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Knockout Mouse
Receptor Biology Section (J.F.C., J.L., K.S.K.), Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709; Laboratory of Reproductive Biology (D.O.B.), Department of Cell Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599; and Departments of Obstetrics and Gynecology and Cell Biology (D.W.S.), Duke University Medical Center, Durham, North Carolina 27710
Address all correspondence and requests for reprints to: Dr. Kenneth S. Korach, Receptor Biology Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, MD B3–02, P.O. Box 12233, Research Triangle Park, North Carolina 27709. E-mail: korach{at}niehs.nih.gov
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(ER)
is present in all three components of the
hypothalamic-pituitary-ovarian axis of the mouse. In contrast, estrogen
receptor-ß (ERß) is easily detectable in ovarian granulosa cells
but is low to absent in the pituitary of the adult mouse. This distinct
expression pattern for the two ERs suggests the presence of separate
roles for each in the regulation of ovarian function. Herein, we
definitively show that a lack of ER in the hypothalamic-pituitary
axis of the ER-knockout (ERKO) mouse results in chronic elevation
of serum LH and is the primary cause of the ovarian phenotype of
polycystic follicles and anovulation. Prolonged treatment with a GnRH
antagonist reduced serum LH levels and prevented the ERKO cystic
ovarian phenotype. To investigate a direct role for ER within the
ovary, immature ERKO females were stimulated to ovulate with
exogenous gonadotropins. Ovulatory capacity in the immature ERKO
female was reduced compared with age-matched wild-type (14.5 ±
2.9 vs. 40.6 ± 2.6 oocytes/animal, respectively);
however, oocytes collected from the ERKO were able to undergo
successful in vitro fertilization. A similar discrepancy
in oocyte yield was observed after superovulation of peripubertal (42
days) wild-type and ERKO females. In addition, ovaries from immature
superovulated ERKO females possessed several ovulatory but
unruptured follicles. Investigations of the possible reasons for the
reduced number of ovulations in the ERKO included ribonuclease
protection assays to assess the mRNA levels of several markers of
follicular maturation and ovulation, including ERß, LH-receptor,
cyclin-D2, P450-side chain cleavage enzyme, prostaglandin synthase-2,
and progesterone receptor. No marked differences in the expression
pattern for these mRNAs during the superovulation regimen were observed
in the immature ERKO ovary compared with that of the wild-type.
Serum progesterone levels just before ovulation were slightly lower in
the ERKO compared with wild-type. These studies indicate that
treatment of ERKO females with a GnRH antagonist decreased the serum
LH levels to within the wild-type range and concurrently prevented
development of the characteristic ovarian phenotype of cystic and
hemorrhagic follicles. Furthermore, a lack of functional ER within
the ovary had no effect on the regulation of several genes required for
follicular maturation and ovulation. However, the reduced numbers of
ovulations following the administration of exogenous gonadotropins in
the ERKO suggests an intraovarian role for ER in follicular
development and ovulation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The majority of documented biological actions of estradiol are mediated
via the estrogen receptor (ER), a class I member of the thyroid/steroid
hormone receptor superfamily of ligand-inducible transcription
factors (reviewed in Ref. 19). Previous studies have reported the
presence of high-affinity and specific estrogen binding sites in the
ovary of the rodent as well as several other species (3, 20, 21).
However, two forms of nuclear ER are now known to exist, the well
described ER, and the newly discovered ERß. Several studies have
demonstrated the presence of the respective mRNAs for both ER and
ERß in the ovaries of the mouse (22, 23), rat (24, 25), cow (26), and
human (27, 28). Furthermore, studies employing in situ
hybridization (24, 25) and immunohistochemistry (26, 29) indicate a
distinct expression pattern for the two ERs in the ovary, in which
ER is highly expressed in the interstitial/thecal compartment,
whereas ERß appears confined to the granulosa cells of growing
follicles.
The dissimilar distribution of the two ERs between and within the
components of the hypothalamic-pituitary-ovarian axis suggests that
separate functions are fulfilled by each. Previous in vivo
investigations of the intraovarian actions of estrogen have relied on
the use of estrogen antagonists or inhibitors of estrogen synthesis.
However, the large amounts of estradiol synthesized in the ovary often
attenuate the ability of these methods to completely block local
estrogen action and thereby complicate the interpretation of
experimental results. Therefore, the estrogen receptor- knockout
(ERKO) mouse, previously described as homozygous for a targeted
disruption of the ER gene (30, 31), provides a unique tool to
clarify the role of each receptor in ovarian function. The conclusions
of several past studies concerning the role of ER in the adult
female were confirmed in the initial descriptions of the ERKO mouse,
including estrogen insensitivity of the reproductive tract (30, 31) and
hypothalamic-pituitary axis (32). The ERKO females exhibit ovaries
characterized by the presence of multiple hemorrhagic and cystic
follicles with no evidence of spontaneous ovulation (30, 33). This
phenotype occurs despite a relatively normal expression pattern for the
ERß gene in the ovaries of the ERKO mice (23, 33). In the ERKO
female, disruption of the negative feedback actions of estradiol in the
hypothalamic-pituitary axis results in elevated levels of the
gonadotropin subunit mRNAs in the pituitary (32) and in serum LH (34).
Therefore, the ERKO ovarian phenotype may be the result of a lack of
ER-mediated action either within the ovary and/or at the level of
the hypothalamic-pituitary axis. Herein, we describe studies
demonstrating the prevention of the adult ERKO ovarian phenotype
when pituitary influence is reduced through the use of a GnRH
antagonist. We also describe the ability of the immature ERKO to
ovulate and exhibit a relatively normal regulation of several genes
critical to folliculogenesis and ovulation when stimulated with
exogenous gonadotropins.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ERKO ovary, wild-type and
ERKO females were treated with a GnRH antagonist. Each treatment
consisted of a single sc injection of 0.15 ml volume of the GnRH
antagonist, Antide (Sigma, St. Louis, MO) (35), suspended
in vehicle (20% propylene glycol in 0.85% saline). Animals were
single-housed and maintained under controlled lighting (12-h light,
12-h dark). Treatments consisted of either vehicle or Antide at 15
µg, 30 µg, or 60 µg between 1200–1300 h every 48 h.
Treatments began when the animals were 28 days of age, before sexual
maturation and the onset of the ERKO ovarian phenotype (33) and
carried out through to 53 days of age, for a total of 12 treatments.
Each group consisted of at least four animals/genotype/dose (except for
ERKO-vehicle = 3). Body weights were monitored throughout the
study with each group showing similar age-related increases regardless
of genotype or treatment (body weight, g; 28 days = 11.9 ±
0.3; 53 days = 18.3 ± 0.2). Animals were killed at 53 days
of age, 18–24 h after the final treatment. Serum was processed from
blood collected from the descending aorta. One ovary was immediately
snap frozen on dry ice for later RNA extraction and the other was fixed
in 10% buffered formalin at 4 C for 6–8 h and then transferred to
70% ethanol at 4 C. For histological analysis, fixed tissues were
paraffin embedded, sectioned at 5 µm and stained with hematoxylin and
eosin according to standard histological procedures.
Superovulation with exogenous gonadotropins
Superovulation assays were carried out on wild-type and ERKO
females at both immature (28 days) and peripubertal (42 days) ages.
Each trial (immature = 5; peripubertal = 2) consisted of a
single sc injection with 2.2 IU PMSG (Sigma) followed
48–52 h later with 3.2 IU human CG (hCG) (Sigma). The
animals were then killed 16–20 h after the hCG injection and the
ovaries and oviduct removed to M-2 medium (Specialty Media, Lavallette,
NJ) supplemented with 0.3% hyaluronidase (Sigma). The
oocyte/cumulus mass was surgically extracted from the oviduct and the
oocytes were counted after enzymatic disassociation from the
surrounding cumulus. The ovary/oviduct was then fixed in 10% buffered
formalin and prepared for histological analysis as described above.
In vitro fertilization assays
For in vitro fertilization assays, immature (28 days)
control (wild-type or heterozygous) and ERKO females were
superovulated as described above. Oocyte/cumulus masses were collected
from the oviducts in M2 medium (Specialty Media) under paraffin oil,
pooled according to genotype, and treated with 0.3% hyaluronidase to
remove the cumulus cells. Cumulus-free oocytes were washed through 2
drops of M2 and transferred to 200 µl droplets of M16 medium
(Specialty Media) with BSA (20 mg/ml) under oil and incubated at 37 C
in 5% CO2. Epididymal sperm were collected from 2 CD-1
males (at least 10 wks of age) per experiment in 500 µl M2 media. The
sperm were diluted 1:10 in M16 medium with BSA (20 mg/ml) under oil for
capacitation at 37 C for 90 min. Oocytes were inseminated with an equal
number of motile sperm in a final droplet volume of 125–200 µl at a
final concentration of 1 x 106 sperm/ml. After 8–10
h of incubation at 37 C, the oocytes were collected and washed through
three drops of M2 medium, fixed in 2.5% glutaraldehyde, transferred to
slides, and stored in 3.7% formaldehyde overnight. The oocytes were
dehydrated with 95% ethanol, stained with acetolacmoid (36), and
scored as fertilized if two pronuclei and a sperm tail were present
within the vitellus. Three separate trials involving animals of each
genotype were carried out.
RNA isolation and ribonuclease protection assay (RPA)
When the mice were killed, tissues were quickly removed and snap
frozen on dry ice, followed by storage at -70 C until processing. All
ovaries were trimmed of oviduct and surrounding tissue before freezing.
Total RNA was isolated using TRIZOL reagent (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer’s
protocol. The concentration of the final preparations were calculated
from an A260 reading using a Beckman Coulter, Inc. DU-640 UV spectrophotometer. An aliquot of all RNA
preparations was then analyzed on a 1% agarose gel to ensure integrity
before further analysis.
We have previously described the generation and use of the antisense riboprobes employed to detect the mRNAs for the mouse ERß (23), LH-R, FSH-R, and PR (33) genes. The antisense riboprobe for PGS-2 mRNA corresponded to bp 175–591 of the mouse PGS-2 cDNA (GenBank no. M64291) and was transcribed from a clone generated via PCR amplification of the fragment from wild-type mouse uterine RNA and cloned into the pCR-Script SK- phagemid (Stratagene Cloning Systems, La Jolla, CA). The antisense riboprobe for the cyclin-D2 mRNA was subcloned from the complete mouse cyclin-D2 cDNA kindly provided by Dr. Charles Sherr, St. Judes Hospital (37), and corresponded to bp 506–864 (GenBank accesion no. M83749). The riboprobe for the mouse P450scc mRNA has been previously characterized (38) and was kindly provided by Dr. Keith Parker, University of Texas-Southwestern. A riboprobe for the mouse cyclophilin mRNA was generated from the pTRI-cyclophilin template (Ambion, Inc. Austin, TX) and included in all assays for normalization between samples. The expected size (nt) of the respective protected fragments were as follows: cyc = 103, cyclin-D2 = 359, ERß = 262, LH-R = 236, P450scc = 558, PGS-2 = 416, PR = 365. RPAs were carried out on 2 µg total RNA from the ovaries of each individual animal using the Hybspeed RPA reagents (Ambion, Inc.) according to the manufacturer’s protocol. For the superovulation studies, a total of eight animals were used for each genotype/treatment/time point (except for the wild-type-4h hCG = 7 animals) and were assayed individually for each marker. Riboprobes were generated and labeled by incorporation of 32P-CTP (Amersham Pharmacia Biotech, Piscataway, NJ) using the Maxiscript Kit (Ambion, Inc.) according to the manufacturer’s protocol. All samples were assayed simultaneously with the same preparation of radio-labeled riboprobe. Hybridization times ranged from 1–1.5 h. All RPA samples were fractionated by electrophoresis on a 1.5 mm thick 6% bis-acrylamide/8.3 M urea/1x Tris-borate gel (National Diagnostics, Atlanta, GA). All gels were fixed and dried in an Easy Breeze Gel Dryer (Amersham Pharmacia Biotech), exposed to a phosphorimager screen and analyzed with a Storm 860 and accompanying ImageQuant Software (Molecular Dynamics, Inc., Sunnyvale, CA), followed by exposure to x-ray film.
Serum LH and progesterone RIAs
Serum was processed from whole blood collected from the
descending aorta when mice were killed and stored at -70 C until
analysis. Serum progesterone was assayed on duplicate 25 µl aliquots
per animal using the Active Progesterone RIA (Diagnostics Systems Laboratories, Inc., Webster, TX) according to the
manufacturer’s protocol. To assess the effectiveness of the GnRH
antagonist treatment regimen, duplicate 100 µl aliquots of serum from
each animal were measured for LH content by RIA as previously described
using materials supplied from the NIDDK (39). These assays have been
previously shown to accurately measure mouse LH (40, 41). Samples that
exhibited undetectable levels of LH were assigned the lower limit of
the assay, 0.1 ng/ml.
Statistical analysis
All data sets were tested for homoscedasticity using Levine’s
test. In cases where data were found to be heteroscedastic, it was
log-transformed before further statistical analysis. Where appropriate,
the Student’s t test was used. In all other cases, a
two-way ANOVA followed by the Bonferroni/Dunn posthoc test was used.
All statistical analysis was carried out using Statview 4.0 Software
(Abacus Concepts, Berkeley, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ERKO female has become a
hallmark phenotype of this model. We have previously described this
phenotype as one that becomes apparent with the onset of sexual
maturity, coinciding with the commencement of increased tonic levels of
serum LH (33). The lack of ER in the hypothalamic-pituitary axis of
the ERKO has resulted in a disruption of the normal feedback
mechanisms of estradiol required for regulation of LH synthesis and
secretion (32, 34). Therefore, ERKO females exhibit chronically
elevated levels of serum LH that are severalfold higher than their
wild-type counterparts, beginning as early as 25 days of age (Fig. 1
). To determine the extent to which the
ERKO ovarian phenotype was due to persistent stimulation by the
heightened LH levels, animals were administered a GnRH antagonist over
the period during which the phenotype is known to develop and worsen.
As described in Materials and Methods, treatments consisted
of either vehicle, or 15, 30, or 60 µg GnRH antagonist (Antide) per
48 h, beginning at the age of 28 days and carried out until 53
days of age.
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shows the effectiveness of the GnRH antagonist treatments in
reducing the serum LH levels in the ERKO females. As expected, the
vehicle-treated ERKO females exhibited serum LH levels that were at
least 10-fold that of the age-matched vehicle-treated wild-types. The
lowest dose of the GnRH antagonist (15 µg/48 h) had no effect,
whereas the two higher doses (30 and 60 µg/48 h) were effective in
reducing the serum LH levels in the ERKO females. However, only at
the highest dose (60 µg/48 h) did the serum LH levels in the ERKO
females no longer differ statistically from those of the wild-type
group. There was no marked effect of the GnRH antagonist on the tonic
levels of serum LH in the wild-type females at any of the doses
used.
Comparative ovarian histology from representative wild-type and ERKO
females at 53 days of age, following 24 days of treatment with vehicle
is shown in Fig. 2, A and B. The
wild-type ovary exhibits indications of normal ovarian function,
including the presence of several follicles at various stages of
maturation, a corpus luteum, and a recently ovulated oocyte in the
oviduct. In contrast, the ERKO ovary illustrates the expected and
previously described phenotype, i.e. few preantral and small
antral follicles, several large and hemorrhagic
cystic follicles, and a lack of corpora lutea. Also shown in Fig. 2 are
representative ovaries from wild-type (C) and ERKO (D) females
following treatment with the GnRH antagonist at 60 µg/48 h for 24
days. The ability of the GnRH antagonist to significantly reduce the
serum LH levels (Fig. 1) and to concurrently prevent the onset of the
ERKO polycystic phenotype is obvious when the ovarian morphology of
the representative treated ERKO animal (Fig. 2D) is compared with
that of the vehicle-treated ERKO (Fig. 2B). The ovaries of the
ERKO females treated with the highest dose of the GnRH antagonist
appeared more like those of the similarly treated wild-type females and
strikingly different than those of the vehicle-treated ERKOs
(compare Fig. 2, B, C, and D). A complete lack of the characteristic
multiple cystic follicles was observed in all of the ovaries of the
ERKO females treated with the highest dose of GnRH antagonist.
Ovaries from ERKO females treated with 15 or 30 µg Antide per
48 h exhibited phenotypes that were intermediate of those treated
with vehicle or 60 µg Antide (data not shown), indicating a
physiological dose-response correlation with the level of circulating
LH (Fig. 1).
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). This is consistent with a previous
report describing the mouse ovary at 20 days after hypophysectomy (42).
However, a distinct phenotype became apparent in the interstitial
compartment of the ERKO ovaries after prolonged treatment with the
highest dose of GnRH antagonist. The interstitial space of ovaries from
wild-type animals following 24 days of treatment with 60 µg Antide/48
h exhibited closely packed cells possessing abundant cytoplasm (Fig. 2C, inset). In contrast, the interstitial compartment of
similarly treated ERKO ovaries was composed of a clearly less dense
cellular population and an increased number of open vacuoles (Fig. 2D, inset).
An observed biochemical effect of the Antide treatment also found to be
unique to the ERKO ovary was a drastic and dose-dependent reduction
in the LH-R mRNA levels when assayed by RPA (Fig. 2F). At the highest
Antide dose, the level of LH-R mRNA in the ERKO ovaries was
significantly reduced (P < 0.001) compared with the level
observed in the ovaries of similarly treated wild-type as well as the
vehicle-treated ERKO females. In contrast, RPAs for FSH-R mRNA
indicated that levels remained relatively stable in the two genotypes
over the increasing doses of GnRH antagonist (Fig. 2E).
Ovulation and in vitro fertilization assays
We have previously described the anovulatory phenotype of the
adult (>100 days old) ERKO female, including an inability to
ovulate when treated with exogenous gonadotropins (33). We therefore
set out to determine if ovulation was possible in immature (28 days)
and peripubertal (42 days) ERKO females, before the onset of the
cumulative effects of the elevated LH described above. As described in
Materials and Methods, superovulation consisted of a single
sc injection with PMSG at 1300–1500 h, followed 48–52 h later with a
single sc injection of hCG. As shown in Table 1, both immature and peripubertal
ERKO females were able to successfully ovulate, although the number
of ova collected from oviducts after superovulation was significantly
reduced (immature = P < 0.001; peripubertal
= P < 0.05) compared with that collected from
age-matched controls. Even the greatest number of oocytes collected
from a single ERKO female was below the wild-type average in each
age group (Table 1A). Three of the 15 immature and 1 of the 9
peripubertal ERKO females tested did not appear to ovulate at all.
The ERKO ova collected exhibited no apparent defect in their ability
to undergo successful in vitro fertilization when using
wild-type sperm, with a combined success rate of 59.7% and 47.6% in
wild-type and ERKO, respectively (Table 1B).
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). The ovaries
from stimulated immature ERKO females also possessed corpora lutea
as well as a consistent presence of multiple preovulatory yet
unruptured follicles (Fig. 3B). Whereas the granulosa cells of some of
the undischarged pre-ovulatory follicles appeared undifferentiated in
the ERKO ovary, in several cases, the oocyte was trapped in a corpus
luteum-like structure, indicating that hCG induced terminal
differentiation of the surrounding granulosa cells was underway.
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ERKO
ovaryERKO ovary suggested a phenotype similar to
that described for other models of targeted gene disruption, including
mice homozygous for disruption of the genes for ERß (44), cyclin-D2
(45), progesterone receptor (PR) (46), and prostaglandin-synthase 2
(PGS-2) (47). We therefore attempted to determine if the reduced
response of the immature ERKO ovary to exogenous gonadotropins was
due to a secondary disruption in the proper regulation of one or more
of the above genes, as well as the genes encoding LH-R and the
P450scc enzyme. For these studies, immature wild-type and
ERKO females were superovulated, and animals selected to be killed
at the following time points during treatment: 48–52 h after vehicle;
48–52 h after PMSG, 48–52 h PMSG + 4 h after hCG, and 48–52 h
PMSG + 20 h after hCG. Total RNA was extracted from whole ovary
and each time point analyzed by RPA for ERß, LH-R, cyclin-D2,
P450scc (Fig. 4); and PGS-2
and PR (Fig. 5). Overall, the ERKO
ovaries exhibited an expression profile for the markers of follicular
maturation and differentiation that was similar to the controls. In
both genotypes, ERß mRNA levels decreased with hCG treatment, in
agreement with that reported in the rat ovary by Byers et
al. (25). Although basal LH-R mRNA levels were significantly
increased in immature ERKO ovaries compared with the wild-type
(P < 0.001), the stimulated levels after PMSG
treatment were similar between the two genotypes. No genotypic
difference in the expression profile of the transcripts encoding
cyclin-D2 and P450scc was observed. As shown in Fig. 5, the
expected increase in PGS-2 and PR mRNAs brought about 4 h after
hCG treatment followed by an equally striking decrease within 20 h
was observed in both the wild-type and ERKO ovaries. As shown in
Fig. 6, serum progesterone peaked 4
h after hCG treatment followed by a measurable decrease within 20
h in both wild-type and ERKO females.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene would be expected to result in dramatic physiological alterations
in the ovary due either to a direct loss of ER within the ovarian
tissue and/or as an indirect effect of alterations in the extra-gonadal
endocrine tissues critical to ovarian function. Herein, we have
definitively demonstrated that the pronounced phenotype of multiple,
enlarged and hemorrhagic cystic follicles in the adult ERKO ovary
are the indirect result of the loss of ER action in the
hypothalamic-pituitary axis. Furthermore, we have provided evidence
that many of the biochemical responses associated with gonadotropin
actions in the ovary are maintained in the immature ERKO female and
do not appear to be related to the reduced ovulatory capacity observed
in this model.
Reduction of serum LH results in a prevention of the ERKO
ovarian phenotype
Both components of the hypothalamic-pituitary axis are known to
express significant levels of ER (48, 49). Accordingly, disruption
of the ER gene results in a loss of the negative regulatory effects
of estradiol in the hypothalamic-pituitary axis, as reflected by the
elevated levels of pituitary gonadotropin subunit mRNAs (32) and serum
LH in the adult ERKO female (Fig. 1). As presented here, the results
of prolonged treatment with a GnRH antagonist in the ERKO female
indicate that: 1) increased serum LH in the ERKO female is primarily
due to the loss of ER mediated actions in the hypothalamus required
for proper regulation of GnRH synthesis and/or secretion; and 2) the
ERKO ovarian phenotype of multiple hemorrhagic follicular cysts is
the result of chronic hyperstimulation by heightened serum LH
levels.
Experimental support for the abnormally high levels of serum LH as the
primary cause of the ERKO ovarian phenotype may be drawn from a
number of studies. Investigations involving prolonged treatment with
antiestrogens over a period of at least 28 days to 6 months have
produced an ovarian phenotype that is similar to that of the ERKO in
both mice (50, 51) and rats (52). However, such studies using estrogen
antagonists are complicated by the possible inhibition of estrogen
action at both the ovarian and hypothalamic-pituitary level, as well as
an inability to discriminate between ER and ERß mediated actions.
Nonetheless, these studies reported that an ERKO-like ovarian
phenotype was produced only after chronic treatment with those
antiestrogens that crossed the blood-brain barrier and concurrently
produced elevated serum LH levels, e.g. ZM-189,154 (52) and
EM-800 (50, 51). In contrast, treatments with tamoxifen had no effect
on serum LH levels and in turn did not produce an ovarian phenotype
similar to the ERKO or to those females treated with ZM-189,154 or
EM-800 (50, 51, 52). Additionally, Risma et al. (53, 54)
provided definitive evidence of the effects of chronic LH exposure on
the ovary via the generation of transgenic mice that overexpress an
LHß transgene in the pituitary. These transgenic females possess a
15-fold increase in physiological levels of LH and exhibit an ovarian
phenotype almost indistinguishable from that of the adult ERKO
female at the morphological level.
The similarity between the ovarian phenotypes described in the above
studies with our observations in the ERKO suggest that ER is not
locally involved in the development of ovarian cysts induced by
hypergonadotropism. However, there are descriptions of at least two
models in which serum LH is chronically elevated but do not exhibit an
ovarian phenotype similar to the ERKO or those induced by
antiestrogens or transgenics as described above. Female mice that are
homozygous for a targeted disruption of the FSHß-subunit gene exhibit
an approximate 5-fold increase in serum LH but do not show indications
of enlarged cystic follicles in the ovary (55), indicating a role for
FSH action in this process as well. Bogovich has also provided
supporting evidence of the need for FSH along with prolonged exposure
to LH to induce follicular cysts in the rat ovary (56). Another
contrasting phenotype is exhibited by the aromatase-knockout (ArKO)
mice, which are homozygous for a targeted disruption of the
P450arom gene, and therefore lack the capacity to
synthesize estradiol (57). Although both serum LH and FSH are elevated
in the ArKO female, folliculogenesis is arrested at the antral stage
and no ERKO-like cystic structures are reported (57). It is possible
that a lack of estradiol synthesis has negated the intraovarian
functions of ERß in the granulosa cells of the ArKO, and thereby
disrupted the pathways conducive to development of the LH-induced
ovarian cysts. Therefore, although the ERKO phenotype may be
triggered by hyperstimulation of the follicles by LH, it is likely
influenced and possibly dependent upon the actions of both FSH and
ERß within the ovary. The preservation of estradiol synthesis (31),
normal serum FSH levels (34), elevated ovarian levels of FSH-R and LH-R
mRNAs (33), and a normal ovarian expression pattern for ERß (23, 33)
in the ERKO female provides further support for this hypothesis.
A unique effect of the GnRH antagonist observed in the ERKO ovary
was a significant dose-dependent reduction in LH-R mRNA levels (Fig. 1F). A possible cause of the drastic reduction in LH-R mRNA levels may
be the apparent decreased cell population in the theca/interstitial
compartments observed in the treated ERKO ovaries (Fig. 1D). In
light of previous reports of GnRH mRNA (58), GnRH receptors (59), and
GnRH regulation of LH-R mRNA and binding-sites (60, 61, 62) in the rodent
ovary, a direct ovarian effect of the GnRH antagonist must also be
considered. However, previous studies have shown that removal of
gonadotropins via hypophysectomy results in a drastic reduction in LH-R
expression in both the theca/interstitial and granulosa cell
compartments of the mouse ovary (42); this effect could be reversed
with treatments of exogenous LH (42) or FSH + estradiol (17).
Therefore, the effect of the Antide in reducing the LH-R mRNA levels in
the ERKO ovary suggest that regulation of the LH-R gene in the
thecal/interstitial cells of the normal ovary may be via both
ER/estradiol and LH-R/LH mediated actions. A decrease in LH-R mRNA
due to lowered serum LH may not be observed in the wild-type ovary
because of the possible compensating role of ER in the
interstitial/thecal cells. In contrast, decreasing levels of
circulating LH due to the effects of the GnRH antagonist in the context
of a loss of ER action may result in the significant reduction in
LH-R mRNA observed in the ERKO ovary. Because the RPA used in this
study was carried out on RNA from whole ovarian tissue, future studies
to localize the decreases in LH-R gene expression among the functional
ovarian compartments in the ERKO ovary are warranted.
Gonadotropin induction of ovulation and follicular markers in the
ERKO
Although the ovaries of the immature ERKO female do not appear
morphologically different than those of age-matched wild-type
littermates (33), a significant reduction in the ovulatory capacity of
the immature ERKO female when superovulated with exogenous
gonadotropins was observed. Similar phenotypes have been described in
mice after targeted disruption of the genes for ERß (44), PR (46),
PGS-2 (47), and cyclin-D2 (45). Another similarity between the ERKO
and those models possessing disruptions of the genes for ERß (44), PR
(46), or cyclin-D2 (63), is the presence of multiple preovulatory yet
unruptured follicles after superovulation. However, quantitative RPAs
for these transcripts, as well as those for LH-R and
P450scc, throughout the time-course of the superovulation
treatment indicate no overall genotypic differences in the expression
pattern of these genes. Therefore, the cause of the reduced ovulatory
capacity observed in the immature ERKO female remains unclear. It is
possible that the ER may play a regulatory role at the translational
level for one or more of the above transcripts; however, this is
doubtful because the successful ovulation of some ova in the ERKO
suggests the presence of the required gene products. Disruption of the
ER gene may have also resulted in the loss of ER/ERß
cooperative functions that may be necessary for proper communication
both within and between the thecal and granulosa cell compartments of
the maturing follicle.
A likely contributing factor to the reduced ovulatory efficiency in the
immature ERKO may be the elevated levels of serum LH that exist in
these females as early as 25 days of age (Fig. 1), the approximate age
at which the superovulation experiments were carried out. Although the
immature ERKO ovary has not yet manifested the cumulative effects of
the chronic LH stimulation, i.e. the multiple cystic
follicles, it is likely that aberrations in ovarian biochemistry
already exist. One indication of such abnormal signaling may be the
significantly elevated levels of LH-R mRNA detected in the immature
ERKO ovary compared with age-matched wild-type females
(P < 0.001; Fig. 4B). We have previously described the
persistence of increased LH-R mRNA levels in both the thecal and
granulosa cell compartments of the ERKO ovary during adulthood and
the manifestation of the polycystic phenotype (33).
Although the studies described herein provide no obvious explanation
for the reduced ovulatory capacity exhibited by the ERKO ovary,
these data do indicate that several biochemical processes that occur
during gonadotropin stimulation in the ovary remain intact in the
absence of functional ER. These include the PMSG-induced
up-regulation of the LH-R gene and simultaneous down-regulation of the
ERß gene. The latter is in agreement with that previously reported in
the rat ovary by Byers et al. (25). Furthermore, we have
provided conclusive evidence that the characteristic rapid and
transient induction of the PR gene in granulosa cells after a single
hCG injection (64) can occur in the absence of ER. These data
support the conclusions of others that induction of the PR gene in the
ovary is a rapid response to the increasing intracellular cAMP levels
induced by the LH-surge (64, 65, 66). Cyclin-D2, another critical component
of follicular maturation, has also been shown to be gonadotropin as
well as estrogen regulated in the granulosa cells of the rat ovary
(63). Although direct estrogen challenges of the ERKO ovary were not
carried out in the current study, our data confirms that gonadotropin
induction of the cyclin-D2 gene can occur in the absence of ER.
Therefore, the data presented here indicate that any estrogen actions
required for full expression of the PR and cyclin-D2 genes, as well as
PGS-2, LH-R, and P450scc expression in the ovulatory
follicle are independent of ER action.
It is worth noting that although the ERKO female is able to ovulate
only at younger ages and only with the administration of
pharmacological levels of gonadotropins, the yielded ova are competent
and able to undergo successful fertilization at a rate similar to that
exhibited by control oocytes. Previous reports of ER mRNA in the
mouse oocyte suggested a direct role for ER in oocyte function (67).
Several studies have also provided evidence of biochemical
communication between the oocyte and surrounding granulosa cells (68, 69), presumably involving the presence of gap junctions that may be
partially estrogen regulated (9). Nonetheless, the loss of ER
mediated action in the mouse ovary does not appear to result in any
alterations in the oocyte that inhibit fertilization.
In summary, we have provided definitive evidence that the hallmark
phenotype of multiple cystic follicles in the ovaries of adult ERKO
females is due to chronic stimulation by heightened levels of serum LH,
and therefore an effect secondary to the lack of ER in the
hypothalamic-pituitary axis rather than the direct loss of receptor in
the ovary. Furthermore, when superovulated before the onset of the
cumulative ovarian phenotype, the immature ERKO female is responsive
to exogenous gonadotropins in terms of successful ovulation and yield
of competent oocytes. However, the average number of ova yielded from
the immature ERKO females was lower than that from their wild-type
counterparts, yet no marked dysregulation of several genes known to be
critical to folliculogenesis and ovulation was observed. These data,
viewed in combination with the significant ovulatory defects reported
in the ERß-knockout mouse (44), strongly suggest that ERß may be
the predominant ER in ovarian physiology or that the combined actions
of ER and ERß are required. In summary, although a critical role
for ER in ovarian function appears to involve the
hypothalamic-pituitary regulation of LH synthesis and secretion, the
inefficient ovulatory capacity of the ERKO female suggests an
intraovarian role for this receptor as well.
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Acknowledgments |
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Footnotes |
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Received April 26, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(ER) and estrogen receptor-ß (ERß) messenger
ribonucleic acid in the wild-type and ER-knockout mouse.
Endocrinology 138:4613–4621 (ER-) and ß (ER-ß) mRNAs in normal ovary, ovarian
serous cystadenocarcinoma and ovarian cancer cell lines:
down-regulation of ER-ß in neoplastic tissues. J Clin Endocrinol
Metab 83:1025–1028 in the rat ovary.
Endocrinology 140:963–971 in the
anterior pituitary gland. Mol Endocrinol 11:674–681
(ER) gene in mice: characterization of ovarian responses and
phenotypes. Endocrinology 140:2733–2744
knockout mice. Endocrinology 139:4092–4101 and ERß) throughout the rat brain: anatomical
evidence of distinct roles of each subtype. J Neurobiol 36:357–78[CrossRef][Medline]
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