RESEARCH ARTICLE

Molecular Reproduction & Development 80:734–743 (2013)

FSH Modulates the Expression of Inhibin-Alpha and theSecretion of Inhibins via Orphan Nuclear Receptor NUR77 inOvarian Granulosa Cells

QIN-YUAN HE,1 LI-JUN DING,1 HAI-XIANG SUN,1 BIN YU,2 AN-YI DAI,1 NING-YUAN ZHANG,1 BIN WANG,1

GUI-JUN YAN,1 PEI-ZHEN XU,2* AND YA-LI HU1*

1 Department of Obstetrics and Gynecology, The Affiliated Drum Tower Hospital of Nanjing University Medical School,Center for Reproductive Medicine, Nanjing, China

2 Key Laboratories of Reproductive Health, Changzhou Maternal and Child Care Hospital, Changzhou, China

SUMMARY

It has been previously reported that follicle-stimulating hormone (FSH) regulates theexpression of inhibin-alpha in human granulosa cells, but the precise molecularpathway remains unknown. In the present study, we investigated the role of theorphannuclear receptor, NUR77, in both the transcriptional regulation of the inhibina-subunit gene and the secretion of inhibins. Our results showed that in a humangranulosa cell tumor-derived cell line (KGN) and in human granulosa-lutein cells(hGL), FSH induced the expression of NUR77 and inhibin-alpha, although inhibin-alpha expression did not increased following FSH treatment if NUR77 was knockeddown. Furthermore, simply overexpressing or reducing NUR77 levels affectedinhibin-alpha expression, while NUR77 overexpression improved the secretion ofinhibin A and B from human granulosa cells. In addition, chromatin immunoprecipita-tion�PCR, avidin�biotin-conjugated DNA precipitation, and luciferase reporterassays confirmed that NUR77 directly regulated the transcription of the inhibin-alphagene through the specific NGFI-B responseelement locatedwithin its promoter. In theovarian granulosa cells of theNur77 knockoutmice, themRNA levels of inhibin-alphawere decreased relative to wild-typemice. These data indicate a role of NUR77 in theregulation of inhibin-alpha in ovarian granulosa cells.

Mol. Reprod. Dev. 80: 734�743, 2013. � 2013 Wiley Periodicals, Inc.

Received 6 March 2013; Accepted 4 June 2013

�Corresponding authors:Department of Obstetrics

and GynecologyThe Affiliated Drum Tower Hospital of

Nanjing University Medical SchoolCenter for Reproductive MedicineZhong Shan Road 321#Nanjing City 210008, China.E-mail: ybcz0519@163.com (P.-Z.X.),yali_hu@hotmail.com (Y.-L.H.).

Q-YHeandL-JDing contributed equally tothis work.

Grant sponsor: Chinese National 973Projects; Grant number:2010CB945104; Grant sponsor:Health Innovation Team Project ofJiangsu Province; Grant numbers:XK201102; LJ201102; RC2011005;Grant sponsor: National NaturalScience Foundation of China;Grant numbers: 81070508; 81070492;30900727, 30900847

Published online 18 June 2013 in Wiley Online Library(wileyonlinelibrary.com).DOI 10.1002/mrd.22206

Abbreviations: FSH, follicle-stimulating hormone; hGL, human granulosa-lutein cells; MOI, modulus of infectivity; NBRE, NGFI-B response element;NUR77, orphan nuclear receptor 77; TGFb, transforming growth factor b

� 2013 Wiley Periodicals, Inc.

INTRODUCTION

Inhibins are part of the transforming growth factor (TGF)superfamily, whose members are closely associated withovarian follicle development (Knight and Glister, 2006).Inhibins are heterodimers composed of an 18-kDa a-sub-unit disulfide-linked to one of two 14-kDa b-subunits (bAand bB), resulting respectively in inhibin A or inhibin B.The b-subunits can also form activins: bA�bA (activin A),bA�bB (activin AB), and bB�bB (activin B) (Lockwoodet al., 1998). Altered levels of the inhibin/activin subunitsduring follicle development suggest that these proteinsmayhave important roles in folliculogenesis, oocytematuration,and corpus lutea function (Knight and Glister, 2001). Inaddition, inhibins are involved in the negative-feedbackcontrol of pituitary follicle-stimulating hormone (FSH)secretion (Muttukrishna et al., 2002). It was previouslydemonstrated that FSH affected inhibin-alpha-subunitexpression as well as the levels of inhibins in granulosacells (Turner et al., 1989; Woodruff and Mayo, 1990; WeckandMayo, 2006), although the precisemechanisms under-lying the FSH regulation of inhibins remains largely un-known. Increasing levels of FSH in the blood are associatedwith female reproductive aging, which also exhibits a sig-nificant decrease in inhibin B levels (Klein et al., 1996;Robertson et al., 2008, 2009). Furthermore, women withprimary ovarian insufficiency have low levels of inhibin Aand B (Petraglia et al., 1998).

The orphan nuclear receptor NUR77 (also knownas NR4A1, NGFI-B, TR3, or NAK-1) is a member ofthe NR4A family. NUR77 is widely expressed in severaltissues, including the ovary, testis, muscle, pituitary,and the central nervous system (Maxwell and Muscat,2006; Jiang et al., 2011). NUR77 can bind to DNAas a monomer through a regulatory element, calledthe NGFI-B response element (NBRE), AAAGGTCA,which is present in a subset of genes (Martin andTremblay, 2008; Martin and Tremblay, 2009; Dai et al.,2012). In addition, NUR77 is expressed in the corporaluteum and cultured ovarian granulosa cells. NUR77 acti-vates steroidogenic gene expression during luteinization,indicating that the stage-specific expression ofNUR77mayhave functional relevance in follicular maturation (Wuet al., 2005).

In the present study, we observed that FSH stimulationcan promote the expression of NUR77 and inhibin-alpha ina human granulosa cell tumor-derived cell line (KGN) andhuman granulosa-lutein cells (hGL). FSH stimulation didnot, however, increase the inhibin-alphamRNA level inKGNcells in which NUR77 was knocked down using Ad-siNUR77 infection. We demonstrated that NUR77 candirectly bind to the NBRE site located in the human inhib-in-alpha promoter region, resulting in increased promoteractivity, inhibin-alpha expression, and secretion of inhibins.In addition, the granulosa cells of Nur77�/� mice displayeda lower level of inhibin-alpha mRNA compared with wild-type control cells. These results suggest the importance ofNUR77 in the regulation of inhibin-alpha following FSHstimulation.

RESULTS

FSH Affected the Expression of NUR77 andInhibin-Alpha in KGN and hGL Cells

To determine if NUR77 and inhibin-alpha were regu-lated by FSH in KGN cells, the mRNA levels of NUR77and inhibin-alpha were analyzed using real-time PCR. Asignificant increase in NUR77 mRNA levels was detectedafter 2 hr following FSH (20 ng/ml) stimulation of KGNcells (about 4.08-fold; Fig. 1A). FSH stimulation wasalso found to increase the level of inhibin-alpha mRNA,showing a 3.28-fold increase at 12 hr post-stimulation(Fig. 1B).

We chose the 2 and 12 hr time points to measure themRNA levels of NUR77 and inhibin-alpha following stimu-lation with different doses of FSH (0�50 ng/ml). FSHincreased the expression ofNUR77, with the maximal levelat 4.16-fold higher than control (0 ng/ml) at the 20 ng/mldose (Fig. 1C). Additionally, the same dose of FSH in-creased the inhibin-alpha mRNA level by approximately2.7-fold compared to control (0 ng/ml) (Fig. 1D).

Protein abundance in whole-cell lysates revealed differ-ent changes of NUR77 in KGN cells after FSH stimulation(see Supporting Information). Following FSH (20 ng/ml)treatment, a significant increase in NUR77 protein wasdetected at 4 hr (Fig. S1-A). Titration of FSH (0�50 ng/ml) showed a similar profile in NUR77 protein abundance4 hr after 20 ng/ml FSH stimulation (Fig. S1-B).

We also analyzed the expression ofNUR77 and inhibin-alpha mRNA level in hGL cells after FSH (20 ng/ml) treat-ment (see Supporting Information). After 2 hr of stimula-tion, NUR77 was 3.13-fold higher than control (Fig. S2-A),then decreased to control levels by 12 hr (Fig. S2-B).Compared to control, there were no significant changeswith inhibin-alpha 2 hr after stimulation (Fig. S2-C),but reached about 2.54-fold at 12 hr after stimulation(Fig. S2-D).

NUR77 Regulated the Expression of Inhibin-Alphaand the Secretion of Inhibins

To assess if NUR77 regulates the expression of inhibin-alpha,weoverexpressedNUR77 inKGNcellswithAd-Flag-NUR77 (modulus of infectivity (MOI) ¼ 10).NUR77mRNAlevels rose about 15.24-fold infection with Ad-Flag-NUR77compared with Ad-LacZ after, as determined by quantita-tive real-time PCR (Fig. 2A). Western blot also showed theincreasedNUR77protein level (Fig. 2B). ThemRNA level ofinhibin-alpha increased by approximately 2.18-fold(Fig. 2C).

To determine the functional significance of NUR77in the regulation of inhibin-alpha expression in KGNcells, we performed loss-of-function studies. EndogenousNUR77 mRNA expression was inhibited by approximately50% in KGN cells infected with Ad-siNUR77 (MOI ¼ 100)for 48 hr (Fig. 2D). Western blot also showed a reductionin NUR77 (Fig. 2E). At the same time, the mRNA level ofinhibin-alpha was down-regulated by approximately 24%following the inhibition of endogenous NUR77 (Fig. 2F).

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We increased the levels of NUR77 in the cell lines abovetheir endogenous quantities (Fig. 3A) by infecting themwithAd-LacZ orAd-Flag-NUR77, and then assayed for inhibin Aand inhibin B secretion. Following an Ad-Flag-NUR77 in-

fection of hGL cells, the mRNA level of NUR77 and inhibin-alpha increased about 24.23- and 2.61-fold, respectively(Fig. 3B,C). As expected, inhibin A and inhibin B secretionrespectively increased by approximately 35% and 40%compared to the control (Fig. 3D).

To test the role of NUR77 in the regulation of inhibin-alpha following FSH stimulation in human granulosacells, we studied KGN cells infected with Ad-siNUR77to knock down endogenous NUR77. At 48 hr post-in-fection, exogenous FSH (20 ng/ml) was added. After the12 hr FSH stimulation, real-time PCR showed that theNUR77 level in Ad-siNUR77-infected cells decreasedabout 60% (Fig. 4A). FSH stimulation increased inhib-in-alpha expression by approximately 2.3-fold in KGNcells infected with Ad-LacZ, but inhibin-alpha mRNAabundance was not significantly changed in cells in-fected with Ad-siNUR77 compared with the control(Fig. 4B).

Figure 2. NUR77 regulated expression of inhibin-alpha in KGNcells. KGN cells were infected with Ad-LacZ or Ad-Flag-NUR77for 48h. mRNA levels of NUR77 (A) and inhibin-alpha (C) weremeasured by quantitative real-time PCR, and is shown as a ratioover control (Ad-LacZ). Measurement of NUR77 (D) and inhibin-alpha (F) mRNA level in KGN cells after NUR77 was knocked downwith Ad-siNUR77 by quantitative real-time PCR analysis (�P < 0.05,��P < 0.01). Western blot analysis of NUR77 protein expressionin KGN cells treated with Ad-NUR77 (B) or Ad-siNUR77 (E) for48 hr.

Figure 1. Different time- and dose- effects of FSH on the NUR77(A,C) or inhibin-alpha (B,D) mRNA level in KGN cells. KGN cells werecultured in a phenol-red free medium (2.5% charcoal/dextran-treatedfetal bovine serum) and treated with 20 ng/ml FSH for 24 hr. In anotherexperiment, KGN cells were cultured in the phenol-free media andtreated with different concentrations of FSH (0�50 ng/ml) for 2 and12 hr. The total RNA of KGN cells was isolated, and themRNA contentwas assessed using quantitative real-time PCR (�P < 0.05,��P < 0.01).

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NUR77 Increased Inhibin-Alpha Expression byDirectly Binding to the Inhibin-Alpha Promotervia the NBRE

Based on the human inhibin-alpha promoter sequencedeposited in the Transcriptional Regulatory Element Data-base (http://rulai.cshl.edu/cgi-bin/TRED/tred.cgi?process¼home), we found an NBRE (TGACCTTT, �2,554/�2,547 bp) within the inhibin-alpha promoter core region.To examine whether or not NUR77 can bind to the inhibin-alpha promoter, a chromatin immunoprecipitation (ChIP)assay was performed. We observed that endogenousNUR77 bound to the region of the inhibin-alphapromoter containing the predicted NBRE site (Fig. 5A).Furthermore, real-time PCR results showed thatchromatin fragments containing the putative NBREwere enriched in the co-precipitations of NUR77 fromlysates of KGN cells. FSH treatment increased this affinity(Fig. 5B).

The wild-type (WT) and mutant (MU) human inhibin-alpha promoter oligonucleotides were prepared to deter-mine if NBREs are required for the binding of NUR77 to theinhibin-alpha promoter. Avidin�biotin conjugate DNA pre-cipitation (ABCD) assays were then performed. Whole-cellextracts from KGN cells were incubated with each of theoligonucleotides immobilized on streptavidin-agarose

beads. The results of these assays demonstrated thatNUR77 could strongly bind to the inhibin-alpha wild-typebut not to the mutant oligonucleotides (Fig. 5C).

The functional role of NUR77 binding to the humaninhibin-alpha promoter was investigated with a luciferasereporter assay in KGN cells. Overexpression of NUR77resulted in increased inhibin-alpha promoter activity ofapproximately 2.45-fold compared with the control. In con-trast, the combination ofmutant inhibin-alpha promoter andoverexpression of NUR77 did not change luciferase activity(Fig. 5D).

Figure 3. Overexpression of NUR77 affected the expression of inhib-in-alpha and the secretion of inhibins in hGL. A: NUR77 proteinexpression was measured by Western blot in cultured hGL andKGN cells. After 48 hr pre-culture, hGL cells were infected with Ad-LacZ or Ad-Flag-NUR77 for 6�8 hr, then the culture medium waschanged to phenol-red free medium with 2.5% charcoal/dextran-treated fetal bovine serum. After 48 hr, mRNA levels of NUR77 (B)and inhibin-alpha (C) were measured by quantitative real-time PCR,and are shown as a ratio over control (Ad-LacZ). Media were collectedand assayed for inhibin A and B (D) levels by ELISA (�P < 0.05,��P < 0.01).

Figure 4. FSH treatment failed to increase the expression of inhibin-alpha after knockdownof NUR77 in KGNcells. KGNcellswere infectedwith Ad-LacZ or Ad-siNUR77 for 48 hr, then the culture medium waschanged to phenol-red free medium (2.5% charcoal/dextran-treatedfetal bovine serum) in which FSH (20 ng/ml) was added. After 12 hrstimulation, mRNA levels of NUR77 (A) and inhibin-alpha (B) weredetected by quantitative real-time PCR, and are shown as a ratio overcontrol (Ad-LacZ) (�P < 0.05, ��P < 0.01).

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The Expression of Inhibin-Alpha Decreased inOvarian Granulosa Cells of Nur77 KnockoutMice

To determine whether or not inhibin-alpha in murineovarian granulosa cells was regulated by Nur77 in vivo,inhibin a-subunit mRNA expression levels in mouse gran-ulosa cells from 3-week-old femaleNur77þ/þ andNur77�/�

mice (Fig. 6A) were analyzed. Both real-time PCR (Fig. 6B)and Western blot (Fig. 6C) assays showed decreasedNur77 levels in Nur77�/� mice. Compared with theNur77þ/þ genotype, the expression levels of inhibin-alpha(Fig. 6D) decreased by approximately 20% in Nur77�/�

granulosa cells.

DISCUSSION

Numerous studies have documented that the NR4Aimmediate-early genes are rapidly induced by a varietyof stimuli, including cAMP, phorbol ester, and growth factors(Herschman, 1991; Maruyama et al., 1998). A member ofthis subfamily, NUR77, is reported to be regulated by FSHin mouse testis and human fetal Sertoli cells (Ding etal., 2011). The expression of NUR77 in both human KGNcells and rat Sertoli cells followed a similar pattern inresponse to the cAMP surge (Wu et al., 2005). Our presentstudy showed that FSH-stimulation induced the expressionofNUR77 in a human ovarian granulosa-like tumor cell line

Figure 5. NUR77 increased the expression of inhibin-alpha by directly binding to the inhibin-alpha promoter via NBRE. A: A chromatinimmunoprecipitation (ChIP)-PCR amplification using primers against the human inhibin-alpha promoter region. PCR products were separatedby agarose gel electrophoresis. Input (non-precipitated) chromatin was used as a positive control for these studies. B: Co-precipitating chromatinwas analyzed using quantitative real-time PCR. C: Avidin�biotin conjugate DNA (ABCD) precipitation assays were performed using either abiotinylated, double-stranded inhibin-alpha wild-type (WT-b) or mutant (MU-b) oligonucleotide and the KGN cell extracts, following an infectionwith Ad-Flag-NUR77. D: KGN cells, transfected with either a human inhibin-alpha wild-type (WT) or mutant (MU) promoter reporter constructsand PCMV-Flag-NUR77, were cultured for 48 hr. Subsequently, the cell lysates were assayed for luciferase activity (�P < 0.05, ��P < 0.01,���P < 0.001).

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(KGN) and in human granulosa cells. It has also beendocumented that FSH and its second messenger, cAMP,stimulate the transcription of many ovarian genes in cul-tured granulosa cells (Taieb et al., 2011). Our present datashowed that FSH stimulation also increased expression ofinhibin-alpha in ovarian granulosa cells.

Inhibin production in granulosa cells can be regulated byendocrine (FSH), paracrine (GDF-9), andautocrine (TGF-band activin) factors (Lanuza et al., 1999; Drummond etal., 2000; Findlay et al., 2001; Kaivo-Oja et al., 2003).Inhibin-alpha is a unique subunit that is essential for theproper function of themammalian ovary. In our study, it wasobserved that FSH-stimulated NUR77 directly bound to thespecific NBRE site in the inhibin-alpha promoter, and thatthis binding is associated with an increase in both theexpression of inhibin-alpha and the secretion of inhibinsin human granulosa cells. As expected, loss-of-functionstudies demonstrated that a reduction in endogenousNUR77 mRNA level also decreased the expression ofinhibin-alpha. These results indicated that NUR77 maybe involved in the FSH/inhibin-alpha pathway, and mayaffect the secretion of inhibins in granulosa cells. In additionto NUR77, multiple effectors mediated the transcriptionalactivation of inhibin-alpha, including CREB, SF-1/LRH-1,beta-catenin, and GATA (Feng et al., 1989, 1998; Ito etal., 2000; Gummow et al., 2003; Weck and Mayo, 2006). Inovarian granulosa cells, phosphorylated CREB activatesthe inhibin-alpha subunit by binding an atypical cAMPresponse element (CRE) in the gene proximal promoter(Mukherjee et al., 1996). NUR77 expression can be medi-ated by CREB after different stimuli, like luteinizing hor-

mone (LH)/human chorionic gonadotropin (hCG) in MA-10cells (Inaoka et al., 2008). Moreover, NUR77 can interactwithCREB to increasegenepromoter activity, including thatof FSHb (Pnueli et al., 2011). GATA-4, which interacts withSmad3, is also a cofactor for TGF-b-mediated activation ofinhibin-alpha (Anttonen et al., 2006). These reports indi-cate that NUR77 may interact with other effectors (CREBand GATA-4) to regulate the transcriptional activation ofinhibin-alpha.

Different types of inhibin-alpha gene mutations areassociated with the prevalence of primary ovarian insuffi-ciency in Indian (Marozzi et al., 2002; Prakash et al., 2010)and Korean (Kim et al., 2011) women. One of the suscepti-bility factors is the reduction of inhibin B bioactivity byinhibin-alpha gene variants (Chand et al., 2007, 2010).Targeted disruption of the inhibin-alpha gene in mice(Inh a�/�) results in an ovarian phenotype of granulosacell tumors that renders the animals infertile (Myers etal., 2009). Furthermore, Inha�/�ovaries cannot be inducedto generate preovulatory follicles, and Inh a�/� ovariangranulosa cells exhibit aberrant expression of many cyto-skeleton genes (Nagaraja et al., 2010). In our currentfindings, there was a lower level of inhibin-alpha in mouseNur77�/� ovarian granulosa cells. This encourages us tostudy the relationship between NUR77 and inhibin-alpha invivo for future research. Since inhibin-alpha was regulatedby NUR77, we suppose that NUR77 may be one of the keyregulators of ovarian follicular development.

In summary, our study demonstrated that in humangranulosa cells, FSH increased NUR77 and inhibin-alphamRNA levels within a certain range. NUR77 regulated thetranscription of inhibin-alpha by directly binding to thespecific NBRE site located in the inhibin-alpha promoter.Additionally, overexpression and reduction of NUR77 af-fected the expression of inhibin-alpha. Overexpression ofNUR77 also enhanced secretion of inhibin A and B in hGLcells. Yet, FSH did not induce the expression of inhibin-alpha in KGNcellswhenNUR77was knocked down. Theseresults enhance our understanding of the molecular mech-anisms of inhibin regulation in ovarian granulosa cells.

MATERIALS AND METHODS

The Preparation of hGL CellsThe study was approved by the Drum Tower Hospital

Medical Ethics Committee. Human granulosa-lutein cellswere obtained on follicle-retrieval day from women under-going in vitro fertilization treatment. After removing theoocytes and cumulus masses, the layers of hGL cellswere resuspended in DMEM/F-12 (Gibco BRL/Invitrogen,Carlsbad, CA) supplemented with 10% fetal bovine serum(FBS, HyClone; Thermo Scientific, Logan, UT), 1 mMpyruvic acid (Gibco BRL/Invitrogen), 2 mM glutamine,and antibiotics (100 IU/ml penicillin, and 100 mg/ml strep-tomycin). hGL cellswere counted, seeded in 24-well cultureplates (1 � 105 cells/well), and incubated in humidified aircontaining 5% CO2, at 378C, for 48 hr before the differenttreatments (Shi et al., 2009, 2011).

Figure 6. Inhibin-alpha mRNA quantity was reduced in primaryNur77�/� mouse granulosa cells. A: Identification of the two geno-types using PCR. RNA was collected from 3-week-old Nur77þ/þandNur77�/� female granulosa cells, and reverse transcribed for real-time PCR. The Nur77 (B) and inhibin-alpha (D) mRNA levelsof the Nur77�/� versus Nur77þ/þmouse ovarian granulosa cellswere measured and expressed as relative quantities (�P < 0.05,��P < 0.01). C: Western blot analysis of Nur77 protein expression inthe Nur77þ/þ and Nur77�/� mGCs.

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Generation of MiceNur77þ/� mice (B6.129S2-Nr4a1tm1Jmi/J) were pur-

chased from Jackson Laboratory (Bar Harbor, ME). Allmice were maintained in the Animal Laboratory Centerof Drum Tower Hospital (Nanjing, China) on a 12:12-hrlight/dark cycle (lights turned off at 19:00 hr) with food andwater available ad libitum. All animal experiments wereperformed according to the guidelines of the ExperimentalAnimals Management Committee (Jiangsu Province,China). According to our observation, development of fol-licles was over-activated at early reproductive stages in theovary of Nur77�/� mice. Then, the number of preovulatoryfollicles decreased at the late reproductive stages, followedby a decrease in fecundity.

Mice were genotyped by PCR assays using thefollowing primer sets: mNur77 common F: 50-CCACGT-CTTCTTCCTCATCC; mNur77 wild-type R: 50-TGAG-CAGGGACTGCCATAGT; and mNur77 mutant R: 50-CAC-GAGACTAGTGAGTG. PCR products were resolved usingagarose gel electrophoresis, and the product sizes weredetermined relative to size markers.

The Construction and Infection of AdenovirusAdenoviruses containing NUR77 fused to a Flag-tag

epitope (Ad-Flag-NUR77), NUR77-targeted siRNA (Ad-siNUR77), or a control Ad-LacZ construct (Ad-LacZ)were generated as described previously (Jiang etal., 2011; Dai et al., 2012). KGN cells were seeded in 60-mm plates to obtain approximately 70% confluency. Aftertransfection for 6�8 hr, the medium was completely re-freshed. After 48 hr transfection, the cells were harvestedfor use in different assays.

Quantitative Real-Time PCRTotal RNA was extracted from tissues and cultured cells

using the TRIzol reagent (Invitrogen). cDNA was synthe-sized from 1 mg of purified total RNA using a PrimeScriptRT reagent kit (Bio-Rad Laboratories, Hercules, CA), ac-cording to the manufacturer’s instructions. The specificprimers used for quantitative polymerase chain reaction(PCR) analysis are listed in Table 1. Each real-time PCR

reaction had the following components: 1 ml of RT product,10 ml of SYBR Green PCR Master Mix (Bio-Rad Laborato-ries), and 500 nM each of the forward and reverse primers.Real-time PCR was performed on a MyiQ Single ColorReal-time PCR Detection System (Bio-Rad Laboratories)for 40 cycles (958C for 10 sec; 608C for 30 sec; 728C for30 sec) following an initial of 3 min incubation at 958C.Each sample was analyzed in triplicate, and the experimentwas repeated three times. The relative transcript abun-dance was determined according to the 2�DDC(t) method(Livak and Schmittgen, 2001), and the fold-change inexpression of eachgenewasnormalized to anendogenouscontrol (18S rRNA).

Western Blot AnalysisKGN cells were lysed using whole-cell lysis buffer

(50.0 mmol/LTris [pH 7.6], 150.0 mmol/L NaCl, 0.1% SDS,1.0% NP-40) and a protease-inhibitor cocktail (Sigma,St. Louis, MO). Then, protein concentrations were quanti-fied using the Bradford assay (Bio-Rad Laboratories).Thirty micrograms of proteins were separated using 10%SDS�PAGE and transferred to polyvinylidene fluoride(PVDF) membranes (Millipore, Billerica, MA). The mem-branes were then blocked in 5% non-fat milk for 1 hr. Immu-noblotting was performed with primary antibodies againstNUR77 (1:1,000 dilution; Cell Signaling Technology, Dan-vers, MA) and Flag-HRP (1:5,000 dilution; Sigma). b-actin(1:5,000 dilution; Bioworld Technology, St. Louis, MO) wasmeasured as an internal control. Immunodetection wasaccomplished using a goat anti-rabbit secondary antibodyand an enhanced chemiluminescence kit (Millipore).

Chromatin Immunoprecipitation (ChIP) AssayA chromatin immunoprecipitation assay kit (Upstate

Biotechnology, Lake Placid, NY) was used, with somemodifications to the protocol (Sun et al., 2009). KGN cells(at 70% confluence) were stimulated with FSH (20 ng/ml)for 8 hr, washed with PBS, and then cross-linked with 1%formaldehyde for 15 min at room temperature. The cross-linking was stopped with the addition of glycine (to a finalconcentration of 0.125 M) for 10 min. Cells were washed

TABLE 1. Oligonucleotide Primer Sequences for Quantitative Real-Time PCR

Species Genes Primers Products size (bp)

Mouse Inhibin-alpha 50-TCCTGGTAGCCCACACTAGG 21850-GAAACTGGGAGGGTGTACGA

Mouse Nur77 50-GCACAGCTTGGGTGTTGATG 18750-CAGACGTGACAGGCAGCTG

Mouse 18S rRNA 50-ATGGCCGTTCTTAGTTGGTG 18350-CGGACATCTAAGGGCATCAC

Human Inhibin-alpha 50-CCAGCTGTGAGGACAAGTCA 18650-CTAGCAGGGGCTCAGAGCTA

Human NUR77 50-ACCCACTTCTCCACACCTTG 20050-ACTTGGCGTTTTTCTGCACT

Human 18S rRNA 50-CGGCTACCACATCCAAGGAA 18650-CTGGAATTACCGCGGCT

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twice with cold PBS, harvested in lysis buffer A (20 mMTris�HCl [pH 8.0], 85 mM KCl, 1 mM EDTA, 0.5 mM EGTA,0.5% Nonidet P40, 1 mM PMSF, 1 mg/ml aprotinin, and1 mg/ml pepstatin A), and then pelleted by centrifugation.The cell pellets were then lysed in nuclear lysis buffer B(50 mM Tris�HCl [pH 8.0], 10 mM EDTA, 1% SDS, andprotease inhibitors, as described above). The cell sampleswere ultra-sonicated on ice to obtain genomic DNA frag-ments of 500�1,000 bps. The pre-cleared sonicates werethen incubated with an antibody to NUR77 (Cell SignalingTechnology) or irrelevant antibody (goat anti-rabbit IgG) ascontrol for 4�6 hr. Then, Protein A/G beads (Upstate Bio-technology) were added and rotated overnight. The collect-ed beads were washed. Following an incubation of thesamples at 658C for 30 min, the immunocomplexes wereeluted at room temperature for 15 min using fresh elutionbuffer (1% SDS; 0.1 M NaHCO3). Crosslinks were re-versed with an incubation of the samples at 658C for5 hr, at a final concentration of 0.3 M NaCl. The eluateswere then incubated with proteinase K, and DNA waspurified using phenol/chloroform extraction followed byethanol precipitation. Finally, we used the purified DNAfragments as a PCR template for the amplification ofNUR77-binding sequences present in KGN cells. Primersdesigned for PCR included: 50-GCACAGCATTCTCT-CCTTCC and 50-GCCGTGCTGGAATGTAAAGT (span-ning �2,695 to �2,340 bp). Quantitative real-time PCRprimers designed to amplify the putative NBRE sites in-cluded: 50-ACCTGGAATCTGAGCACCTG and 50-CTCAT-CATCCACAGGCTGAA (spanning �2,609 to �2,456 bp).

AvidinBiotin Conjugate DNA Precipitation (ABCD)Assay

For this assay, double-stranded oligonucleotides weredesigned. The sequence of these primers included 50-biotin-CACGGGCCAGCTGACCTTTGACTGTGACGT forhuman inhibin-alpha wild-type (WT)-F (from �2,565 to�2,536 bp); and 50-biotin-CACGGGCCAGCTGAATTCT-GACTGTGACGT for human inhibin-alpha mutant (MU)-F.All of these primers were labeled with biotin at the 50-end ofeach sense strand.

KGN cells (at 70% confluence) were infected with Ad-LacZ and Ad-Flag-NUR77 (10 MOI) for 48 hr, and thenlysed with a lysis buffer (10 mM Tris�HCl [pH 7.8], 1 mMEDTA, 150 mM NaCl, 0.1% NP-40) containing proteaseinhibitors at 48C for 1 hr. The cell extracts were incubatedwith 500 pmol of each double-stranded DNA immobilizedon streptavidin-agarose beads in a binding buffer (10 mMTris, [pH 8.0], 150 mM NaCl, 0.5% Triton X-100, 0.5 mMDTT, 0.5 mM EDTA, 10% glycerol, and 20 mg/ml poly [dI�dC]) containing protease-inhibitor cocktail for 4 hr at 48C.Following protein transfer onto polyvinylidine fluoride(PVDF) membranes (Millipore), the Flag-conjugated pro-teins were detected using an anti-Flag-HRP antibody(1:3,000; Sigma), which were subsequently bound bygoat anti-rabbit secondary antibodies and visualized usingan enhanced chemiluminescence kit (AmershamBioscien-ces, Piscataway, NJ).

Luciferase Reporter AssayThe human inhibin-alpha subunit promoter sequence

(�2,703/þ161) was amplified by PCR using the primers:50-CGGGGTACCCCACTAGCAGCACAGCATTCTCTCCT-TCC and 50-CGA AGATCTGCCCACACTGTACTGGGA-GAAGGACGGGA.

Furthermore, we used an overlap extension PCR meth-od to amplify the inhibin-alpha promoter mutant sequence.The primers were: 50-GCCAACGTCACAGTCAgAatT-CAGCTGGCCCGTGGG and 50-GGGCCAGCTGAatTcT-GACTGTGACGTTGGCCACGG.

The resulting 2.8-kb PCR products were subcloned intopGL3-basic (Promega, Madison, WI) using the Kpn I andBgl II sites, and sequenced to confirm the resulting vectors.

KGN cells (at 70% confluence) in 12-well plates weretransfected with PCMV-Flag-NUR77 or PCMV-empty vec-tor (PCMV-EV) and the human inhibin-alpha subunit or themutant firefly and the Renilla luciferase reporter plasmidusing the Sofast transfection reagent (Xiamen SunmaBiotechnology Co., Ltd., Xiamen, China). After 48 hr trans-fection, the luciferase activity was assayed using a dualLuciferase Assay System (Promega).

Inhibin AssaysDimeric inhibin levels in the spent culture media were

quantified with specific enzyme-linked immunosorbent as-say (ELISA) kits, including inhibin A (Westang, Shanghai,China) and inhibin B (Beckman Coulter, Brea, CA). Theinhibin A kit has a sensitivity of 10 pg/ml, with intra- andinter-observer variation coefficients of 7.5% and 9.5%. Theinhibin A kit has a sensitivity of 7 pg/ml, with intra- and inter-observer variation coefficients of 3.5% and 6.2%. All hor-mone measurements were performed in duplicate. Secret-ed hormone levels were normalized to total cellular proteincontent.

Statistical AnalysisAll values are expressed as themeans � standard error

of the mean. The differences between the mean values forthe two groups were analyzed using a two-tailed Student’st-test. One-way ANOVA followed by Student�Newman�Keuls multiple comparison tests were performed for com-parison differences among more than two groups. Valueswere determined to be significant when P < 0.05.

ACKNOWLEDGMENTS

We thankXinZhen for the preparation of adenovirus andFei Yu for the help in the animal experiments.

REFERENCES

Anttonen M, Parviainen H, Kyr€onlahti A, Bielinska M, Wilson DB,Ritvos O, Heikinheimo M. 2006. GATA-4 is a granulosa cellfactor employed in inhibin-a activation by the TGF-b pathway. JMol Endocrinol 36:557�568.

NUR77 REGULATES INHIBIN-ALPHA

Mol. Reprod. Dev. 80:734–743 (2013) 741

Chand AL, Ooi GT, Harrison CA, Shelling AN, Robertson DM.2007. Functional analysis of the human inhibin alpha subunitvariant A257Tand its potential role in premature ovarian failure.Hum Reprod 22:3241�3248.

ChandAL, Harrison CA, Shelling AN. 2010. Inhibin and prematureovarian failure. Hum Reprod Update 16:39�50.

Dai A, Yan G, He Q, Jiang Y, Zhang Q, Fang T, Ding L, Sun J,Sun H, Hu Y. 2012. Orphan nuclear receptor nur77 regulatesandrogen receptor gene expression in mouse ovary. PLoSONE 7:e39950.

Ding LJ, YanGJ,GeQY, YuF, ZhaoX,Diao ZY,WangZQ, YangZZ,Sun HX, Hu YL. 2011. FSH acts on the proliferation of type Aspermatogonia via Nur77 that increases GDNF expression inthe Sertoli cells. FEBS Lett 585:2437�2444.

Drummond AE, Dyson M, Thean E, Groome NP, Robertson DM,Findlay JK. 2000. Temporal and hormonal regulation of inhibinprotein and subunit mRNA expression by post-natal and imma-ture rat ovaries. J Endocrinol 166:339�354.

Feng ZM, Li YP, Chen CL. 1989. Analysis of the 50-flankingregionsof rat inhibin-a and -bB-subunit genes suggests two differentregulatory mechanisms. Mol Endocrinol 3:1914�1925.

Feng ZM, Wu AZ, Chen CL. 1998. Testicular GATA-1 factor up-regulates the promoter activity of rat inhibin alpha-subunit genein MA-10 Leydig tumor cells. Mol Endocrinol 12:378�390.

Findlay JK, Drummond AE, Dyson M, Baillie AJ, Robertson DM,Ethier JF. 2001. Production and actions of inhibin and activinduring folliculogenesis in the rat. Mol Cell Endocrinol 180:139�144.

Gummow BM, Winnay JN, Hammer GD. 2003. Convergenceof Wnt signaling and steroidogenic factor-1 (SF-1) ontranscription of the rat inhibin a gene. J Biol Chem 278:26572�26579.

HerschmanHR. 1991.Primary responsegenes inducedbygrowthfactors and tumor promoters. Annu Rev Biochem 60:281�319.

Inaoka Y, Yazawa T, UesakaM,Mizutani T, Yamada K,Miyamoto K.2008. Regulation of NGFI-B/Nur77 gene expression in therat ovary and in leydig tumor cells MA-10. Mol Reprod Dev75:931�939.

Ito M, Park Y, Weck J, Mayo KE, Jameson JL. 2000. Synergisticactivation of the inhibin a-promoter by steroidogenic factor-1and cyclic adenosine 30,50-monophosphate. Mol Endocrinol14:66�81.

Jiang Y, Hu Y, Zhao J, Zhen X, Yan G, Sun H. 2011. The orphannuclear receptor Nur77 regulates decidual prolactin expressionin human endometrial stromal cells. Biochem Biophys ResCommun 404:628�633.

Kaivo-Oja N, Bondestam J, K€am€ar€ainen M, Koskimies J, Vitt U,Cranfield M, Vuojolainen K, Kallio JP, Olkkonen VM, HayashiM, Moustakas A, Groome NP, ten Dijke P, Hsueh AJ, Ritvos O.2003. Growth differentiation factor-9 induces Smad2 activa-tion and inhibin B production in cultured human granulosa-luteal cells. J Clin Endocrinol Metab 88:755�762.

KimH,ChunS,GuBS, KuSY, KimSH,Kim JG. 2011. Relationshipbetween inhibin-a gene polymorphisms and premature ovarianfailure in Korean women. Menopause 18:1232�1236.

Klein NA, Illingworth PJ, Groome NP, McNeilly AS, Battaglia DE,Soules MR. 1996. Decreased inhibin B secretion is associatedwith themonotropic FSH rise in older, ovulatory women: A studyof serum and follicular fluid levels of dimeric inhibin A and B inspontaneous menstrual cycles. J Clin Endocrinol Metab81:2742�2745.

Knight PG, Glister C. 2006. TGF-beta superfamily members andovarian follicle development. Reproduction 132:191�206.

Knight PG, Glister C. 2001. Potential local regulatory functions ofinhibins, activins and follistatin in the ovary. Reproduction121:503�512.

Lanuza GM, Groome NP, Bara~nao JL, Campo S. 1999. Dimericinhibin A and B production are differentially regulated by hor-mones and local factors in rat granulosa cells. Endocrinology140:2549�2554.

Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expres-sion data using real-time quantitative PCR and the 2�DDC(T)

method. Methods 25:402�408.

Lockwood GM, Muttukrishna S, Ledger WL. 1998. Inhibins andactivins in human ovulation, conception and pregnancy. HumReprod Update 4:284�295.

Martin LJ, Tremblay JJ. 2008. Glucocorticoids antagonize cAMP-induced Star transcription in Leydig cells through the orphannuclear receptor NR4A1. J Mol Endocrinol 41:165�175.

Martin LJ, Tremblay JJ. 2009. The nuclear receptors NUR77 andSF1 play additive roles with c-JUN through distinct elements onthe mouse Star promoter. J Mol Endocrinol 42:119�129.

Marozzi A, Porta C, Vegetti W, Crosignani PG, Tibiletti MG, DalprÁL,Ginelli E. 2002.Mutationanalysis of the inhibin alphagene in acohort of Italian women affected by ovarian failure. HumReprod17:1741�1745.

Maruyama K, Tsukada T, Ohkura N, Bandoh S, Hosono T, Yama-guchi K. 1998. The NGFI-B subfamily of the nuclear receptorsuperfamily. Int J Oncol 12:1237�1243.

Mukherjee A, Park-Sarge OK, Mayo KE. 1996. Gonadotropinsinduce rapid phosphorylation of the 30,50-cyclic adenosinemonophosphate response element binding protein in ovariangranulosa cells. Endocrinology 137:3234�3245.

Muttukrishna S, Sharma S, Barlow DH, Ledger W, Groome N,Sathanandan M. 2002. Serum inhibins, estradiol, progester-one and FSH in surgical menopause: A demonstration ofovarian pituitary feedback loop in women. Hum Reprod17:2535�2539.

Myers M, Middlebrook BS, Matzuk MM, Pangas SA. 2009. Lossof inhibin alpha uncouples oocyte-granulosa cell dynamicsand disrupts postnatal folliculogenesis. Dev Biol 334:458�467.

Nagaraja AK, Middlebrook BS, Rajanahally S, Myers M, Li Q,Matzuk MM, Pangas SA. 2010. Defective gonadotropin-

Molecular Reproduction & Development HE ET AL.

742 Mol. Reprod. Dev. 80:734–743 (2013)

dependent ovarian folliculogenesis and granulosa cell geneexpression in inhibin-deficient mice. Endocrinology 151:4994�5006.

Petraglia F, HartmannB, Luisi S, Florio P, Kirchengast S, SantuzM,Genazzani AD, Genazzani AR. 1998. Low levels of seruminhibin A and inhibin B in women with hypergonadotropic amen-orrhea and evidence of high levels of activin A in women withhypothalamic amenorrhea. Fertil Steril 70:907�912.

Pnueli L, Luo M, Wang S, Naor Z, Melamed P. 2011. Calcineurinmediates thegonadotropin-releasinghormoneeffect onexpres-sion of both subunits of the follicle-stimulating hormone throughdistinct mechanisms. Mol Cell Biol 31:5023�5036.

Prakash GJ, Ravi Kanth VV, Shelling AN, Rozati R, Sujatha M.2010. Mutational analysis of inhibin alpha gene revealed threenovel variations in Indian women with premature ovarian failure.Fertil Steril 94:90�98.

RobertsonDM, HaleGE, Fraser IS, Hughes CL, Burger HG. 2008.A proposed classification system for menstrual cycles in themenopause transition based on changes in serum hormoneprofiles. Menopause 15:1139�1144.

Robertson DM, Hale GE, Jolley D, Fraser IS, Hughes CL, BurgerHG. 2009. Interrelationships between ovarian and pituitaryhormones in ovulatory menstrual cycles across reproductiveage. J Clin Endocrinol Metab 94:138�144.

Shi FT,CheungAP, LeungPC.2009.Growth differentiation factor 9enhances activin a-induced inhibin B production in humangranulosa cells. Endocrinology 150:3540�3546.

Shi FT, Cheung AP, Huang HF, Leung PC. 2011. Growth differen-tiation factor 9 (GDF9) suppresses follistatin and follistatin-like 3production in human granulosa-lutein cells. PLoS ONE 6:e22866.

Sun H, Chen L, YanG,Wang R, Diao Z, Hu Y, Li C. 2009. HOXA10suppresses p/CAF promoter activity via three consecutive TTAT

units in human endometrial stromal cells. BiochemBiophys ResCommun 379:16�21.

Taieb J, Grynberg M, Pierre A, Arouche N, Massart P, Belville C,Hesters L, FrydmanR, Catteau-Jonard S, Fanchin R, Picard JY,Josso N, Rey RA, di Clemente N. 2011. FSH and its secondmessenger cAMP stimulate the transcription of human anti-Müllerian hormone in cultured granulosa cells. Mol Endocrinol25:645�655.

Turner IM, SaundersPT, Shimasaki S, Hillier SG. 1989.Regulationof inhibin subunit gene expression by FSH and estradiol incultured rat granulosa cells. Endocrinology 125:2790�2792.

Woodruff TK, Mayo KE. 1990. Regulation of inhibin synthesis inthe rat ovary. Annu Rev Physiol 52:807�821.

Weck J, Mayo KE. 2006. Switching of NR5A proteins asso-ciated with the inhibin alpha-subunit gene promoter afteractivation of the gene in granulosa cells. Mol Endocrinol20:1090�1103.

Maxwell MA, Muscat GE. 2006. The NR4A subgroup: Immediateearly response genes with pleiotropic physiological roles. NuclRecept Signal 4:e002.

Wu Y, Ghosh S, Nishi Y, Yanase T, Nawata H, Hu Y. 2005.The orphan nuclear receptors NURR1 and NGFI-B modulatearomatase gene expression in ovarian granulosa cells:A possible mechanism for repression of aromatase expres-sion upon luteinizing hormone surge. Endocrinology 146:237�246.

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NUR77 REGULATES INHIBIN-ALPHA

Mol. Reprod. Dev. 80:734–743 (2013) 743