficient to
direct tissue-specific expression in vivo. In the present
study we found expression of a GnRH receptor fusion gene containing
1900 bp of the 5'-flanking region predominantly in the pituitary gland
of transgenic mice, and this is consistent with pituitary expression of
the endogenous GnRH receptor gene. Clearly, a primary role for GnRH receptors is to mediate pituitary responsiveness to GnRH. However, the presence of these receptors in a number of extrapituitary sites suggests a broader physiological role for GnRH. In particular, GnRH receptors have been detected in the central nervous system, gonad, and placenta of multiple species (1). Thus, expression of the GnRH receptor fusion gene in brain and testes of transgenic mice was not unexpected. In regard to the latter, GnRH receptors have been localized to Leydig cells in adult testis and may mediate the paracrine effects of GnRH on steroid hormone production (1, 29). Although ovarian expression of GnRH receptors has also been demonstrated (1, 29, 30), we were not able to detect luciferase activity significantly above background in the ovary. Although these data were based on ovaries obtained from sexually immature mice, we have similarly been unable to detect luciferase activity in the ovaries of adult transgenic mice (data not shown).
In addition to its endocrine and potential paracrine roles, GnRH has long been implicated as a potential neurotransmitter or neuromodulatory peptide within the central nervous system (31, 32). Consistent with this is the presence of GnRH receptors in multiple regions of the brain (31, 32). Thus, expression of the GnRH receptor fusion gene in the brain, like the pituitary gland and testis, appears to reflect a normal site of expression of the endogenous GnRH receptor gene. If correct, the promoter fragment used in the present study may represent a useful tool for targeting gene expression to specific sets of GnRH-responsive brain neurons.
In regard to hormonal regulation of the transgene, we find that
treatment with a GnRH antagonist or AS leads to a reduction in
pituitary expression of the transgene. Additionally, replacement of
antiserum-treated animals with a noncross-reacting GnRH analog prevents
the decline in pituitary transgene expression seen in AS-treated
animals. Thus, we suggest that homologous regulation of GnRH receptor
gene expression in the pituitary gland is partially mediated by one or
more cis-acting elements residing within 1900-bp proximal
5'-flanking region in the murine GnRH receptor gene. Furthermore, as
gonadotropes are the only cell type in the pituitary gland known to
harbor GnRH receptors, these data provide indirect evidence for
gonadotrope-specific expression of the transgene. Interestingly, we
have not been able to detect any regulation of this same 1900-bp
promoter fragment by GnRH in T3–1 cells (unpublished data).
Similarly, Albarracin et al. (15) were unable to demonstrate
GnRH regulation of a slightly shorter (1200-bp) promoter region in
T3–1 cells. Thus, the mechanisms underlying the transcriptional
response of the GnRH receptor gene may be nonfunctional in the T3–1
cell line. It is important to point out, however, that homologous
up-regulation of GnRH receptors in T3–1 cells has been reported to
occur independent of any change in the level of GnRH receptor mRNA (33, 34). Therefore, it is quite possible that GnRH regulation of GnRH
receptor numbers in the pituitary gland may occur through both
transcriptional and posttranscriptional mechanisms. Finally, we were
not able to detect any effect of GnRH immunoneutralization on
testicular or brain expression of the transgene. To conclude, however,
that the transgene is not responsive to GnRH in these tissues assumes
an endocrine mechanism for GnRH regulation at these sites. That is, if
homologous regulation of GnRH receptors within the brain or testis is
occurring via autocrine or paracrine mechanisms, then peripheral
immunoneutralization of GnRH may have little or no effect (29, 35).
A stimulatory action of 17ß-estradiol to increase GnRH receptor mRNA and numbers in the pituitary gland has been well established in many species (6, 8, 14). However, in the present study we were unable to demonstrate estradiol regulation of the GnRH receptor transgene. Perhaps the most likely explanation is simply that 1900 bp of proximal promoter lacks the necessary regulatory elements for estrogen responsiveness. Consistent with this idea is the absence of a canonical estrogen response element within the proximal promoter of the murine GnRH receptor gene (15, 17). Furthermore, we have been unable to detect high affinity binding of estrogen receptor within this region of the murine GnRH receptor gene promoter (unpublished data). It is, of course, possible that estrogen regulation of the GnRH receptor gene is mediated by a noncanonical response element(s) or is occurring through an indirect mechanism that does not require binding of estrogen receptor to the GnRH receptor gene itself. Unfortunately, our ability to differentiate among these potential mechanisms awaits identification of an estrogen-responsive promoter region of the GnRH receptor gene and definition of an appropriate model system to assess physiological relevance. In regard to the latter, the present data suggest that transgenic mice may represent such a model. Finally, for two reasons it is unlikely that the absence of an estrogen response was due to an inadequate dose of 17ß-estradiol. First, in a previous study pellets containing 10-fold less estrogen resulted in serum 17ß-estradiol levels of 300 pg/ml (23). Second, a physiological consequence of estrogen treatment was evident, in that the average uterine weight of estrogen-treated mice (126 ± 0.2 mg) was significantly higher than that of ovx nonestrogen-treated mice (27 ± 0.1 mg).
In summary, approximately 1900 bp of proximal promoter from the murine GnRH receptor gene are sufficient to direct expression of luciferase to the pituitary gland, brain, and testes of transgenic mice. This tissue-specific pattern of luciferase expression is in accordance with established sites of expression of the endogenous GnRH receptor gene. Thus, this line of mouse represents an essential first step in defining the molecular requirements for tissue-specific expression of the GnRH receptor gene in vivo. Furthermore, attenuation of luciferase expression after GnRH immunoneutralization suggests that one or more elements capable of conferring GnRH responsiveness are located within 1900 bp of the proximal 5'-flanking region of the murine GnRH receptor gene and confirms the utility of a transgenic mouse model for studying the molecular mechanisms underlying hormonal regulation of GnRH receptor gene expression.
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Acknowledgments |
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Received January 13, 1997.
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-subunit genes is regulated by distinct
cis-acting elements. Mol Endocrinol 6:1745–1755T3–1 cells. Mol
Cell Endocrinol 87:95–103[CrossRef][Medline]
-subunit gene
despite the absence of a high affinity binding site for estrogen
receptor. Mol Endocrinol 5:725–733T3-1 cells. Endocrinology 136:1128–1136[Abstract]
T3-1 cells is associated with unchanged receptor
messenger RNA (mRNA) levels and altered mRNA activity. Mol Endocrinol 7:1625–1633This article has been cited by other articles:
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D. L. Duval, A. R. Farris, C. C. Quirk, T. M. Nett, D. L. Hamernik, and C. M. Clay Responsiveness of the Ovine Gonadotropin-Releasing Hormone Receptor Gene to Estradiol and Gonadotropin-Releasing Hormone Is Not Detectable in Vitro But Is Revealed in Transgenic Mice Endocrinology, March 1, 2000; 141(3): 1001 - 1010. [Abstract] [Full Text] [PDF]
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D. L. Duval, S. E. Nelson, and C. M. Clay The Tripartite Basal Enhancer of the Gonadotropin-Releasing Hormone (GnRH) Receptor Gene Promoter Regulates Cell-Specific Expression Through a Novel GnRH Receptor Activating Sequence Mol. Endocrinol., November 1, 1997; 11(12): 1814 - 1821. [Abstract] [Full Text]
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0.02) from the corresponding control value.
B, Time scale for experimental protocol.
