Chronic Hypersecretion of Luteinizing Hormone in Transgenic Mice Selectively Alters Responsiveness of the
-Subunit Gene to Gonadotropin-Releasing Hormone and Estrogens
Rula A. Abbud,
Rebecca K. Ameduri,
J. Sunil Rao,
Terry M. Nett and
John H. Nilson
Department of Pharmacology (R.A.A., R.K.A., J.H.N.) Department
of Biostatistics (J.S.R.) Case Western Reserve University
Cleveland, Ohio 44106
Animal Reproduction and Biotechnology
Laboratory (T.M.N.) Colorado State University Fort Collins,
Colorado 80523
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ABSTRACT
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Steroid hormones can act either at the level of
the hypothalamus or the pituitary to regulate gonadotropin subunit gene
expression. However, their exact site of action remains controversial.
Using the bovine gonadotropin
-subunit promoter linked to an
expression cassette encoding the ß-subunit of LH, we have developed a
transgenic mouse model where hypersecretion of LH occurs despite the
presence of elevated ovarian steroids. We used this model to determine
how hypersecretion of LH could occur when steroid levels are
pathological. During transition from the neonatal period to adulthood,
the endogenous LHß subunit gene becomes completely silent in these
mice, whereas the
-directed transgene and endogenous
-subunit
gene remain active. Interestingly, gonadectomy stimulates expression of
the endogenous
and LHß subunit genes as well as the transgene;
however, only the endogenous LHß gene retains responsiveness to
17ß-estradiol and GnRH. In contrast, LH levels remain responsive to
negative regulation by androgen. Thus,
-subunit gene expression, as
reflected by both the transgene and the endogenous gene, has become
independent of GnRH regulation and, as a result, unresponsive to
estradiol-negative feedback. This process is accompanied by a decrease
in estrogen receptor
gene expression as well as an increase in the
expression of transcription factors known to regulate the
-subunit
promoter, such as cJun and P-LIM. These studies provide in
vivo evidence that estrogen-negative feedback on
and LHß
subunit gene expression requires GnRH input, reflecting an indirect
mechanism of action of the steroid. In contrast, androgen suppresses
-subunit expression in both transgenic and nontransgenic mice. This
suggests that androgens must regulate
-subunit promoter activity
independently of GnRH. In addition to allowing the assessment of site
of action of sex steroids on
-subunit gene expression, these studies
also indicate that chronic exposure of the pituitary to LH-dependent
ovarian hyperstimulation leads to a heretofore-undescribed pathological
condition, whereby normal regulation of
, but not LHß, subunit
gene expression becomes compromised.
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INTRODUCTION
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Regulation of LH synthesis and secretion involves an integrated
hypothalamic-pituitary-gonadal axis. Pulsatile release of GnRH from the
hypothalamus stimulates synthesis and secretion of LH (1, 2, 3). In
females, LH stimulates thecal cell production of androstenedione, which
diffuses to granulosa cells and undergoes conversion to estrogen by
aromatase. FSH controls aromatase activity and also induces appearance
of LH receptors in granulosa cells, further augmenting production of
estrogen (4).
There is little doubt that sustained levels of androgens or estrogens
feed back to inhibit secretion of LH and suppress levels of
and
LHß mRNAs both in animal models and in in vitro studies
(see Refs. 2, 5, 6 for review). The site of steroid action,
however, remains unknown. Numerous studies in rodents support a
hypothalamic site of action for steroid feedback, namely regulation of
GnRH secretion. Others propose that estrogens feed back at both the
hypothalamus and pituitary, while androgens act only at the
hypothalamus (5). At the level of the pituitary, estrogen appears to
modulate GnRH receptors to alter the responsiveness of the pituitary to
GnRH (5). Finally, many of the discrepancies noted between these
studies may reflect significant species-specific variation. In primates
and humans, direct action of steroid hormones at the level of the
pituitary has been reported (5, 7). Thus, the site of steroid action in
control of LH synthesis and secretion still remains controversial.
Genetic evidence suggests that steroid receptors are critical for
controlling gonadotropin gene expression. Elevated levels of LH are
found in Tfm mice that lack functional androgen receptors
(ARs) (8). This is a male-limited genetic trait as AR resides on the X
chromosome (9, 10), and LH hypersecretion only occurs with complete
loss of AR. Similarly, estrogen receptor knockout (ERKO) studies
reported by Korach and colleagues (11, 12, 13) provide new evidence for the
importance of ERs in controlling LH secretion. Female ERKO mice secrete
elevated levels of LH and develop cystic ovaries. While these
observations provide evidence for the importance of androgen feedback
in the male and estrogen feedback in the female, they can not delineate
the exact site of action of the steroids.
If sex steroids act at the level of gonadotropes to regulate secretion
of LH, then gonadotropin subunit promoters must be regarded as likely
targets. Numerous reports of steroid suppression of gonadotropin gene
expression further increase this likelihood (see Refs. 2, 5 for
review). Indeed, we demonstrated recently that AR represses activity of
the
-subunit promoter in
T31 cells through a mechanism that
requires the DNA-binding domain of the receptor but not its direct
binding to the 5'-flanking region (14, 15). In contrast to AR, we were
unable to detect a high-affinity binding site for ER within the human
-subunit gene or to observe a negative transcriptional effect of ER
in transfection assays (16). While another group observed binding of ER
to a 29-bp region within the proximal
-subunit promoter (17),
transcriptional regulation occurred only in the presence of thyroid
hormone receptor in GH3 cells.
With regard to the LHß gene, Shupnik and Rosenzweig (18)
reported a high affinity-binding site for ER between -1174/-1159 bp
of the 5'-flanking region of the rat gene. Estrogen, however, conferred
a positive, rather than negative, response when this element was tested
for activity by transfection assays in GH3 cells. Subsequently, we
observed that a truncated bovine LHß promoter (780 bp) directs
expression of reporter genes specifically to gonadotropes in transgenic
mice (19). This promoter also conferred positive responsiveness to GnRH
and negative responsiveness to androgens and estrogens. Ironically,
this steroid-responsive promoter lacked high-affinity binding sites for
both AR and ER, suggesting that steroid receptors repress transcription
of the bovine LHß gene, and possibly other mammalian LHß genes,
through an indirect mechanism.
Recently, we constructed a new transgenic mouse model in which elevated
serum levels of a chimeric LH heterodimer were maintained chronically
without requiring multiple injections, pharmacological dosing, or
artificial dampening of the hypothalamic-pituitary-gonadal axis (20, 21). The transgene contained a bovine
-subunit promoter fused to the
coding region of a chimeric LHß subunit. The
-subunit promoter
ensured that transgene expression occurred only in gonadotropes (20, 22, 23). The LHß chimera contained the carboxyl-terminal peptide
(CTP) of the human CG ß-subunit linked to the carboxyl terminus of
bovine LHß. Addition of the CTP moiety increased the serum half-life
of heterodimers containing the chimeric ß-subunit (20). The
CTP-containing chimera was expressed only in gonadotropes of transgenic
mice, causing elevated levels of LH. These mice were infertile,
ovulated infrequently, maintained a prolonged luteal phase, and
developed pathological ovarian changes including polycystic
ovaries, marked enlargement of ovaries, and granulosa cell
tumors (20, 21, 24). In addition, testosterone and estradiol
(E2) levels were increased compared with
nontransgenic littermates with an overall increase in the ratio of
testosterone to E2 (20). In short, the biochemical and
morphological features of these mice bear a striking resemblance to
those of women with functional ovarian hyperandrogenism (25).
Our transgenic model demonstrates, for the first time, a direct
association between abnormal secretion of LH, development of functional
ovarian hyperandrogenism, and infertility. Since the transgene is the
only variable in this animal model, expression of transgenic LH must be
the initiating step for all subsequent pathological events. However,
hypersecretion of LH occurred in female but not male mice. In female
transgenic mice, the high levels of LH suggest that the gonadotropin
genes, including the transgene, must manifest a resistance to
steroid-negative feedback. Thus, this model provides a unique
opportunity to explore developmentally how high levels of LH can be
maintained in the presence of a chronically hyperstimulated ovary. The
work reported herein reveals several unexpected findings that are
relevant to both site of action of gonadal steroids and the dependency
of their action on GnRH.
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RESULTS
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Hypersecretion of LH Occurs Despite the Presence of Apparent
Gonadal Feedback
To establish a frame of reference for understanding how
hypersecretion of LH occurs in transgenic females in the presence of
elevated gonadal steroids, we measured serum LH in intact and
ovariectomized (OVX) animals. Mean serum levels of LH in intact
transgenic mice are elevated compared with their nontransgenic
counterparts (Fig. 1
). In fact, levels of
LH in transgenic females are comparable to those in OVX nontransgenic
mice (Fig. 1
). Transgenic mice, however, do not lack gonadal feedback,
because serum levels of LH increase to even higher levels after
ovariectomy. This suggests that the high levels of sex steroids in the
intact transgenic animal are at least partially capable of suppressing
LH levels.

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Figure 1. Hypersecretion of LH in Female Transgenic Mice Is
Not Due to the Lack of Gonadal Feedback
Serum LH levels in female transgenic and nontransgenic (control) mice
in the presence or absence of the gonads. Age-matched female transgenic
(Intact, n = 5; OVX, n = 4) and nontransgenic (Intact, n
= 7; OVX, n = 6) mice were killed in the morning hours at random
stages of the estrous cycle. a, P <
0.05 when compared with Intact of the same genotype.
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Partial responsiveness to gonadal feedback could reflect changes in
gonadal regulation of LHß encoded by the transgene, the endogenous
gene, or both. To begin to address these possibilities, we used an
RNAse protection assay to measure the relative concentration of the
endogenous mouse LHß (mLHß) and the bLHßCTP transgene-encoded
mRNAs in total RNA prepared from individual mouse pituitaries. This
assay allowed us to distinguish between the bLHßCTP transgene and
endogenous mLHß gene. In intact transgenic females, bLHßCTP mRNA is
readily detectable, whereas the endogenous mLHß mRNA is not
discernible (Fig. 2
). Upon gonadectomy,
the endogenous mLHß mRNA becomes apparent and bLHßCTP mRNA appeared
to increase even further. In contrast, endogenous mLHß mRNA is
readily detectable in nontransgenic animals and increases after
ovariectomy. These results indicate that serum LH in intact, transgenic
adults is comprised of ß-subunit produced solely by the bLHßCTP
transgene. Furthermore, whereas gonadal factors completely suppress the
endogenous LHß gene in transgenic animals, they only partially
suppress the bLHßCTP transgene. Since the LHß-encoding transgene is
linked to the heterologous
-subunit promoter, we posit that the
and LHß promoters respond differentially to gonadal feedback.

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Figure 2. Reversible Suppression of Endogenous Mouse LHß
mRNA Levels in Transgenic Mice
RNAse protection assays were performed to detect mLHß, transgene, and
actin mRNA levels in individual mouse pituitaries from transgenic and
nontransgenic mice. Quantitative analysis of mLHß and transgene mRNA
expressed relative to actin in transgenic and nontransgenic (Control),
OVX, and ovarian-intact (Intact) pituitaries. aP < 0.05 when compared with Intact. bP < 0.05 when compared with Control.
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Partial Escape of Transgene Expression from Suppression by Elevated
Ovarian Steroids Becomes Evident at the Onset of Puberty in Female
Transgenic Mice
We reported previously that hypersecretion of LH occurred as early
as 14 days of age even though androgens and estrogens were at levels
higher than those seen in an adult nontransgenic mouse (21). To study
the relative contribution of the bLHßCTP transgene and the endogenous
LHß gene to the total LHß mRNA pool during early pre- and postnatal
development in female transgenic mice, we used both double-label
in situ hybridization and RNAse protection assays. This
analysis further confirmed that the transgene is expressed specifically
in cells that express the
-subunit of LH (data not shown).
Expression of the transgene was detected with an
35S-labeled probe, whereas a digoxigenin-labeled probe was
used to assess expression of the endogenous LHß gene. Expression of
endogenous mLHß mRNA was observed from embryonic day 19 up to
postnatal day 14 in transgenic animals (Fig. 3
, panels BD) and was similar to that
observed in nontransgenic littermates (data not shown). Activity of the
bLHßCTP transgene was also detected in the pituitary at these early
stages of development (Fig. 3A
and data not shown). In contrast, little
staining for endogenous mLHß mRNA was observed in pituitary sections
from adult female transgenic mice, especially when compared with
nontransgenic siblings (Fig. 3
, E and F). As expected, adult transgenic
pituitaries expressed high levels of the transgene (data not
shown).

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Figure 3. Endogenous mLHß Gene Expression Becomes
Suppressed after Puberty
A, Pattern of transgene expression in the anterior pituitary at
embryonic day 19. Dark field image showing the results from in
situ hybridization using 35S-labeled transgene
probe on coronal sections from an embryo at embryonic day 19. The
transgenic signal appears as silver grains in the developing anterior
pituitary, Magnification, x40. BF, Endogenous mLHß gene expression
in transgenic female mice at E19 (B, x40), P-1 (C, x40), P-14 (D,
x40), and adult (E, x400) stages of development. The mLHß signal
appears as a dark stain using digoxigenin-labeled mLHß riboprobe for
in situ hybridization. Note that endogenous gene
expression becomes completely suppressed in the adult pituitary. F,
Pattern of mLHß gene expression in adult nontransgenic mouse
pituitaries using in situ hybridization with
digoxigenin- labeled mLHß riboprobe.
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To determine when endogenous LHß gene expression becomes suppressed,
we performed RNAse protection assays on single pituitaries from animals
killed at 3, 4, and 6 weeks of age. As shown in Fig. 4
, endogenous mLHß mRNA levels
progressively declined, becoming completely suppressed by 6 weeks of
age. At 3 weeks of age both the transgene and endogenous gene
contribute equally to pituitary LHß mRNA, coincident with precocious
follicular maturation as evidenced by early antral formation (24).
Complete suppression of endogenous gene expression occurs at 6 weeks of
age, a time normally associated with the onset of puberty. In
nontransgenic animals, gonadal regulation of pituitary gonadotropin
levels becomes fully mature at puberty, around 6 weeks of age (26).
This is clearly not the case for the transgenic mice because the
transgene is demonstrating partial gonadal feedback insensitivity.
Thus, secreted LH remains elevated and ultimately causes the demise of
ovarian function.

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Figure 4. Endogenous mLHß but Not bLHßCTP Transgene
Expression Becomes Suppressed by 6 Weeks of Age
Pattern of transgene and mLHß gene expression expressed relative to
actin in female transgenic pituitaries at 3 (n = 3), 4 (n =
6), and 6 (n = 7) weeks of age as determined by RNAse protecion
assay. a P < 0.05 when compared with 3
weeks.
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Selective Resistance to Estrogen-Negative Feedback Causes
Hypersecretion of LH
To further understand how transgene expression becomes partially
insensitive to gonadal feedback in adult females, we evaluated levels
of serum LH in OVX animals before and after selective steroid hormone
replacement. Figure 5
shows that
replacement with 5
-dihydrotestosterone (DHT), a nonaromatizable
analog of T, suppressed LH levels to the same extent in both transgenic
and nontransgenic female mice (mean OVX transgenic: 64.6 ± 1.2
ng/ml; OVX nontransgenic: 5.3 ± 0.9 ng/ml; OVX + DHT transgenic:
9.9 ± 4.6 ng/ml; OVX + DHT nontransgenic: 1.3 ± 0.3 ng/ml).
Indeed, mean serum LH levels in DHT-treated females were
indistinguishable from the levels observed in intact female transgenic
mice (data not shown). This suggests that androgens may be the primary
agent for gonadal feedback in transgenic females. In further support of
this notion, 17ß-estradiol suppressed LH levels less effectively in
transgenic mice than in nontransgenic littermates (mean OVX transgenic:
68.3 ± 6.6 ng/ml; OVX nontransgenic: 6.5 ± 1.4 ng/ml; OVX +
E transgenic: 25.8 ± 2.28 ng/ml; nontransgenic: 0.6±0.1 ng/ml).
Therefore, hypersecretion of LH appears to reflect transcriptional
resistance to estrogen-negative feedback. Since the bLHßCTP transgene
is the primary source of secreted LH in intact females, it follows that
the
-subunit promoter must be the target of this partial
resistance.

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Figure 5. Suppression of the Postovariectomy Rise in LH by
E2, but Not DHT, Is Compromised in Female Transgenic Mice
Serum levels of LH are expressed relative to the levels obtained after
ovariectomy in animals implanted with either E2 or DHT
pellets. Both transgenic and nontransgenic animals were OVX for several
weeks before administration of the steroid implants. OVX + DHT,
Nontransgenic: n = 5; OVX + DHT, transgenic: n = 6; OVX +
E2, nontransgenic: n = 6, OVX + E2,
transgenic: n = 6. a P < 0.05
when compared with nontransgenic.
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The rise in serum LH in transgenic animals after ovariectomy
results from contributions by both the endogenous LHß gene and the
chimeric bLHßCTP transgene. To determine whether altered
responsiveness to E2 is a unique property of the transgene,
we measured the effect of E2 implants after ovariectomy on
mLHß and bLHßCTP transgene mRNA levels. Whereas both endogenous
mLHß and bLHßCTP transgene mRNA levels increased after ovariectomy,
only the endogenous mLHß gene was suppressed by 17ß-estradiol
implants (Fig. 6
). These results suggest
that the hypersecretion of LH in adult female transgenic mice could be
attributed to resistance of the transgene to negative feedback by
E2. Interestingly, although the transgene remains
responsive to androgen regulation, androgens in the intact transgenic
female alone are insufficient to prevent LH hypersecretion.

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Figure 6. The Transgene Is Unresponsive to
E2-Negative Feedback in Female Transgenic Mice
mLHß and transgene mRNA levels in intact (n = 9), OVX (n =
13), and OVX + E (n = 9) were determined by RNAse protection
assay, normalized relative to actin mRNA levels, and expressed relative
to OVX levels. a P < 0.05 when
compared with OVX, b P > 0.05 when
compared with OVX.
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Expression of the Endogenous
-Subunit Gene Also Becomes
Resistant to Estrogen-Negative Feedback in Response to Chronic LH
Hypersecretion
The inability of E2 to regulate transgene expression
is surprising in light of previous work demonstrating that this same
bovine
-subunit promoter responds to estrogen-negative feedback when
linked to chloramphenicol acetyltransferase (CAT) in transgenic mice
(14, 16). This suggests that chronic hypersecretion of LH causes the
truncated
-subunit promoter to become unresponsive to
estrogen-negative feedback. To determine whether this was a unique
property of the transgene or whether a fundamental change had occurred
in the normal endocrine feedback loop, we assessed the ability of
E2 to regulate the endogenous
-subunit gene. As shown in
Fig. 7
, expression of the endogenous
mouse
-subunit mRNA is compromised in transgenic mice when compared
with nontransgenic littermates. While it is still responsive to
ovariectomy, it fails to respond to E2 implants. Therefore,
the endogenous mouse
-subunit promoter becomes dysregulated in the
same manner as the transgene. These findings suggest that a
pathological change has occurred that affects appropriate regulation of
but not LHß subunit gene expression.

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Figure 7. The Endogenous -Subunit Becomes Unresponsive to
E2-Negative Feedback
Mouse mRNA was determined by RNAse protection assay and expressed
relative to actin. Data shown are from nontransgenic (Control) and
transgenic, intact (Control, n = 5; Transgenic, n = 6), OVX
(Control, n = 5; Transgenic, n = 3), and OVX + E (Control,
n = 5; Transgenic, n = 4) mice. aP < 0.05 when compared with OVX. bP > 0.05 when compared with OVX.
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The Loss of
-Subunit Responsiveness to
E2 Correlates with a Loss of Regulation by
GnRH
Estrogen-negative feedback on gonadotropin gene expression can
occur either at the level of the hypothalamus, pituitary, or both (1, 2). If the repressive effect of estrogen on
-subunit promoter
activity occurs predominately at the hypothalamus by diminishing
production of GnRH, then administration of a GnRH antagonist should
block the postcastration rise in
-subunit mRNA. Surprisingly,
treatment of OVX transgenic females with antide, a GnRH antagonist, had
no impact on transgene mRNA, whereas it was much more effective in
blocking the postcastration rise in endogenous LHß mRNA (Fig. 8
). This suggests that expression of the
transgene is independent of GnRH and its postovariectomy rise is due to
regulation at the level of the pituitary rather than the hypothalamus.
This independence of GnRH regulation is selective for the
-subunit
promoter since the endogenous LHß mRNA retains responsiveness to
antide. Furthermore, GnRH levels must be quite low in intact transgenic
females since treatment with antide had no impact on serum levels of LH
(data not shown). However, this treatment had no measurable effect in
nontransgenic randomly cycling females because circulating levels of LH
in these animals are at the limits of detection by RIA. Consequently,
treatment with antide has a measurable effect only when levels of LH
are first elevated by ovariectomy, which causes a dramatic increase in
GnRH. The inability of antide to block the postcastration rise in
transgenic bLHßCTP mRNA, but not endogenous LHß mRNA, provides
evidence for the reprogramming of the responsiveness of the
-subunit
promoter. In addtion, it indicates that the
-subunit promoter
retains appreciable activity even in this environment of low GnRH. More
importantly, since E2 fails to suppress transgene activity
in this altered endocrine milieu, the steroid must normally exert its
effect on the
-subunit promoter through an indirect mechanism that
involves regulation of GnRH.

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Figure 8. Transgene Expression Becomes Independent of GnRH in
Transgenic Mice
Transgene and endogenous (mLHß) mRNA levels expressed relative to
actin in transgenic and nontransgenic (Control) mice that were OVX and
treated either with saline (ovx + saline) or antide (ovx + antide).
n = 47 animals per treatment group. aP < 0.05 when compared with OVX + saline.
b P > 0.05 when compared with OVX +
saline.
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Increased Expression of trans-Activators of the
-Subunit
Promoter in the Pituitaries of Female Transgenic Mice: Possible
Mechanism of
-Subunit Autonomy from GnRH and
E2
To further understand how
but not LHß becomes independent of
GnRH regulation and refractory to estrogen-negative feedback, we
measured the expression of GnRH (GnRH-R), androgen (AR), and the
-isoform of estrogen (ER) receptors using reverse Northern assays.
We chose to study the
- but not ß-isoform of ER because ERß does
not appear to be expressed in the pituitary (27). In addition, ERKO
mice, which express ERß but not ER
, have elevated levels of LH
despite high levels of E2 (13). Thus,
E2-negative feedback on LH appears to be mediated by ER
rather than ERß. In addition, we examined the expression pattern of
several transcription factors that are known to regulate both
and
LHß subunit gene expression. These include steroidogenic factor-1
(SF-1) (28, 29, 30); P-Otx/Ptx1 (31, 32); P-LIM (33, 34), cJun (35), and
cAMP response element binding protein (CREB) (35). SF-1 and
P-Otx/Ptx1 act synergistically to regulate the LHß promoter (31).
P-Otx/Ptx1 also acts in synergy with another factor, P-LIM, to regulate
-subunit gene expression (32). In the pituitary, expression of the
-subunit promoter in the transgene and endogenous
-subunit gene
depends on a variant cAMP response element sequence (CRE). CREB
binds this sequence in pituitary cell lines but at a low affinity.
However, cJun and ATF-2 also bind this sequence but with high affinity
in the pituitary (35). Furthermore, we analyzed the expression of the
protooncogenes cFos, ras, and c-myc.
Statistical analysis of the reverse Northern data required inverse log
transformation. Figure 9
shows the
difference between the inverse log net counts in transgenic and
nontransgenic samples. Only three factors showed statistical difference
in their expression pattern between transgenic and nontransgenic
pituitaries. ER expression was significantly decreased, while cJun and
P-LIM expression was increased in transgenic mouse pituitaries. Changes
in the expression pattern of the other genes were not significantly
different. Thus, chronic exposure to high levels of LH affected the
expression of the ER, cJun, and P-LIM. These changes might be involved
in the pathological events that alter regulation of the
-subunit
promoter by GnRH and E2.

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Figure 9. Quantitative Analysis of Reverse Northern Slot-Blot
Analysis Revealing Differential Expression of an Array of Genes in
Transgenic and Nontransgenic Mice
Data were inverse log transformed and the difference between transgenic
and nontransgenic values was plotted. aP < 0.05 statistical difference.
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DISCUSSION
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The data reported herein indicate that chronic hypersecretion of
LH renders the
-subunit promoter independent of GnRH and, as a
result, unresponsive to estrogen-negative feedback. This same promoter
appeared to be normally regulated by androgens since DHT suppressed the
postcastration rise in LH in these animals. In contrast, the endogenous
LHß gene remains fully regulated by GnRH and steroid hormones. It is
not unexpected to see the two subunits of LH responding differently
under these pathological conditions because both promoters have been
previously reported to be differentially regulated by GnRH (36, 37, 38).
However, it is surprising that the
-subunit promoter has become
unresponsive to the postovariectomy rise in GnRH and, as a result,
unresponsive to E2-negative feedback.
With chronic exposure to elevated LH, expression of the
and LHß
genes vary dramatically in their requirement for GnRH. Expression of
the LHß gene depends almost entirely on GnRH, whereas activity of the
-subunit promoter has a significant component of independence. From
a teleological point of view, this makes sense since production of the
defining glycoprotein hormones for at least three cell types
(gonadotropes, thyrotropes, and trophoblasts) requires expression of
the
-subunit gene. Since only one of these cell types, gonadotropes,
has a GnRH-R with clearly defined functional significance, it follows
that activity of the
-subunit promoter should only partially depend
on GnRH. In contrast, expression of the LHß subunit gene, one of the
defining characteristics of the gonadotrope, appears to fundamentally
require appropriate, sustained, pulses of GnRH.
Our strongest evidence for the differential requirement of
and
LHß gene expression for GnRH comes from the antide treatment studies
carried out in OVX females (Fig. 8
). Castration removes steroids and
releases the hypothalamus from their tonic suppressive effect. The
ability of antide to block the postcastration rise in LHß mRNA
provides indirect evidence for both the presence of, and requirement
for, GnRH. The inability of the transgenic
-subunit promoter to
respond to the antide paradigm suggests that the
-subunit has
significant residual activity in the presence of high steroids. Since
GnRH release is likely suppressed under these conditions, the
-subunit promoter must function in the absence of GnRH and has lost
its ability to respond to its restoration upon castration.
Loss of
-subunit promoter responsiveness to GnRH and E2
in transgenic mice is probably mediated by the pathological effects of
exposure to high levels of LH early in life. In previous transgenic
studies with a CAT reporter gene, we found that the same bovine
-subunit promoter used in this study confers responsiveness to both
GnRH and E2 (16). Thus, a fundamental change has occurred
in these animals as a result of placing LHß subunit gene expression
under control of the
-subunit promoter. This change is probably
caused by a complex sequelae of events. During the infantile period,
both the bLHßCTP transgene and endogenous LHß gene are expressed at
a time when androgens and estrogens are elevated over that seen in
nontransgenic control mice (21). Repression of endogenous LHß gene
expression was evident during the prepubertal period and became
complete at puberty, which is precocious in these mice. This is most
likely caused by a reduction of GnRH due to excessive steroid levels.
However, coincident with the suppression of the mLHß subunit gene,
the endogenous
-subunit gene and the transgene develop independence
of GnRH and insensitivity to E2.
It is apparent from these studies that the use of the
-subunit
promoter to target expression of the chimeric LHß subunit was key for
the success of our LH hypersecretion model. Exposure to elevated levels
of LH results not only in the escape of the transgenic
-subunit
promoter from GnRH and E2 regulation, but also alters the
regulation of the endogenous
-subunit gene. Thus, the observed
effects on the
-subunit promoter are due to chronically elevated
levels of LH rather than some intrinsic property unique to the
transgene. The LHß subunit promoter would not have been able to
recapitulate this phenotype, since it retained responsiveness to
E2 and GnRH.
Loss of GnRH responsiveness must have occurred through reprogramming of
key signal transduction cascades. This may involve concerted changes in
several transcription factors, including steroid hormone receptors,
that normally regulate the
-subunit promoter. These changes appear
permanent, suggesting that activity of the promoter is reprogrammed
either early in development or later after the onset of puberty. As a
first approach to understanding the molecular mechanisms underlying
altered
-subunit promoter regulation, we focused on transcription
factors that are known to be expressed in the pituitary and regulate
gonadotropin gene expression (Fig. 9
). It is clear that chronic
hypersecretion of LH causes changes in ER, cJun, and P-LIM gene
expression. These changes could be occurring in any cell type in the
pituitary. However, we can speculate on the significance of these
changes since all three genes are known to be involved in the
regulation of gonadotropin subunit gene expression. P-LIM and cJun are
known to regulate basal expression of the
-subunit gene by binding
to cognate sites in the proximal promoter region (32). The site of
P-LIM binding in the mouse and human
-subunit promoters, which is
called PGBE, has been shown to mediate GnRH responsiveness.
Furthermore, GnRH stimulates the JNK-signaling pathways in cell lines
of the gonadotrope lineage (39). Thus, increased expression of both
cJun and P-LIM may convey significant promoter activity in the
transgenic mouse pituitaries in the absence of GnRH. In addition, the
decrease in ER gene expression may reflect an attempt to up-regulate
endogenous mLHß gene expression. While the functional impact of these
changes awaits further analysis, it is plausible that changes in their
expression pattern may allow a compensatory mechanism for the
-subunit promoter to lose E2 responsiveness and retain
appreciable activity even in the absence of GnRH.
One of the strengths of our pathophysiological model of LH
hypersecretion is that the absence of GnRH permitted clear
identification of the site of action regarding estrogen suppression of
-subunit promoter activity. In female transgenic mice, GnRH input
appears to be suppressed, presumably by the high levels of estrogen.
Thus, in the absence of GnRH, estrogen has no effect on residual levels
of
-subunit because there is no pituitary mechanism that enables
estrogen to regulate activity of the
-subunit promoter. Indeed, in
previous studies we demonstrated that a larger human
-subunit
promoter lacks a high affinity-binding site for ER and cannot respond
to estrogen in cotransfection studies with human ER (16). Like the
bovine promoter (16, 23), this human
-subunit promoter also confers
responsiveness to GnRH and estrogen in a transgenic setting. Taken
together, these studies provide compelling evidence that estrogen
suppresses activity of the
-subunit promoter through an indirect
mechanism that occurs at a distal site of action, most likely the
hypothalamus.
Synthesis of LH involves expression of the
and LHß subunit genes.
For the
-subunit, our data strongly suggest that estrogen exerts its
action solely at the level of the hypothalamus, where it regulates
production of GnRH. In contrast, activity of the LHß promoter is
completely dependent on continuous pulses of GnRH. With no GnRH (due to
hypothalamic estrogen-negative feedback), the endogenous LHß gene is
completely suppressed in female transgenic mice. Because the LHß
promoter is silent, we cannot rule out additional effects of estrogen
that utilize transcriptional pathways within a gonadotrope. What we can
say is that for promoters such as the
-subunit, where
appreciable activity remains when GnRH is absent, resistance to
estrogen-negative feedback is most likely linked directly to escape
from GnRH regulation. For promoters like LHß that are completely
dependent on GnRH, there may be additional sites of estrogen action
that include effects on GnRH receptor and components of its downstream
signaling pathway.
Thus, when considering resistance of LH to steroid-negative feedback,
there are two primary genes (
and LHß) and two cellular sites of
action (gonadotropes and GnRH neurons) that together present a
combinatorial array of targets that, if any single one is affected,
could result in hypersecretion of LH. Our work singles out one
mechanism that illustrates how the GnRH independence of
-subunit
allows appreciable expression to occur when higher than normal
concentrations of E2 abound. This finding provides an
important paradigm for understanding how hypersecretion of LH could
occur in women. Our observations in mice have been recently confirmed
in women with polycystic ovary syndrome who appear to have elevated
levels of LH as a result of resistance to steroid-negative feedback
(40). However, the mechanism involved in the escape of LH from
steroid-negative feedback remains unclear. Since ovarian
hyperstimulation will suppress secretion of GnRH, either GnRH neurons
must become resistant to steroid-negative feedback or the dependence of
the LHß promoter on GnRH must be relieved presumably through
alterations in gonadotropes that give the promoter some transcriptional
autonomy.
In contrast to estrogen, androgens appear to suppress activity of the
- subunit promoter through a direct mechanism. Thus, androgen action
can be distinguished from estrogen action by its ability to regulate
the
-subunit promoter in the absence of GnRH. Our earlier AR
cotransfection studies indicated that androgens repress activity of the
human
-subunit promoter through a mechanism that does not require
binding of AR directly to DNA (14, 15). Instead, AR responsiveness maps
to two elements required for
-subunit promoter basal activity [CRE
and
BE; (41)] suggesting some form of transcriptional interference.
In the end, while chronic hypersecretion of LH renders the
-subunit
promoter refractory to GnRH and estrogen, responsiveness to androgen
remains intact. Our next challenge will be to map the complete pathway
that permits androgen regulation of the
-subunit promoter
independent of GnRH.
 |
MATERIALS AND METHODS
|
---|
Animals
Female transgenic mice carrying the bLHßCTP transgene were
obtained by breeding male transgenic mice with CF-1 female
nontransgenic mice and were at least seven generations from the founder
animal. Mice were genotyped by PCR of tail DNA using primers specific
for the
-subunit promoter and the bovine LHß subunit gene as
reported earlier (20). In addition, the sex of the embryos used in
these studies was determined by PCR using primers for Sry:
5'-GAGAGCATGGAGGGCAT-3' and 5'-CCACTCCTCTGTGACACT-3'. All mice were
housed under controlled light (12 h light, 12 h dark) and
temperature (25 C) conditions. All experimental procedures were
approved by the Case Western Reserve University Institutional Animal
Care and Use Committee.
Developmental Studies
Female transgenic mice and nontransgenic littermates were killed
by decapitation at different stages of development: embryonic day 19
(n = 2 per group); postnatal day 1 (n = 2 per group);
postnatal day 14 (n = 2 per group); 3 weeks of age (n = 3); 4
weeks of age (n = 6); and 6 weeks of age (n = 7). Pituitaries
or heads (for younger animals) were immediately frozen and stored at
-80 C until used in RNAse protection assays or in situ
hybridization. Ovaries were inspected for obvious phenotypes. Trunk
blood was collected and serum LH levels were determined by a previously
validated RIA (20).
Treatment Paradigms
Adult female transgenic and nontransgenic littermates were OVX
under Avertin (1:1 Tribromoethanol:tertiary-amylalcohol stock,
diluted 1:40 in normal saline) anesthesia. Orbital sinus blood samples
were obtained under anesthesia before OVX or treatment. The effect of
estrogen-negative feedback was tested in chronically OVX mice (24
weeks) implanted subcutaneously with 17ß-estradiol constant-release
pellets (0.25 mg/pellet; Innovative Research of America,
Toledo, OH) for 23 weeks (1315 animals per group). This pellet
results in serum E2 levels of approximately 100 pg/ml (19).
DHT pellets (7.5 mg/pellet, Innovative Research of America) were similarly implanted after ovariectomy to obtain a
serum concentration of approximately 5 ng/ml (56 animals per group).
Control animals were implanted with placebo pellets. For the
ovariectomy/antide treatment paradigm, we used the same protocol that
has been previously used in our laboratory with subcutaneous injections
of antide (60 µg in 20% propylene glycol in normal saline per
animal, once every other day for 10 days; n = 47 animals per
group) (19). At the end of each treatment, animals were killed by
decapitation and trunk blood was collected. Pituitaries were
immediately frozen and stored at -80 C. Total RNA was isolated using
the Trizol method (Life Technologies, Inc., Gaithersburg,
MD).
Riboprobes Used For Both in Situ Hybridization and
RNAse Protection Assay
For double-label in situ hybridization, the
endogenous
and LHß probes were labeled with digoxigenin and the
bLHßCTP probe was 35S labeled. The mouse
cDNA was
obtained from Dr. David Gordon, University of Colorado Health Science
Center (Denver, CO). It is a 460-bp PstI insert in
pGEM3Zf+ (Promega Corp., Madison, WI). After linearization
with EcoRI the riboprobe was synthesized using SP6 RNA
Polymerase in the presence of 80% Digoxigenin-11-UTP (Roche Molecular Biochemicals, Indianapolis, IN). The mouse LHß cDNA
is a 250-bp fragment that was cloned in the SmaI site of
BSK- (Stratagene, La Jolla, CA) (42).
Antisense digoxigenin-labeled riboprobe was generated after
linearization of the plasmid with HindIII and using T7 RNA
Polymerase. bLHßCTP cDNA (520bp) inserted in the
KpnI/BamHI site of BKS- was used for
transgene probe synthesis. The transgene probe was generated after
linearization with StuI and using T7 RNA Polymerase. All
modifying enzymes were obtained from Life Technologies, Inc..
RNase Protection Assay and in Situ Hybridization
For the RNAse protection assays, 32P-labeled
riboprobes for mLHß, m
, and bLHßCTP were synthesized using the
same cDNA templates mentioned above. As a control, a riboprobe for
mouse ß-actin was also generated. The mouse ß-actin template is
obtained by digesting the pTRI-B-Actin-Mouse vector (Ambion, Inc., Austin, TX) with DdeI. The mLHß, bLHßCTP,
and actin probes are 250 bp, 387 bp, and 160 bp, respectively, allowing
detection of each RNA in the same sample. For m
, four protected
bands were observed. The signal from all four bands with background
subtracted was expressed relative to actin.
RNAse protection assays were performed on total RNA using the RPAII kit
(Ambion, Inc.). Total RNA from individual pituitaries was
denatured and coprecipitated with 32P-labeled riboprobes
for mLHß, bLHßCTP, and actin (7.3 x 104 cpm of
each probe). After overnight hybridization at 42 C, samples were
treated with RNAse A/TI (1:100 dilution) and precipitated according to
kit instructions. Pellets were resuspended in nondenaturing loading
buffer (16% sucrose, 0.05% bromophenol blue, 0.05% xylene cyanol)
and run on nondenaturing 4.5% polyacrylamide gels. Gels were dried and
exposed to x-ray film overnight at -80 C. A standard curve was
generated for each probe using in vitro transcribed sense
RNA (data not shown). Quantitative analysis of the signal from each
band was performed using the Ambis Radioanalytical Imaging system
(AMBIS, Inc., San Diego, CA). The background was subtracted from the
signal for each band. Data were expressed relative to actin as mean
values ± SEM, and statistical analysis was performed
using the unpaired Students t test with unequal
variance.
Double-label in situ hybridization, using
digoxigenin-labeled probe for mLHß and 35S-labeled probe
for bLHßCTP, was performed on fresh frozen 10-µm sections as
described earlier (42, 43). For all the RNA work, sense probes were
used to control for specificity of the hybridization signal.
Reverse Northern Assays
Reverse Northern slot blot assays (n = 3) were performed
using a modified version of previously published protocols (44, 45, 46).
The cDNA clones used in this experiment were as follows: human ER (47),
mouse GnRH-R (48), human AR (49), SF-1 (28), P-Otx/Ptx1 (32), H-ras
(50), c-myc (51), P-LIM (32), cJun (52), CREB (53), BSK-CAT
(42), and Fos (54). Briefly, cDNA clones (1 µg) were spotted in
duplicate on two identical Nylon membranes
(HybondTM-N+; Amersham Pharmacia Biotech, Cleveland, OH) and hybridized overnight at 65 C in
Churchs buffer with either transgenic or nontransgenic probes. Pooled
(n = 1015) total pituitary RNA (20 µg) was used for
first-strand cDNA synthesis using Superscript II (Life Technologies, Inc.) in the presence of 60 µCi of
[
-32P]dCTP, nonlabeled dGTP, dATP, dTTP, and random
primers (Life Technologies, Inc.). Blots were washed in
2xX SSC, 0.1% SDS (15 min). Data analysis was performed using the
Ambis Radioanalytical Imaging System. Each lane on the blot was
analyzed separately, and the computer was allowed to identify peaks and
determine appropriate background. Net counts from each peak were used
in the statistical analysis. To avoid problems with changes in the
expression of the housekeeping genes, statistical analysis was
performed without normalization of the data. Comparison of transgenic
and nontransgenic control was made using a multiway ANOVA. In this
analysis, the gene expression response was first transformed using a
1/log(exp) transformation to satisfy normality and variance
stabilization assumptions of the analysis. The ANOVA allowed for
controlling variation in expression due to different genes, different
experiments, and replications within experiments. Interactions of these
factors with treatment were also explored. Further comparisons between
transgenic and control for each gene were made using specific contrasts
derived from the ANOVA. This was done after first examining from the
ANOVA whether an overall transgenic effect was present and whether
there were any differences in expression between genes. All tests were
run at a significance level of P = 0.05 and were two
sided.
 |
ACKNOWLEDGMENTS
|
---|
The authors would like to acknowledge the contributions of the
following people whose assistance made this work possible: Dr. M. Susan
Smith, Dr. Richard Hanson, Dr. Joe Nadeau, Dr. Kimberly Risma, Dr. Ruth
Keri, Rachel Adamek, Kit Sutherland, and David Peck.
 |
FOOTNOTES
|
---|
Address requests for reprints to: John H. Nilson, Ph.D., Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio 44106.
This work was supported by an NIH training grant in metabolism,
5T32DK-07319 (to R.A.), NICHD Grant HD-34032, and NIDDK Grant DK-28559
(to J.H.N.).
Received for publication December 21, 1998.
Revision received May 25, 1999.
Accepted for publication June 7, 1999.
 |
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