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-Knockout Mice
Women’s Health Research Institute, Wyeth-Ayerst Research, Radnor, Pennsylvania 19087
Address all correspondence and requests for reprints to: Dr. Paul J. Shughrue, Women’s Health Research Institute, Wyeth-Ayerst Research, 145 King of Prussia Road, Radnor, Pennsylvania 19087. E-mail: shughrp{at}war.wyeth.com
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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knockout (ERKO)
mice. The present study used in vivo autoradiography to
evaluate the binding of 125I-estrogen, a compound with a
similar affinity for both ERs to ascertain whether ERß mRNA is
translated into biologically active receptor. Mice were injected with
125I-estrogen, and sections were mounted on slides and
opposed to emulsion. After exposure, labeled cells were seen in ERKO
brain regions where ERß is expressed (preoptic and paraventricular
nuclei of the hypothalamus; bed nucleus of the stria terminalis;
amygdala; entorhinal cortex; and dorsal raphe). Competition studies
with 17ß-estradiol eliminated binding in the ERKO brain, whereas
16IE2, an ER selective agonist and
dihydrotestosterone had no effect. In contrast, competition studies
with 16IE2 in wild-type mice eliminated
125I-estrogen binding to ER and resulted in a pattern of
residual binding comparable to that seen in the ERKO brain. The
results demonstrate that residual estrogen binding sites are present in
regions of the ERKO brain where ERß is expressed, brain regions
that were also seen after eliminating binding to ER in wild-type
mice. These data provide the first evidence that ERß mRNA is
translated into a biologically active protein in the rodent brain. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), it was
thought that these actions were mediated through a putative membrane
receptor, activation of second messenger systems, or through
interneuronal connections (4, 7, 8).
The recent isolation of a second nuclear estrogen receptor (ERß; 9,
10) and localization in brain (11) has provided new sites for estrogen
action. In situ hybridization histochemical studies have
detected ERß messenger RNA (mRNA) in some rat brain regions where
ER was seen, but also in regions, such as the olfactory bulb,
cortex, paraventricular, and supraoptic hypothalamic nuclei where ER
was sparse or absent (11). This observation suggested that many of the
actions of estrogen in regions previously thought to lack ER may be
attributed to ERß.
In an attempt to further understand the importance of ERß in
the rodent brain, we must first ascertain whether ERß mRNA is
translated into biologically active protein in vivo. The
present studies used in vivo steroid autoradiography to
evaluate the presence and distribution of estrogen binding sites in the
wild-type and estrogen receptor- knockout (ERKO) mouse brain. A
new radiolabeled ligand (125I-estrogen; 12), that binds
equally to ER and ERß, was used to evaluate the distribution of
estrogen binding sites in the ERKO brain and compare these sites
with the known distribution of ERß mRNA (13). In addition, an ER
selective agonist was used as a competitor in these studies to
demonstrate that the residual binding seen in the ERKO brain was due
to ERß. Together, the results of these studies provide the first
evidence that a biologically active ERß protein is present in the
brain of wild-type and ERKO mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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or ERß modified with a
carboxyl-terminal flag-epitope were expressed in Escherichia
coli BL21(DE3) by IPTG induction at 25 C for 2 h. A crude
lysate was prepared in 50 mM Tris-Cl, 150 mM
NaCl (pH 7.4) using a French press, and insoluble material was removed
by centrifugation. The binding reactions were performed in EG&G Wallac
(Gaithersburg, MD) high binding 96-well plates containing 2
nM [3H] 17ß-estradiol (New England Nuclear, Boston, MA), unlabeled compound and 1 µg crude
lysate in Dulbecco’s PBS supplemented with 1 mM EDTA.
After incubation at room temperature for 5 h, unbound material was
removed by rinsing, and bound DPMs were determined by liquid
scintillation counting.
Animals
C57BL/6J x 129 ERKO mice (14) were genotyped using PCR
analysis of tail samples to assess the presence of the neomycin
resistance and/or ER mRNAs (15). Pups exhibiting only the neomycin
resistance mRNA were considered homozygous mice, whereas animals
that express ER were wild types. The ERKO and wild-type mice used
in these studies were 28-day-old postnates. This age was selected
because it allowed us to take advantage of the animals low body weight,
using less radiolabeled ligand, while still obtaining information about
the developed brain. The studies described in this paper were reviewed
and approved by the Wyeth-Ayerst Research, Animal Care and
Use Committee.
In vivo binding studies
On postnatal day 21, ERKO and wild-type mice were
ovariectomized and weaned from their mothers. Seven days after surgery,
the ovariectomized mice were sc injected in the dorsal cervical region
with 100 µl of vehicle (50% DMSO, 40% PBS, and 10% ethanol; n
= 5/group) or 250 µg/kg BW of 17ß-estradiol (n = 2/group),
16-iodo-17ß-estradiol (16IE2; a gift from Dr. R.
Hochberg, Yale University, New Haven, CT, see 16; n = 3/group),
the androgen dihydrotestosterone (Sigma Chemical Co.;
n = 1/group) or a synthetic progestin R5020 (NEN; n =
1/group). Thirty minutes after the initial injection, mice where again
injected with the same compound as well as 2 µg/kg BW of
17-iodovinyl-11ß-methoxyestradiol (125I-estrogen;
specific activity 2200 Ci/mM). The
125I-estrogen was obtain from the iodination (New England Nuclear custom iodination) of
E-17-tributylstannyvinyl-11ß-methoxy estradiol (RAXL Enterprises,
Inc., Newton, MA) as described previously (12). One hour after
injection of 125I-estrogen, the brains were collected,
frozen and 16 µm coronal cryostat sections thaw-mounted onto
gelatin-coated slides. Section-mounted slides were apposed to x-ray
film (Eastman Kodak, Rochester, NY; BMR-1) for 16 h
and then processed for emulsion coating (17). Briefly, section-mounted
slides were washed for 5 min in 4 C PM buffer (3 mM
MgCl2, 1 mM KH2PO4, pH
6.8), postfixed for 5 min in ice-cold 4% paraformaldehyde, washed
3 x 5 min in 4 C PMTX buffer (PM buffer containing 0.1% Triton
X-100), washed 2 x 5 min in 4 C PM buffer, dipped in water, and
allowed to dry at room temperature. The slides were then dipped in
liquid nuclear emulsion (Kodak, NTB-2, diluted 1:1 with
water), air dried and stored at 4 C in light-tight desiccator slide
boxes. After 10–30 days of exposure, the slides were developed,
stained with cresyl violet and coverslipped. Brain autoradiograms were
scanned at low magnification with a light microscope to determine the
regional distribution of estrogen target cells in the forebrain regions
of wild-type and ERKO mice. Brain regions where ERß mRNA is
expressed were further evaluated to determine the relationship between
the distribution of ERß mRNA and the residual binding seen in the
ERKO mice as well as the effect of competition with 17ß-estradiol,
16IE2, dihydrotestosterone, or R5020.
In situ hybridization
The distribution of ERß mRNA was also evaluated in the ERKO
mouse brain with in situ hybridization histochemistry as
previously described (13). Briefly, female ERKO mice were
ovariectomized for 5–7 days, euthanized, and their brains collected
and frozen on dry ice. Twenty micron coronal cryostat sections were
mounted on gelatin-coated slides, processed for in situ
hybridization, and hybridized with 200 µl of an antisense or sense
(control) 35S-labeled riboprobe (8 x 106
DPM/probe/slide) -50% formamide hybridization mixture containing a
cocktail of two unique riboprobes for ERß mRNA (ERß 558 and ERß
600; see Ref. 13). The section-mounted slides were incubated overnight
at 55 C in a humidified chamber, treated with RNase A, and stringently
washed at 67 C in 0.1 x SSC to remove nonspecific label. Slides
were then dehydrated, apposed to x-ray film for 5 days, and dipped in
NTB2 nuclear emulsion. The slides were exposed for 2–4 weeks,
photographically processed, stained in cresyl violet, and
coverslipped.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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KO mice
was the identification of a radiolabeled ligand that had the same
binding affinity for both ERs. As shown in Fig. 1
, early competition studies with
16-iodo-17ß-estradiol (16IE2) and
[3H]17ß-estradiol on ER and ERß revealed that
16IE2 was a very selective ER agonist, with an
IC50 of 5.03 nM ± 1.33 (mean +
SEM; n = 4) on ER and 146.7 nM ± 31.6
(mean + SEM; n = 5) on ERß. In contrast, the binding
affinity of 17-iodovinyl-11ß-methoxyestradiol
(125I-estrogen), a new radiolabeled estrogen, was similar
on both ERs (Fig. 1), with an IC50 of 9.34 nM
± 0.54 (mean + SEM; n = 2) on ER and 13.3
nM ± 2.2 (mean + SEM; n = 2) on ERß. In
this assay, 17ß-estradiol also had a similar IC50 on
ER and ERß (3.9 nM and 5 nM, respectively;
data not shown).
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KO brain, the present study used
125I-estrogen because of its similar affinity for both ERs
(Fig. 1
). When 125I-estrogen was injected into wild-type
mice (Figs. 2 and 3), cells with a nuclear uptake and
retention of 125I-estrogen were seen in brain regions that
predominantly contain ER (the ventromedial and arcuate nuclei, Fig. 2E), ERß (the paraventricular nucleus, Fig. 2C; medial tuberal
nucleus; entorhinal cortex; and dorsal raphe) or similar levels of both
ERs (such as the preoptic area, Fig. 3A; bed nucleus of the stria
terminalis, Fig. 3A; and amygdala). In contrast, the distribution of
125I-estrogen binding sites seen in the ERKO brain (see
Figs. 2–5) was more restricted than in wild-type animals, although the
degree of nuclear labeling seen in the wild-type and ERKO brain was
similar. Cells with nuclear label were seen in the ERKO preoptic
area (Figs. 3E, 4B), paraventricular
nucleus of the hypothalamus (Fig. 2B), bed nucleus of the stria
terminalis (Figs. 3E and 4, B–D), medial amygdala (Fig. 4F),
entorhinal cortex (Fig. 5D), hippocampus,
and dorsal raphe (Fig. 5B). A comparison of the distribution of ERß
mRNA in the ERKO brain (Figs. 2A; 4, A and E; and 5, A and C) with
the distribution of 125I-estrogen-labeled cells (Figs. 2B;
4, B and F; and 5, B and D), revealed that binding was detected in
subregions of the brain where ERß was expressed. Moreover, there was
a positive correlation between the degree of 125I-estrogen
binding and the intensity of ERß hybridization signal, with areas
such as the bed nucleus of the stria terminalis (Fig. 4A),
paraventricular nucleus (Fig. 2A), medial amygdala (Fig. 4E), and
dorsal raphe (Fig. 5A) containing the highest levels of ERß mRNA and
125I-estrogen binding. Competition studies with
17ß-estradiol (Fig. 3, D and F), but not dihydrotestosterone (Fig. 3B) and R5020, eliminated the nuclear uptake and retention of
radiolabeled ligand seen in the ERKO (Fig. 3E) and wild-type (Fig. 3A) brain, thus demonstrating the specificity of
125I-estrogen for ER. Because 16IE2 is an
ER selective agonist (Fig. 1), this compound was used as a
competitor to further characterize the binding of
125I-estrogen in wild-type and ERKO mice. When ERKO
mice were injected with a 125-fold excess of 16IE2
before 125I-estrogen, only a slight attenuation in binding
was observed. In contrast, injection of wild-type animals with
16IE2 before 125I-estrogen eliminated ER
binding sites (Figs. 2, E vs. F; and 3, A vs. C)
and resulted in a pattern of binding that was comparable to the ERKO
brain (Figs. 2, B vs. D; and 3, C vs. E). In
particular, brain regions such as the ventromedial and arcuate nuclei
(Fig. 2E) contained little or no 125I-estrogen binding,
whereas labeling in the paraventricular nucleus (Fig. 2D) and medial
amygdala were not markedly attenuated.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ERß, to assess the distribution of residual binding sites
in the ERKO brain. Nuclear uptake and retention of
125I-estrogen was seen in regions of the ERKO mouse
brain where ERß is expressed, including the preoptic area, bed
nucleus of the stria terminalis, paraventricular nucleus, amygdala,
entorhinal cortex, and dorsal raphe (13). In wild-type animals, binding
was also abundant in these same regions as well as the arcuate and
ventromedial nuclei, areas where ER is concentrated in rat (11). In
an attempt to characterize the nature of the estrogen binding site(s)
seen in the ERKO and wild-type brain, animals were treated with an
ER selective agonist (16IE2) before the injection of
125I-estrogen. Competition with 16IE2
markedly reduced the distribution of binding sites seen in the
wild-type brain and eliminated binding in brain regions where ER is
predominantly expressed. The resulting pattern of binding seen in the
wild-type brains was similar to that seen in ERKO animals. In the
ERKO mouse brain, 16IE2 slightly attenuated the
degree of binding seen in the brain but had no effect on the topography
of labeled cells. The attenuation in labeling intensity seen in the
ERKO brains was probably due to the amount of compound administered
(250 µg/kg), a dose that was intended to eliminate ER binding but
may have also eliminated some ERß binding. Additional competition
studies showed that all binding in the ERKO and wild-type brains was
eliminated with estradiol, but not DHT or R5020, demonstrating that the
nuclear concentration of radiolabeled estrogen was specific for an
estrogen binding site.
A previous autoradiographic study using MIE2 as a ligand
and ERKO mice (18) detected only a few weakly labeled cells in the
medial preoptic nucleus, bed nucleus of the stria terminalis, and
amygdala after long exposure times (4–6 weeks). Although the pattern
of distribution was similar to the localization of ERß mRNA (13), the
degree of binding was markedly attenuated considering the similar level
of ERß expression in ERKO and wild-type mice. This discrepancy
suggested that the radiolabeled estrogen used in these studies
(MIE2) had a weak affinity for ERß or that the residual
binding seen in the ERKO brain was due to the interaction with a
splicing variant of ER (15, 18). Subsequent in vitro
binding studies (data not shown) revealed that MIE2 had a
higher affinity for ER than for ERß, suggesting that the weak
labeling seen in the ERKO brain (18) was due to the lower affinity
of MIE2 for ERß. This hypothesis is supported by the
present finding that 16IE2, an ER selective agonist,
eliminated binding in regions of the wild-type brain were ER is
concentrated (such as the ventromedial and arcuate nuclei), but was
unable to eliminate the residual binding seen in regions of the ERKO
brain where ERß is expressed. This finding clearly illustrates that
the nuclear uptake and retention of 125I-estrogen in
certain regions of the ERKO brain was due to the interaction with
ERß. However, the results of these studies cannot discount the
possibility that an active splicing variant of ER may also exist in
the ERKO brain. A recent study by Maffart et al. (19) has
detected very weak ER immunoreactivity in several hypothalamic
regions of the ERKO brain including the ventromedial, arcuate, and
preoptic nuclei and shown that the treatment of ovariectomized ERKO
mice with estrogen increase the number of progesterone receptor
immunoreactive cells in these same brain regions. Together, these
observations suggest that the presence of an active splicing variant of
ER in the ERKO brain cannot be discounted at this time. It is
also important to realize that the radiolabeled estrogen used in these
studies could specifically interact with other proteins, such as
additional undiscovered ERs. Thus, although we feel that the present
data demonstrate the presence of ERß, which is capable of binding
radiolabeled ligand, it is also possible that a portion of the binding
seen may represent other yet undiscovered ER(s).
A recent in situ hybridization histochemistry study
evaluated the anatomical distribution of ERß mRNA in the ERKO
mouse forebrain (13). The results of that study and the present results
found that ERß mRNA was abundant in the preoptic area, bed nucleus of
the stria terminalis, paraventricular nucleus, amygdala, and dorsal
raphe. Additional hybridization signal was also detected in the
suprachiasmatic nucleus, dorsomedial nucleus, medial tuberal nucleus,
and entorhinal cortex, whereas only weak signal was seen in the
olfactory bulb, septum, cerebral cortex, and Ammon’s horn of the
hippocampus. The present study observed 125I-estrogen
binding in many of the same regions. In particular, areas such as the
preoptic area, bed nucleus of the stria terminalis, paraventricular
nucleus, medial amygdala, and dorsal raphe had the highest level of
ERß mRNA and 125I-estrogen binding, whereas additional
regions where the receptor expression was low, had little or no
specific binding. Moreover, an alignment of the receptor localization
and estrogen binding sites further indicate that the number of cells,
subregional distribution, and topography are in good agreement,
suggesting that 125I-estrogen was binding to ERß in the
ERKO mouse brain.
Together, the present results provide the first evidence that a
biologically active ERß protein is present in the brain of wild-type
and ERKO mice. These observations also indicate that estrogen can
modulate the expression of genes via ERß as well as ER, although
other nonreceptor mediated mechanisms are also possible (7, 8). Because
there are some differences in the distribution of the ERs in the rodent
brain (11), it will be important to ascertain which genes are regulated
by ER vs. ERß. In addition, the finding that the two
ERs heterodimerize in vitro (20) and are colocalized in
neurons of the rat preoptic area, bed nucleus of the stria terminalis,
and amygdala (21), further suggests that genes may be differentially
regulated by estrogens depending on the ERs present in that particular
neuron.
The discovery of ERß and demonstration that it is biologically active
in vivo also provides many new insights about estrogen
action in the brain. Before the discovery of ERß, investigators
noticed that estrogen regulated many physiological parameters in brain
regions that lacked the classical ER. In the absence of a nuclear
ER, it was thought that these actions were mediated through a putative
membrane receptor, activation of second messenger systems, or by way of
interneuronal connections (4, 7, 8). For example, it was known that the
levels of oxytocin and vasopressin mRNA in the magnocellular neurons of
the paraventricular and/or supraoptic nuclei changed over the estrus
cycle as well as during pregnancy and lactation (22), even though these
regions lacked the classical ER. The finding that many of these
neurons also express ERß mRNA (23) and are capable of binding
125I-estrogen (Shughrue, P. J., and I. Merchenthaler,
unpublished observations), suggests that estrogen may directly regulate
these genes via ERß, although this hypothesis has yet to be verified
experimentally. Perhaps the most intriguing sites of estrogen action
are the brain regions associated with learning and memory, where
estrogen has been shown to regulate many parameters, including the
expression of nerve growth factors and their receptors (24), choline
acetyltransferase (24), synaptogenesis, and dendritic arborization (6).
The presence of ERß in the cortex (13, data reported herein), an area
with no ER in the adult rat brain (11), suggests that estrogen may
regulate genes in these regions via ERß. Future studies are clearly
needed to elucidate the role of ERß in these brain regions and to
determine if ERß-dependent pathways are involved in the cognitive
improvements seen in postmenopausal women taking estrogen replacement
therapy (25, 26, 27).
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Acknowledgments |
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Received November 19, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß mRNA in the
rat central nervous system. J Comp Neurol 388:507–525[CrossRef][Medline]
-iodovinyl-11ß methoxyestradiol in the immature female rat.
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knock-out mouse. Endocrinology 138:5649–5652-disrupted mice. Proc Natl Acad Sci USA 94:11008–11012 gene-disrupted mice. J Neurosci 18:9556–9563. Mol Endocrinol 11:1486–1496 in neurons of the rat forebrain. Endocrinology 139:5267–5270This article has been cited by other articles:
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