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Knockout Mice
Receptor Biology Section, Laboratory of Reproductive and Developmental Toxicology (J.L., J.F.C., K.S.K.) and Hormone Action Group (W.C.W.), Laboratory of Signal Transduction, National Institute for Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709; the Department of Psychiatry and Behavioral Sciences, Duke University (W.C.W.), Durham, North Carolina 27710; the Reproductive Toxicology Division, Environmental Protection Agency (T.S., R.C.), Research Triangle Park, North Carolina 27711
Address all correspondence and requests for reprints to: Dr. Kenneth S. Korach, Laboratory of Reproductive and Developmental Toxicology, National Institute for Environmental Health Science, 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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(ER), and estrogen receptor-ß (ERß)
in mediating feedback effects of sex steroids on reproductive
neuroendocrine function, we have determined the effects of castration
and steroid replacement therapy on hypothalamic GnRH content, pituitary
LHß and FSHß messenger RNA (mRNA) levels, and serum gonadotropins
in male wild-type (WT) and estrogen receptor- knockout (ERKO) mice.
Hypothalami from intact WT and ERKO males contained similar amounts of
GnRH, whereas castration significantly reduced GnRH contents in both
genotypes. Replacement therapy with estradiol (E2),
testosterone (T), or dihydrotestosterone (DHT) restored hypothalamic
GnRH content in castrated (CAST) WT mice; only the androgens were
effective in CAST ERKOs. Analyses of pituitary function revealed that LHß mRNA and serum LH levels in intact ERKOs were 2-fold higher than those in intact WT males. Castration increased levels of LHß mRNA (1.5- to 2-fold) and serum LH (4- to 5-fold) in both genotypes. Both E2 and T treatments significantly suppressed LHß mRNA and serum LH levels in CAST WT males. However, E2 was completely ineffective, and T was only partially effective in suppressing these two indexes in the CAST ERKO males. DHT treatments stimulated a 50% increase in LHß mRNA and serum LH levels in WT males, whereas serum LH was significantly suppressed in DHT-treated ERKO males.
Although the pituitaries from intact ERKO males contained similar amounts of FSHß mRNA, serum FSH levels were 20% higher than those in the intact WT males. Castration increased FSHß mRNA levels only in WT males, but significantly increased serum FSH levels in both genotypes. Both E2 and T treatments significantly suppressed serum FSH in CAST WT males, whereas only E2 suppressed FSHß mRNA. DHT treatments of CAST WT mice stimulated a small increase in serum FSH, but failed to alter FSHß mRNA levels. None of the steroid treatments exerted any significant effect on FSHß mRNA or serum FSH levels in CAST ERKOs.
These data suggest that hypothalamic GnRH contents can be maintained
solely through AR signaling pathways. However, normal regulation of
gonadotrope function requires aromatization of T and activation of
ER signaling pathways in the gonadotrope. In addition, serum FSH
levels in male ERKOs appear to be regulated largely by nonsteroidal
testicular factors such as inhibin. Finally, these data suggest that
hypothalamic ERß may not be involved in mediating the negative
feedback effects of T on serum LH and FSH in male mice.
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Introduction |
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Besides examining hypothalamic sites of action, a number of
investigators have also examined feedback directly on the pituitary. T,
dihydrotestosterone (DHT), or estradiol (E2) can suppress
GnRH-stimulated LH secretion from pituitary cultures (20, 21), whereas
T treatments can increase basal FSH secretion and intrapituitary FSH
levels (22). Furthermore, molecular analyses of the promoter regions of
the gonadotropin genes have revealed that some of the feedback effects
of E2 or T may be mediated through androgen receptor (AR)
or estrogen receptor (ER) interactions with response elements and other
transcription factors that regulate expression of the -gonadotropin
subunit (GSU), and the FSHß and LHß subunits (1, 2, 23).
Although these data support both hypothalamic and pituitary sites of
steroid feedback, it is unclear whether the effects of T are primarily
mediated directly through the AR or indirectly via aromatization and
activation of ER. Support for AR-mediated feedback is found in studies
in which treatments with DHT (a nonaromatizable androgen) effectively
suppressed serum LH and steady state levels of GSU and LHß mRNA in
rats (24, 25), androgen blockade with flutamide (an antiandrogen)
resulted in elevations of serum LH (26), and AR-mediated suppression of
the GSU gene expression occurred through enhancer elements in the
promoter region of that gene (23). Convincing arguments for the role of
AR are also found in models of receptor-based androgen resistance, the
testicular feminized mouse (Tfm) (27) and human androgen insensitivity
syndromes (28), in which serum LH can be significantly elevated. By
contrast, other studies have demonstrated robust suppression of serum
LH and FSH, and gonadotropin mRNAs by exogenous E2
treatment (1, 2) and elevations in serum LH after treatments with
aromatase inhibitors (29). The roles of estrogens and ER are further
supported clinically by the elevated serum FSH levels in an
estrogen-resistant patient (30) and in aromatase-deficient humans
(31).
The question of AR- vs. ER-mediated negative feedback on
serum gonadotropins is further complicated by recent characterizations
of a second nuclear ER, termed ERß (32, 33). Although ERß mRNA
levels are very low in adult mouse pituitaries (34), there are reports
of ERß protein and mRNA in hypothalamic nuclei of both rats and mice
(34, 35, 36). Thus, androgens could regulate neuronal activity directly
through AR signaling pathways or indirectly through aromatization and
activation of either ER or ERß signaling pathways.
The recent development of an ER knockout (ERKO) mouse line (37)
provides a unique opportunity to explore the roles of AR, ER, and
ERß in regulating hypothalamic and pituitary function. To better
define the roles of these steroid receptors in mediating the negative
feedback effects of T on hypothalamic GnRH, pituitary gonadotropin gene
expression, and serum levels of gonadotropins, we have conducted
castration and steroid replacement experiments using male wild-type
(WT) and ERKO mice. The findings from our study indicate that both
ER and AR signaling pathways effectively regulate GnRH content, but
that aromatization and activation of ER enhance the negative
feedback effects of T on serum gonadotropins. In addition, these
findings suggest that hypothalamic ERß does not play an obvious role
in regulating GnRH content or gonadotrope physiology in adult male
mice.
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Materials and Methods |
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Three weeks after implantation, the males were weighed and killed. Blood samples were collected, and pituitaries and hypothalami were frozen on dry ice for subsequent analysis of steady state levels of gonadotropin mRNA and hypothalamic GnRH content. The preoptic-hypothalamic regions were removed as a tissue block, as previously described (38). Seminal fluid was expressed, and the seminal vesicles were weighed to gauge the efficacy of the exogenous androgen treatments. This study was replicated three times, and the samples were assayed as described below. All studies were conducted according to the principles and procedures outlined by the NIH Guidelines for the Care of Experimental Animals and under an approved animal study protocol from the NIEHS animal care and use committee.
Preparation of SILASTIC capsules
Steroid capsules were prepared by cutting SILASTIC tubing (0.62
id x 0.125 od inches) into 15-mm lengths, sealing one end with
SILASTIC adhesive, and filling the capsule with either crystalline DHT
or T (Steraloids, Wilton, NH) to a length of 1 cm or with 20 µl
E2 (Sigma Chemical Co., St. Louis, MO) suspended in sesame
oil (1.0 µg/µl). The tubes were then sealed with SILASTIC adhesive.
Immediately before implantation, capsules were rinsed using 70%
ethanol and washed with sterile saline.
Gonadotropin, GnRH, and steroid RIAs
Serum LH and FSH were quantified by RIA. The assays were
performed using the following materials supplied by the NIDDK: 1) for
LH, iodination preparation I-9, reference preparation RP-3, and
antiserum S-11; and 2) for FSH, iodination preparation I-8, reference
preparation RP-3, and antiserum S-11. These RIAs have been previously
described and used in the measuring of mouse gonadotropins (39, 40).
Fifty-microliter aliquots of sera were assayed in duplicate for both LH
and FSH. Due to limited sample volumes, some samples were assayed for
FSH as single tubes. In cases where LH and FSH levels were below the
limit of detection, the minimum detectable concentration was assigned
to that sample. The lower limits of detection for LH and FSH were 0.12
and 1.6 ng/ml, respectively. The intraassay variations were 5% and 9%
for the LH assays and 2% and 4% for the FSH assays. Interassay
variations were approximately 10% and 5% for LH and FSH assays,
respectively.
In samples in which sufficient serum remained after gonadotropin assays, circulating T, DHT, and E2 levels were determined using direct serum RIA kits (Diagnostic Systems Laboratories, Webster, TX). Limited sample volumes prevented measurement of E2, T, and DHT in all treatment groups. Therefore, serum E2 was measured in E2-treated CAST males, and serum T levels were measured in both intact and T-treated CAST males. Serum DHT was measured only in the DHT-treated CAST males. Due to limited sample volumes, some of the samples were assayed as single tubes for steroid levels. The sensitivities of the T, DHT, and E2 assays were 80, 4, and 5 pg/ml, respectively. All samples were assayed within a single assay, and the intraassay variations were approximately 8%, 4%, and 5% for the T, DHT, and E2 assays, respectively.
Hypothalami were sonicated in 0.1 N acetic acid, boiled for 5 min, and centrifuged at 14,000 rpm in a microfuge at 4 C, and the resulting supernatant was lyophilized overnight. The crude protein pellet was saved for subsequent protein determinations and normalization of GnRH values (41). The lyophilized supernatants were resuspended in assay buffer and subjected to RIA for GnRH as described previously (42). Intra- and interassay variations were approximately 6% and 9%, respectively.
RNA isolation and Northern blot analysis
Total pituitary RNA was isolated from single pituitaries using a
modification of the Trizol reagent protocol (Life Technologies,
Gaithersburg, MD). Briefly, a single pituitary was homogenized in 200
µl Trizol reagent, incubated at room temperature for 5 min, and
extracted with 40 µl chloroform. After thorough mixing, the tubes
were centrifuged at 14,000 x g for 15 min at 4 C.
Glycogen (10 µg/tube) was added to increase the yield of RNA. The
aqueous phase was removed, and RNA was precipitated using 1 vol
isopropanol for 10 min at room temperature. RNA was centrifuged at
14,000 x g for 15 min at 4 C, washed with 70%
ethanol, and resuspended in 40 µl ribonuclease-free water, and the
concentration was determined by UV spectrophotometry.
Aliquots of total RNA (1.0 µg) were suspended in 20 µl RNA loading buffer (Ambion, Austin, TX), fractionated on formaldehyde gels, and transferred overnight to Hybond nylon membranes (Amersham, Arlington Heights, IL) using 10 x SSC (1.5 M sodium chloride-0.15 M sodium citrate). RNA was cross-linked by UV exposure in a Stratalinker (Stratagene, La Jolla, CA). Blots were prehybridized for a minimum of 6 h in 12 ml Northern Max hybridization solution (Ambion) containing salmon sperm DNA. Antisense riboprobes for LHß and PL7 were generated using a Maxiscript kit (Ambion), purified on a Nuc-Trap column (Stratagene), counted, and added to the hybridization buffer at 2 x 106 cpm/ml for both LHß and PL7 probes. After an overnight hybridization at 65 C, blots were washed for 30 min at low stringency (2 x SSC-0.1% SDS, at room temperature), followed by a 30-min wash at high stringency (0.1% SSC-0.1% SDS, at room temperature). Blots were exposed to PhosphorImager screens, and mRNA levels were quantified using a PhosphorImager Storm 860 (Molecular Dynamics, Sunnyvale, CA). Blots were then exposed to Hyperfilm (Amersham) for autoradiography and photography.
Ribonuclease protection assays (RPA)
Antisense riboprobes for the mouse FSHß and cyclophilin genes
were generated using Ambion Maxiscript kits, and RPAs were conducted
using a Hybspeed RPA kit (Ambion). Briefly, total RNA (0.5 µg) was
ethanol precipitated with 50,000 cpm each of the FSHß and cyclophilin
riboprobes, resuspended in 10 µl Hybspeed hybridization buffer (95
C), and incubated at 65 C for 30 min. After hybridization, samples were
treated with a 1:250 dilution of ribonuclease A/T1 mix (Ambion) for 30
min at 37 C. At the end of this period, stop solution was added, and
the RNA was precipitated for a minimum of 1 h at -80 C. Precipitates
were centrifuged at 14,000 rpm for 15 min at 4 C, the supernatant was
carefully removed, and the pellets were resuspended in 8 µl RNA
loading buffer (Ambion). The samples were heated at 95–100 C for 3
min, placed on wet ice, and electrophoresed on a denaturing, 6%
bis-acrylamide gel (8.3 M urea) at 275 V. Gels were fixed
twice for 15 min each time in 10% glacial acetic acid-0.5% glycerol
and dried overnight before exposure to PhosphorImager screens and
Hyperfilm (Amersham).
Statistical analyses
Data for GnRH content, serum LH and FSH, LHß and FSHß mRNA,
and steroid levels were analyzed using Levene’s test for homogeneity
of variance. All of the variables, except the serum DHT levels,
exhibited statistically significant heteroscedascticity. Therefore,
although raw data are graphically presented in the figures, all data
(except for serum DHT) were log transformed for the statistical
analyses. Data were analyzed using two-way ANOVA, and in most cases,
significant interactions between genotype and treatment were noted (see
figure legends). Post-hoc analyses were conducted using a
Student Newman-Keuls test. In all cases, statistical significance was
accepted at P < 0.05.
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Results |
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). No effect was observed with
E2 treatment. Based on SVWs, there were no obvious
genotypic differences in peripheral responses to the androgens.
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Hypothalamic GnRH content
As sex steroids are known to exert feedback effects on LHRH
neurons (5, 7, 9, 11, 45, 46, 47), hypothalamic GnRH contents were
examined. There were no significant differences between mean GnRH
contents of intact WT and ERKO males, and castration significantly
reduced these concentrations by 42% and 33%, respectively (Fig. 2). Treatment with E2
restored GnRH contents in CAST WT males to levels comparable to those
found in intact WT males, but it failed to have any effect in CAST ERKO
males. However, T treatments restored GnRH content to precastration
levels in both WT and ERKO castrates. Interestingly, DHT treatment
completely reversed the castration effect on GnRH content in ERKO
males, but it only produced a partial reversal (51%) of the castration
effect in WT males.
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, A and B). Long term castration
caused 2.0-fold (P > 0.05) and 1.4-fold
(P > 0.05) increases in steady state levels of LHß
mRNA in WT and ERKO males, respectively. Treatments of CAST WT males
with E2 and T suppressed levels of LHß mRNA to 29%
(P < 0.05) and 23% (P < 0.05),
respectively, of the levels found in CAST WT controls (Fig. 3, A and
B), whereas DHT treatments resulted in a 47% increase in LHß mRNA
(P > 0.05). Unlike steroid treatments of CAST WT
males, neither E2, T, nor DHT treatment of CAST ERKOs
resulted in significant changes in steady state levels of LHß mRNA
(Fig. 3, A and B).
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). This increased level of serum LH
agrees roughly with the 2-fold higher levels of LHß mRNA found in
intact ERKO pituitaries. Although castration did not significantly
alter LHß mRNA levels in either WT or ERKO males, castration resulted
in 5-fold (P < 0.05) and 4-fold (P <
0.05) elevations in serum LH in WT and ERKO males, respectively.
Treatments of WT castrates with E2 and T suppressed serum
LH to 14% and 6% (P < 0.05), respectively, of the
values in CAST WT males (Fig. 4). However, E2 treatments of
CAST ERKO males failed to suppress serum LH. On the other hand, T
treatments of CAST ERKO animals suppressed serum LH by 32%
(P > 0.05) relative to levels in CAST ERKO controls.
Interestingly, DHT treatment resulted in a 52% increase
(P > 0.05) in serum LH values in CAST WT while
producing a 58% decrease (P < 0.05) in ERKO
males.
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).
Castration resulted in a 1.7-fold increase (P < 0.05)
in FSHß mRNA levels in WT males (Fig. 5), but it failed to have any
appreciable effect in ERKO males (Fig. 5). Compared with CAST WT
controls, E2 treatments of CAST WT males decreased steady
state levels of FSHß mRNA by approximately 50% (P <
0.05), whereas T and DHT treatments had no effect. In contrast to WT
males, neither castration nor steroid replacement therapy significantly
altered FSHß mRNA levels in ERKO males.
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). Whereas castration elevated FSHß
mRNA only in WT males, it resulted in significant increases (
68%)
in serum FSH in both WT and ERKO males. E2 and T treatments
of CAST WT males suppressed serum FSH by 36% (P <
0.05) and 47% (P < 0.05), respectively, whereas DHT
resulted in a small increase in serum FSH (P > 0.05).
Despite the fact that castration significantly increased serum FSH in
ERKO males, none of the steroid treatments significantly altered serum
FSH in ERKO males (Fig. 6). Thus, in WT males, the
E2-induced suppression of serum FSH is paralleled by a
suppression of FSHß mRNA levels, whereas the T-induced suppression of
serum FSH is not accompanied by a similar suppression of FSHß mRNA
levels. Furthermore, in ERKO males, the postcastration rise in serum
FSH levels without accompanying FSHß mRNA increases is probably due
to the removal of testicular inhibins.
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Discussion |
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are coexpressed in many hypothalamic nuclei and in
gonadotropes (48, 49). Recent data also indicate that the hypothalamus
in rats and mice contains both ERß mRNA and protein (34, 35, 36). To
explore the roles of AR, ER, and ERß in mediating the negative
feedback effects of sex steroids on the hypothalamus and gonadotropes,
we examined the effects of castration and steroid replacement on GnRH
content, gonadotropin gene expression, and serum gonadotropins in WT
and ERKO mice.
Hypothalamic GnRH content
Many studies have documented that GnRH plays a major role in
determining both the synthesis and release of LH from gonadotropes.
However, the exact roles of T and E2 in regulating GnRH
synthesis and secretion remain somewhat controversial. Indeed, studies
have demonstrated that castration can increase (5, 6, 7), decrease (50),
or have no effect (51) on GnRH mRNA levels, whereas exogenous androgens
can either decrease (5, 7) or increase (50) levels of GnRH mRNA. Some
of these differences may be explained by differences in experimental
designs. In several species, castration leads to a rapid increase in
the frequency of GnRH pulses (16, 17, 18, 19), which can be suppressed by T
treatment (17, 19). Although GnRH pulse frequencies increase rapidly
and persist for weeks after castration, a number of studies have also
demonstrated that long term castration results in diminished GnRH
content and that either E2 or androgen replacement can
restore GnRH contents to intact levels (11, 12, 13). Indeed, one study has
demonstrated that in long term castrate guinea pigs, hypothalamic GnRH
contents are diminished, whereas GnRH pulse frequencies are increased
(16). As GnRH content reflects the net effects of changes in
transcription, translation, pro-GnRH processing, GnRH secretion, and
degradation, we measured GnRH content as an initial step in addressing
the roles of AR, ER, and ERß in regulating GnRH neuronal
function.
In these studies, we found that GnRH contents were similar in intact WT
and ERKO males despite the fact that castrated ERKO males were
completely insensitive to exogenous E2. As ERß mRNA
levels appear unaltered in ERKO mice (34, 35, 36), this suggests that
hypothalamic ERß may not play an obvious role in mediating T or
E2 regulation of GnRH content in male mice, and that in the
absence of functional ER, physiological levels of T can effectively
regulate GnRH content directly through AR signaling pathways. However,
given the in vitro evidence that ERß and ER form
heterodimers (52), it is possible that disruption of ER has ablated
any role of ERß in regulating GnRH content. This possibility will
need to be addressed by determining the extent to which ER and ERß
are colocalized in neurons. Future development of ERß-specific
agonists and antagonists and ERß knockout models will also allow
further delineation of any roles that ERß may play in regulating the
GnRH content in male mice.
The fact that both T and DHT treatments of castrated ERKO mice
restored the hypothalamic GnRH content to levels observed in intact
ERKO animals confirms that AR signaling pathways can effectively
regulate the neuronal GnRH content. Although DHT treatments of WT males
were less effective than that of CAST ERKO males, both DHT and T
treatments of CAST WT males also resulted in significant elevations of
GnRH content. The ability of both E2 and DHT to regulate
GnRH content in WT males suggests that both AR and ER signaling
pathways can regulate hypothalamic GnRH contents in male WT mice.
However, the increased effectiveness of DHT in ERKO male mice suggests
that ER disruption has increased the sensitivities of hypothalamic
target cells to androgens. This raises the possibility that the ER
disruption may have resulted in developmental perturbations that lead
to increased sensitivity to DHT.
These experiments measured only GnRH content, which is a reflection of
the net rates of pro-GnRH synthesis, processing, and secretion. Thus,
it is possible that AR and ER receptor systems may regulate
different steps in the synthesis and secretion of GnRH that yield
similar results in terms of GnRH contents. Indeed, it will be
interesting to determine the effects of these treatments on GnRH mRNA
levels and GnRH secretion patterns in both WT and ERKO males.
Nonetheless, these data are consistent with studies suggesting that
both AR and ER signaling pathways can regulate hypothalamic GnRH
content (5, 11, 12, 13).
LHß mRNA expression and serum LH
In addition to regulating GnRH synthesis and secretion, various
investigators have shown that E2 and androgens can suppress
LHß mRNA and serum LH levels (1, 2, 20, 21, 23). Despite complete
E2 insensitivity, we found that both LHß mRNA and serum
LH levels in intact male ERKOs were only 2-fold higher than those in
intact WT males. As GnRH contents were comparable, these data suggest
that the slightly higher levels of LHß mRNA and serum LH in intact
ERKO males stem from estrogen insensitivity at the level of the
gonadotrope. However, this hypothesis must be examined by testing for
genotypic differences in GnRH release patterns and in vitro
challenges of primary pituitary cell cultures with steroids and
GnRH. Nonetheless, it is apparent that ER disruption results in
relatively small elevations in LHß mRNA and serum LH levels in male
ERKO mice compared with the 8- to 10-fold increase in LHß mRNA and
serum LH levels in ERKO females (53, 54). This suggests that ER
plays a more dominant role in regulating LHß mRNA and serum LH levels
in females, whereas AR signaling pathways are relatively effective in
regulating LH synthesis and secretion in intact male mice. This
hypothesis is supported by the fact that intact Tfm mice have
significantly elevated serum LH values that are not further elevated by
castration (27).
Long-term castration of both WT and ERKO males induced small (1.5- to 2-fold) elevations in LHß mRNA levels compared with the effects of castration in male rats (1, 2). The discrepancy between the data from this study and those from rat studies may reflect species differences, as an earlier mouse study reported similar elevations of LHß mRNA after castration (27). Alternatively, as reported for male rats, it is possible that LHß mRNA is rapidly induced after castration and subsequently decreases with increasing length of castration. Interestingly, the large increases in serum LH levels (4- to 5-fold) in castrated male WT and ERKO mice were of much greater magnitude than the increases in LHß mRNA levels. This suggests that alterations in pulsatile GnRH secretion and/or gonadotrope sensitivity to GnRH may be primarily responsible for the elevated serum LH observed in long term castrated WT and ERKO males. This interpretation is consistent with experiments demonstrating that castration leads to increased GnRH pulse frequency and elevated serum LH in several species (16, 17, 18, 19).
Both E2 and T significantly suppressed LHß mRNA and serum
LH levels in WT castrates while exerting no significant effects in
castrated ERKOs. Thus, it appears that aromatization and activation of
ER enhance the feedback effects of T on LH synthesis and secretion
in male mice. It is possible that the lower serum T levels found in
T-treated ERKO mice may account for this genotypic difference in the
effects of T. However, the range of serum T levels found in T-treated
CAST ERKOs overlapped the range of serum T values in intact ERKOs.
Furthermore, despite the fact that intact ERKOs have 2-fold higher
serum T levels than intact WT males, serum LH values remain 2-fold
higher in intact ERKO males. Thus, the genotypic difference in the
ability of exogenous T to suppress serum LH may be primarily due to the
ER disruption. In theory, ER disruption could compromise
T-induced suppression of GnRH pulse frequency or alter gonadotrope
sensitivity to GnRH and rates of transcription of the gonadotropin
subunits. We are presently addressing these possibilities with
experiments that examine GnRH pulse frequencies and responses of
pituitary cell cultures to steroid and GnRH challenges. Studies have
suggested that direct effects of E2 and T on gonadotropes
may include the regulation of GnRH receptor numbers (55, 56, 57),
intracellular responses to GnRH (56, 57), or transcriptional activity
of gonadotropin subunit genes (23, 58). It will be interesting to
determine the extent to which ER vs. AR signaling
pathways regulate GnRH receptor levels, basal levels of transcription
of the GSU or LHß genes, and gonadotrope responsivity to GnRH.
Despite data indicating that aromatization and activation of ER
greatly enhance the feedback effects of T on LHß mRNA and serum LH
levels, it is unclear why T treatments of CAST ERKO males did not
suppress serum LH closer to the levels found in intact ERKO males. Some
studies have demonstrated that the duration of castration and steroid
therapy or steroid dosages may influence the magnitudes of change in
LHß mRNA and serum LH responses (1). Thus, it is possible that
different lengths of castration and T treatments or different dosages
of T might result in a more robust suppression of serum LH in ERKO
mice. An intriguing possibility is that AR signaling pathways may
suffice to maintain negative feedback control over serum LH levels, but
that ER signaling pathways are more potent and, therefore, are
better able to reinstate negative feedback control over highly elevated
serum LH levels found in CAST males. Finally, it is also possible that
T was not as effective in male ERKOs because, in the absence of
functional ER, nonsteroidal testicular factors may play an enhanced
role in mediating LH synthesis and secretion.
Based on experiments demonstrating that exogenous DHT suppressed GnRH
mRNA levels (7) and serum LH in rats (24), and that serum LH is
elevated in Tfm mice (27), we had anticipated that DHT would
effectively suppress serum LH in both WT and ERKO males. Surprisingly,
we found that DHT treatments consistently resulted in small increases
in serum LH and LHß mRNA levels in CAST WT males while significantly
suppressing serum LH in CAST ERKO males. Furthermore, DHT was also less
effective in raising GnRH content in CAST WT males. It is possible that
DHT failed to suppress serum LH levels in CAST WT because of
differences in experimental design (chronic castration and DHT
treatments) or because of a pharmacological DHT effect. Nonetheless,
these possibilities do not address the observation that identical DHT
treatments exerted opposite effects on serum LH levels in male WT and
ERKO mice. Perhaps the most parsimonious explanation of this phenomenon
would be genotypic differences in metabolism and clearance that
resulted in different levels of circulating DHT. However, RIA confirmed
that serum DHT levels were similar in DHT-treated WT and ERKO males.
Thus, it appears that the absence of functional ER has altered
hypothalamic and/or pituitary sensitivities to DHT. Such altered
sensitivities may be due to developmental changes in patterns of AR
expression, expression of steroid-metabolizing enzymes, or
developmental selection for cells that are more sensitive to androgens.
It is also possible that ER disruption has simply increased the
availability of transcription factors common to both ER and AR
signaling pathways. Recent studies indicate no obvious genotypic
difference in the levels of hypothalamic AR found by
immunohistochemistry (59) or in the levels of AR mRNA in the
pituitaries of WT and ERKO mice (our unpublished data). We are
presently determining whether ER disruption has altered patterns of
expression of steroid-metabolizing enzymes in androgen target cells or
altered availability of transcription cofactors common to both ER
and AR signaling pathways.
Although the ERKO mouse has provided some interesting insights into the
nuclear receptor systems mediating the feedback effects of T on serum
LH, it is important to recognize the possibility that ablation of ER
signaling pathways has led to the development or enhancement of
alternative feedback mechanisms. Indeed, the genotypic differences in
the effects of DHT suggest that this may have occurred and that,
therefore, ER signaling pathways may play a more important role in
WT mice. However, studies of Tfm mice indicate that serum LH levels are
much higher than those in ERKO males and that, unlike ERKO males,
castration of Tfm mice does not lead to a further increase in serum LH
levels (27). Thus, comparisons of these two models of steroid
insensitivity support the general conclusion that AR signaling pathways
are more dominant than ER signaling pathways in regulating serum LH
levels in male mice.
FSHß mRNA expression and serum FSH
The synthesis and secretion of FSH are stimulated by GnRH and
activins and suppressed by inhibins and estrogens. However, the
feedback effects of T are more complex. For instance, some studies
demonstrate that in vivo T treatments can suppress serum FSH
(1), whereas other studies illustrate that T can stabilize or stimulate
levels of FSHß mRNA, FSH secretion, and serum FSH when endogenous
GnRH stimulation is eliminated through the use of GnRH antagonists
in vivo (60) or primary pituitary cultures (1). Given the
absence of functional ER and elevated serum T levels in ERKO males,
it was anticipated that FSHß mRNA levels might be elevated in ERKO
males. However, FSHß mRNA levels were comparable in intact WT and
ERKO mice. This suggests that estrogen insensitivity at the level of
the pituitary does not have a significant impact on basal levels of
FSHß mRNA in pituitaries of intact ERKO males. In contrast, ER
disruption in female mice leads to a 7- to 8-fold increase in FSHß
mRNA levels (53). This suggests that a sex difference exists in the
importance of ER in regulating FSHß mRNA synthesis.
Consistent with earlier rodent studies (1, 2), we found that castration
increased FSHß mRNA levels in WT males and that E2
treatments suppressed this increase in WT males. The absence of an
androgen effect on FSHß mRNA levels agrees with several studies
suggesting that androgens may stabilize FSHß mRNA levels (1, 2). In
contrast to WT males, FSHß mRNA levels in ERKO males were not
significantly altered by any of the treatments, and therefore, it
appears that FSHß mRNA levels are relatively static in long term
castrated and steroid-replaced ERKO males. However, it is unclear why
the large androgen-induced changes in GnRH content did not induce
changes in steady state levels of FSHß mRNA. These data suggest that
either the changes in hypothalamic GnRH content in ERKO mice are not
accompanied by changes in GnRH secretion patterns or, more likely, that
ER-mediated effects on the gonadotrope are required for GnRH
regulation of FSHß mRNA levels.
In agreement with the normal levels of FSHß mRNA in intact ERKO pituitaries, we found that serum FSH levels were only slightly elevated in ERKO males. In addition, castration significantly increased serum FSH levels in both WT and ERKO males, whereas FSHß mRNA levels were increased only in WT males. This indicates that the postcastration rise in serum FSH in ERKO males is independent of changes in steady state levels of FSHß mRNA. Furthermore, as none of the steroid treatments exerted any significant effect on serum FSH levels in ERKO males, we believe that the postcastration rise in serum FSH levels in ERKO males is due to removal of nonsteroidal testicular factors such as inhibin.
Whereas inhibins may play a dominant role in regulating serum FSH
levels in ERKO males, it is clear that both E2 and T can
effectively suppress serum FSH in WT males. Interestingly, only the
E2 treatments of CAST WT males were effective at
suppressing postcastration increases in FSHß mRNA, whereas both
E2 and T suppressed the postcastration rise in serum FSH.
This indicates that mechanisms underlying T suppression of serum FSH in
WT males are separate and distinct from the effects of T on steady
state levels of FSHß mRNA. It is possible that T treatments
stabilized or increased FSHß mRNA levels as reported in rats (1, 60),
but suppressed serum FSH in WT males through a reduction in GnRH pulse
frequency. However, T treatments elevated GnRH content but failed to
suppress serum FSH in male ERKOs. Thus, in WT mice, it is likely that T
is aromatized and acts through gonadotrope ER to modulate the
translation and/or secretion of FSH independently of changes in FSHß
mRNA levels. The fact that T treatments failed to suppress serum FSH in
ERKO males lends further support to this hypothesis.
In summary, we have employed the ERKO mouse, a genetic model of
estrogen insensitivity, to explore the roles of AR, ER, and ERß in
regulating gonadotropins. The data presented here indicate that ER
is the predominant signaling pathway involved in mediating
E2 suppression of GnRH content, serum gonadotropins, and
gonadotropin subunit mRNA expression in male mice. Thus, it does not
appear that hypothalamic ERß plays an obvious role in mediating
negative feedback regulation of gonadotropins in male mice. Comparisons
of serum LH data in this paper with serum LH values in Tfm mice also
indicate that AR signaling pathways are the dominant signaling pathways
by which T suppresses serum LH in male mice. Indeed, in the absence of
functional ER, androgens can effectively act through AR signaling
pathways to regulate GnRH content, and this may be the predominant
pathway by which androgens regulate serum LH in ERKO males.
Nonetheless, it does appear that aromatization and activation of
gonadotrope ER are required to achieve the levels of LHß mRNA and
serum LH found in WT males. Interestingly, the ability of both E and T
to suppress serum FSH in WT mice is clearly dependent on the presence
of ER. It also appears that T suppresses serum FSH levels through
ER-mediated posttranscriptional effects that are independent of
steady state levels of FSHß mRNA. Finally, in the absence of
functional ER, nonsteroidal testicular factors (i.e.
inhibins) may effectively regulate serum FSH levels in male mice.
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Acknowledgments |
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Received March 20, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit gene by androgen receptor occurs
independently of DNA binding but requires the DNA-binding and
ligand-binding domains of the receptor. Mol Endocrinol 11:1497–1506 (ER) and estrogen receptor-ß (ERß) mRNA
in the wild-type and ER-knockout mouse. Endocrinology 138:4613–4621-reductase
activity in pituitaries of male rhesus macaques. J Clin Endocrinol
Metab 66:1251–1258 and ß form heterodimers on DNA. J Biol
Chem 272:19858–19862 in the
anterior pituitary gland. Mol Endocrinol 11:674–681T3–1 cells. Mol Cell
Endocrinol 87:95–103[CrossRef][Medline]
-subunit gene
despite the absence of high affinity binding site for estrogen
receptor. Mol Endocrinol 5:725–733This article has been cited by other articles:
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|
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|
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|
|
|
|
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|
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|
|
|
|
|
|
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|
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|
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|
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|
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|
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|
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|
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J. F. Couse and K. S. Korach Estrogen Receptor Null Mice: What Have We Learned and Where Will They Lead Us? Endocr. Rev., June 1, 1999; 20(3): 358 - 417. [Abstract] [Full Text]
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