Androgens, Progestins, and Glucocorticoids Induce Follicle...

Androgens, Progestins, and Glucocorticoids InduceFollicle-Stimulating Hormone �-Subunit GeneExpression at the Level of the Gonadotrope

Varykina G. Thackray,* Shauna M. McGillivray,* and Pamela L. Mellon

Departments of Reproductive Medicine and Neurosciences, the Biomedical Sciences GraduateProgram and the Center for Reproductive Science and Medicine, University of California, San Diego,La Jolla, California 92093-0674

FSH is produced by the pituitary gonadotrope toregulate gametogenesis. Steroid hormones, in-cluding androgens, progestins, and glucocorti-coids, have all been shown to stimulate expres-sion of the FSH� subunit in primary pituitary cellsand rodent models. Understanding the molecularmechanisms of steroid induction of FSH� hasbeen difficult due to the heterogeneity of theanterior pituitary. Immortalized L�T2 cells are amodel of a mature gonadotrope cell and expressthe endogenous steroid receptor for each of thethree hormones. Transient transfection of eachreceptor, along with ligand treatment, stimulatesthe mouse FSH� promoter, but induction is se-verely diminished using receptors that lack theability to bind DNA, indicating that induction islikely through direct DNA binding. All three ste-roid hormones act within the first 500 bp of the

FSH� promoter where six putative hormone re-sponse elements exist. The �381 site is criticalfor FSH� induction by all three steroid hormones,whereas the �197 and �139 sites contribute tomaximal induction. Interestingly, the �273 and�230 sites are also necessary for androgen andprogestin induction of FSH�, but not for glu-cocorticoid induction. Additionally, we find thatall three receptors bind the endogenous FSH�promoter, in vivo, and specifically bind the �381site in vitro, suggesting that the binding of thereceptors to this element is critical for the induc-tion of FSH� by these 3-keto steroid hormones.Our data indicate that androgens, glucocorti-coids, and progestins act via their receptors todirectly activate FSH� gene expression in thepituitary gonadotrope. (Molecular Endocrinology20: 2062–2079, 2006)

FSH AND LH are produced in the gonadotrope cellsof the anterior pituitary. These hormones act on

the gonads to regulate critical aspects of reproduc-tion, including steroidogenesis, gametogenesis, andovulation (1–3). FSH and LH are heterodimeric glyco-proteins composed of a shared �-subunit and aunique �-subunit (4). The �-subunit confers the bio-logical specificity of FSH and LH, and synthesis of thissubunit appears to be the rate-limiting step for pro-duction of FSH and LH (5, 6). Both peptide and steroidhormones regulate FSH and LH synthesis to producethe hormonal pattern necessary for the estrous cycleand normal reproductive function. The hypothalamicneuropeptide, GnRH, mediates the synthesis and se-cretion of both FSH and LH (7). In addition, the mod-ulation of FSH� gene expression by the activin/inhibin/

follistatin system has been well characterized (8, 9).Recent data show that activin can also regulate LH�gene expression (10–14).

A fundamental concept in the study of reproductionis the regulation of the hypothalamic-pituitary-gonadalaxis by steroid hormone feedback. In addition to ex-erting effects at the level of the hypothalamus, steroidscontrol the transcription of FSH� and LH� subunitgenes at the level of the pituitary, although their mech-anisms of action have not been as well characterizedas GnRH and activin. More specifically, androgens,progestins, and glucocorticoids have all been shownto induce FSH� gene expression in the anterior pitu-itary [recently reviewed by Burger et al. (15)]. Althoughandrogens and progestins are produced by the go-nads, and glucocorticoids are produced by the adre-nal glands, all of these 3-keto steroids possess a sim-ilar chemical structure and mechanism of action.Moreover, all three of their respective receptors, theandrogen receptor (AR), progesterone receptor (PR),and glucocorticoid receptor (GR), are related membersof the class I steroid receptor family (16, 17) and bindDNA directly at response elements containing the half-site TGTTCT organized as 15-bp inverted repeats with3-bp spacers (18, 19). In the classical mechanism ofaction, the receptors homodimerize upon ligand bind-ing and bind their response elements causing trans-

First Published Online May 4, 2006* V.G.T. and S.M.M. contributed equally to this work.Abbreviations: AR, Androgen receptor; ChIP, chromatin

immunoprecipitation; DHT, dihydrotestosterone; ER, estro-gen receptor; GnRH-R, GnRH receptor; GR, glucocorticoidreceptor; HRE, hormone response element; PR, progester-one receptor; PRE, progesterone response element; SDS,sodium dodecyl sulfate.

Molecular Endocrinology is published monthly by TheEndocrine Society (http://www.endo-society.org), theforemost professional society serving the endocrinecommunity.

0888-8809/06/$15.00/0 Molecular Endocrinology 20(9):2062–2079Printed in U.S.A. Copyright © 2006 by The Endocrine Society

doi: 10.1210/me.2005-0316

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activation of specific genes (20). More recently, it hasbeen recognized that the steroid receptors can alsofunction in a nonclassical manner to modulate tran-scription through indirect DNA binding via interactionswith other transcription factors such as activation pro-tein 1 (21–23).

Several lines of evidence indicate that androgensregulate the levels of FSH� and LH� mRNAs in theanterior pituitary. Studies in castrated, GnRH antago-nist-treated rats have shown a selective increase inFSH� mRNA upon treatment with testosterone (24–27). Subsequent studies in primary rat pituitary cellsconfirmed that the activation of FSH� expression bytestosterone occurs at the level of the pituitary (28–30).In contrast to FSH�, androgens repressed LH�-sub-unit gene expression in both castrated, GnRH antag-onist-treated rats (26, 27) and primary pituitary cellculture (29). Additionally, the effects of androgens ontranscription of the LH� subunit have been studied ingonadotrope-derived immortalized cell lines. Jor-gensen and Nilson (31) found that androgens sup-press bovine LH� subunit gene expression throughprotein-protein interaction between ligand-bound ARand steroidogenic factor 1. Using a �617/�44 rat LH�promoter, Curtin et al. (32) determined that androgentreatment suppressed GnRH-induced LH� subunitgene transcription as well. However, they found thatthe suppression occurred primarily as a consequenceof direct protein-protein interaction between AR andSp1.

Progesterone also appears to mediate FSH� geneexpression at the level of the pituitary. FSH� mRNAlevels were increased in rats treated with estrogen andprogesterone (33). The presence of estrogen and pro-gesterone also further augmented the increase inFSH� mRNA expression in response to GnRH stimu-lation (34). Furthermore, antiprogestins blocked FSHsecretion and mRNA expression during the preovula-tory FSH surge (35) as well as blocking the secondaryFSH surge (36, 37). Both the rat and ovine FSH�promoters have been examined for progesterone re-sponsiveness. Reporter genes containing either theproximal rat or ovine FSH� promoter responded toprogestin treatment in primary rat or ovine pituitarycultures, respectively (38, 39). Within the ovine FSH�promoter, six progesterone response elements (PREs)were shown to bind PR (38), whereas three PREsbound PR in the rat promoter (39), although the func-tional role of the individual elements was not deter-mined. Less is known about the effects of progestinson LH� mRNA expression, although no effect wasreported on LH� mRNA levels in ovariectomized ratstreated with estrogen and progestin (34) or on LH�mRNA levels in rat primary pituitary cells treated withboth estrogen and progestin (40).

Similar to androgens and progestins, glucocorti-coids affect FSH� expression at the level of the pitu-itary. Several studies have demonstrated a selectiveincrease in FSH� gene expression in response to glu-cocorticoids in both intact animals (41, 42) as well as

in primary pituitary cells (30, 43, 44). The effect of theglucocorticoids on FSH� appears to be at the level oftranscription as no effect on FSH� mRNA half-life wasdetected (43). Unlike FSH�, a direct effect of glucocor-ticoids on LH� expression has not been observed(41–43), although Rosen et al. (45) did demonstratethat LH� induction by GnRH was inhibited byglucocorticoids.

Given the importance of androgens, progestins, andglucocorticoids in the regulation of FSH� gene expres-sion, the purpose of this study was to investigate themolecular mechanisms of this regulation at the level ofthe gonadotrope. The identification of PREs in theproximal rat and ovine FSH� promoters almost a de-cade ago by O’Conner et al. (39) and Webster et al.(38) suggested that there could be a direct mechanismof action. Additionally, because PR contains a DNA-binding domain (DBD) that is highly conserved withother members of the steroid receptor superfamily,including AR and GR, these researchers hypothesizedthat all of these receptors could bind to the FSH�promoter and mediate its transcription. However, dueto the fact that gonadotropes constitute only about10% of the total secretory cells in the anterior pituitary(46) and that a majority of these cells express steroidreceptors (47–50), the mechanism of action awaitedthe availability of an appropriate gonadotrope cellmodel system.

The development of the L�T2 gonadotrope-derivedimmortalized cell line provided a model system inwhich to study gonadotropin gene expression in apure population of gonadotrope cells. The L�T2 cellline endogenously expresses many markers of a ma-ture gonadotrope including FSH�, LH�, GnRH recep-tor (GnRH-R), activin, activin receptor, follistatin, andinhibin (13, 51, 52). It has also been shown to endog-enously express the estrogen receptor (ER) (53) andAR (54). These properties make the L�T2 cell line anexcellent model system for directly studying the reg-ulation of gonadotropin gene expression by steroids.

Previously, L�T2 cells have been used to study howandrogens repress the induction of LH� gene tran-scription by GnRH (31, 32). More recently, Spady et al.(55) found that an ovine FSH� reporter gene was ac-tivated in response to testosterone in the L�T2 cellline. In the current study, we used the L�T2 cell line todetermine whether steroid hormones directly regulatethe expression of the murine FSH� gene at the level ofthe gonadotrope and whether this regulation occursthrough binding of the steroid receptor to the FSH�promoter. In particular, we used transient transfec-tions with a FSH�-luciferase reporter gene to examinethe responsiveness of the proximal murine promoterto steroid regulation and to analyze the role of putativehormone response elements (HREs). We also usedchromatin immunoprecipitation (ChIP) and gel-shiftanalysis to determine whether the steroid hormonereceptors can bind to the proximal mouse FSH� pro-moter in vivo and in vitro. Overall, we show that steroidhormone regulation of the FSH� promoter can occur

Thackray et al. • AR, PR, and GR Regulate FSH� Mol Endocrinol, September 2006, 20(9):2062–2079 2063

at the level of the gonadotrope through direct DNAbinding of these steroid receptors to specific HREs.

RESULTS

Androgens, Progestins, and GlucocorticoidsMediate Transcription of the Murine FSH� Gene

Gonadotrope cells in the anterior pituitary of rodentshave been shown to express the receptors for andro-gens, progestins, and glucocorticoids (56–59), and theL�T2 cell line has been shown previously to expressAR (54). We tested whether the L�T2 cells also ex-press PR and GR to determine whether these cellsconstitute a relevant model system for studying themechanisms of steroid responsiveness. Oligonucleo-tide primers encompassing 550 bp of AR, 406 bp ofPR, and 298 bp of GR were used to amplify the re-spective receptors from cDNA generated by reversetranscription of mRNA from the L�T2 cell line. All threereceptors were amplified by PCR at the expectedsizes, and no bands were seen in the control lanes thatlacked reverse transcriptase (Fig. 1A). Next, ChIP withantibodies specific to AR, PR, and GR was used todetermine whether the endogenous steroid receptorscould bind the mouse FSH� promoter in vivo in L�T2cells. The results shown in the upper panel of Fig. 1Bdemonstrate that all three ligand-bound receptorsbind to the endogenous FSH� promoter (Fig. 1B, up-per panel; lanes 3–5). The strong signal for PR in theChIP assay does not necessarily indicate that PR ismore abundant than AR or GR in the L�T2 cells be-cause quantitative PCR was not performed. Instead, itmay reflect the affinity of the PR antibody for thereceptor. In contrast, there was no precipitation ofFSH� promoter DNA with a nonspecific mouse IgGcontrol (lane 2). The FSH� promoter was also ampli-fied from the input chromatin (lane 1) as a positivecontrol for genomic DNA preparation and PCR condi-tions. As a control for specificity, primers encompass-ing part of the downstream coding region of the FSH�gene were also used in PCR (Fig. 1B, lower panel).Although these primers amplified FSH� from the inputchromatin as expected (lane 1), no bands were ampli-fied from the precipitated DNA (lanes 2–5).

To monitor changes in promoter activity upon ste-roid treatment, L�T2 cells were transiently transfectedwith the proximal 1000 bp of the mouse FSH� 5�-regulatory region linked to a luciferase reporter gene(�1000FSH�luc). Because the L�T2 cells are murinein origin, we investigated whether androgen regulationoccurs in a similar manner on the murine promoter asit does on the ovine promoter (55). In previous studies,androgen induction of the ovine FSH� promoter in thepresence of endogenous AR resulted in a modeststimulation of transcription (1.5- to 1.9-fold) (55). Giventhis relatively weak induction, we amplified the steroidhormone response on the FSH� promoter by trans-fecting the cells with 200 ng of rat AR.

Treatment of the cells with 100 nM testosterone for24 h resulted in a 9-fold induction of the mouse FSH�promoter (Fig. 1C). Dihydrotestosterone (DHT) wasalso tested to determine whether it could induce FSH�because DHT cannot be aromatized to estrogen.Treatment with 100 nM DHT activated FSH� to a sim-ilar extent as testosterone, implying that estrogenicactivity does not play a role (Fig. 1C). To determinewhether other 3-keto steroid hormones such as pro-gestins or glucocorticoids could also regulate murineFSH� gene expression, the cells were transfected with200 ng of rat PRB or GR. Interestingly, progesteroneand corticosterone also activated FSH� transcription.Treatment of 100 nM progesterone resulted in a 27-fold induction, whereas 100 nM corticosterone acti-vated FSH� 4-fold (Fig. 1C).

To further ascertain whether estrogen can modulatetranscription of FSH�-subunit gene expression, theL�T2 cells were transfected with ER� or ER� andtreated with 100 nM 17�-estradiol for 24 h. Estrogendid not stimulate transcription of the �1000FSH�lucreporter gene compared with the vehicle control in thepresence of either ER� or ER� under these conditions(Fig. 1C). Moreover, estrogen did not positively regu-late FSH� gene expression in the presence of bothERs (data not shown). As a control, a luciferase re-porter gene driven by a consensus estrogen responseelement inserted upstream of a minimal Herpes virusthymidine kinase promoter was induced under theseconditions (data not shown). Our results agree withprevious experiments in rats in which estrogen did notalter the expression of FSH� mRNA in ovariectomizedrats treated with a GnRH antagonist (60) or in femalerat pituitary fragments (61).

Androgens, Progestins, and GlucocorticoidsStimulate FSH� Gene Expression in a Receptor-and Dose-Dependent Manner

In addition to investigating hormone responsive-ness, we examined whether the induction of FSH�by androgens, progestins, and glucocorticoids wasdependent upon receptor concentration. L�T2 cellswere transfected with increasing concentrations ofthe receptor and then treated for 24 h with 100 nM ofa synthetic analog of the relevant steroid hormones.There was a trend toward a positive induction withthe endogenous receptors, and the addition of even100 ng exogenous AR, PR, or GR all significantlyinduced FSH� gene expression (Fig. 2). The smallinduction with the endogenous receptors likely re-flects titration of the receptors by the transfectedreporter genes. For each receptor, the level of FSH�induction correlated with the amount of exogenousreceptor, and the induction did not reach saturationeven with the addition of 800 ng of receptor. Theseresults indicate that the steroid receptors are nec-essary for transcriptional activation of FSH� by an-drogens, progestins, and glucocorticoids and thatthe L�T2 cells are highly responsive to the presence

2064 Mol Endocrinol, September 2006, 20(9):2062–2079 Thackray et al. • AR, PR, and GR Regulate FSH�

of additional receptor. Furthermore, these data sug-gest that the steroid responsiveness is not likely dueto an artificially high level of steroid receptors be-cause the receptor is limiting in the cells.

To test whether the induction of FSH� occurs via ahormone-dependent mechanism, the L�T2 cells weretransfected with AR, PR, or GR and then treated withincreasing concentrations of the respective hormone.For each of the three hormones, treatment of the cellswith a synthetic steroid analog stimulated FSH� pro-moter activity in a dose-dependent manner (Fig. 3).Treatment with 100 pM of R1881, a synthetic andro-gen, activated FSH� 4-fold, with a maximal inductionof 12-fold at the 100 nM dose (Fig. 3A). Treatment withthe synthetic progestin, R5020, resulted in activationof the FSH� promoter at 100 pM with a maximal in-duction of 40-fold at 10 nM (Fig. 3B). Similarly, treat-ment with dexamethasone, a synthetic glucocorticoid,activated FSH� with a concentration of 1 nM (3-fold)with saturation occurring at 100 nM (14-fold) (Fig. 3C),demonstrating that the steroid hormone regulation ofFSH� occurs in a saturable, dose-dependent mannerand that physiological concentrations of hormone canactivate the FSH� gene promoter.

Induction by Steroid Hormone Receptors IsGene Specific

To determine whether the transcriptional activation bythe steroid hormone receptors is specific to the FSH�

gene, the effects of androgens, progestins, or glu-cocorticoids on the regulation of the LH�-subunitgene were examined. In this set of experiments, theproximal 1.8 kb of the rat LH� 5�-regulatory regionlinked to a luciferase reporter gene (1.8LH�luc) wastransiently transfected into L�T2 cells. The cells werealso transfected with 200 ng of AR, PR, or GR. Treat-ment of the cells with 100 nM R1881 repressed theLH� promoter by 17% (Fig. 4A) whereas treatmentwith the progestin, R5020, caused a 48% decrease inLH� gene expression (Fig. 4B). Treatment with dexa-methasone also repressed LH� by 32% (Fig. 4C).Thus, our results demonstrate that ligand-bound AR,PR, or GR can suppress LH� gene transcription,whereas they induce FSH� mRNA levels. This sug-gests that androgens, progestins, and glucocorticoidsdifferentially regulate LH� and FSH� subunit gene ex-pression at the level of the gonadotrope.

Fig. 1. Androgens, Progestins, and Glucocorticoids InduceFSH� Gene Expression in Immortalized Gonadotropes

A, RT-PCR was used to detect expression of the steroidreceptors in L�T2 cells. Amplified AR is visible at 550 bp, PRat 406 bp, and GR at 298 bp. In reverse transcription of totalRNA isolated from L�T2 cells, � indicates the presence ofreverse transcriptase, and � indicates no reverse transcrip-tase. B, ChIP was performed using cross-linked protein/chro-matin from L�T2 cells and antibodies directed against AR,PR, and GR or, as a negative control, against nonspecific IgG.Upper panel, PCR primers encompassing the proximal pro-moter of FSH� were used to detect precipitation of genomicDNA. Lower panel, PCR primers encompassing the down-stream FSH� coding region were used as a control for spec-ificity. PCR amplification was performed on 0.2% chromatininput (lane 1), and chromatin was precipitated with eithermouse IgG (lane 2), AR (lane 3), PR (lane 4), or GR (lane 5)antibodies. C, The �1000FSH�luc reporter gene was tran-siently transfected into L�T2 cells along with 200 ng of therespective steroid receptor expression vectors. After over-night starvation in serum-free media, the cells were treatedwith 100 nM testosterone, DHT, progesterone, corticoste-rone, or 17�-estradiol for 24 h. Luciferase activity was nor-malized to �-galactosidase activity and set relative to theempty reporter vector. The results represent the mean � SEM

of at least three experiments performed in triplicate and arepresented as fold induction of hormone treatment relative tothe vehicle control (ethanol). For cells transfected with AR,

and treated with testosterone or DHT, * indicates significantlydifferent from the vehicle-treated control, using one-wayANOVA followed by Tukey’s post hoc test. For L�T2 cellstransiently transfected with PR, GR, ER�, or ER� and treatedfor 24 h with progesterone, corticosterone, or 17�-estradiol,respectively, * indicates significantly different from the re-spective vehicle-treated control using Student’s t test. RT,Reverse transcriptase.


DNA Binding by Steroid Hormone Receptors IsNecessary for Transcriptional Activation of theFSH� Gene Promoter

To determine whether direct DNA binding by AR, PR,or GR plays a critical role in the transactivation ofFSH� by steroid hormones, we transfected L�T2 cellswith steroid receptor mutants deficient in DNA bind-ing. These mutant receptors included an AR C562Gmutation in the DNA binding domain (DBD) (62), a PRC577A mutation in the DBD (63), and a GRdim4 mu-tation containing the dim1 mutation, A458T, which has

been previously described (22) as well as three othermutations (N454D, R460D, D462C) that further ensureloss of dimerization ability (21, 64), and thereby pre-vent DNA binding by the mutant receptor. To ensurethat the mutant receptors were expressed at similarlevels as the wild-type receptors, we overexpressedthe mutant receptors in either L�T2 cells, in the case ofmutant AR, or in Cos-1 cells, in the case of mutant PRand GR. For PR and GR, Cos-1 cells were used be-cause overexpression of either wild-type or mutantreceptors could not be detected by Western blot inL�T2 cell extracts, perhaps due to a lack of stability of

Fig. 2. Androgens, Progestins, and Glucocorticoids InduceTranscription of FSH� in a Receptor-Dependent Manner

The �1000FSH�luc reporter gene was transiently trans-fected into L�T2 cells along with increasing receptor concen-trations ranging from 0–800 ng/well, as indicated. After over-night starvation in serum-free media, the cells were treatedfor 24 h with the indicated hormone. The results represent themean � SEM of at least three experiments performed in trip-licate and are presented as fold induction relative to thevehicle control. A, L�T2 cells were transiently transfectedwith increasing amounts of AR and then treated with 100 nM

R1881. B, L�T2 cells were transiently transfected with in-creasing amounts of PR and then treated with 100 nM R5020.C, L�T2 cells were transiently transfected with increasingamounts of GR and then treated with 100 nM dexamethasone(Dex).

Fig. 3. Steroid Hormones Mediate Transcription of FSH� ina Dose-Dependent Manner

The �1000FSH�luc reporter gene was transiently trans-fected into L�T2 cells along with the respective receptorexpression vector indicated on the graph. After overnightstarvation in serum-free media, the cells were treated for 24 hwith the indicated hormone concentrations. The results rep-resent the mean � SEM of at least three experiments per-formed in triplicate and are presented as fold induction rela-tive to the vehicle control. A, L�T2 cells were transientlytransfected with the AR expression vector and then treatedwith R1881 concentrations ranging from 10 pM to 100 nM. B,L�T2 cells were transiently transfected with the PR expres-sion vector and then treated for 24 h with 10 pM to 100 nM

R5020. C, L�T2 cells were transiently transfected with the GRexpression vector and then treated with 100 pM to 1 �M

dexamethasone (Dex).


the full-length receptor or lower transfection efficiencythan Cos-1 cells. As demonstrated in Fig. 5, bothwild-type and mutant receptors were expressed atsimilar levels (Fig. 5, A, C, and E, arrowhead). Equalamounts of protein were loaded as indicated by thesimilar level of expression of nonspecific bands in allthree lanes of the gels. Although both wild-type andmutant receptors were expressed at similar levels, weobserved no significant induction of the FSH� pro-moter with the AR C562G mutant compared with the

induction with the wild-type receptor upon treatmentwith 100 nM R1881 (Fig. 5B). Activation of the FSH�promoter by the PR C577A mutant was also dimin-ished after treatment with 100 nM R5020 (Fig. 5D).Additionally, no FSH� induction with the GRdim4 mu-tation was seen upon treatment with 100 nM dexa-methasone (Fig. 5F). These data indicate that AR, PR,and AR all need to bind DNA directly to stimulatetranscription of the FSH� gene in the presence of theappropriate steroid hormone.

HREs in the Proximal Promoter Are Critical forFSH� Induction by Androgens, Progestins, andGlucocorticoids

To map the promoter elements required for steroidhormone responsiveness in the mouse FSH� genepromoter, truncation/deletion analysis was used. L�T2cells were transiently transfected with truncations ofthe mouse FSH� gene ranging from 1526 to 95 bpupstream of the transcription start site, and the abilityof steroids to induce FSH� was measured (Fig. 6).Truncation of the promoter to �95 resulted in a loss ofresponsiveness for each of the three hormones. Al-though some response to androgens was found in theupstream regions of the FSH� promoter, a consider-able degree of responsiveness was conferred in thefirst 500 bp of the FSH� promoter (Fig. 6A). Interest-ingly, progestins and glucocorticoids did not appear toconfer any additional response to FSH� after the first500 bp of the promoter (Fig. 6, B and C). Althoughdifferences in steroid responsiveness may exist on theFSH� promoter, all three steroid receptors studied canact within the first 500 bp of the FSH� promoter,implying that all three regulate the FSH� promoterdirectly at the level of the gonadotrope.

Because the responses to androgens, progestins,and glucocorticoids mapped to a proximal region ofthe FSH� promoter and the responses also required areceptor capable of binding DNA, we examined themouse promoter sequence in this region for putativeHREs. This region contains putative HREs at �381/�367, �230/�216, and �139/�125 of the mousepromoter (see Fig. 7). These elements contain three orfour of the G/C residues that have been shown to becritical for high-affinity binding of steroid receptors toDNA. These elements are also analogous to elementsin the proximal rat and ovine FSH� promoters thatwere identified as PR-binding sites, although the func-tional role of these sites in progesterone responsive-ness was not previously determined (38, 39). Threeadditional elements were originally identified in the ratand ovine promoters. These correspond to putativeHREs at �273/�259, �197/�183, and �175/�161 ofthe mouse promoter but are less conserved than theprevious three elements.

To investigate the importance of the individual ele-ments, we created mutations in the six putative HREsby site-directed mutagenesis in the context of the�1000FSH�luc reporter gene. To disrupt binding of

Fig. 4. Androgens, Progestins, and Glucocorticoids Down-Regulate LH� Gene Expression

The 1.8LH�luc reporter gene was transiently transfectedinto L�T2 cells along with the indicated receptor expressionvector. After overnight starvation in serum-free media, thecells were treated for 24 h with hormone. The results repre-sent the mean � SEM of at least three experiments performedin triplicate and are presented as fold induction relative to thevehicle control. *, Indicates that the hormone treatment wassignificantly different from the vehicle-treated control usingStudent’s t test. A, L�T2 cells were transiently transfectedwith the AR expression vector and then treated with 100 nM

R1881. B, Cells were transiently transfected with the PRexpression vector and then treated with 100 nM R5020. C,Cells were transiently transfected with the GR expressionvector and then treated with 100 nM dexamethasone (Dex).


the steroid receptors to the putative HREs, mutationswere made in the G/C residues critical for high-affinitybinding (Fig. 7). Not surprisingly, none of the mutationsaffected basal expression of the FSH� gene (data notshown). Mutation of the �381 site completely pre-vented the response to androgen (100 nM R1881),whereas the androgen responsiveness with mutationsin the �273, �230, �197, and �139 HREs was di-minished to approximately 46%, 55%, 42%, and 65%,respectively, of the wild-type FSH� reporter gene (Fig.8A). Interestingly, the �381 and �197 HRE mutationsreduced the response to progesterone (100 nM R5020)to approximately 26% and 29%, respectively,

whereas the �273, �230, and �139 HRE mutationsdecreased the response to progesterone to 55%,83%, and 76%, respectively, of wild type (Fig. 8B). The�381 HRE mutation also completely abrogated re-sponsiveness of the FSH� reporter gene to glucocor-ticoid (100 nM dexamethasone). Induction by dexa-methasone was inhibited with the �197 and �139HRE mutations to approximately 21% and 59%, re-spectively, but, in contrast to the other two hormones,no significant inhibition was seen with mutation of the�273 and �230 HREs (Fig. 8C). Mutation of the �175HRE had no functional effect on the androgen, pro-gestin, or glucocorticoid responsiveness of the FSH�

Fig. 5. AR, PR, and GR Require DNA Binding to Facilitate FSH� Gene ExpressionA, C, and E, Wild-type and mutant steroid receptors were overexpressed, and protein levels were analyzed by Western blot

to ensure that wild-type and mutant receptors were expressed at a similar level. The arrowhead denotes the band specific for therespective steroid receptors, and the nonspecific bands demonstrate equal protein loading. A, WT AR, mutant AR (ARC562G),or empty vector control (pSG5) were overexpressed in L�T2 cells. B, WT PR, mutant PR (PRC577A) or empty vector control(pCMV5) were overexpressed in Cos-1 cells. C, WT GR, mutant GR (GRdim4) or empty vector control (pSG5) were overexpressedin Cos-1 cells. B, D, and F, �1000FSH�luc reporter gene was transiently transfected into L�T2 cells along with the wild-type ormutant receptor as indicated. After overnight starvation in serum-free media, the cells were treated for 24 h with hormone. Theresults represent the mean � SEM of at least three experiments performed in triplicate and are presented as fold induction relativeto the vehicle control. *, Indicates that the hormone treatment was significantly different from the vehicle-treated control usingStudent’s t test. B, L�T2 cells were transiently transfected with the wild-type or mutant AR (ARC562G) expression vector and thentreated with 100 nM R1881. D, Cells were transiently transfected with wild-type or mutant PR (PRC577A) expression vector andthen treated with 100 nM R5020. F, Cells were transiently transfected with wild-type GR or GRdim4 mutant expression vector andthen treated with 100 nM dexamethasone (Dex). WT, Wild type.


promoter (Fig. 8). Clearly, the �381 and �197 HREsplay a prominent role in the response to all three of thesteroid hormones. The �139 element also appears tobe necessary for induction by all three hormones and,whereas the �273 and �230 elements play a role inandrogen and progesterone responsiveness, they donot appear to be necessary for glucocorticoidresponsiveness.

AR, PR, and GR Bind to the �381 HRE in theMouse FSH� Promoter

We used an EMSA to assess whether AR, GR, and PRcan bind to the HREs in the proximal mouse FSH�promoter. Although we did not detect steroid receptorbinding using L�T2 nuclear extracts, we observedbinding of AR, PR, and GR from baculovirus-infectedinsect whole-cell extracts to the wild-type �381 ele-ment (Fig. 9, lane 3), whereas no binding to the �381HRE mutant was seen (lane 4). For each of the recep-tors, the resulting complex on the �381 oligonucleo-tide was supershifted by a receptor-specific antibody(lane 5) but not by IgG (lane 6). The complexes also

Fig. 6. Induction of FSH� by Steroid Hormone Receptors Maps toa Region Between �500 and �95 bp of the Proximal Promoter

The �1526FSH�luc, �1000FSH�luc, �500FSH�luc, or�95FSH�luc reporter genes were transiently transfected into L�T2cells along with the indicated receptor expression vector. Afterovernight starvation in serum-free media, the cells were treated for24 h with hormone. The results represent the mean � SEM of atleast three experiments performed in triplicate and are presentedas fold induction relative to the vehicle control. Significantly differ-ent levels of hormone induction among truncations are indicatedby differing letters, a, b, or c as determined by one-way ANOVAfollowed by Tukey’s post hoc test. A, L�T2 cells were transientlytransfected with the reporter genes and the AR expression vectorafter which the cells were treated with 100 nM R1881. B, L�T2 cellswere transiently transfected with the reporter genes and the PRexpression vector and then the cells were treated with 100 nM

R5020. C, L�T2 cells were transiently transfected with the reportergenes and the GR expression vector after which the cells weretreated with 100 nM dexamethasone (Dex).

Fig. 7. Conservation of Putative HREs in the Proximal FSH�Promoter

Putative HREs from the mouse, rat, ovine, and humanFSH� promoters are aligned 5� to 3�. The 5�-start of the HREin each respective species is given on the left. Bold lettersindicate homology to the mouse sequence. Boxes highlightconserved G/C residues. References for the studies in whichthe respective HREs have been characterized are listed onthe right.


showed self-competition (lane 7), failed to competewith a mutant probe (lane 8), and showed evidence ofcompetition with a consensus HRE (lane 9). Theseresults confirm that AR, PR, and GR can all bind di-rectly and specifically to the �381 HRE in the mouseFSH�-subunit gene. In agreement with the results ob-

tained by Spady et al. (55), using the ovine FSH�promoter, AR also bound to the �230 element (Fig.9A, lane 2) albeit to a much lesser degree than the�381 element.

To determine whether AR, PR, or GR could bind theother functional HREs, the EMSA was repeated withprobes encompassing the �273 and �197 sites as

Fig. 8. Multiple HREs Play Roles in the Induction of FSH� byAR, PR, and GR

The wild-type �1000FSH�luc reporter gene, or one of thesix mutants, was transiently transfected into L�T2 cells alongwith the indicated receptor expression vector. After overnightstarvation in serum-free media, the cells were treated for 24 hwith hormone. The results represent the mean � SEM of atleast three experiments performed in triplicate and are pre-sented as fold induction relative to the vehicle control. *,Indicates that the hormone treatment was significantly differ-ent from the vehicle-treated control using Student’s t test. #,Indicates that the induction of the mutant reporter gene issignificantly different from the induction of the wild-type re-porter gene using one-way ANOVA followed by Tukey’s posthoc test. A, L�T2 cells were transiently transfected with theindicated reporter gene and the AR expression vector andthen treated with 100 nM R1881. B, L�T2 cells were tran-siently transfected with the indicated reporter gene and thePR expression vector and then treated with 100 nM R5020. C,L�T2 cells were transiently transfected with the indicatedreporter gene and the GR expression vector and then treatedwith 100 nM dexamethasone (Dex). WT, Wild type.

Fig. 9. Ligand-Bound AR, PR, and GR All Bind Specificallyto the �381 HRE

Whole-cell extracts containing overexpressed AR, PR, orGR from baculovirus-infected insect cells were incubatedwith the �139, �230, or �381 probe and tested for complexformation in EMSA. The relevant steroid receptor-DNA com-plex on the �381 element is shown in lane 3 and on themutated �381 element (mutation as in Fig. 8), whereas theantibody supershift is shown in lane 5, IgG control (lane 6),self-competition (lane 7), mutant competition (lane 8), andcompetition with a consensus HRE (HRE) (lane 9). A, Probeswere incubated with overexpressed AR. B, Probes were in-cubated with overexpressed PR. C, Probes were incubatedwith overexpressed GR. Ab, Antibody; Comp, competition.


well as the �230 and �139 sites. As seen in Fig. 10, anantibody specific for PR produced a supershift whenthe �197 probe was incubated with baculovirus-in-fected insect whole-cell extracts overexpressing PR(arrowhead), whereas no shift was seen with the neg-ative nonspecific IgG control. This supershift was de-tectable after a much longer exposure than the EMSAin Fig. 9B. These results show that PR binds weakly tothe �197 HRE compared with the �381 site and con-trast with previous gel shifts that used HREs from therat and ovine promoters (38, 39). In addition, we saw avery weak supershift on the �230 and �139 sites thatcould indicate potential binding at these sites by PR aswell. We did not observe binding to the other func-tional HREs by AR or GR under these EMSA condi-tions (data not shown) with the exception of the bind-ing of AR to the �230 HRE as shown in Fig. 9. Theseresults indicate that, although these HREs play a func-tional role in hormone responsiveness, they do notbind steroid receptors with high affinity on their own.

Because AR, PR, and GR did not bind to the otherfunctional HREs in a convincing fashion (except PRbinding to the �197 site), we tested whether otherproteins from L�T2 nuclear extracts bound the �273,�230, �197, or �139 sites. Although no differencewas seen between vehicle-treated and hormone-treated L�T2 nuclear extracts (data not shown), basalprotein complexes bound all four probes (Fig. 11).Arrows denote specific complexes that were com-peted by unlabeled-self but not by unlabeled-mutant,

competitor, using the �273 probe (Fig. 11A), the �230probe (Fig. 11B), the �197 probe (Fig. 11C), and the�139 probe (Fig. 11D). Competition with mutantprobes containing the same mutations used in thetransient transfection experiments was used to deter-mine whether any of the basal complexes were sen-sitive to those specific mutations. As can be seen,each of the gel shifts contained at least one complexthat was sensitive to a mutation in the functional HRE,indicating that these basal proteins may also contrib-ute to steroid binding or induction although, as men-tioned previously, mutation of any of the HREs did notalter basal activity of the FSH� promoter (data notshown) nor did steroid treatment alter these EMSAcomplexes.

DISCUSSION

Gonadal steroid hormone feedback is a crucial com-ponent of the control of gonadotropin synthesis in thepituitary gonadotrope (15). In addition, although expo-sure to stress hormones such as corticosterone ordexamethasone can inhibit ovulation in primates androdents (45, 65–70), there is evidence that glucocorti-coids play a physiological role in activating FSH� geneexpression. For instance, both progestin and glu-cocorticoid levels peak during proestrus (71, 72), andthis coincides with increased FSH� mRNA expression

Fig. 10. PR Binds the �197 HREWhole-cell extracts containing overexpressed PR from baculovirus-infected insect cells were incubated with the �273, �230,

�197, or �139 probes and tested for complex formation in EMSA. Antibodies (Ab) are indicated with the left lane containing noantibody (�), the middle lane containing PR antibody (PR), and the right lane containing a nonspecific IgG control antibody (IgG).The antibody supershift is marked with an arrowhead.


and the secondary FSH� surge that occurs in themorning of estrus in rodents (73). Furthermore, glu-cocorticoids, as well as progestins, appear to be nec-essary for the secondary FSH� surge (37, 74). Thesestudies complement the numerous experiments in ratsand mixed rat pituitary cells that found induction ofFSH� mRNA after treatment with androgens, proges-tins, or glucocorticoids (24, 26, 28, 29, 33, 34, 41–43).

In the current study, we characterized the specificmolecular mechanisms of steroid hormone regulationof the FSH� gene in L�T2 cells. Our results demon-strate that the proximal murine FSH� promoter is ac-tivated by androgens, progestins, and glucocorticoids(as well as their synthetic analogs) in the context ofimmortalized gonadotropes in culture (Figs. 1 and 3) incontrast to the lack of estrogen responsiveness on themurine FSH� promoter (Fig. 1). We also show, for thefirst time, that progestins and glucocorticoids, like an-drogens, can suppress transcription of the LH�-sub-unit gene at the level of the gonadotrope (Fig. 4),although glucocorticoids have previously been shownto inhibit GnRH induction of LH� (45). However, be-cause no obvious HREs exist in the LH� promoter, it islikely that the mechanism(s) of action are indirect, sim-ilar to the aforementioned studies concerning the sup-pression of LH� by androgens. The study of the mech-anism(s) of LH� suppression by progestins andglucocorticoids is currently ongoing in our laboratory.Although FSH and LH are produced in the same celltype and both of their �-subunits are positively regu-lated by GnRH and activin (10, 75–77), the expressionprofiles of FSH� and LH� have been shown to differover the menstrual cycle (73, 78). It is intriguing topostulate that some of the observed differential regu-lation of the gonadotropin �-subunits may be due tothese three 3-keto steroid hormones.

Androgens, progestins, and glucocorticoids couldaffect FSH� synthesis directly or by indirect meanssuch as altering the balance of the autocrine activin/inhibin/follistatin system (30, 79). Our data indicatethat induction of FSH� by androgens, progestins, andglucocorticoids is dependent on the presence of theappropriate hormone-bound steroid receptor becauseexpression of the murine FSH�luc reporter gene de-pends on the receptor levels (Fig. 2) and hormoneconcentration (Fig. 3). We also provide evidence thatthe murine FSH� promoter is directly bound and reg-ulated by all three steroid hormone receptors. UsingChIP, we show that the endogenous AR, PR, and GRcan bind the FSH� promoter in vivo in L�T2 cells (Fig.1B). Moreover, the use of mutant steroid receptorslacking the ability to bind DNA demonstrated that thesteroid hormone receptors must bind DNA directly tomodulate FSH� gene expression (Fig. 5). We alsoshow that all three of the steroid hormone receptorscan bind to specific sites within the FSH� promoter invitro using gel-shift analysis (Figs. 9 and 10). Further-more, mutation of putative HREs in the context of the�1000FSH�luc reporter gene abolished the respon-siveness of FSH� to androgens, progestins, and glu-

Fig. 11. Specific Protein Complexes from L�T2 Nuclear Ex-tracts Bind the �273, �230, �197, and �139 HREs

Nuclear extracts prepared from L�T2 cells were incu-bated with the �273, �230, �197, or �139 probes andtested for specific complex formation in EMSA. Unlabeledcompetitors (500�) (Comp) were added to test the spec-ificity of complex formation as indicated. A, �273 probewith no competition (�), self-competition (273), or mutantcompetition (273m). B, �230 probe with no competition(�), self-competition (230), or mutant competition (230m).C, �197 probe with no competition (�), self-competition(197), or mutant competition (197m). D, �139 probe withno competition (�), self-competition (139), or mutant com-petition (139m). Complexes competed by self-competition,but not by mutant competition, are indicated with arrows.


cocorticoids (Fig. 8). Thus, this is the first demonstra-tion that AR, PR, and GR can all directly induce murineFSH� gene expression.

It has been suggested previously that steroids (an-drogens in particular) can induce the expression of theGnRH-R (32, 55) and that this regulation may be one ofthe contributing factors to induction of FSH� by ste-roids. However, our results demonstrating the sup-pression of the LH� gene by steroids would argueagainst a role of the GnRH-R. Furthermore, Bedecar-rats and Kaiser (80) have shown that up-regulation ofGnRH-R by overexpression or a higher-pulse fre-quency of GnRH inhibits FSH� expression. These re-sults all point to direct regulation of the gonadotropin�-subunit genes by steroid hormones.

In addition to establishing that androgens, proges-tins, and glucocorticoids directly modulate FSH� tran-scription, we characterized the roles played by theHREs contained within the FSH� promoter. Becausethe responsiveness to steroid hormones mappedwithin the first 500 bp of the murine FSH� promoter(Fig. 6), we concentrated on the putative HREs in thisregion of the promoter. The six elements characterizedhad all been shown to bind PR in earlier studies usingthe rat and ovine promoters (38, 39), although therelative importance of these sites had not been eval-uated previously. In agreement with the data obtainedusing the rat promoter, the �381 HRE specificallybound PR as well as binding AR and GR (Fig. 9). Ourgel-shift experiments suggest that the �381 HRE maybe critical for direct regulation of the murine FSH�promoter. This hypothesis is further substantiated bythe fact that mutation of this site greatly reduced orabolished FSH� responsiveness to androgens, pro-gestins, and glucocorticoids (Fig. 8). Interestingly, the�381 HRE is an 11/12 match for a direct repeat ele-ment as opposed to a classical palindromic invertedrepeat. This type of element has been reported toselectively confer specificity to AR on several enhanc-ers and promoters [recently reviewed by Verrijdt et al.(81)]. However, in the context of the FSH� promoter,this sequence appears to be recognized by all threesteroid receptors.

This raises the question of whether there is a spe-cific response to these 3-keto steroid hormones on themurine FSH� promoter because androgens, proges-tins, and glucocorticoids have such different physio-logical effects. Much of the specificity may be pro-vided by different levels of the steroid hormones. Forinstance, there is a circadian variation in glucocorti-coid levels that can be affected by internal or externalstress. Additionally, progesterone levels vary dramat-ically over the estrous cycle, and high levels are pro-duced during pregnancy. Testosterone levels also in-crease in the male at puberty and, after peaking overthe following decade, progressively decline with age.Factors such as temporal expression of the steroidreceptors or subtle sequence preferences in the mul-tiple HREs present in the FSH� promoter or in residuesflanking the HREs, which result in different conforma-

tional changes in the receptors and, thus, differentialcofactor recruitment or protein-protein interactionswith adjacent DNA-binding proteins, could also havean effect on specificity.

Because the magnitude of steroid hormone re-sponse imparted by each HRE is often weak, multipleHREs are often found in close proximity in the pro-moters of steroid-responsive genes (82). This situationalso appears to apply to the murine FSH� promoterbecause mutation of the �273, �230, �197, and�139 HREs all reduce induction of FSH� by bothandrogens and progestins, suggesting that they areneeded for the full induction. Glucocorticoid respon-siveness seems to be less sensitive to mutation of theHREs although mutation of the �197 and �139 HREsdid have a considerable effect. In addition to binding ofAR, PR, and GR to the �381 HRE, we observed weakbinding by PR to the �197 HRE and AR to the �230site (Figs. 9 and 10). These data suggest several pos-sibilities. First, these elements (with the exception ofthe �381 HRE) may have a low affinity for steroidreceptors, and binding is difficult to detect using gel-shift analysis although binding could be aided by high-affinity binding at the �381 site. Second, nuclear pro-teins present in the L�T2 cells may be necessary forthe steroid receptors to bind to these elements. Fi-nally, these sites could be required for the binding ofother transcription factors that would alter steroid hor-mone responsiveness. In support of the last two pos-sibilities, we observed basal protein complex forma-tion on the �297, �230, �197, and �139 elementsusing L�T2 nuclear extracts that were sensitive tomutations in these HREs (Fig. 11) although mutationsin the HREs did not affect basal activity of the FSH�promoter nor did steroid treatment of the cells alter thecomplexes (data not shown).

All of the functional HREs we characterized in themouse promoter have a degree of conservation acrossmultiple species, suggesting that they may play rolesin steroid regulation of other mammalian species (Fig.7). Not surprisingly, the putative HRE with the leastconservation, the �175 element, had no functionaleffect in androgen, progestin, or glucocorticoid re-sponsiveness of the murine FSH� promoter (Fig. 8).Although the HREs in the proximal FSH� promoter arewell conserved in mammals, there appear to be spe-cies-specific differences in the regulation of the FSH�promoter by steroids. Our experiments with the murineFSH� promoter in L�T2 cells and previous studiesexamining endogenous FSH� expression in rodentsand in rat pituitary cells contrast with the regulation ofFSH� by androgen and progestins observed in sheepand primates. In particular, androgens have beenshown to repress FSH secretion at the level of thepituitary in rams and male rhesus monkeys (83, 84) aswell as in mixed pituitary cell cultures from transgenicmice containing 10 kb of the human FSH� promoter(85). Moreover, FSH� mRNA levels have been shownto decrease after progestin treatment in ovine mixedpituitary cell cultures (86) and in ovariectomized ewes


pretreated with estrogen and treated with progester-one that received a hypothalamic-pituitary disconnec-tion (87). Progesterone treatment also repressed theluciferase activity in pituitary cells derived from atransgenic mouse containing 4.7 kb of the ovine FSH�promoter linked to a luciferase reporter gene (88). Cu-riously, this same FSH�luc reporter gene was acti-vated by progestins when transiently transfected intoovine mixed pituitary culture (38) and induced by an-drogens when transfected into L�T2 cells (55).

One possibility is that the results obtained in ro-dents, as compared with sheep and primates, differdue to species-specific steroid effects on the FSH�promoter. For instance, the �381 HRE that we foundto be essential for hormone responsiveness on themurine FSH� promoter is conserved between miceand rats but not present in sheep. However, species-specific differences would not explain why the ovineFSH�luc reporter gene was activated by progestins(38) whereas the endogenous gene was repressed(86). It is also plausible that a factor present in sheepand primate gonadotropes is necessary for the nega-tive regulation; however, this is difficult to addressbecause the available immortalized gonadotrope-de-rived cell lines and transgenic animal models arebased on the mouse. Finally, it is credible that para-crine influences from other secretory cell typespresent in the anterior pituitary could be responsiblefor the negative regulation observed with the ovine andprimate promoters. For example, androgens havebeen shown to regulate follistatin mRNA levels (27, 30,89) and thus could indirectly mediate transcription ofFSH� mRNA by impacting the autocrine/paracrine ac-tivity of activin in the gonadotrope. In addition, follicu-lostellate cells in the pituitary have been shown tosecrete follistatin and overgrow mixed pituitary cellcultures (89). Clearly, further investigation is needed todiscern whether steroids play distinct roles in the reg-ulation of FSH� mRNA levels in different mammalianspecies.

In summary, we have demonstrated that androgens,progestins, and glucocorticoids via their receptors di-rectly induce expression of the murine FSH�-subunitgene at the level of the gonadotrope and that thisregulation occurs through binding of the receptors tothe �381 HRE in the proximal FSH� promoter. Addi-tionally, this activation is specific for FSH�, becauseLH� gene expression is repressed by androgens, pro-gestins, and glucocorticoids. Although our studies donot exclude the possibility of steroidal regulation ofGnRH synthesis as a central regulator of gonadotropinproduction, they do suggest that in endocrine disor-ders, such as polycystic ovary syndrome and Cush-ing’s disease with excess androgens and glucocorti-coids, respectively, fertility could be impacted byaltered FSH� and LH� gene expression directly at thelevel of the gonadotrope, as well as the hypothalamus.Further examination of androgen, progesterone, andglucocorticoid regulation of the gonadotropin genes inthe anterior pituitary is needed to better understand

the physiological role these steroid hormones play inthe maintenance of reproductive fitness. To begin toaddress their roles, selective ablation of the individualsteroid hormone receptors in the gonadotrope cellsneeds to be performed using the Cre-LoxP system inmice. These conditional knockouts will determinewhether AR, PR, and GR are necessary for gonado-tropin regulation or whether there is a more dominantsteroid regulation of the gonadotrope through afferentcommunication with the GnRH neuron. Evidence sug-gests that, individually, each of these steroids contrib-utes to reproductive fitness through their cognate re-ceptors. However, it is likely that complex interactionsamong them and other regulators of the hypothalamic-pituitary-gonadal axis such as GnRH and activin existand are also critical for reproduction.

MATERIALS AND METHODS

Hormones

Testosterone, dihydrotestosterone, 17�-estradiol, progester-one, corticosterone, and dexamethasone were obtained fromSigma-Aldrich (St. Louis, MO). Promegestone (R5020) andmethyltrienolone (R1881) were purchased from NEN Life Sci-ences (Boston, MA).

Construction of Reporter Plasmids

Three luciferase reporter gene plasmids were generated byPCR amplification of the mouse FSH� promoter from agenomic clone kindly provided by Malcolm Low. Each PCRproduct was ligated into a pGL3 luciferase reporter plas-mid (Promega Corp., Madison, WI) that had been digestedwith KpnI and HindIII. Initially, a luciferase reporter plasmiddriven by 1526 bp of the mouse FSH� promoter(�1526FSH�luc) was constructed. Two other reporterplasmids, �1000FSH�luc and �500FSH�luc were gener-ated in a similar manner. Construction of the �95FSH�lucplasmid was described previously (90). We confirmed thesequences of all promoter fragments with dideoxynucle-otide sequencing by the DNA Sequencing Shared Re-source, University of California San Diego Cancer Center.

The steroid receptor expression vectors used in thesestudies all contained rat cDNAs and were: AR, pSG5-rAR(62); PR, pCMV5-rPRB (provided by Benita Katzenellenbo-gen); GR, pSG5-rGR (provided by Keith Yamamoto); and ER,pcDNA3.1-rER�, and ER� (91).

Mutagenesis

We used the QuikChange Site-Directed Mutagenesis Kit(Stratagene, La Jolla, CA) to generate mutations in six puta-tive HREs in the proximal FSH� promoter. The mutagenesiswas performed using the �1000FSH�luc plasmid and theappropriate oligonucleotides according to the manufactur-er’s protocol. The following oligonucleotides were used formutagenesis: �139HREmut [5�-CATTTAGACTGCTTTGC-CGAGGCTTCATGTCCCTGTCCGT-3�], �175HREmut [5�-A-AACCATCATCACTGATAGGATTTTCTGGTCTGTGGCATTT-AGAC-3�], �197HREmut [5�-ATATCAGATTCGGTTTCTAGA-GAAACCATCATCACTGATAGCATTTTC-3�], �230HREmut[5�-TAATTTACAAGGTGAGCGAGTGGGTCTGGTGCCATAT-CAGATTCG-3�], �273HREmut [5�-AAGATCAGAAACAATA-GTCTAGAGTCTAGAGTCACATTTAATTTAC-3�], and �381-


HREmut [5�-TTCATACACTTGGAGTCTTGAGTCTCTTGTTG-GATCAATTAAGAC-3�]. The mutated residues are underlined.

We also used the QuikChange Site-Directed MutagenesisKit to generate mutant PRs and GRs unable to bind to DNA.The PRC577A mutant is analogous to a C587A mutation inthe human PRB that prevents DNA binding (63). The GRdim4mutant contains four point mutations that prevent dimeriza-tion of the receptor and thus DNA binding: N454D, A458T,R460D, and D462C (64). The mutagenesis was performedusing the pCMV5-rPRB or the pSG5-rGR plasmid, respec-tively, and the appropriate oligonucleotides according to themanufacturer’s protocol. The following oligonucleotides wereused for mutagenesis: PRC577Amut [5�-TCACTATGGTGT-GCTTACCTGTGGGAGCGCCAAGGTCTTCTTTAAGAGGG-3�] and GRdim4mut [5�-AAGGACAGCACGATTACCTTTG-TACTGGAGATAACTGTTGCATCAT-3�]. As with the reporterplasmids, all mutations were confirmed by dideoxynucleotidesequencing. Construction of the pSG5-rARC562G mutantwas described previously (62).

Cell Culture and Transient Transfection

All of the transient transfection experiments were performedwith the L�T2 cell line (13). L�T2 cells were maintained in10-cm diameter dishes in DMEM (Cellgro, Mediatech, Hern-don, VA) supplemented with 10% fetal bovine serum (OmegaScientific, Inc., Tarzana, CA) at 37 C with 5% CO2. One daybefore transfection, 3 � 105 cells per well were plated into12-well plates. Transient transfection was performed usingFugene 6 reagent (Roche Molecular Biochemicals, Indianap-olis, IN) following the manufacturer’s instructions. Each wellwas transfected with 0.4 �g of reporter plasmid. Additionally,the cells were transfected with 0.2 �g of the appropriatesteroid receptor (unless otherwise noted). As an internal con-trol for transfection efficiency, we transfected the L�T2 cellswith 0.2 �g of a reporter plasmid containing �-galactosidasedriven by the Herpes virus thymidine kinase promoter (tk-�-gal) or the Rous sarcoma virus promoter (RSV-�-gal). Thecells were switched to serum-free DMEM supplemented with0.1% BSA, 5 mg/liter transferrin, and 50 nM sodium selenite6 h after transfection. The following day, the cells weretreated with ethanol (vehicle control) or hormone for 24 h. Thecells were washed once with 1� PBS and then lysed with 0.1M K-phosphate buffer, pH 7.8, containing 0.2% Triton X-100.After lysing the cells, we assayed the luciferase activity usinga buffer containing 100 mM Tris-HCl, pH 7.8, 15 mM MgSO4,10 mM ATP, and 65 �M luciferin. �-Galactosidase activity wasmeasured using the Galacto-light assay (Tropix, Bedford,MA) according to the manufacturer’s protocol. Both lucif-erase and �-galactosidase activities were measured using anEG&G Berthold Microplate Luminometer (PerkinElmer Corp.,Norwalk, CT).

RT-PCR

L�T2 cells were grown to 80% confluency on 6-cm plates,and total RNA was extracted from L�T2 cells with TRIzolReagent (Invitrogen, Carlsbad, CA) following the manufactur-er’s protocol. Contaminating DNA was removed with DNA-free reagent (Ambion, Inc., Austin, TX). RNA (2.5 �g) wasreverse-transcribed using the SuperScript III First-StrandSynthesis System (Invitrogen) according to the manufactur-er’s protocol. The following oligonucleotides were used forPCR: AR sense [5�-GAGAACCCATTGGACTACG-3�] and ARantisense [5�-TGAAGAAGACCTTGCAGC-3�]; PR sense [5�-CTAAATGAGCAGAGGATGAAGGAG-3�] and PR antisense[5�-TGGGGCAACTGGGGCAGCAATAAC-3�]; GR sense [5�-TGCTATGCTTTGCTCCTGATCTG-3�] and GR antisense [5�-TGTCAGTTGATAAAACCGCTGCC-3�] under the followingconditions: 95 C for 2 min followed by 32 cycles of 95 C for45 sec, 52 C for 1 min, and 72 C for 1 min. The PCR containedeither 2 �l of reverse-transcribed L�T2 RNA or, as a negative

control, 2 �l of L�T2 RNA that was not reverse-transcribed.PCR products were run on a 1% acrylamide gel stained withethidium bromide.

ChIP

L�T2 cells were grown to confluency in 15-cm plates, treatedwith 100 nM hormone, and proteins were cross-linked to DNAby the direct addition of 1% formaldehyde to the cell medium.The nuclear fraction was obtained, and chromatin was son-icated to an average length of 1 kb in sonication buffer [50 mM

HEPES, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1%Na-deoxycholate, and 0.1% sodium dodecyl sulfate (SDS)].The lysate was diluted with ChIP dilution buffer (0.01% SDS,1.1% Triton, 1.2 mM EDTA, 16.7 mM Tris pH 8, 167 mM NaCl)to a total of 3.5 ml and precleared with 100 �l Protein A/GPLUS-Agarose beads (Santa-Cruz Biotechnology, Inc.,Santa Cruz, CA). Protein-DNA complexes were incubatedovernight with the C-19x AR polyclonal antibody (Santa CruzBiotechnology, Inc.), the 1294 PR mouse monoclonal anti-body (provided by Dean Edwards), the N499 GR rabbit poly-clonal antibody (donated by Keith Yamamoto), or a nonspe-cific IgG control (Santa Cruz) and precipitated with ProteinA/G beads (Santa Cruz). A fraction of the protein-DNA wasnot precipitated but set aside as the input. The agarosebeads were washed in the following order: low-salt washbuffer [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris(pH 8), 150 mM NaCl], high-salt wash buffer [0.1% SDS, 1%Triton X-100, 2 mM EDTA, 20 mM Tris (pH 8), 500 mM NaCl],LiCl wash buffer [250 mM LiCl, 1% Nonidet P-40, 1% Na-deoxycholate, 1 mM EDTA, 10 mM Tris (pH 8)] and twice withTris-EDTA buffer. The protein-DNA complexes were elutedwith elution buffer (1% SDS, 0.1 M NaHCO3), and the cross-links were reversed with the addition of 200 mM NaCl andincubation at 65 C for 4 h. The DNA was phenol-chloroformextracted, precipitated, and then resuspended in 50 �l ofwater. The primers used in PCR for the FSH� promoter were5�-GGTGTGCTGCCATATCAGATTCGG-3� and 5�-GCAT-CAAGTGCTGCTACTCACCTGTG-3� and spanned the280-bp sequence in the mouse FSH� gene from �223 to�57. The primers for the FSH� coding region were 5�-GC-CGTTTCTGCATAAGC-3� and 5�-CAATCTTACGGTCTCG-TATACC-3�. The following PCR conditions were used: 4 minat 95 C, followed by 26 cycles consisting of 1 min at 95 C, 1min at 60 C, and 1 min at 72 C, and an extension of 10 minat 72 C. The PCR product was labeled by including[�-32P]dATP in the nucleotide mix and run on a 5% acryl-amide gel in 0.5� Tris-borate-EDTA buffer. The gels weredried and subjected to autoradiography.

Preparation of Protein Extracts

Full-length, human AR containing a Flag epitope tag (92),human Flag-PRB (93), or Flag-GR (kindly provided by SteveNordeen) was overexpressed in Sf9 insect cells via a bacu-lovirus expression system by the University of Colorado Can-cer Center Tissue Culture Core Facility. The Sf9 cells wereinoculated with virus at a multiplicity of infection of 1.0 andgrown for an additional 48 h at 27 C. Cells containing AR, PR,or GR were treated for the last 24 h before harvest with 1 �M

DHT, 200 nM R5020, or 500 nM triamcinolone acetonide (finalconcentration), respectively. The cells were harvested bycentrifugation at 1500 rpm for 15 min, washed once in TGbuffer (10 mM Tris-HCl, pH 8.0; and 10% glycerol) and frozenas a pellet at �80 C. We lysed the Sf9 cells in a homogeni-zation buffer [20 mM Tris-HCl (pH 7.5), 350 mM NaCl, 1 mM

dithiothreitol, 10% glycerol, 0.5 �g/ml leupeptin, 10 �g/mlbacitracin, 2 �g/ml aprotinin, 1 �g/ml pepstatin]. All proce-dures were done at 0�4 C. The cell lysate was centrifuged at40,000 rpm for 30 min, and the supernatant was taken as asoluble whole-cell extract.


Nuclear extracts were prepared from L�T2 and Cos-1 cellsas previously described (94).

Western Blot Analysis

Wild-type and mutant steroid receptors, as well as their re-spective empty vector controls, were overexpressed in L�T2cells (AR, ARC562G, and pSG5) or Cos-1 cells (PR,PRC577A, pCMV5 and GR, GRdim4, pSG5) by transientlytransfecting 10 �g of DNA using Fugene 6 into 10-cm platesof cells and treating the cells with 100 nM R1881, R5020, ordexamethasone, respectively. Cells were harvested 24 h laterby incubating cells in a lysis buffer [20 mM Tris-HCl (pH 7.4),140 mM NaCl, 0.5% Nonidet P-40, 0.5 mM EDTA, 10 �g/mlaprotinin, 10 �g/ml pepstatin, 10 �g/ml leupeptin, and 1 mM

phenylmethylsulfonyl fluoride] for 30 min. The protein con-centration was determined with Bradford reagent (Bio-RadLaboratories, Inc., Hercules, CA), and an equal amount ofprotein per sample was loaded on a SDS-PAGE gel. After theproteins were resolved by electrophoresis and transferred toa polyvinylidene fluoride membrane, they were probed withspecific antibodies for AR, PR, and GR. Specifically, theC-19x AR polyclonal antibody (Santa Cruz Biotechnology,Inc.), the 1294 PR mouse monoclonal antibody (provided byDean Edwards), or the N499 GR rabbit polyclonal antibody(donated by Keith Yamamoto) were used. The bands weredetected with secondary antibodies linked to horseradishperoxidase and enhanced chemiluminescence reagent (Am-ersham Biosciences, Piscataway, NJ).

EMSA

To determine whether 3-keto steroid hormone receptorscould bind to HREs in the proximal mouse FSH� promoter,whole-cell extracts containing either AR, PR, or GR wereincubated with 1 fmol of 32P-labeled oligonucleotide at 4 Cfor 30 min in a DNA-binding buffer [10 mM HEPES (pH 7.8), 50mM KCl, 5 mM MgCl2, 0.1% Nonidet P-40, 1 mM dithiothreitol,2 �g polydeoxyinosinic deoxycytidylic acid, and 10% glyc-erol]. The oligonucleotides were end-labeled with T4 DNApolymerase and [�-32P]ATP. After 30 min, the DNA bindingreactions were run on a 5% polyacrylamide gel (30:1 acryl-amide-bisacrylamide) containing 2.5% glycerol in a 0.5�Tris-acetate-EDTA buffer. The C-19x AR polyclonal antibody,the 1294 PR mouse monoclonal antibody, and the N499 GRrabbit polyclonal antibody were used to supershift AR, PR,and GR, respectively. We used rabbit IgG or mouse IgG as acontrol for nonspecific binding. A 500-fold excess of therelevant oligonucleotide was used for competition. The fol-lowing oligonucleotides were used for EMSA: �139HRE, 5�-AGACTGCTTTGGCGAGGCTTGATCTCCCTGTCCGT-3�;�197HRE, 5�-AGATTCGGTTTGTACAGAAACCATCAT-CACTGATA-3�; �230HRE, 5�-TACAAGGTGAGGGAGT-G G G T G T G C T G C C A T A T C A G - 3 � ; � 2 7 3 H R E , 5 � -AAGATCAGAAAGAATAGTCTAGACTCTAGAGTCAC-3�;�381HRE, 5�-ACACTTGGAGTGTTCAGTCTGTTCTTG-GATCAATT-3�; �139mut, 5�-AGACTGCTTTGCCGAGCT-TCATGTCCCTGTCCGT-3�; �197mut, 5�-AGATTCGGTT-TCTAGAGAAACCATCATCACTGATA-3�; �230mut, 5�-TACAAGGTGAGCGAGTGGGTCTGGTGCCATATCAG-3�;�273mut, 5�-AAGATCAGAAACAATAGTCTAGAGTCTA-GAGTCAC-3 � ; �381mut, 5 �-ACACTTGGAGTCTT-GAGTCTCTTGTTGGATCAATT-3�; and the consensus HRE,5�-ACGGGTGGAACGCGGTGTTCTTTTGGC-3�.

Statistical Analyses

Transient transfections were performed in triplicate, and eachexperiment was repeated at least three times. The data werenormalized for transfection efficiency by expressing lucif-erase activity relative to �-galactosidase activity and relative

to the empty pGL3 plasmid to control for hormone effects onthe vector DNA. The data were analyzed by Student’s t testfor independent samples or one-way ANOVA followed bypost hoc comparisons with the Tukey-Kramer honestly sig-nificant difference (HSD) test using the statistical packageJMP 5.0 (SAS Institute, Inc., Cary, NC). Significant differ-ences were designated as P � 0.05.

Acknowledgments

We thank Djurdjica Coss, Janice Sue Bailey, and othermembers of the Mellon lab for helpful discussions and com-ments; Scott Kelley for his suggestions and critical reading ofthe manuscript; Malcolm Low for providing the mouse FSH�genomic clone; Jorma Palvimo for the pSG5-rAR and pSG5-rARC562G plasmids; Benita Katzenellenbogen for thepCMV5-rPRB plasmid; and Keith Yamamoto for the pSG5-rGR plasmid and the N499 GR rabbit polyclonal antibody. Wealso thank Margaret Shupnik for providing the pcDNA3.1-rER� and ER� plasmids; Mark Lawson and Marit Kreidel forthe 1.8LH�luc plasmid; Elizabeth Wilson for the Flag-AR bac-ulovirus; Steve Nordeen for the Flag-PRB and Flag-GR bacu-loviruses; and Dean Edwards for the 1294 PR mouse mono-clonal antibody. We also thank the University of ColoradoCancer Center Tissue Culture Core Facility for baculovirusproduction and the University of California San Diego CancerCenter DNA Sequencing Shared Resource for dideoxynucle-otide sequencing.

Received August 2, 2005. Accepted April 25, 2006.Address all correspondence and requests for reprints to:

Pamela L. Mellon, Ph.D., Department of ReproductiveMedicine, University of California at San Diego, 9500 Gil-man Drive, La Jolla, California 92093-0674. E-mail:[email protected].

This work was supported by National Institute of ChildHealth and Human Development/National Institutes of Health(NIH) through a cooperative agreement (U54 HD012303) aspart of the Specialized Cooperative Centers Program in Re-production Research (to P.L.M.). This work was also sup-ported by NIH Grant R37 HD020377 (to P.L.M.). V.G.T. wassupported by NIH Grants F32 DK065437 and T32 HD007203.S.M.M. was partially supported by NIH Grant T32 DK07541.

Disclosure statement: The authors have nothing todisclose.

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