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The Journal of Neuroscience, November 15, 1998, 18(22):9556-9563
Induction of Progestin Receptors by Estradiol in the Forebrain of
Estrogen Receptor- Gene-Disrupted Mice
C. A.
Moffatt1,
E. F.
Rissman2,
M. A.
Shupnik3, and
J. D.
Blaustein1
1 Neuroscience and Behavior Program and Center for
Neuroendocrine Studies, University of Massachusetts, Amherst,
Massachusetts 01003, and Departments of 2 Biology and
3 Internal Medicine, University of Virginia,
Charlottesville, Virginia 22903
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ABSTRACT |
Mice, rats, and humans have two types of estrogen receptors,
estrogen receptor- (ER) and estrogen receptor- (ER).
Estrogen receptor- gene-disrupted (ER-disrupted) mice bear two
nonfunctional copies of the ER gene. This mutation blocks the
synthesis of full-length ER, renders the animals infertile, and
inhibits the induction of female sexual behaviors by estradiol and
progesterone. It is likely that many of the processes contributing to
the regulation of sexual receptivity by estradiol and progesterone are
compromised in ER-disrupted mice. However, given the importance of
progesterone in the regulation of sexual receptivity and given the
importance of progestin receptors (PRs) in mediating the
responses of females to progesterone, we investigated the effects of
ER disruption on the induction of PRs by estradiol in the forebrain.
We hypothesized that estradiol would induce PRs in wild-type mice but
not in ER-disrupted mice. Ovariectomized wild-type and
ER-disrupted mice were implanted with either estradiol-filled capsules or empty capsules for 5 d, after which their brains were processed for the immunocytochemical detection of PR. Estradiol increased the number of PR-immunoreactive cells in both wild-type and
ER-disrupted mice. The residual responsiveness of ER-disrupted mice to estradiol could be accounted for by an ER-dependent
mechanism or another as yet unidentified estrogen receptor; however,
because ER-immunoreactivity and PCR product representing the 3' end
of ER mRNA were found in at least one PR-containing region of the ER-disrupted mice, an ER splice variant may also mediate the induction of PR-immunoreactivity in ER-disrupted mice.
Key words:
estrogen receptors; progestin receptors; ventromedial
hypothalamus; ovariectomy; estradiol; progesterone
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INTRODUCTION |
Activation of hypothalamic progestin
receptors (PRs), particularly PRs in the ventromedial
hypothalamus (VMH), influences many neuroendocrine-controlled processes
in female rats, including preovulatory surges in gonadotropin secretion
and female sexual behaviors (Blaustein and Olster, 1989 ; Rubin and
Barfield, 1983a,b; Kalra, 1993). Presumably, the initiation of these
events is a consequence of activated progestin receptors binding to PR
response elements and facilitating gene transcription, as well as
protein synthesis (Blaustein and Olster, 1989; Pfaff et al., 1994).
Blockade of PR activation, via the use of progesterone antagonists or
antisense oligonucleotides to PR mRNA, disrupts PR-modulated processes
and, as a consequence, disrupts the PR-dependent facilitation of sexual behavior (Brown and Blaustein, 1984; Bittencourt et al., 1992; Mani et
al., 1994; Ogawa et al., 1994; Lydon et al., 1995).
Although the activation of PRs contributes to the regulation of female
sexual behaviors, treatment with progesterone by itself does not
facilitate sexual receptivity. For progesterone to facilitate sexual
behaviors, female rats must first be exposed to estradiol (Boling and
Blandau, 1939). The reason for this may lie with the manner in which
PRs are regulated in the areas of the brain that control female sexual
behaviors. Neural PRs can be divided into two populations: a population
whose synthesis is induced by estradiol and a population whose
synthesis is not (MacLusky and McEwen, 1978). It is the former
population, the estradiol-induced PRs, that is believed to be involved
in the regulation of female sexual receptivity (Blaustein and Feder,
1979). When the concentration of estradiol-induced hypothalamic PRs is
high, females exhibit sexual behavior in response to treatment with
progesterone (Blaustein and Olster, 1989; Pfaff et al., 1994).
Conversely, when the concentration of estradiol-induced PRs is low,
typical doses of progesterone do not facilitate sexual behaviors
(Blaustein and Feder, 1980; Blaustein and Olster, 1989).
Recently, a strain of mice has been developed that lacks functional
copies of the estrogen receptor- (ER) form of the estrogen receptor (Lubahn et al., 1993; Couse et al., 1995). Female ER gene-disrupted (ER-disrupted) mice show a variety of reproductive deficits, including a lack of sexual behavior (Ogawa et al., 1996), even after hormone treatments that are sufficient to induce sexual behavior in wild-type (WT) mice of the same strain (Rissman et al.,
1997). It is unlikely that the lack of behavioral responsiveness to
estradiol and progesterone can be attributed to the disruption of a
single ER-regulated process. Instead, the impairment in behavior is
more likely to be the product of a constellation of neuroendocrine
disruptions. With this caveat in mind, we investigated whether
ER-disrupted mice showed impairments in one component of the
mechanism that regulates sexual behavior in female mice, the induction
of hypothalamic PRs in response to treatment with estradiol. In
particular, we focused on the induction of PRs in the VMH, an area of
the brain that is crucial for mediating the effects of estradiol and
progesterone on sexual receptivity and which had not previously been
examined for responsiveness to estradiol in ER-disrupted mice.
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MATERIALS AND METHODS |
Experiment 1: immunocytochemical detection of PRs
Animals. Animals were produced by heterozygotic
breeding pairs. Each member of the pair had one normal and one
disrupted ER gene. All offspring were screened by PCR amplification
of tail DNA (Lubahn et al., 1993). The breeding colony is maintained at the University of Virginia and was founded using mice provided by
Dennis Lubahn (University of Missouri), which were heterozygotic for
disruption of the ER gene. Twenty adult ovariectomized mice, eight
of which were ER-disrupted and 12 of which were WT littermates, were
used in the immunocytochemistry experiment. One month after all the
animals were ovariectomized, four of the ER-disrupted and eight
wild-type mice were implanted subcutaneously with SILASTIC (Dow Corning, Midland, MI) capsules [1.98 mm inner diameter
(i.d.) × 3.17 mm outer diameter (o.d.)] containing 50 µg of
estradiol dissolved in 25 µl of sesame oil; the remaining animals
(four ER-disrupted and four WT mice) were implanted with empty
capsules. Five days after the capsules were implanted, the mice were
perfused with 2% acrolein in a 0.1 M phosphate buffer, and
the brains were removed and immersed overnight in a 0.1 M
phosphate buffer containing 20% sucrose. Serial coronal sections (30 µm) were made through the forebrain. The sections were collected into
cryoprotectant (Watson et al., 1986) and then stored at  20°C until
every fourth section was processed for the immunocytochemical detection
of either PRs or ER.
Immunocytochemistry. The sections were removed from
cryoprotectant and rinsed three times in 0.5 M
Tris-buffered saline (TBS), pH 7.6. These rinses were followed by a 10 min incubation in a 1% sodium borohydride solution made in TBS. After
rinsing again, the tissue was incubated for 20 min in TBS containing
20% normal goat serum, 1% bovine serum albumin, and 0.1% hydrogen
peroxide. The sections were then transferred directly into a primary
incubation buffer containing 1% gelatin, 0.02% sodium azide, 1%
normal goat serum, and 0.5% Triton X-100 in TBS, pH 7.6, at 4°C.
Added to this buffer was either an antibody directed against the hinge region of the PR (H-928, 0.2 µg/ml; StressGen Biotechnologies Corp.,
Victoria, British Columbia, Canada) or an antibody directed against the
last 14 amino acids of the rat ER (C1355, 1:5000) (Friend et al.,
1997). The last 14 amino acids of ER has no homology to the last 14 amino acids of ER, so C1355-immunoreactivity should reflect the
presence of only ER. The specificity of the PR antibody H928 for the
immunocytochemical detection of PR-immunoreactivity has been
established previously in rats (Auger et al., 1996). Using females that
were heterozygous with respect to the disruption of the ER gene, the
specificity of the C1355 ER antibody was assessed by
preadsorbing it for 24 hr at 4°C, with a 10 M excess of the peptide against which it was generated and by omitting C1355
from the primary incubation buffer. The tissue was incubated with the
appropriate primary antibody for ~72 hr at 4°C. After this
incubation, the tissue was rinsed three times in TBS, pH 7.6, at 23°C
and incubated for 90 min in TBS containing 1.5% normal goat serum,
1.5% Triton X-100, and 3 µg/ml either biotinylated goat anti-mouse
IgG (Jackson ImmunoResearch, West Grove, PA) to label
H928-immunoreactivity or biotinylated goat anti-rabbit IgG to label
C1355-immunoreactivity. The sections were rinsed again and incubated
for 90 min in TBS containing 1% Avidin DH-biotinylated horseradish
peroxidase H complex (Vectastain Elite; Vector Laboratories, Burlingame, CA). After three more rinses in TBS, the tissue was incubated for ~5 min in TBS containing 0.05% diaminobenzidine and
0.05% H2O2, rinsed again, mounted on
gelatin-covered slides, and coverslipped with Permount (Fisher
Scientific, Fair Lawn, NJ) before analysis of PR-immunoreactivity.
Image analysis. Computer-assisted analysis of
PR-immunoreactivity was performed using a Leitz Dialux 20 microscope (Leitz, Wetzler, Germany), fitted with an MTI CCD72
camera (Dage MTI, Michigan City, MI) and connected to a Macintosh
Quadra 700 computer using the public domain image analysis program
developed by National Institutes of Health, Image 1.55 (available at
http://rsb.info.nih.gov/nih-image/).
Briefly, the microscope was adjusted for Kohler illumination and
focused on a black circle that had been affixed to a coverslipped slide. The camera gain and black levels were adjusted so that the blank
portions of the slide produced gray levels of approximately five units,
whereas the black circle produced gray levels of 254 units. Cells were
considered to be PR-immunoreactive (PR-IR) if they satisfied two
criteria. The putative PR-IR cells had to be >10 pixels but <200
pixels in area, and the optical density of the pixels corresponding to
putative PR-IR cells had to exceed the average optical density of the
surrounding tissue by a predetermined number of SDs. The number
of SDs varied according to the area that was analyzed, because the
difference in optical density between PR-IR cells and the surrounding
tissue varied between areas. For a specific area, however, the same
criterion was used for every section analyzed. The areas analyzed in
the forebrain are depicted in Figure 1
and were chosen because they were known to contain large numbers of
estradiol-induced PRs in WT mice.
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Figure 1.
Drawings illustrating the regions of the brain
examined for PR-immunoreactivity. Although the shaded
regions indicate where the majority of PR-IR cells are located, PR-IR
cells are not restricted to these regions. The regions
outlined by thick black lines are the
regions in which PR-IR cells were counted. 3V, Third
ventricle; Arc, arcuate nucleus; BNST,
bed nucleus of the stria terminalis; cVMH, caudal
ventromedial hypothalamus; DMH, dorsomedial
hypothalamus; f, fornix; ic, internal
capsule; LSV, ventrolateral septum; LV,
lateral ventricle; MePD, posterodorsal medial amygdala;
MPO, medial preoptic nucleus; MT, medial
tuberal nucleus; opt, optic tract; ox,
optic chiasm; rVMH, rostral ventromedial hypothalamus;
sm, stria medullaris.
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Experiment 2: detection of estrogen receptor- and ER mRNA in
the VMH using PCR
Animals. Brains from four ovariectomized
ER-disrupted females and four ovariectomized WT females were used
for the detection of ER and estrogen receptor- (ER) mRNA. Two
animals of each genotype were implanted with steroid (5 mm of a 1:1
mixture of estradiol/cholesterol) in a SILASTIC capsule
(1.02 mm i.d. × 2.16 mm o.d.). Five days after implants were received,
all females were killed by an overdose injection of anesthesia. Brains
were quickly removed and frozen on dry ice. Frozen tissue was sectioned in a cryostat. Four sections, 180 µm each, were collected
through the rostral to caudal VMH. A 26 gm stylus was used to punch the frozen tissue, which was collected in a microfuge tube on dry ice.
PCR. Total RNA was purified from the VMH punches using an
established procedure (Friend et al., 1997). To perform reverse transcription (RT)-PCR amplifications, a single RT-reaction was performed, and the resultant cDNA product was partitioned into four PCR
reactions to amplify products for -actin (equivalent of 0.5 µg of
RNA) or (equivalent of 1 µg of RNA each) the 5' end of mouse
ER (exons 1-3), the 5' end of mouse ER (exons 1-3), or the 3'
end of mouse ER (exons 4-7). RT-PCR reactions were performed as
described previously, except that only one set of amplifications (35 cycles of 95°C for 1 min, annealing temperature for 1 min, 72°C for
1 min) was performed for the PCR amplification. The annealing reactions
for mouse ER were performed at 49°C, but all other annealing
reactions were performed at 55°C. The primers used to amplify the
mouse ER product (351 bp) were ERBST: 5'-CTATGACATTCTACAGTCC-3' and
ERB3: 5'-GTAATGATACCCAGAGCA-3'. Primers for the 5' end of ER
(638 bp for intact ER) were RERC: 5'-GACCATGACCATGACCCT-3' and MER4:
5'-CTTGCAGCCTTCACAGGAC-3'. Primers to amplify the 3' end of the coding
region of ER (441 bp) were SERRB: 5'-GATCCTTCTAGACCCTTC-3' and
MER7: 5'-CTTGTCCAGGACTCGGTGGAT-3'. After amplification, products
were displayed on a 1% agarose gel containing ethidium bromide as
described previously (Friend et al., 1997).
Statistics. The effects of genotype and hormone treatment on
PR-IR cell numbers were assessed using an ANOVA procedure (Systat Version 6.1). When the analysis indicated that there was a
significant interaction between treatment effects, post hoc
comparisons were made between group means using the Newman-Keuls
procedure. It was decided a priori to compare the number of
PR-IR cells in oil-treated and estradiol-treated ER-disrupted mice
in each of the areas of the brain that were examined. These comparisons
were made using t tests or nonparametric tests where appropriate.
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RESULTS |
Experiment 1
The ability of estradiol to induce PRs in this experiment was
strongly influenced by the genotype of the animals (Figs.
2, 3). That
is, although treatment with estradiol significantly increased PR-IR
cell number in each of the areas examined (p < 0.05 in all cases), post hoc comparisons revealed that WT
animals treated with estradiol had significantly more PR-IR cells than
estradiol-treated ER-disrupted animals (p < 0.05 in all cases). Nevertheless, estradiol-treated ER-disrupted
mice did have more PR-IR cells than oil-treated ER-disrupted mice in
the caudal VMH (cVMH), arcuate nucleus (Arc), and posterodorsal medial
amygdala (MePD) (p < 0.05 in all cases). There
was also a large difference between the two groups in the medial
preoptic nucleus (MPO), but the difference did not reach statistical
significance (p > 0.05). No differences were
observed between oil-treated WT and oil-treated ER-disrupted mice
with respect to PR-IR cell number (p > 0.05).
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Figure 2.
Representative photomicrograph (20×
magnification) of PR-IR cells in the cVMH of an estradiol-treated WT
mouse (A), an oil-treated WT mouse
(B), an estradiol-treated ER-disrupted mouse
(C), and an oil-treated ER-disrupted mouse
(D).
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Figure 3.
The number of PR-IR cells (+SEM) counted in WT and
ER-disrupted mice after being implanted with either an
estradiol-filled capsule or empty capsule for 5 d. See Figure 1
for nomenclature.
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In addition to quantifying PR-IR cell numbers, we determined whether
there were differences in the proportion of darkly immunostained cells
found in the MePD, Arc, and cVMH of estradiol-treated WT and
ER-disrupted mice (Fig. 4).
Contrasting the proportion of darkly immunostained cells (cells with
optical densities >225 units) in each group in the Arc and cVMH, we
found that estradiol-treated WT mice had a greater proportion of darkly
immunostained cells than did ER-disrupted mice
(p < 0.05 in all cases). Because there were few
cells in the MePD that had optical densities >225 units, the threshold
was adjusted, and contrasts in this area were made between cells having
optical densities >160 units. Again, estradiol-treated WT mice had a
significantly greater proportion of darkly immunostained cells than
estradiol-treated ER-disrupted mice (p < 0.05).
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Figure 4.
The proportion (+SEM) of darkly immunostained
PR-IR cells counted in WT and ER-disrupted mice after being
implanted with either an estradiol (E2)-filled or empty capsule for
5 d. See Figure 1 for nomenclature.
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Although ER-disrupted mice had fewer ER-IR cells than WT mice
(p < 0.05 in all cases), ER-IR cells were
observed in the Arc, cVMH, MePD, and ventrolateral septum of
both groups (Figs. 5,
6). In contrast, ER-IR cells were not
seen in the rostral VMH and medial tuberal nucleus of the
ER-disrupted mice. Although ER-IR cells were also detected in the
MPO (189 cells) and bed nucleus of the stria terminalis (BNST) (23 cells) of ER-disrupted mice, insufficient numbers of well matched
sections were available for contrasts with WT animals. Preadsorption of
the C1355 antibody with the peptide against which it was raised and
omission of the C1355 antibody from the incubation buffer eliminated
all ER immunostaining (Fig. 7).
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Figure 5.
Representative photomicrograph (20×
magnification) of ER-IR cells in the cVMH of a WT mouse
(top) and an estradiol-treated ER-disrupted mouse
(bottom).
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Figure 6.
The number of ER-IR cells (+SEM) counted in WT
mice (black bars) and ER-disrupted mice
(gray bars). See Figure 1 for nomenclature.
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Figure 7.
Representative photomicrographs (20×
magnification) of the cVMH showing ER immunostaining produced by
C1355 (A), immunostaining when C1355 was
preadsorbed with the peptide against which it was raised
(B), and immunostaining in the absence of C1355
(C).
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Experiment 2
All mRNA samples taken from the VMH showed PCR products for actin
mRNA, ER mRNA, and the 3' end of ER mRNA. Only the WT females
showed an appropriately sized exon 1-3 PCR product
corresponding to the 5' end of the full-length ER mRNA (Fig.
8). The estradiol-treated animals had
more than one band corresponding to PCR products for the ER mRNA.
These latter ER PCR products have not been characterized, but they
are similar in size to products that would lack exon 2. Such products
have been characterized in human pituitary tumors (M. Shupnik,
unpublished data), but the protein product of such mRNAs would
only correspond to exon 1 and are unlikely to be bioactive.
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Figure 8.
PCR products representing ER mRNA in tissue of WT
and ER-disrupted female mice. A shows 5' ER
product, B shows 3' ER product, C
depicts actin present in all samples, and D shows ER
product. Control lanes run without PCR product are
designated as C. Marker 1 (M1) (a
ligation ladder of progressive lengths of 123 bp DNA) was used for gels
A, B, and D. In gel
C, the marker used (M2) is DNA from
bacteriophage  digested with Bstel restriction endonuclease.
In D, the first lane contains marker DNA,
and the last lane is the negative control.
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DISCUSSION |
Disruption of the ER gene suppressed, but did not completely
inhibit, the induction of progestin receptors in ER-disrupted mice.
More specifically, although ER-disrupted mice had fewer estradiol-induced progestin receptors than WT mice, treatment with
estradiol increased PR-IR cell number in the MePD, Arc, and cVMH of
ER-disrupted mice. When the intensity of the PR immunostaining was
analyzed, it was found that the proportion of cells that were darkly
immunostained in ER-disrupted mice was smaller than the proportion
that was darkly immunostained in WT mice. Differences in the optical
density or intensity of immunostaining can be attributed to differences
that exist between cells in the relative amount or concentration of
antigen they contain (Auger and Blaustein, 1995). With this in mind,
one interpretation of our results is that, although estradiol caused an
increase in PR-IR cell number in ER-disrupted mice, the relative
number of PRs induced in these cells was smaller in ER-disrupted
mice than in WT mice. The apparent difference in PR concentration
between WT and ER-disrupted mice is consistent with the results of
the in situ hybridizations performed by Shughrue and
colleagues (1997b) and suggests that PR synthesis in
ER-disrupted mice is impaired even in those cells that exhibit estradiol-induced PR-immunoreactivity.
The pattern of estradiol-induced PR expression we observed in
ER-disrupted mice is very similar to the pattern of
estradiol-induced PR mRNA described in a previous report for sites that
both studies examined (Shughrue et al., 1997b). In the previous
study, PR mRNA levels were found to increase in the BNST and preoptic
area (POA) of ER-disrupted mice after treatment with
estradiol, but changes in more caudal regions of the forebrain, areas
such as the MePD, Arc, and cVMH, were not examined in that study.
Responsiveness to estradiol in ER-disrupted mice is not limited only
to the brain; it is also evident in other systems and tissues. For
instance, estradiol is equally effective in ER-disrupted mice and WT
mice at inhibiting the thickening of arterial walls after arterial injury (Iafrati et al., 1997). What remains to be identified is the
mechanism that mediates this responsiveness to estradiol in ER-disrupted mice.
One possibility is that the induction of progestin receptors in
ER-disrupted mice is mediated via an estrogen receptor that is
distinct from ER. ER, for instance, is a novel estrogen receptor that was initially identified in rat tissue and then subsequently found
to exist in mice and humans (Kuiper et al., 1996, 1997; Mosselman et
al., 1996; Tremblay et al., 1997). Analysis of the ligand-binding
properties of ER and ER has shown that the two receptors bind
estradiol with equal affinity (Kuiper et al., 1997). In addition, the
DNA-binding domains of ER and ER are quite similar to one
another, having a homology of 97% (Tremblay et al., 1997). Although it
has not been determined whether ER regulates PR synthesis, the
homology that exists in the DNA-binding domains of ER and ER is
consistent with this possibility. ER regulates expression of the PR
gene via its interactions with estrogen response elements (EREs) within
the 5'-flanking region of the PR gene (Kraus et al., 1994). Given that
ER binds to these EREs, then it might be possible for ER to
regulate the expression of PR (Paech et al., 1997). Consistent with
this hypothesis is the observation that ER mRNA and protein are
present in many PR-containing forebrain regions of female rats and
ER-disrupted mice (Shughrue et al., 1996, 1998; Li et al.,
1997).
The hypothesis that ER mediated the induction of PRs by estradiol in
ER-disrupted mice is consistent with our observation that a PCR
product corresponding to ER mRNA is present in the VMH of
ER-disrupted mice. Previous reports of the distribution of ER in
the hypothalamus have, however, described ER as absent in VMH of
rats (Shughrue et al., 1996, 1997a) and "sparse or absent" in the
VMH of ER-disrupted mice (Shughrue et al., 1998). The discrepancy
between the results of the present study and those of previous studies
may be attributed to methodological differences between the studies.
Shughrue and colleagues (1996, 1997a, b, 1998) used in
situ hybridization to map the distribution of ER mRNA in the
brains of rats and ER-disrupted mice, whereas PCR (using primers
having different sequences than the probes used by Shughrue and
colleagues) was used in the present study. In situ
hybridization, although providing excellent neuroanatomical localization of ER mRNA, may not be as sensitive as PCR in detecting mRNA that occurs in low abundance. Despite the detection of PCR products corresponding to ER mRNA in the VMH, it is still very important to determine whether ER protein is present in the VMH and
whether ER has the capacity to mediate the effects of
estradiol on PR synthesis. Until such time as the role of ER
in PR regulation is determined, alternative hypotheses must be considered.
An alternative mechanism that might contribute to the induction of
PR-immunoreactivity in ER-disrupted mice is the activation of mutant
ER or ER splice variants by estradiol. The ER gene in
ER-disrupted mice was disrupted by inserting the
Neo gene into exon 2 of the gene encoding ER, the
portion of the gene that encodes the N-terminal region of ER
protein. This insertion disrupts the reading frame of the ER gene
and introduces multiple stop codons (Lubahn et al., 1993).
Consequently, the tissues of ER-disrupted mice contain no
full-length ER mRNA. However, despite this disruption of the ER
gene, portions of it are transcribed. The detection of PCR products in
the VMH of ER-disrupted mice that correspond to the 3' end of ER
mRNA not only confirms a previous report describing the presence of
this portion of ER mRNA in the hypothalamus of ER-disrupted mice
(Couse et al., 1997) but also bolsters the hypothesis that an ER
splice variant could mediate the effects of estradiol in the VMH. The
results of our PCR analysis also validate previous and current reports of ER-IR in the VMH of ER-disrupted mice. In a previous study, H222, an antibody that recognizes the ligand-binding region of ER,
was used to detect ER-IR (Rissman et al., 1997). Using an antibody,
ER21, which recognizes the N-terminal region of ER, Ogawa and
colleagues (1997) failed to detect ER-IR in the VMH of
ER-disrupted mice; however, in the same study, they did report sparse or residual ER-IR in the medial POA. The lack of
ER-IR detected in the VMH using the ER21 antibody is consistent with the results of the PCR analysis undertaken in the present study.
Couse and colleagues (1995) have assessed the biological activity of
two ER splice variants detected in the uterus of ER-disrupted mice. The smaller of the two splice variants they detected, ER2, would
produce a protein that probably binds neither DNA nor estradiol, whereas the larger splice variant, ER1, is capable of coding a protein
that binds estradiol. ER1 is transcriptionally active in
vitro, but despite its presence in the uterus, the uterus of ER-disrupted mice is not responsive to estradiol (Couse et al., 1995). This does not mean, however, that a splice-variant such as ER1
could not mediate the responsiveness of hypothalamic cells to
estradiol. Rather, the induction of PRs in the hypothalamus may be a
more sensitive indicator of transcriptional activity of ER1 in
vivo than is uterine responsiveness. This hypothesis is consistent
with the results of a number of studies showing cell-specific
differences, even among cells that contain estrogen receptors, in the
ability of estradiol to induce changes in the translation of proteins
and transcription mRNAs (i.e., Shupnik et al., 1989; Ing and Tornesi,
1997). Whether or not the PCR products corresponding to the 3'
ER mRNA detected in the present study corresponds to the ER1
transcript detected by Couse and colleagues (1995) remains to be
determined. However, the presence of an ER splice variant in the VMH
of ER-disrupted mice raises the possibility that an ER splice
variant mediates the induction of PRs by estradiol.
Alternatively, as yet unidentified nongenomic estradiol-sensitive
mechanisms could mediate the responses of ER-disrupted mice to
estradiol. Among the candidate mechanisms are membrane-bound estrogen
receptors (Ramirez et al., 1996). The function of this type of receptor
has yet to be well characterized in the brain, but it has been
suggested that membrane-bound receptors that bind steroid hormones may
influence neuroendocrine function (DeBold and Frye, 1994; Frye et al.,
1996). As well, the possibility must be considered that other estrogen
receptors, estrogen receptors distinct from ER and ER, may
mediate the effects of estradiol in the CNS.
In summary, it is clear that ER-disrupted mice retain a residual
responsiveness to estradiol. This responsiveness is sufficient to
regulate PR-immunoreactivity but is insufficient to induce sexual
behaviors under the conditions tested (Rissman et al., 1997). One
interpretation of this pattern of responsiveness is that ER splice
variants act in conjunction with other mechanisms, possibly ER, to
induce progestin receptors and regulate sexual behaviors. Although this
is an exciting hypothesis, we cannot yet discard the possibility that
it is an ER splice variant or an alternate mechanism that mediates
the responses of ER-disrupted mice to estradiol.
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FOOTNOTES |
Received March 10, 1998; revised Aug. 18, 1998; accepted Sept. 3, 1998.
This research was supported by National Institutes of Health
Grants HD08181 (C.A.M.), MH56187 (J.D.B.), K05-MH01312 (J.D.B.), NS35429 (E.F.R.), KO2-MH 01349 (E.F.R.), and HD25719 (M.A.S.). We thank
Dr. Dennis Lubahn for providing EFR with the heterozygotic ER-disrupted mice used to set up our colony and for expert technical and scientific consultation. We also thank Robin Lempicki and Xia Li
for their excellent technical assistance.
Correspondence should be addressed to C. A. Moffatt, Department of
Biology, San Francisco State University, 1600 Holloway Avenue, San
Francisco, CA 94132.
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Abstract
Introduction
