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Molecular Endocrinology 18(4):925–940
Copyright © 2004 by The Endocrine Society
doi: 10.1210/me.2003-0115
Androgen Regulates Follicle-Stimulating Hormone ␤
Gene Expression in an Activin-Dependent Manner in
Immortalized Gonadotropes
THOMAS J. SPADY, RANA SHAYYA, VARYKINA G. THACKRAY, LISA EHRENSBERGER,
JANICE S. BAILEY, AND PAMELA L. MELLON
Departments of Reproductive Medicine and Neurosciences, Center for the Study of Reproductive
Biology and Disease, University of California, San Diego, La Jolla, California 92093-0674
Little is known about the molecular mechanisms of
androgen regulation of the FSH␤ gene; however,
studies suggest that it consists of a complex feedback loop that involves multiple mechanisms acting at both the level of the hypothalamus and the
pituitary. In the present study, we address androgen regulation of the FSH␤ gene in immortalized
gonadotrope cells and investigate the roles of activin and GnRH in androgen action. Using transient
transfection assays in the FSH␤-expressing mouse
gonadotrope cell line, L␤T2, we demonstrate that
androgens stimulate expression of an ovine FSH␤
reporter gene in a dose-dependent manner. Mutation of either of two conserved androgen response
elements at ⴚ245/ⴚ231 and ⴚ153/ⴚ139 within the
proximal region of the ovine FSH␤ gene promoter
abolishes this stimulation, and androgen receptor
binds directly to the ⴚ244 ARE in vitro. Androgen
induction of the FSH␤ reporter gene is also dependent upon the activin autocrine loop present in the
L␤T2 cells, as well as an activin-response element
at ⴚ138/ⴚ124 of the FSH␤ gene. However, activin
regulation of other genes remains unaffected by
androgens. In addition, androgens stimulate expression of a mouse GnRH receptor reporter gene,
and thus may indirectly augment the response of
the FSH␤ gene to GnRH. Taken together, these
data demonstrate that, in mouse gonadotropes,
androgens act directly on the ovine FSH␤ gene to
stimulate expression by a mechanism that is dependent upon activin, as well as acting indirectly,
potentially through a second mechanism that may
be dependent upon induction of GnRH receptor.
(Molecular Endocrinology 18: 925–940, 2004)
A
in GnRH antagonist-treated rats (6) and rat primary
pituitary cell cultures (7, 8). Recently, two different
molecular mechanisms by which androgen receptor
(AR) represses LH␤ gene expression have been elucidated using the immortalized mouse gonadotrope cell
line, L␤T2 (9, 10). Both studies show that repression is
dependent upon GnRH stimulation and that the mechanisms are indirect, requiring protein-protein interactions with Sp1 (9) or steroidogenic factor 1 (10), rather
than through AR binding to the LH␤ gene.
In contrast to LH␤ regulation, the mechanisms
through which androgens modulate FSH␤ expression
at the level of the pituitary are unknown. A growing
body of evidence indicates that androgens act at the
level of the pituitary to stimulate, rather than repress,
FSH␤ gene transcription in rodents. Previous studies
demonstrated that testosterone (T) increases FSH␤
mRNA levels by 2-fold in GnRH antagonist-treated
rats, whereas ␣-GSU and LH␤-subunit mRNA levels
are decreased (11, 12). Furthermore, T selectively induces FSH␤ mRNA in both male and female primary
rat pituitary cell cultures (7, 8, 13). In addition to these
actions of androgen on FSH␤ transcription, androgens
have been shown to modulate levels of follistatin (FS)
in the rat pituitary in vivo and both activin and FS in
cultured rat pituitary cells (8, 14, 15), indicating that the
mechanism of androgen action on FSH␤ might be
indirect, through modulation of the activin/FS system in
NDROGENS ARE A CLASS of sex steroids that
play important roles in sexual development and
reproduction in both males and females. Androgen
action at both the hypothalamus and the anterior pituitary regulates synthesis and secretion of the heterodimeric gonadotropin hormones LH and FSH. At
the level of the hypothalamus, androgens reduce
steady-state levels of GnRH mRNA (1, 2) and GnRH
peptide (3, 4) and modulate processing of the GnRH
prohormone (5) in rats, all resulting in dampened
GnRH stimulation of LH and FSH. At the level of the
pituitary gland, androgen action differs in a speciesand gonadotropin subunit gene-specific manner. Androgens repress both ␣-subunit of the glycoprotein
hormones (␣-GSU) and LH␤-subunit gene expression
Abbreviations: AR, Androgen receptor; ARE, androgen response element; CMV, cytomegalovirus; DHT, dihydrotestosterone; FBS, fetal bovine serum; FS, follistatin; GnRH-R,
GnRH receptor; GRAS, GnRH receptor-activating sequence;
GRE, glucocorticoid response element; ␣-GSU, ␣-subunit
of the glycoprotein hormones; HPG, hypothalamic-pituitarygonadal; MMTV, mouse mammary tumor virus; PRE, progesterone response element; RSV, Rous sarcoma virus; SBE,
Smad binding element; Smad, Sma- and Mad-related protein; T, testosterone; TK, thymidine kinase.
Molecular Endocrinology is published monthly by The
Endocrine Society (http://www.endo-society.org), the
foremost professional society serving the endocrine
community.
925
926 Mol Endocrinol, April 2004, 18(4):925–940
the pituitary. Furthermore, GnRH receptor (GnRH-R)
within the pituitary of rodents is modulated by androgens
(16–20), which in turn may alter responsiveness of FSH␤
to GnRH, and thus act as another indirect mechanism
through which androgens regulate FSH␤ expression at
the level of the pituitary. Because androgens stimulate
GnRH-R expression in mouse pituitary (9, 21), in contrast
to the inhibitory effect most commonly observed in rats
(18–20), androgens may stimulate mouse FSH␤ gene
expression through multiple mechanisms, including via
enhancing responsiveness of FSH␤ to GnRH.
Historically, the lack of an appropriate gonadotrope
cell culture model has impeded efforts to characterize
the molecular mechanisms of steroid regulation of
FSH␤ gene expression. In this study, the L␤T2 mouse
gonadotrope cell line serves as a model system to
discriminate androgen action at the level of the isolated gonadotrope from that of the whole pituitary
gland by eliminating the variables associated with
whole-animal studies or complex primary pituitary cultures. The L␤T2 cells express GnRH, androgen, and
activin receptors, and produce activin and FS, providing a model that allows investigation of the interactions
of these hormones on the expression of gonadotropin
genes (22, 23). We find that androgens act directly
within gonadotropes to stimulate FSH␤ gene transcription independent of GnRH, and that this androgen stimulation occurs through two conserved androgen response elements (AREs) within the FSH␤ gene,
at least one of which specifically binds to AR in vitro.
Furthermore, we demonstrate that androgen regulation is dependent upon the activin autocrine loop in the
gonadotrope and specifically on an element in the
FSH␤ gene that is also required for activin responsiveness. However, androgens do not alter the activin
responsiveness of an activin response element from
the GnRH-R gene, indicating that the mechanism of
androgen action is not through global modulation of
the activin/FS system. Furthermore, activin and androgen act synergistically to induce FSH␤ gene expression, as do androgen and GnRH. Finally, androgens
stimulate mouse GnRH-R gene expression in gonadotropes, revealing a potential mechanism by which androgens might enhance the response of the ovine
FSH␤ gene to GnRH. Thus, we demonstrate that androgen stimulation of FSH␤ gene transcription at the
level of the gonadotrope is dependent upon activin
action and requires both AREs and elements conferring activin response, providing insight into the mechanisms of androgen regulation of FSH.
RESULTS
Androgens Stimulate FSH␤ Transcription in a
Dose-Dependent Manner
The ovine FSH␤ gene regulatory region drives gene
expression and is hormonally regulated in the L␤T2
mouse gonadotrope cell line (22). Furthermore, andro-
Spady et al. • Androgen Induction of FSH␤
gen regulation of the rat and bovine LH␤ subunit genes
has been studied in this model (9, 10). Here, we have
used this cell line model system to address the question of whether androgens stimulate FSH␤ gene expression by acting directly and solely within gonadotropes. We chose to utilize the ovine (o) FSH␤
promoter for three reasons. First, it is the longest
mammalian FSH␤ promoter clone available (⫺4.7 kb).
Second, the accuracy of the targeting and hormonal
responses of the ovine gene has been demonstrated
in transgenic mice (24). Finally, the ovine gene is regulated by activin and GnRH in L␤T2 cells (22), and the
signal transduction of the GnRH response has been
elucidated (25).
L␤T2 cells were transiently transfected with a luciferase reporter gene in which expression was driven by
approximately 5.5 kb of the ovine FSH␤ gene regulatory region with the first exon and the first intron
(oFSH␤-Luc), deprived of steroids [10% charcoalstripped fetal bovine serum (FBS)] for 24 h and then
treated with 0.1% ethanol vehicle or T at a range of
concentrations for 24 h. T treatment stimulates FSH␤
promoter activity in a dose-dependent manner, with
statistically significant induction starting at 100 pM T
Fig. 1. Androgen Stimulates oFSH␤ Transcription in L␤T2
Cells
L␤T2 cells were transfected with 4.7 kb oFSH␤-luciferase
reporter plasmid, deprived of steroids for 24 h, and then
treated with either ethanol vehicle or various concentrations
of T (top panel) or DHT (bottom panel) for 24 h. Data were
pooled from three independent experiments (n ⫽ 3 replicates
per treatment group for each experiment) and expressed as
the mean ⫾ SEM of the ratio of luciferase/CMV-␤-galactosidase activity, normalized to the vehicle-treated mean. * Indicates a statistically significant difference (P ⬍ 0.05) from
vehicle-treated mean. ** Indicates a highly significant difference (P ⬍ 0.0001) from the vehicle-treated mean.
Spady et al. • Androgen Induction of FSH␤
(P ⫽ 0.006) and a maximal 2-fold stimulation (P ⬍
0.0001) at 100 nM (Fig. 1). The nonaromatizable androgen, dihydrotestosterone (DHT), similarly stimulates oFSH␤-Luc transcriptional activity when administered for 24 h, with a significant induction at 1 nM
(P ⬍ 0.05), and peak induction at 10 nM DHT (P ⬍
0.0001; Fig. 1). These data demonstrate that androgens are capable of stimulating FSH␤ gene transcription in a dose-dependent manner by acting solely and
directly upon gonadotropes. Furthermore, the stimulation of FSH␤ is likely due to androgenic rather than
estrogenic activity, because both the nonaromatizable
(DHT) and the more physiological, aromatizable (T)
androgens have identical stimulatory effects on FSH␤
promoter activity. Taken together, these data indicate
that physiologically relevant concentrations of androgens stimulate FSH␤ gene expression by acting directly within gonadotropes. This androgen regulation
of FSH␤ occurs in a specific and saturable, dosedependent manner typical of classic steroid hormone
action.
Two AREs in the Proximal Promoter Are
Required for Androgen Response
To further elucidate the mechanism(s) through which
androgens stimulate the FSH␤ gene, we mapped regions of the oFSH␤ gene promoter that confer androgen response using truncation-deletion analysis. L␤T2
cells were transiently transfected with a series of truncations of the oFSH␤ gene regulatory region ranging in
length from 982–105 bp upstream of the transcription
start site, and the ability of 100 nM DHT, administered
for 24 h, to induce FSH␤ gene transcription was tested
(Fig. 2). DHT significantly induced reporter gene activity by 1.9- (P ⬍ 0.0001), 1.7- (P ⬍ 0.0001), and 1.8-fold
(P ⬍ 0.0001), respectively, in transfections of reporter
Mol Endocrinol, April 2004, 18(4):925–940
927
genes containing 982, 561, or 401 bp upstream of the
oFSH␤ gene mRNA start site. The degree of DHT
stimulation of these three promoters was identical to
that of the 4.7-kb flanking region of the oFSH␤ gene
(see Fig. 1). Further truncation to ⫺105 resulted in a
loss of the ability to respond to DHT (P ⫽ 1.0). These
data demonstrate that androgen response is conferred by the 296 bp region of the oFSH␤ gene-proximal promoter between ⫺401 and ⫺105 bp.
In examining the ⫺401 to ⫺105 region of the oFSH␤
gene promoter for candidate AREs, homology searches
revealed three candidate sites, located at ⫺245/⫺231,
⫺212/⫺198, and ⫺153/⫺139 (Fig. 3A). These three elements have homology to known AREs (Fig. 3A), including a shared consensus ARE/glucocorticoid response
element (GRE)/progesterone response element (PRE)
from the mouse mammary tumor virus (MMTV) long terminal repeat (26, 27), consensus AREs from various androgen responsive genes (reviewed in Ref. 28), and an
experimentally deduced sequence proven to be highly
selective and specific for AR (hasp-ARE) (29). Of the
three candidate AREs, only the one at ⫺245/⫺231 contains a perfect consensus half-site (TGTTCT). Overall, the
⫺245/⫺231 and ⫺153/⫺139 putative AREs exhibit the
highest homology to previously characterized AREs (Fig.
3A). In contrast, the putative ARE at ⫺212/⫺198 has
higher homology to the shared ARE/GRE/PRE of MMTV
(71%) than do either the ⫺245/⫺231 (47%) or the ⫺153/
⫺139 (47%) sites, and a lower homology (66%) than
either the ⫺245/⫺231 (73%) or ⫺153/⫺139 (80%) sites
to the hasp-ARE. These data implicate these three sites
as potential targets for AR action and suggest the ⫺245/
⫺231 and ⫺153/⫺139 sites may have higher specificity
for AR and thus are most likely to be AREs.
This entire 211-bp proximal region of the oFSH␤
gene (⫺248 to ⫺137), encompassing all three putative
ARE motifs, is remarkably well conserved among nu-
Fig. 2. Localization of the Androgen-Responsive Region in the oFSH␤ Regulatory Region
L␤T2 cells were transfected with a series of truncated oFSH␤ luciferase reporter plasmids (⫺982, ⫺561, ⫺401, and ⫺105),
deprived of steroids for 24 h, and then treated with ethanol vehicle or 100 nM DHT for 24 h. Data were pooled from five
independent experiments (n ⫽ 4–6 replicates per treatment group for each experiment) and expressed as the mean ⫾ SEM of the
ratio of luciferase to CMV-␤-galactosidase activity, normalized to the vehicle-treated mean within the same plasmid group. *
Indicates a statistically significant difference (P ⬍ 0.0001) from the vehicle-treated mean within the same plasmid group.
928 Mol Endocrinol, April 2004, 18(4):925–940
Spady et al. • Androgen Induction of FSH␤
Fig. 3. Sequence Comparison to Consensus AREs and Evolutionary Conservation of Three Regions in the oFSH␤ Promoter
A, The three putative ARE motifs within the proximal region of the oFSH␤ gene promoter were examined for sequence
homology to known ARE motifs from three different androgen-responsive genes, as well as a computer-generated high-affinity
and specific consensus ARE (hasp-ARE). The ARE motif of the MMTV gene is a shared ARE/GRE/PRE, whereas the PSA, C3, and
hasp-ARE motifs are more specific for the AR than the related GR and PR. Boxes outline the critical conserved C and G bases
of the consensus AREs. Percent homology of each ARE is compared at right. B, The proximal promoter region of the oFSH␤ gene
from ⫺248 to ⫺137 nucleotides was aligned to sequences from the FSH␤ gene of various mammalian species, and the relative
conservation of sequence homology was determined. Conserved sequences with ARE motifs (ARE 1–3) are shadowed in gray.
Minus symbols (⫺) indicate a deletion compared with alignment with the oFSH␤ gene. Asterisks indicate the site of insertion of
an additional G nucleotide, only present in the mouse and rat genes. An inverted triangle indicates the site of insertion of a
trinucleotide repeat (CAT), present only in the mouse, rat, and human genes.
merous mammalian species (Fig. 3B). The ⫺245 and
⫺153 AREs are the most highly conserved sites, and
the C/G bases known to be critical for function of the
consensus ARE/GRE/PRE are perfectly conserved in
all species examined. The less than perfect overall
conservation homology of the middle ARE (⫺212/
⫺198) site is due to insertions of a single guanidine
base in FSH␤ of rodent species and of a CAT duplication in the mouse, rat, and human FSH␤ genes
compared with the sheep, cow, and pig FSH␤ genes
(Fig. 3B). Previously, Webster et al. (30) identified
these three same sites within the oFSH proximal promoter to be PREs that bind PR in vitro and confer
progesterone response when present as multimers on
a heterologous promoter. We hypothesized that one or
more of these three PREs may also act as AREs,
because both AR and PR are known to bind the same
DNA elements within certain genes, including the
long-terminal repeat of the MMTV gene promoter (26).
To determine the role of these putative regulatory
elements in androgen stimulation of FSH␤, we exam-
ined the ability of DHT treatment (24 h) to stimulate
promoter activity when the sequences from the ⫺245/
⫺231, ⫺212/⫺198, or ⫺153/⫺139 candidate ARE
motifs were disrupted by site-directed mutagenesis
(mutations shown in Fig. 4A). As shown in Fig. 2,
expression of the wild-type ⫺982 FSH␤ reporter was
stimulated 1.9-fold by DHT (P ⬍ 0.0001). A 2-bp mutation (GT to CC) within the conserved ⫺236/⫺231
half-site of the putative ARE at ⫺245/⫺231 completely
abolished DHT stimulation of reporter gene expression
in the context of the ⫺985 FSH␤ promoter (P ⫽ 1.0)
(Fig. 4B). Similarly, a 2-bp mutation (GA to CC) of the
⫺144/⫺139 half-site of the putative ARE at ⫺153/
⫺139 also completely abolished the ability of DHT to
stimulate the FSH␤ promoter (P ⫽ 1.0). In contrast,
mutation of 2 critical C/G base pairs within the putative
ARE at ⫺212/⫺198, one in each half-site, had no
deleterious effect on the ability of DHT to induce FSH␤
promoter activity (1.65-fold compared with 1.9-fold
induction of ⴚ985 FSH␤; P ⫽ 0.453). Similarly, a CA to
CC mutation of the ⫺202/⫺198 half-site of the puta-
Spady et al. • Androgen Induction of FSH␤
Mol Endocrinol, April 2004, 18(4):925–940
929
human AR protein by overexpressing it in baculovirusinfected Sf9 insect cells using a Flag-tagged AR baculovirus (kindly provided by E. Wilson). Cells were
treated with androgens (1 mM DHT final concentration)
for 24 h before harvest. Soluble whole-cell extract was
incubated with a 29-bp probe to the conserved ⫺245
oFSH␤ ARE and analyzed by EMSA (Fig. 5). The resulting complex was supershifted by anti-AR antibody
but not by IgG, showed self-competition, failed to
compete with a mutant probe containing the same
mutations noted in Fig. 4, and was competed by an
ARE consensus sequence (Materials and Methods).
We conclude that AR can bind directly and specifically
to the ⫺245 ARE element in the oFSH␤ subunit gene.
However, under these conditions, we were unable to
demonstrate AR binding to the ⫺153/⫺139 ARE in
EMSA. This might be due to the fact noted above, that
only the ⫺245/⫺231 ARE contains a perfect consensus half-site (TGTTCT) for AR binding.
Fig. 4. Mutagenesis of AREs in the oFSH␤ Promoter
A, Three candidate AREs identified within the ⫺401 to
⫺105 bp fragment of the oFSH␤ promoter were individually
disrupted by site-directed mutagenesis. Base pairs in the
region of the consensus are in uppercase and others are in
lowercase. Mutated bases are underlined. Because the C
nucleotide chosen for mutation in the ⫺245 and ⫺153 sites
was not conserved with the consensus within the ⫺212 site,
two different base pairs, one in each half-site, which were
conserved with the consensus, were mutated as shown. B,
L␤T2 cells were transfected with the wild-type ⫺982 oFSH␤luciferase reporter plasmid or one of three mutant oFSH␤luciferase reporter plasmids with mutations in the ⫺245 ARE,
⫺212 ARE, or ⫺153 ARE, deprived of steroids for 24 h, and
then treated with vehicle or 100 nM DHT for 24 h. Data were
pooled from three to five independent experiments (n ⫽ 4–6
replicates per treatment group for each experiment) and expressed as the mean ⫾ SEM of the ratio of luciferase to
CMV-␤-galactosidase activity, normalized to the vehicletreated mean. * Indicates a statistically significant difference
(P ⬍ 0.0001) from the vehicle-treated mean within the same
plasmid group.
tive ARE at ⫺212/⫺198 also had no effect on DHT
stimulation (data not shown). These data indicate that
the ⫺245/⫺231 and ⫺153/⫺139 ARE motifs, but not
the ⫺212/⫺198 ARE, are crucial for androgen response. The lack of activity from the central ARE is
consistent with its lack of evolutionary conservation.
Thus, we have identified two functional AREs within
the oFSH␤ subunit gene, the sequences of which are
conserved in other mammalian species and thus are
likely to be functional in those species as well.
AR Binds to the ⴚ245 ARE
To determine whether AR can bind directly to an ARE
in the oFSH␤ subunit gene, we obtained full-length
Fig. 5. Ligand-Bound AR Binds Specifically to an Oligonucleotide Containing the ⫺245 ARE in the oFSH␤ Promoter
Extracts of AR-baculovirus-infected insect cells were incubated with the ⫺245 probe and tested for complex formation in EMSA. The AR-DNA complex is apparent in lane 1,
anti-AR antibody supershift (lane 2), IgG control (lane 3),
self-competition (lane 4), mutant competition (mutation as in
Fig. 4) (lane 5), and ARE consensus competition (Materials
and Methods) (lane 6).
930 Mol Endocrinol, April 2004, 18(4):925–940
Androgen Stimulation is Dependent on the
Activin Autoregulatory Loop
In addition to elucidating the mechanism through
which androgens act within gonadotropes to directly
stimulate the oFSH␤ gene, we also examined whether
androgens act within the gonadotrope through additional mechanisms to indirectly regulate FSH␤ gene
expression. Previous studies in rats in vivo and in
cultured rat pituitary cells have shown that androgens
can modulate the levels of activin and FS in the pituitary (8, 14, 15, 31, 32). To determine whether the
activin autocrine loop, the key regulatory system of
FSH synthesis and secretion, plays a role in androgen
stimulation of FSH␤ transcription, we examined the
effects of coadministration of FS or activin on androgen stimulation of FSH␤ expression. L␤T2 cells were
transiently transfected with the 4.7 kb oFSH␤-Luc reporter, incubated overnight in DMEM containing 10%
charcoal-treated FBS and 250 ng/ml FS to block endogenous activin action, followed by treatment with
vehicle or 100 nM DHT ⫾ 250 ng/ml FS or 10 ng/ml
activin-A for 24 h (Fig. 6). As expected, treatment with
either DHT or activin alone induced FSH␤ promoter
Fig. 6. Androgen Induction of the oFSH␤ Gene Is Blocked
by FS and Synergistic with Activin (Act)
L␤T2 cells were transfected with 4.7 kb oFSH␤-Luc reporter plasmid, incubated overnight in DMEM containing
10% charcoal-treated FBS and 250 ng/ml FS, and treated for
24 h with vehicle or 100 nM DHT ⫾ 250 ng/ml FS or 10 ng/ml
activin A. Data were pooled from four independent experiments (n ⫽ 4 replicates per treatment group for each experiment) and expressed as the mean ⫾ SEM of the ratio of
luciferase to RSV-␤-galactosidase activity, normalized to the
vehicle-treated mean. * Indicates a statistically significant
difference (P ⬍ 0.001) from the vehicle-treated mean. † Indicates a statistically significant difference (P ⬍ 0.001) from the
activin only-treated mean.
Spady et al. • Androgen Induction of FSH␤
activity by 1.5-fold (P ⫽ 0.0015) and 11-fold (P ⫽
0.000003), respectively. Interestingly, addition of FS
abolished the ability of DHT to stimulate FSH␤ promoter activity, whereas addition of activin with DHT
resulted in induction of FSH␤ promoter activity by
19-fold, significantly greater than either DHT (P ⫽
0.00007) or activin treatment alone (P ⫽ 0.0039). Using
the methodology as described by Slinker (33), analysis
of the data by two-way ANOVA reveals a synergistic
increase upon activin and androgen cotreatment that
is significantly (P ⬍ 0.0001) different than an additive
effect. Therefore, these data indicate that there is a
synergistic interaction between androgen and the activin/FS system in the regulation of the FSH␤ promoter
as well as a complete dependence upon activin as a
permissive agent for androgen action. Thus, the endogenous activin secretion by the gonadotrope plays
a crucial role in androgen regulation of FSH␤ gene
expression.
The Activin Autoregulatory Loop Does Not Affect
Androgen Action Globally, nor Vice Versa
To determine whether the interaction between androgen and activin is specific to the FSH␤ gene or involves a general effect of the activin system on AR
function or vice versa, we examined both the effect of
FS on androgen induction of the highly androgensensitive MMTV promoter and the effect of androgen
on FS or activin regulation of the highly activin-sensitive GnRH receptor-activating sequence (GRAS) element of the mouse GnRH-R gene promoter (34). In the
first series of experiments, L␤T2 cells were transiently
transfected with the MMTV-Luc reporter plasmid, pretreated overnight with DMEM containing 10% charcoal-treated FBS and 250 ng/ml FS, and treated with
vehicle or 100 nM DHT ⫾ 250 ng/ml FS for 24 h (Fig.
7A). DHT stimulated MMTV promoter activity by 2.56fold (P ⫽ 0.024). FS treatment did not inhibit MMTV
promoter activity, nor did FS diminish the ability of
DHT to stimulate the MMTV promoter. These data
indicate that FS blockade of androgen action is gene
specific, rather than due to a global perturbation of AR
function in the gonadotrope.
A route for androgen regulation of FSH␤ gene expression in pituitary that has been previously suggested is androgen regulation of activin system components such as FS (14, 15). To determine whether
androgens were globally affecting activin action, L␤T2
cells were transiently transfected with the activinsensitive GRAS-Luc reporter plasmid containing the
activin response element from the GnRH-R gene (34,
35), pretreated overnight with DMEM containing 10%
charcoal-treated FBS and 250 ng/ml FS, and treated
with vehicle or 100 nM DHT ⫾ 10 ng/ml activin-A ⫾ 250
ng/ml FS for 24 h (Fig. 7B). FS treatment inhibited
GRAS-Luc reporter activity by 70% (P ⬍ 0.00000001),
whereas activin treatment stimulated reporter activity
by 6-fold (P ⫽ 0.0000000001). DHT alone had no
effect and DHT in combination with FS or activin had
Spady et al. • Androgen Induction of FSH␤
Mol Endocrinol, April 2004, 18(4):925–940
931
no effect on either FS repression or activin stimulation
of GRAS promoter activity. These data suggest that
the stimulatory effect of androgens on FSH␤ gene
expression is not due to modulating bioavailable levels
or activity of activin, its receptors, FS, or other activinregulatory system components present in gonadotropes. Taken together, these data show that androgen regulation of FSH␤ gene expression is not due to
an indirect action on the activin/FS system, but likely
involves interaction with the activin system specifically
within the context of the FSH␤ gene promoter.
Androgen Regulation Is Dependent upon an
Activin-Responsive Element in the FSH␤ Gene
Fig. 7. Activin (Act) Does Not Globally Affect Androgen Action and Vice Versa
A, L␤T2 cells were transfected with the androgen-sensitive
MMTV-Luc reporter plasmid, deprived of steroids for 24 h,
and then treated with ethanol vehicle or 100 nM DHT ⫾ 250
ng/ml FS for 24 h. Data were pooled from three independent
experiments (n ⫽ 4 replicates per treatment group for each
experiment) and expressed as the mean ⫾ SEM of the ratio of
luciferase/RSV-␤-galactosidase activity, normalized to the
vehicle-treated mean within the same plasmid group. B, L␤T2
cells were transfected with the activin-sensitive GRAS-Luc
reporter plasmid, incubated overnight in DMEM containing
10% charcoal-treated FBS and 250 ng/ml FS, and treated for
24 h with vehicle or 100 nM DHT ⫾ 250 ng/ml FS ⫾ 10 ng/ml
activin A. Data were pooled from seven independent experiments (n ⫽ 4 replicates per treatment group for each experiment) and expressed as the mean ⫾ SEM of the ratio of
luciferase to RSV-␤-galactosidase activity, normalized to the
vehicle-treated mean. * Indicates a statistically significant
difference (P ⬍ 0.001) from the vehicle-treated mean within
the same plasmid group.
To investigate potential interactions of the androgenand activin-regulatory systems on the FSH␤ gene, we
addressed the potential role of an activin response
element in the FSH␤ gene in androgen action. Recent
data from our laboratory indicate that a putative Smaand Mad-related protein (Smad) binding element
(SBE) located at ⫺138/⫺124 of the oFSH␤ gene promoter is crucial for activin regulation of transcription
(35a). In the current study, we used an activin-insensitive ⫺138/⫺124 mutant oFSH␤-Luc reporter to further elucidate the molecular mechanism of interaction
between androgen and the activin loop in the regulation of FSH␤ transcription. L␤T2 cells were transiently
transfected with either the ⫺982 truncation of oFSH␤Luc, the ⫺982 truncation of oFSH␤-Luc with the mutation in the ⫺245/⫺231 ARE, or the ⫺982 truncation
of oFSH␤-Luc with a 2-bp mutation in the putative
SBE half-site (Fig. 8A), pretreated overnight with 10%
charcoal-treated FBS and treated with vehicle or 100
nM DHT ⫾ 10 ng/ml activin-A for 24 h. These experiments were performed in serum-free conditions, and
we did not include a FS pretreatment to lower the
endogenous activin; hence, the magnitude of the activin induction is smaller than in Fig. 6. This is not due
to differences in the activin responsiveness of the
⫺982 vs. the ⫺4.7-kb oFSH␤ promoter (35a). As
shown in Fig. 8B, DHT or activin treatment alone each
stimulated activity of the ⫺982 oFSH␤-Luc reporter by
approximately 2-fold (P ⫽ 0.001 and P ⬍ 0.000001,
respectively), and combined DHT plus activin treatment stimulated reporter activity by 4-fold (P ⬍
0.000001). The oFSH␤-Luc reporter with the ARE mutation was not affected by DHT alone (P ⬍ 0.64) and
was induced 1.7-fold by activin alone (P ⫽ 0.00004),
and the cotreatment of DHT with activin did not significantly increase reporter activity above that of activin treatment alone (P ⫽ 0.28; 1.9-fold vs. 1.7-fold).
As expected, the oFSH␤-Luc reporter in which the
⫺138/⫺124 putative SBE half-site has been disrupted
by a double-point mutation was not significantly induced by activin treatment alone (1.2-fold; P ⫽ 0.213).
Interestingly, the ⫺138/⫺124 activin nonresponsive
mutant of the oFSH␤-Luc reporter was completely
insensitive to DHT treatment alone (0.98-fold vs. vehicle; P ⫽ 0.11) or DHT enhancement of activin induc-
932
Mol Endocrinol, April 2004, 18(4):925–940
Spady et al. • Androgen Induction of FSH␤
Fig. 8. Androgen Induction of the FSH␤ Gene Is Dependent upon an Activin-Responsive Element
A, Base pairs in the region of the consensus sequences for the ⫺245 ARE and ⫺138 SBE are in uppercase and others are in
lowercase. Mutated bases are underlined. B, L␤T2 cells were transfected with the wild-type, ARE mutant, and SBE mutant
FSH␤-Luc reporter plasmids, incubated overnight in DMEM containing 10% charcoal-treated FBS, and treated for 24 h with
vehicle or 100 nM DHT ⫾ 10 mg/ml activin A. This simplified protocol does not include the FS pretreatment as compared with the
protocols for Figs. 6 and 7, but the activin stimulation remains significant. Data were pooled from seven independent experiments
(n ⫽ 3–4 replicates per treatment group for each experiment) and expressed as the mean ⫾ SEM of the ratio of luciferase to
RSV-␤-galactosidase activity, normalized to the vehicle-treated mean. * Indicates a statistically significant difference (P ⬍ 0.05)
from the vehicle-treated mean. † Indicates a statistically significant difference (P ⬍ 0.05) from the activin only-treated mean. mut,
Mutant.
tion (0.90-fold vs. vehicle and activin; P ⫽ 0.446) of
FSH␤ reporter gene activity. Therefore, androgen induction of FSH␤ requires both the ⫺245/⫺231 ARE
and ⫺138/⫺124 SBE sites, whereas activin response
requires the ⫺138/⫺124 SBE site, but may be slightly
dampened by loss of the ⫺245/⫺231 ARE. These data
provide strong evidence in support of the activin dependence of androgen stimulation demonstrated in
Fig. 7. Moreover, they suggest that an interaction between AR and the activin-regulatory system occurs
through at least two distinct cis elements in the proximal oFSH␤ promoter.
Androgen Enhances GnRH Stimulation of FSH␤
Gene Expression
In addition to modulating the activin/FS system in the
pituitary, androgens have been shown to regulate
GnRH sensitivity via alterations in the level of
GnRH-Rs (16–20). In fact, in the case of the LH␤ gene,
GnRH stimulation was required to observe androgen
repression in the L␤T2 cell model (9, 10). We hypothesized that androgens may act upon the gonadotrope
to alter the responsiveness of the FSH␤ gene to GnRH
and thereby indirectly regulate its expression. To test
this hypothesis, L␤T2 cells were transfected with the
4.7-kb oFSH␤-luciferase reporter and the ability of
10⫺12 to 10⫺8 M DHT administered for 24 h to affect
GnRH (10 nM for 6 h) induction of oFSH␤ promoter
activity was examined (Fig. 9). Treatment for 24 h with
10 nM DHT induced FSH␤ promoter activity by 1.6-fold
(P ⫽ 0.032), and 6 h treatment with 10 nM GnRH
induced promoter activity by 1.7-fold (P ⬍ 0.015).
These results were expected because both androgens
(Fig. 1) and GnRH (22, 25) are each able to independently stimulate FSH␤ expression in L␤T2 cells. Interestingly, when increasing concentrations of DHT were
coadministered with 10 nM GnRH, the oFSH␤ promoter was increasingly stimulated in a DHT dosedependent manner. The 1 nM DHT concentration,
when coadministered with GnRH, resulted in a 2.75fold induction of FSH␤ promoter activity, which was
significantly greater than either 10 nM DHT (P ⬍
0.0001) or 10 nM GnRH (P ⬍ 0.001) treatment alone.
The maximal stimulation of 3-fold was observed in
cells cotreated with 10 nM DHT and GnRH. FSH␤
promoter activity of this treatment group was also
significantly greater than that of cells treated with either DHT (P ⬍ 0.0001) or GnRH (P ⬍ 0.0001) alone.
Analysis of the data by two-way ANOVA reveals a
Spady et al. • Androgen Induction of FSH␤
Mol Endocrinol, April 2004, 18(4):925–940
933
mouse GnRH-R promoter activity (Fig. 10A). Statistically significant induction of GnRH-R gene expression
was observed at 1 (P ⫽ 0.002), 10 (P ⬍ 0.0001), and
100 nM (P ⬍ 0.0001) DHT concentrations, with maximal stimulation at 10 nM. These data indicate that at
Fig. 9. DHT and GnRH Act Synergistically to Stimulate
oFSH␤ Expression
L␤T2 cells were transfected with 4.7 kb oFSH␤-luciferase
reporter plasmid, deprived of serum for 20 h, pretreated for
18 h with vehicle or various concentrations of DHT, and
treated an additional 6 h with the same concentration of DHT
in the presence or absence of 10 nM GnRH. Data were pooled
from six independent experiments (n ⫽ 3–4 replicates per
treatment group for each experiment) and expressed as the
mean ⫾ SEM of the ratio of luciferase/TK-␤-galactosidase
activity, normalized to the vehicle-treated mean. * Indicates a
statistically significant difference (P ⬍ 0.05) from the vehicletreated mean. † Indicates a statistically significant difference
(P ⬍ 0.0001) from the DHT only-treated mean. ‡ Indicates a
statistically significant difference (P ⬍ 0.0001) from the GnRH
only-treated mean.
synergistic increase upon cotreatment with GnRH and
androgen that is significantly (P ⫽ 0.001) different than
an additive effect (33). These data suggest that androgens, in addition to directly stimulating FSH␤ expression through novel AREs, may also be enhancing the
ability of GnRH to stimulate FSH␤ expression. To test
this hypothesis, we examined whether androgens are
able to modulate responsiveness of gonadotropes to
GnRH by inducing the GnRH-R gene.
Androgen Stimulates GnRH-R Gene Expression in
a Time- and Dose-Dependent Manner
One mechanism through which androgens could enhance response of gonadotropes to GnRH is by stimulating expression of the GnRH-R. We examined the
effects of androgen treatment on expression of the
mouse GnRH-R gene in L␤T2 cells transfected with a
luciferase reporter plasmid the expression of which
was driven by 1.2 kb of the mouse GnRH-R gene
promoter. L␤T2 cells were deprived of steroids for
24 h, and then incubated for 24 h with media containing either 0.1% ethanol vehicle alone or increasing
concentrations of DHT. Treatment for 24 h with DHT
resulted in a gradual dose-dependent increase in
Fig. 10. The GnRH-R Gene Is Induced by Androgens
A, L␤T2 cells were transfected with a 1.2-kb mouse GnRHR-luciferase reporter plasmid, deprived of steroids for 20 h,
and then treated with vehicle or various concentrations of
DHT for 24 h. Data were pooled from five independent experiments (n ⫽ 5–6 replicates per treatment group for each
experiment) and expressed as the mean ⫾ SEM of the ratio of
luciferase to RSV-␤-galactosidase activity, normalized to the
vehicle-treated mean. * Indicates a statistically significant
difference (P ⬍ 0.0001) from the vehicle-treated mean. B,
L␤T2 cells were transfected with the mouse GnRH-R-luciferase expression vector, deprived of steroids for 20 h, and
then treated with 10 nM DHT for 6, 12, 24, or 48 h. Data were
pooled from three independent experiments (n ⫽ 4–6 replicates per treatment group for each experiment) and expressed as the mean ⫾ SEM of the ratio of luciferase to
RSV-␤-galactosidase activity, normalized to the vehicletreated mean of the same treatment time. * Indicates a statistically significant difference (P ⬍ 0.0001) from the vehicletreated mean of the same treatment time.
Spady et al. • Androgen Induction of FSH␤
934 Mol Endocrinol, April 2004, 18(4):925–940
doses physiologically relevant to adult male mice, androgen stimulates expression of the mouse GnRH-R
gene in a dose-dependent manner.
To further characterize the mechanism of androgen
stimulation of GnRH-R gene expression, we treated
transfected L␤T2 cells with either ethanol vehicle or 10
nM DHT for 6, 12, 24, or 48 h (Fig. 10B). DHT (10 nM)
treatment for 6 h had no effect on GnRH-R promoter
activity. As the length of exposure to DHT was increased, the induction of promoter activity increased.
DHT treatment for 12 h resulted in a modest 1.2-fold
induction of promoter activity but was not statistically
significant (P ⫽ 0.15). As expected from the results of
Fig. 10A, 24 h of 10 nM DHT resulted in a statistically
significant 1.4-fold (P ⬍ 0.0001) induction of GnRH-R
promoter activity. DHT treatment for 48 h resulted in
an even greater 1.9-fold (P ⬍ 0.0001) induction of
GnRH-R reporter gene expression. These data indicate that a physiologically relevant dose of androgen
stimulates GnRH-R gene expression in a time-dependent manner.
Androgen Enhancement of GnRH Stimulation
Persists after Abrogation of Direct Androgen
Stimulation of FSH␤ Expression
We have demonstrated that androgen stimulates
GnRH-R gene expression in the mouse gonadotrope;
however, these data do not discount that the effect of
DHT and GnRH treatment in the stimulation of FSH␤
promoter activity might still be due to cross-talk between the androgen and GnRH signaling pathways
rather than due to androgen causing sensitization of
the gonadotrope to GnRH. To test this alternate hypothesis, we examined the ability of DHT and GnRH to
stimulate promoter activity of a 4.7-kb oFSH␤-luciferase expression plasmid that had been rendered insensitive to direct androgen stimulation. The ⫺245/
⫺231 ARE was mutated in the same manner as in Fig.
4, but in the context of the 4.7-kb oFSH␤ 5⬘-flanking
region. As described in Fig. 4, this same mutation
completely abolished androgen stimulation in the context of the ⫺982 oFSH␤ promoter. As expected, 24 h
of 10 nM DHT failed to result in stimulation of reporter
gene activity in L␤T2 cells transiently transfected with
the 4.7-kb oFSH␤-luciferase plasmid containing the
ARE mutation (P ⫽ 0.85; Fig. 11). This indicates that
mutation of this ARE alone is sufficient to block androgen induction even in the context of the ⫺4.7 kb
promoter. GnRH (10 nM) for 6 h induced mutant FSH␤
promoter activity by 2.2-fold compared with vehicletreated controls (P ⬍ 0.0001). Interestingly, when 10
nM DHT was coadministered with GnRH, the activity of
the ARE mutant oFSH␤ promoter was still significantly
increased above that of GnRH treatment alone by
1.78-fold (P ⬍ 0.0001). These data indicate that androgens act indirectly through GnRH to induce FSH␤
promoter activity, even when androgens are no longer
able to directly induce FSH␤ expression. Taken together with the data from Figs. 9 and 10, these results
Fig. 11. DHT Enhances GnRH Stimulation of an AndrogenInsensitive Mutant of FSH␤
The ⫺245/⫺231 ARE was disrupted in the 4.7 kb oFSH␤luciferase reporter plasmid by site-directed mutagenesis, and
the ability of 10 nM DHT for 24 h, 10 nM GnRH for 6 h, or the
combination of both DHT (24 h) and GnRH (final 6 h) treatments to stimulate the mutant FSH␤ promoter activity was
examined. Data were pooled from four independent experiments (n ⫽ 4 per group in each experiment) and expressed as
the mean ⫾ SEM of the ratio of luciferase to TK-␤-galactosidase, normalized to the vehicle-treated mean. * Indicates a
statistically significant difference (P ⬍ 0.0001) from the vehicle-treated mean. † Indicates a statistically significant difference (P ⬍ 0.0001) from GnRH only-treated mean.
support the hypothesis that androgens also act in the
gonadotrope to indirectly promote transcription of the
FSH␤ gene by stimulating GnRH-R expression and
thereby enhancing responsiveness of the gonadotrope and FSH␤ gene to GnRH. Alternatively, it is also
possible that androgen could be affecting the sensitivity of the L␤T2 cell to GnRH by augmenting the
signal transduction pathways through which GnRH is
acting on the FSH␤ gene.
DISCUSSION
Accumulating evidence indicates that the steroid hormone feedback regulation of gonadotropin synthesis
involves multiple mechanisms, with distinct actions at
the levels of both the hypothalamus and pituitary
gland. Very little is known about how androgenic steroids regulate the FSH␤ gonadotropin subunit gene at
the level of the pituitary gland, the molecular mechanisms involved, or even whether the gonadotrope itself
is the direct target of androgen. The key impediment to
such investigations has been the lack of an appropriate FSH␤-expressing gonadotrope model system.
Recent studies using highly sensitive RT-PCR (22),
immunohistochemistry (36), and RNase protection assays (37) have demonstrated that the L␤T2 mouse
Spady et al. • Androgen Induction of FSH␤
gonadotrope cell line expresses endogenous FSH␤,
and that expression of FSH␤ in L␤T2 cells is regulated
by the same factors known to regulate FSH␤ in vivo,
such as GnRH, activin, and FS (22). Previous studies
had reported that L␤T2 cells, like normal gonadotropes, express ER (38) and AR (23). The data presented in the current study confirm that endogenous
AR is functional and active in L␤T2 cells, demonstrating the relevance of the L␤T2 cell line as a model
system to study the action of androgenic steroid
hormones.
Previous literature has demonstrated that androgen
regulation of FSH␤ gene expression appears to be
species specific and involves actions at both the hypothalamus and pituitary gland. In rats, for example,
androgens act through at least two opposing mechanisms to regulate FSH synthesis, i.e. an inhibitory action at the hypothalamic level that is GnRH dependent
and a stimulatory action at the pituitary level that is
independent of GnRH (7, 11–13). In primates, as in
rats, androgens act both at the hypothalamic level in a
GnRH-dependent manner and at the pituitary level in a
GnRH-independent manner. However, unlike the rat,
both mechanisms of androgens appear to be inhibitory to FSH␤ expression in primates (39, 40). In the
current study, we elucidate the activin-dependent,
GnRH-independent, molecular mechanism through
which androgens stimulate expression of the oFSH␤
gene at the level of the pituitary gonadotrope. Furthermore, we identify a GnRH-dependent mechanism of
androgen stimulation that potentially functions
through the induction of GnRH-R gene expression.
Several investigators have demonstrated, using either GnRH antagonist-treated rats or primary cultures
of rat pituitary cells, that at the level of the pituitary
gland, androgens increase steady-state levels of
FSH␤ mRNA (7, 8, 11–13) and primary transcript (41)
and enhance stability of FSH␤ transcripts (11) through
unknown mechanisms independent of GnRH. These
studies, however, were not able to address whether
androgen regulation of FSH␤ expression occurs by
direct actions on the FSH-producing gonadotrope or
is indirectly mediated by one of the other many distinct
populations of the anterior pituitary. This is an important distinction because gonadotropes comprise only
a small minority (5–15%) of the total secretory cell
types within the anterior pituitary (42), and several
anterior pituitary cell types express AR in vivo (43–45).
Moreover, the different pituitary cell populations communicate with each other through paracrine, juxtacrine, and endocrine mechanisms (46). The data presented herein specifically address whether the
androgen regulation of FSH␤ expression observed at
the level of the whole pituitary gland also occurs at the
level of the gonadotrope. Taken together, these data
indicate that physiologically relevant concentrations of
androgens stimulate FSH␤ gene expression by acting
directly within gonadotropes. This androgen regulation of FSH␤ occurs in a specific and saturable, dosedependent manner typical of classic steroid hormone
Mol Endocrinol, April 2004, 18(4):925–940
935
action. Furthermore, androgen induction of FSH␤ transcription occurs within 24 h, a physiologically relevant
time frame when compared with the diurnal changes in
androgen levels that occur in many different animal
genera, including rodents (47, 48).
Our finding that two of the three sites within the
proximal oFSH␤ promoter shown to act as PREs by
Webster et al. (30) also act as AREs, demonstrates that
PR and AR likely use distinct mechanisms with shared
components. Because disruption of the ⫺212/-198
PRE does not interrupt androgen stimulation of FSH␤,
it must not be crucial to the mechanism of androgen
regulation. Although the necessity of the three PREs
for progesterone stimulation was not examined in the
studies by Webster et al. (30), all three PREs were
shown to specifically bind PR in gel shift assays and
confer progesterone response when multimerized on a
heterologous promoter, demonstrating their sufficiency as PREs. In contrast, we have shown that two
of these elements (⫺245/⫺231 and ⫺153/⫺139) are
individually required for androgen induction of the
oFSH␤ gene, i.e. that neither is sufficient when the
other is mutated. Notably, only one of these elements
could be demonstrated to bind to AR protein in vitro
(⫺245/⫺231). This may indicate that the ⫺153/⫺139
element is low affinity despite the fact that its mutation
ablates androgen responsiveness. Perhaps the ⫺153/
⫺139 element is required to coordinate accessory or
interacting proteins that provide context for the action
of the AR binding at the ⫺245/⫺231 element or perhaps other binding proteins present in the L␤T2 cell
nuclei are required to allow AR to bind to the ⫺153/
⫺139 sequence.
Interestingly, two of the three PRE/ARE sequences
within the oFSH␤ gene proximal promoter are highly
conserved among mammalian species, including pigs,
cows, rats, mice, and humans (Fig. 4). The high degree
of conservation of these sequences would suggest
that they play important roles in androgen and/or progesterone regulation of FSH␤ in a wide variety of species. However, the significance of this high degree of
sequence conservation in mammals is complicated by
observations of androgen inhibition of FSH synthesis
in primate model systems. In castrated rhesus macaques treated with GnRH antagonist, administration
of T significantly reduced the levels of serum FSH,
suggesting an inhibitory role of androgen in FSH synthesis at the level of the pituitary gland in nonhuman
primates (39). Similarly, in primary pituitary cell cultures from hypogonadal (hpg), GnRH-deficient, human
FSH␤ promoter-containing transgenic mice, administration of testosterone propionate or DHT for 24 h
reduced steady-state levels of human FSH␤ transgene
mRNA (40). The authors postulated that differences in
the FSH␤ gene between humans and rats were a
possible reason for the contradiction of their data to
that observed in rats. Our finding that both sequences
responsible for androgen action within the oFSH␤
gene are extremely well conserved among mammals
does not support such a hypothesis, and therefore, the
936
Mol Endocrinol, April 2004, 18(4):925–940
relative differences in the responses of the rat and
sheep gene to androgen (stimulatory) compared with
that of the human gene (inhibitory) are probably not
due to sequence differences. We cannot, however,
disregard the possibility that subtle sequence differences in regions flanking the highly conserved 110-bp
region of the proximal FSH␤ promoter examined could
affect sensitivity to androgens. Alternatively, the two
ARE sequences identified in the current study may be
crucial to the actions of AR in FSH␤ expression for
both sheep and humans, but interaction of AR with
different combinations of coactivators or corepressors
on the FSH␤ gene may ultimately determine whether
androgens will stimulate or repress expression in a
given species.
The dependence of androgen activation of the
oFSH␤ gene on the activin autocrine loop indicates a
potential mechanism for species differences. For example, both Smad 3 and Smad 4 are known to bind AR
in vitro, and overexpression of AR affects expression
of Smad 3/4-sensitive genes and vice versa (49–51).
This is of physiological importance because Smads
are downstream mediators of the activin receptor. Activin is a key physiological regulator of FSH synthesis
and secretion and is believed to be a principal mechanism through which FSH is regulated differentially
from LH (52). Thus, the dependence of androgen induction on the presence of endogenous activin indicates that activin acts as a permissive agent for the
regulation of this gene by androgen.
Androgens have been hypothesized to act in the
anterior pituitary through modulation of the components of the activin and FS system (8, 14, 15, 31, 32).
Our finding that FS blocks the action of androgens on
the FSH␤ promoter might seem to support this hypothesis. However, the further demonstration that
androgens have no effect on activin induction of an
activin-response element from the GnRH-R gene indicates that the role that the activin/FS system plays in
androgen action is gene specific, not a global alteration in the bioavailability of activin or FS or in the level
or responsiveness of the activin receptors. Furthermore, the removal of activin by FS tends to increase,
rather than decrease, the response of MMTV to androgens, indicating that activin is not necessary for the
action of AR on expression of other genes. Finally,
mutation of only one ARE in the FSH␤ promoter prevents androgen regulation despite the presence of the
intact activin-response elements and activin responsiveness indicating that androgen is not regulating this
promoter by altering the levels of activin or FS. This
finding is supported by a recent study by Burger et al.
(41) in which androgen was shown to induce FSH␤
primary transcript independent of pituitary FS mRNA
regulation. The mechanism of the activin dependence
is further elucidated by the demonstration that mutation of an element required for activin action on the
oFSH␤ gene abrogates androgen induction despite
the presence of both intact AREs. This indicates the
potential for an interaction between these elements
Spady et al. • Androgen Induction of FSH␤
perhaps through AR/SMAD protein-protein interactions, as has been demonstrated in other systems
(49–51).
In addition to species-specific and activin-dependent effects of direct androgen action on FSH␤, the
current study also supports a species-specific effect
of indirect androgen action on FSH␤. Studies using
rats have most commonly demonstrated that the levels of GnRH-R and GnRH-R gene expression are reduced by androgen treatment (18–20). However, this
does not appear to be the case in the mouse. Naik et
al. (21) determined by radioligand binding studies that
castration reduced the number of GnRH binding sites
(GnRH-Rs) within the pituitary gland of male mice by
approximately 50%, and that T replacement at the
time of castration completely prevented the castration-mediated decline in the number of GnRH-Rs.
More recently, Curtin et al. (9) demonstrated that the
levels of GnRH-R mRNA are increased approximately
1.7-fold by 24 h of 1 nM DHT in L␤T2 cells. Our data
confirm the findings of these earlier studies that androgens stimulate GnRH-R gene expression in the
mouse gonadotrope. Furthermore, our demonstration
of the dose and time dependency of androgen stimulation of a mouse GnRH-R reporter gene in mouse
gonadotropes indicates the specificity of this apparent
species-specific androgen action. Although our data
demonstrate that androgens synergistically enhance
GnRH stimulation of FSH␤ gene expression, and that
this same regimen of androgen treatment induces
GnRH-R gene expression, these data do not prove
that the mechanism through which androgens act synergistically with GnRH to stimulate FSH␤ gene expression depends on an increase in GnRH-R number. It
remains possible that this synergism and the remaining effects of androgen on the ARE-mutated FSH␤
gene are due to androgen effects on the GnRH signaling cascade. Gonadotropes possess a reservoir of
spare GnRH-Rs (53), and increasing the number of
cell-surface GnRH-Rs does not necessarily increase
the responsiveness to GnRH (54, 55). Furthermore,
studies have shown that GnRH can regulate the level
of FS (56, 57) in rat pituitary, providing another possible mechanism for the interaction of GnRH and androgen on the FSH␤ gene. However because GnRH
was shown to induce FS levels (56, 57), this would be
counter to our finding of GnRH induction of the FSH␤
gene. Nevertheless, our data are strongly coincidental,
and the hypothesis that the two androgen-regulated
events are linked is an alluring one.
The physiological significance of the opposing
mechanisms of androgen-positive and -negative feedback regulation of FSH␤ at the pituitary and hypothalamic levels, respectively, may seem paradoxical or
counterproductive. However, these two opposing
mechanisms of androgen feedback regulation of
FSH␤ have potential benefits. First, the contrary effects of androgen at the pituitary and hypothalamic
levels provide a means through which a single hormone, androgen, may differentially regulate synthesis
Spady et al. • Androgen Induction of FSH␤
of the two gonadotropin hormones, LH and FSH, by
acting differently at only one hypothalamic-pituitarygonadal (HPG) axis site (gonadotrope) without requiring different actions on the other axis sites (hypothalamus and gonads). Thus, expression of the two
gonadotropin genes could be altered differentially in a
subtle yet complex manner using relatively simple
mechanisms. This is further supported by our suggestion that androgens may stimulate responsiveness of
the gonadotrope to GnRH and, in turn, further enhance
FSH␤ transcription. In this manner, androgens could
selectively stimulate FSH␤ gene transcription by two
mechanisms occurring at the level of the gonadotrope.
Additionally, the dependence on activin of androgen
regulation of the FSH␤ further distinguishes the responses of the two gonadotropin genes. Another advantage of opposing mechanisms of androgen regulation of FSH is that in males, which have sufficient
concentrations of androgen to stimulate FSH expression, the opposing mechanisms of androgen action in
the context of the entire HPG axis would result in
relatively stable expression of FSH at a time of dramatic and precipitous decline in LH. Androgen acts at
the hypothalamus to reduce GnRH availability, which
by itself results in reduction of both LH and FSH␤
transcription (due to loss of GnRH stimulation). Simultaneously at the level of the gonadotrope, androgen
stimulates the FSH␤ gene directly and may also increase the number of GnRH-Rs, which would result in
each gonadotrope being more sensitive to the GnRH
that is still available. When the results of each mechanism within the context of the entire HPG axis are
added together, they balance each other out so that
net FSH␤ expression is unchanged or perhaps even
modestly increased. The increased expression of
GnRH-R would not, however, enhance GnRH induction of LH, because androgen interferes with the binding of downstream GnRH-R-induced signal transducers to the LH␤ gene itself (9, 10). Because AR
repression of LH␤ expression at the level of the gonadotrope appears to be downstream of the GnRH-R,
increasing GnRH-R does not alter the inhibitory effect
of androgen on LH, resulting in inhibition of GnRH
stimulation of LH by androgen. The physiological relevance of these combined opposing mechanisms are
applicable both to nonseasonal animals like rats,
which would require steady FSH levels to maintain
spermatogenesis throughout the year, and to seasonal
animals like sheep, in which seasonally increasing androgen levels could result in seasonal net increases in
FSH and thereby dramatically increase spermatogenesis during the breeding season. Interestingly, FSH
levels in male rats do not fluctuate substantially once
maturity is reached, whereas both androgen and LH
levels surge diurnally (48, 58, 59). In male sheep, androgen and FSH levels rise simultaneously at the beginning of the breeding season (60). The physiological
significance of the findings of the current study, that
androgens act directly through AR binding to ARE(s)
on FSH␤ in an activin-dependent manner, and may act
Mol Endocrinol, April 2004, 18(4):925–940
937
indirectly through stimulation of GnRH-R expression
at the level of the gonadotrope to stimulate FSH␤ gene
expression, is likely relevant to the underlying physiological mechanisms of steroid hormone feedback regulation of reproduction in a variety of both seasonal
and nonseasonal breeding animal species.
MATERIALS AND METHODS
Hormones
T and DHT were purchased from Sigma Chemical Co. (St.
Louis, MO). All steroid stock solutions were prepared by
dissolving crystalline hormone in 100% ethanol at a 10 mM
concentration and stored in lightproof borosilicate glass vials
at 4 C. Before each experiment, fresh steroid treatment preparations were made by diluting the 10 mM stock in 100%
ethanol to 103-fold higher concentration than the final target
concentration, and subsequently, 1 ␮l of diluted steroid was
added to 1 ml total volume of media to achieve the final target
concentration of steroid in 0.1% ethanol vehicle. The human
recombinant activin A was obtained from Calbiochem (San
Diego, CA). Recombinant mouse FS 288 was kindly provided
by Shunichi Shimasaki. Both were resuspended in PBS with
0.1% BSA. GnRH was obtained from Sigma.
Hormone Treatments
For all transient transfection experiments, 8 h after transfection, the L␤T2 cells were preincubated in steroid-free DMEM
supplemented with 10% charcoal/dextran-treated FBS for
20 h, followed by the appropriate treatment protocol. In the
steroid experiments, fresh DMEM with 10% charcoal/
dextran-treated FBS containing T or DHT was added, and the
cells were incubated for 6–48 h as indicated in the figure
legends. For the activin/FS experiments in Figs. 6 and 7, the
preincubation media was DMEM with 10% charcoal/dextrantreated FBS containing 250 ng/ml (final concentration) recombinant human FS. After 20 h of steroid-free FS pretreatment, fresh DMEM with 10% charcoal/dextran-treated FBS
containing 100 nM DHT and/or 250 ng/ml FS or 10 ng/ml
recombinant human activin-A was added, and the cells were
incubated for 24 h. In the activin/DHT experiments in Fig. 8,
the transfection and treatment procedure was the same as
that of Figs. 6 and 7, except that DMEM supplemented with
10% charcoal/dextran-treated FBS without FS was used for
the 20-h preincubation before treatment for 24 h with 100 nM
DHT and/or 10 ng/ml activin A. In the GnRH/DHT experiments, steroid-free DMEM with 10% charcoal/dextrantreated FBS was used as preincubation media. After 20 h of
steroid deprivation, the media were changed to serum-free
DMEM supplemented with 0.1% BSA and 5 ng/ml transferrin
containing DHT, and the cells were incubated for 24 h. Six
hours before harvest, additional serum-free DMEM (with
BSA/transferrin supplement) containing DHT and/or GnRH
was added, and the cells were incubated for the remaining
6 h until harvest. The cells were then harvested, and luciferase and ␤-galactosidase assays were performed.
Construction of Plasmids for Transfection
A 5.5-kb region of the oFSH␤ gene (oFSH␤) encompassing
4741 bp of the promoter and 759 bp downstream from the
⫹1 transcription start site was subcloned into the pGL3Basic luciferase promoter plasmid (Promega Life Science,
Madison, WI) as described previously (22). The cloning of the
⫺982 truncation was described previously (25). Cloning of
Spady et al. • Androgen Induction of FSH␤
938 Mol Endocrinol, April 2004, 18(4):925–940
the ⫺750 truncation was performed by inserting a SacI to SalI
fragment of oFSH␤ into the SacI to XhoI sites of the pGL3Basic vector. Cloning of the ⫺561 truncation was performed
by inserting a NdeI (filled in with Klenow fragment enzyme) to
BglII fragment of oFSH␤ into the SmaI to BglII sites of the
pGL3-Basic vector. Cloning of the ⫺105 truncation was performed by inserting a HindIII fragment of oFSH␤ into the
HindIII site of the pGL3-Basic vector. The ⫺401 truncation
was made by PCR amplification using a 5⬘-primer: 5⬘-CTCT
GCTA GTTTT TCAA TCTA CC- 3⬘ and a 3⬘-primer: 5⬘-CTGC
AGCA GATT GCTCTCC-3⬘, and the amplified fragment was
cloned into the SmaI site of the pGL3-Basic vector. Mutagenesis was performed using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) according to the
manufacturer’s protocol. All plasmids were sequenced to
confirm the fidelity of the sequence, the junctions, and the
mutations.
Plasmid DNA was prepared from overnight bacterial cultures using DNA plasmid columns according to the manufacturer’s protocol (QIAGEN, Chatsworth, CA) or a cesium
chloride protocol adapted from Sambrook et al. (61).
The MMTV-pGL3 basic luciferase promoter plasmid was
kindly provided by Jeff Miner of Ligand Pharmaceuticals. The
GRAS-luciferase reporter gene contains four repeats of the
GRAS element (⫺340 to ⫺315 from the mouse GnRH-R
gene) upstream of a minimal ⫺81 thymidine kinase (TK) promoter in pGL3. The cytomegalovirus (CMV)-␤-galactosidase,
TK-␤-galactosidase, and Rous sarcoma virus (RSV)-␤-galactosidase reporter plasmids were prepared as described previously (22, 25, 62, 63).
Cell Culture and Transient Transfection
L␤T2 cells were maintained in 100-mm diameter dishes in
DMEM (Cellgro, Mediatech, Inc., Herndon, VA) supplemented
with 10% FBS (Omega Scientific, Inc., Tarzana, CA) at 37 C
with 5% CO2. Charcoal-treated FBS was also obtained from
Omega Scientific, Inc. When cells reached 70–80% confluency, they were trypsinized and 2 ⫻ 105 cells were plated per
well into 24-well plates (Nunclon) in DMEM supplemented
with 10% FBS. Transient transfections were performed using
FuGENE 6 transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN), following the manufacturer’s protocol.
Unless otherwise noted in the figure legends, CMV-␤-galactosidase was used as an internal transfection efficiency control for androgen experiments, RSV-␤-galactosidase was
used for an internal control in activin/androgen cotreatment
experiments, and TK-␤-galactosidase was used as an internal control for GnRH/androgen cotreatment experiments.
None of the hormone treatments had significant effects on
expression of the internal control plasmids (data not shown).
Luciferase and ␤-Galactosidase Assays
L␤T2 cells were washed once with 1⫻ PBS and then 40 ␮l of
lysis solution (Galacto-light assay system, Tropix, Bedford,
MA) was added to each well of the 24-well plates. Cells were
then incubated at room temperature on a plate shaker for 5
min to detach and lyse cells. The contents of the wells were
then transferred to microcentrifuge tubes on ice and centrifuged at 12,300 rpm for 8 min at 4 C. Lysed sample (20 ␮l)
was assayed for luciferase activity, and 10 ␮l were aliquoted,
incubated at 48 C for 1 h to heat inactivate endogenous
␤-galactosidase, and then assayed for ␤-galactosidase activity from the reporter gene. Luciferase and ␤-galactosidase
activity were measured using an EG&G Berthold Microplate
Luminometer (PerkinElmer Corp., Norwalk, CT) as described
previously (22).
EMSA
Full-length, human AR containing a Flag epitope tag was
overexpressed in Sf9 cells via a baculovirus expression system (64). The Sf9 cells were inoculated with virus at a multiplicity of infection of 1.0 and allowed to grow for an additional
48 h at 27 C. They were treated for the last 24 h before
harvest with 1 mM DHT (final concentration). Cells were harvested by centrifugation at 1500 rpm for 15 min, washed
once in TG buffer (10 mM Tris-HCl, pH 8.0; and 10% glycerol)
and frozen as a pellet at ⫺80 C. The Sf9 cells were lysed in
a homogenization buffer (20 mM Tris-HCl, pH 7.5; 350 mM
NaCl, 1 mM dithiothreitol, 10% glycerol, 0.5 ␮g/ml leupeptin,
10 ␮g/ml bacitracin, 2 ␮g/ml aprotinin, 1 ␮g/ml pepstatin). All
procedures were done at 0⫺4 C. The cell lysate was centrifuged at 40,000 rpm for 30 min, and the supernatant was
taken as a soluble whole-cell extract. The ability of AR to bind
an oligonucleotide from the oFSH␤ promoter was determined
by EMSA. AR was incubated with 1 fmol of 32P-labeled
oligonucleotide at 4 C in a DNA binding buffer (10 mM HEPES,
pH 7.8; 50 mM KCl, 5 mM MgCl2, 0.1% Nonidet P-40, 1 mM
dithiothreitol, 2 ␮g poly(dI-dC), and 10% glycerol) in the
absence or presence of the C-19x polyclonal antibody to AR
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA), rabbit IgG
control, or 1000-fold excess of the ⫺245, ⫺245 mutant or
ARE consensus oligonucleotides. The 245 oligo is 5⬘-CAAGGTAAAGGAGTGGGTGTTCTACTATA-3⬘; the 245 mutant is
5⬘-CAAGGTAAAGGAGTGGGTCCTCTACTATA-3⬘; and the
ARE consensus is 5⬘-ACGGGTGGAACGCGGTGTTCTTTTGGC-3⬘. The oligonucleotide was end-labeled with T4 DNA
polymerase and [␥-32P]ATP. After 30 min, the DNA binding
reactions were electrophoresed on a 5% polyacrylamide gel
(40:1 acrylamide-bisacrylamide) containing 2.5% glycerol in
a 0.25⫻ TBE buffer (20 mM Tris-borate, pH 8; 20 mM boric
acid; 0.5 mM EDTA).
Acknowledgments
We thank Djurdjica Coss, Suzanne B. R. Jacobs, Mark A.
Lawson, Flavia Pernasetti, and Vyacheslav Vasilyev for helpful discussions, providing plasmids, and/or reading the
manuscript. We thank Shunichi Shimasaki for the gift of FS.
We thank Laura Neely and Rachel White for technical assistance. We also thank Elizabeth Wilson for providing the Flagtagged AR baculovirus and the University of Colorado Cancer
Center Tissue Culture Core facility for baculovirus production.
Received April 2, 2003. Accepted December 23, 2003.
Address all correspondence and requests for reprints to:
Pamela L. Mellon, Ph.D., University of California, San Diego,
Department of Reproductive Medicine 0674, 9500 Gilman
Drive, La Jolla, California 92093-0674. E-mail: pmellon@
ucsd.edu.
This work was supported by National Institute of Child
Health and Human Development/National Institutes of Health
through cooperative agreement (U54 HD12303) as part of the
Specialized Cooperative Centers Program in Reproduction
Research (P.L.M.). This work was also supported by NIH
Grant R37 HD20377 (to P.L.M.). T.J.S. was supported by NIH
NRSA F32 HD08686 and NIH T32 DK07044; R.S. was supported by a Summer Medical Student Fellowship from NIH
T32 HL07491; V.G.T. was supported by NIH T32 HD07203;
L.E. was supported in part by a Howard Hughes Medical
Institute Summer Fellowship; and J.S.B. was supported in
part by NIH T32 GM08666.
Present address for T.J.S.: Center for Reproduction of
Endangered Species, Zoological Society of San Diego, PO
Box 120551, San Diego, California 92112-0551.
Spady et al. • Androgen Induction of FSH␤
Mol Endocrinol, April 2004, 18(4):925–940
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