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Endocrinology, doi:10.1210/en.2003-1141
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Endocrinology Vol. 145, No. 7 3424-3433
Copyright © 2004 by The Endocrine Society

Forkhead L2 Is Expressed in the Ovary and Represses the Promoter Activity of the Steroidogenic Acute Regulatory Gene

Margareta D. Pisarska, Jeehyeon Bae, Cynthia Klein and Aaron J. W. Hsueh

Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, California 94305-5317

Address all correspondence and requests for reprints to: Aaron J. W. Hsueh, Stanford University School of Medicine, Department of Obstetrics and Gynecology, 300 Pasteur Drive, Stanford, California 94305-5317. E-mail: aaron.hsueh{at}stanford.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Premature ovarian failure in a subgroup of women with blepharophimosis-ptosis-epicanthus inversus type 1 syndrome has been associated with nonsense mutations in the gene encoding a Forkhead transcription factor, Forkhead L2 (FOXL2). However, the exact function of FOXL2 in the ovary is unclear. We investigated the expression of FOXL2 in the mouse ovary during follicular development and maturation by RT-PCR and in situ hybridization. The FOXL2 mRNA is expressed in ovaries throughout development and adulthood and is localized to the undifferentiated granulosa cells in small and medium follicles as well as cumulus cells of preovulatory follicles. FOXL2 belongs to a group of transcription factors capable of interacting with specific DNA sequences in diverse gene promoters. With the presence of multiple putative forkhead DNA consensus sites, the promoter of the human steroidogenic acute regulatory (StAR) gene was used to test for regulation by FOXL2. Cotransfection studies revealed that wild-type FOXL2 represses the activity of the StAR promoter, and the first 95 bp upstream of the transcriptional start site of the StAR gene is sufficient for FOXL2 repression. EMSAs confirmed that FOXL2 interacts directly with this region. Analyses using FOXL2 mutants also demonstrated the importance of the entire alanine/proline-rich carboxyl terminus of FOXL2 for transcriptional repression. Furthermore, these mutations produce a protein with a dominant-negative effect that disables the transcriptional repressor activity of wild-type FOXL2. Dominant-negative mutations of FOXL2 could increase expression of StAR and other follicle differentiation genes in small and medium follicles to accelerate follicle development, resulting in increased initial recruitment of dormant follicles and thus the premature ovarian failure phenotype.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PREMATURE OVARIAN FAILURE is defined as a condition causing amenorrhea, hypoestrogenism, and elevated gonadotropins in women under 40 yr of age (1). By age 30 yr, 0.1% of women are affected by this disease, whereas 1% of women are affected by 40 yr of age (2). Premature ovarian failure can be associated with failure to endow the follicle pool or an early loss of the fixed follicle pool after excess follicle recruitment and/or atresia.

Although the etiology of premature ovarian failure remains largely unknown, a genetic basis has been determined for selective cases. The X chromosome plays a significant role, yet autosomal gene disorders also are associated with premature ovarian failure (3). Within the gene locus on chromosome 3q22–23, blepharophimosis-ptosis-epicanthus inversus (BPES) type I is a syndrome in patients exhibiting premature ovarian failure in association with characteristic eyelid dysplasia, blepharophimosis, ptosis, and epicanthus inversus (4). Ovaries from patients with this disorder are variable in their histological appearance, ranging from the presence of some primordial follicles with atretic follicles to complete absence of follicles and scarring of the ovaries (5). Crisponi et al. (6) cloned and characterized a gene encoding a putative forkhead transcription factor, Forkhead L2 (FOXL2), that maps to the locus BPES on chromosome 3q22–23. FOXL2 is expressed in the ovary and developing eyelid and is mutated in patients with BPES. Mutations in individuals with BPES type I create premature stop codons in FOXL2, presumably producing a truncated protein lacking the carboxyl terminus (6, 7, 8, 9).

FOXL2 is a member of the forkhead (FKH)/hepatocyte nuclear factor 3 (HNF3) gene family of transcription factors (6) that is highly conserved and contains members that are essential in embryogenesis (10, 11), tumorigenesis (12, 13, 14), and cell differentiation (15, 16, 17). The FKH/HNF3 family of transcription factors has a characteristic conserved winged helix domain important for DNA binding to a common DNA motif in the promoter of target genes. Outside the winged helix domain, FKH/HNF3 family members have transactivation or transrepression domains that are divergent (18, 19, 20, 21). The carboxyl terminus of FOXL2 contains a region rich in alanine and proline residues (6). In a number of Forkhead family proteins, alanine/proline-rich regions have been attributed to transcriptional repression, particularly in genes affecting differentiation (22, 23, 24). Thus, FOXL2 may contain a putative repressor domain that is lost in mutations of the FOXL2 gene found in patients with BPES type 1.

Forkhead transcription factors bind to a 7-bp core recognition motif 5'[(G/A (T/C) (C/A) A A (C/T) A]-3' in the promoter of target genes (19, 25, 26, 27, 28). This recognition motif was found in the promoter of the human steroidogenic acute regulatory (StAR) gene. StAR is a 30-kDa protein that controls the rate-limiting step in steroidogenesis. It transports cholesterol from the outer to the inner mitochondrial membrane for conversion to pregnenolone and other downstream steroid hormones (29, 30, 31, 32). Extensive studies (33, 34) have been done in the transcriptional regulation of the StAR gene. Major positive regulators include steroidogenic factor 1 (32, 35, 36, 37, 38), the sterol regulatory element binding proteins (39, 40, 41), the CCAAT/enhancer-binding proteins (32, 42, 43, 44), and a cAMP response element-binding protein family member (cAMP response element-binding protein/cAMP response element modulator family) (45). In contrast, dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X-chromosome (46, 47), c-Fos (48), and yin yang 1 (39, 49) are transcriptional repressors of StAR. Our analysis of the human StAR promoter reveals that there are putative binding sites for the FOXL2 protein, suggesting that FOXL2 could function as a regulator of the StAR promoter, a marker of granulosa cell differentiation.

Because FOXL2 mutations are associated with premature ovarian failure in human BPES type I, we investigated FOXL2 expression during ovarian development using a rodent model. Based on the presence of a putative transcription repressor region in FOXL2, and the existence of forkhead DNA binding sites in the StAR gene promoter, we tested transcription repression of the StAR gene by FOXL2 to delineate the potential role of FOXL2 in follicular differentiation that may lead to the ovarian failure phenotype.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal treatment
Adult female Swiss Webster outbred mice (Simonsen Laboratories, Inc., Gilroy, CA) were housed under 12-h light, 12-h dark lighting conditions (lights-on at 0700 h). On 17.5 d post conception (dpc), pregnant mice were killed to obtain fetal ovaries. Neonatal and adult mice were also killed at various ages. Immature female mice at 21 d of age were primed with 4 IU pregnant mare serum gonadotropin (PMSG; Calbiochem, La Jolla, CA) via ip injection. After 48 h of PMSG priming, mice were killed immediately or at 6 or 16 h after ip injection with 5 IU human chorionic gonadotropin (hCG, Schein Pharmaceuticals, Florham Park, NJ). Ovaries were dissected free from adherent tissues and collected for either RT-PCR or in situ hybridization. All animal protocols were approved by the Administrative Panel on Laboratory Animal Care at Stanford University and experiments were conducted in accordance with the International Guiding Principles for Biomedical Research Involving Animals.

RT-PCR
Ovaries were snap frozen in a dry-ice ethanol bath and stored at –80 C. Total RNA from whole ovaries were extracted using the RNeasy minikit (Qiagen, Valencia, CA). Reverse transcription of total RNA from mouse ovaries was performed using oligo (dT)18 primer and recombinant Moloney-murine leukemia virus reverse transcriptase as described in the manufacturer’s protocol (Clontech, Palo Alto, CA). For PCR amplification of FOXL2 cDNAs, 10 µl of the reverse transcription reaction was used in a 100 µl PCR. Two primers (5'-AAGCCCCCGTACTCGTACGTGGCGCTCATC-3' and 5'-GTAGTTGCCCTTCTCGAACATGTC-3') were used to amplify mouse FOXL2 cDNA fragments based on GenBank sequences (accession no. AF060873). Using the HotStarTaq polymerase (Qiagen), the PCR cycling profile consisted of an initial denaturation step at 95 C for 15 min followed by five cycles (94 C for 30 sec, 72 C for 3 min), five cycles (94 C for 30 sec, 70 C for 3 min), and 25 cycles (94 C for 30 sec, 68 C for 3 min). After PCR, the amplified product was subjected to electrophoresis on a 1.5% agarose gel stained with ethidium bromide to visualize the anticipated 241-bp fragment. As a control, glyceraldehyde-3-phosphate dehydrogenase was amplified in a similar fashion using the primers 5'-TGAAGGTCGGTGTGAACGGATTTGGC-3' and 5'-CATGTAGGCCATGAGGTCCACCAC-3' to produce a 983-bp product. The FOXL2 band was eluted and purified using a QIAquick gel extraction kit (Qiagen) and confirmed by sequencing. A T7 polymerase promoter was ligated to the FOXL2 cDNA fragment in an orientation-specific manner to produce cDNA for in situ hybridization studies using a Lig’n Scribe kit (Ambion, Austin, TX).

In situ hybridization studies
Ovaries were fixed at 4 C for 6 h in 4% paraformaldehyde in PBS, followed by immersion in 0.5 M sucrose in PBS overnight. Cryostat sections (7 mm thick) of fixed ovaries were mounted on microscope slides (Sigma Chemical Co., St. Louis, MO), fixed in 4% paraformaldehyde in PBS, and stored at –80 C before the hybridization procedure. As previously described (50), sections were pretreated serially with 0.2 M HCl, 2x saline sodium citrate, pronase E (0.125 mg/ml), 4% paraformaldehyde, and acetic anhydride in triethanolamine before dehydration in ascending grades of ethanol. The antisense and sense probes were generated and labeled with [35S]uridine 5-triphosphate (1000 Ci/mmol, Amersham Pharmacia Biotech, Piscataway, NJ) by in vitro transcription using the Riboprobe System-T7 (Promega, Madison, WI). The sections were hybridized overnight at 45 C in 50% formamide, 0.3 M NaCl, 10 mM Tris-HCl, 5 mM EDTA, 1 x Denhardt’s solution, 10% dextran sulfate, 1 µg/ml carrier transfer RNA, and 10 mM dithiothreitol. After ribonuclease A (20 µg/ml) treatment at 37 C for 30 min, posthybridization washing was performed to a final stringency of 0.1 x saline sodium citrate. Slides were dipped into NTB-2 emulsion (Eastman Kodak Co., Rochester, NY) and exposed at 4 C for 1 wk before development. The slides were subsequently stained with hematoxylin and eosin and mounted with DPX Mountant (Electron Microscopy Sciences, Ft. Washington, PA). Photographs of the slides were taken using a 35-mm camera and microscope (Zeiss, Oberkochen, Germany) with bright- and dark-field illumination. To allow for direct comparison among the ovarian sections from different experimental groups, all slides were processed simultaneously and under identical conditions.

Cloning of wild-type and mutant FOXL2 cDNAs
The full-length coding sequence of human FOXL2 was amplified using HotStarTaq polymerase with Q solution according to the manufacturer’s protocol (Qiagen). The primers used were 5'-ACGGAATTCATGGCCAGCTACCCCGAGCCC-3' and 5'-CTAGGATCCTCAGAGATCGAGGCGCGAATG-3' based on the coding region of human FOXL2 (GenBank accession no. AF301906). The PCR product was electrophoresed, eluted, and purified using QIAquick gel extraction kit (Qiagen) and directionally subcloned into the BamH1 and EcoR1 sites of the pcDNA3 expression vector (Invitrogen, Carlsbad, CA). Mutated human FOXL2 constructs with a premature stop codon corresponding to those found in families with BPES type I (6, 7) were amplified from full-length pcDNA3-FOXL2 using the following primers: 5'-ACGGAATTCATGGCCAGCTACCCCGAGCCC-3' and 5'-CTAGGATCCCTAGCAGGAGGCATAGGGCAT-3' for the derivation of the 218-amino acid protein and 5'-ACGGAATTCATGGCCAGCTACCCCGAGCCC-3' and 5'-CTAGAATTCTCACTTCTCGTAGAACGGGAA-3' for the 93-amino acid protein. All PCR products were subcloned into the pcDNA3 expression vector and confirmed by sequencing.

Cell transfection and target gene promoter activity assay
Chinese hamster ovary (CHO) cells (1.5 x 105/well) were cultured in DMEM/F12 supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine. One day later, cells were transfected using Lipofectamine 2000 (Invitrogen) with the pcDNA3 expression vector with or without different human FOXL2 cDNAs and with 0.5 µg of different human StAR promoter luciferase constructs (36). The indicator plasmid pCMV-ß-galactosidase was used to estimate transfection efficiency. The total DNA concentration was maintained at 1 µg/200 µl by the inclusion of the empty pcDNA3 vector. Cells were incubated with plasmids in a serum-free medium (Opti-MEM media, Invitrogen) for 4 h, followed by replacement with fresh media containing 10% fetal bovine serum for 24 h. Cells were lysed with reporter lysis buffer (Promega) and freeze/thawing before determination of luciferase activities in cell lysates with the luciferase assay system (Promega) using the Lumimark luminometer (Bio-Rad Laboratories, Hercules, CA). Results are reported as relative luminescence units and normalized with ß-galactosidase activity.

EMSAs
We used two methods to obtain functional human FOXL2 proteins for EMSAs. Nuclear extracts of CHO cells transfected with either the human FOXL2 cDNA or the empty vector were obtained using a rapid micropreparation technique (51). Protein concentrations were determined using the BCA protein assay (Pierce Biotechnology, Rockford, IL). To generate in vitro-translated human recombinant FOXL2 protein, the TnT quick coupled transcription/translation system (Promega) was used.

For EMSA studies, the StAR promoter probe (–95/+39) was prepared by PCR amplification of the human StAR promoter luciferase reporter (36) using HotStarTaq polymerase with Q solution according to the manufacturer’s protocol (Qiagen). The primers used were 5'-CCCTTCCTTTGCACAGTGAGTGATG-3' and 5'-TCGCCTCTGAGTCGCCTCTGAGTCC-3' based on the coding region of human StAR promoter (GenBank accession no. U29098). Double-stranded DNA probes were end labeled with {gamma}-32ATP using T4 polynucleotide kinase (Invitrogen). For studies using nuclear extracts, protein-DNA binding reactions were performed using 10 µg protein in a 20-µl volume of 5 x binding buffer [20 mM HEPES (pH 7.9), 5 mM EGTA, 7.5 mM MgCl2, 250 mM KCL, 50% glycerol], the radiolabeled probe (50,000 cpm), 20 mM dithiothreitol, and 2 µg poly(dI)·(dC). After 20 min incubation at room temperature, samples were loaded on a 5% polyacrylamide gel and run at 170 V at 4 C for 3 h. For studies using in vitro-translated proteins, protein-DNA binding reactions were performed using 4 µl protein in a 10-µl volume of binding buffer [20 mM HEPES (pH 7.5), 0.1 mM EDTA, 0.1 mg/ml BSA, 150 mM NaCl, 1 mM DDT], the radiolabeled probe (50,000 cpm), and 1 µg of poly(dI)·(dC). After a 15-min incubation on ice, 5 µl of loading dye containing 50% glycerol and bromphenol blue was added and the samples fractionated on a 5% polyacrylamide gel at 130 V for 3 h. The gels were dried and autoradiographed using XAR-5 film (Eastman Kodak). Competition experiments were performed in the presence of 10- to 200-fold molar excess of unlabeled probes that were added 15 min before the labeled probe.

Generation of FOXL2 antibodies and immunoblot analysis of FOXL2
The FOXL2 polyclonal antibody was generated specifically against the human FOXL2 peptide sequence, (C)TGRTVKEPEGPPS-COOH. This peptide sequence was selected using the MacVector software package (Accelrys, Burlington, MA) based on its hydrophilicity, surface probability, flexibility, and antigenic index. It does not have significant homology to other proteins as determined by a BLAST search. The peptide was conjugated to KLH (keyhole limpet hemocyanin) by conjugation through the N terminus cysteine residue. Rabbits were primed with the peptide-KLH conjugate emulsified in complete Freund’s adjuvant and boosted with the same peptide-KLH conjugate emulsified in incomplete Freund’s adjuvant at 3-wk intervals. A peptide-specific ELISA was performed to determine the specific antibody titers and the antiserum was purified through an affinity gel coupled with the antigen (Zymed Laboratories, South San Francisco, CA).

For immunoblotting of wild-type and mutant human FOXL2 proteins, CHO cells (3 x 105/well) were cultured and transfected with different expression vectors. The cells were washed once on ice with chilled PBS followed by lysis in radioimmunoprecipitation assay buffer. Protein concentration was determined using the BCA protein assay (Pierce Biotechnology). Nupage sample reducing agent (Invitrogen) and loading buffer were added to the samples, sonicated on ice for 15 sec with an MSE sonicator (Sanyo Corp., Osaka, Japan), and boiled for 3 min. Proteins were separated on 12% Tris-HCL ready gels (Bio-Rad Laboratories) before transferring onto Immobilon-P PVDF membranes (Millipore, Bedford, MA). Membranes were blocked overnight at room temperature in Tris-buffered saline 0.1% Tween 20 with 10% fat-free dry milk, incubated with the FOXL2 antibody (1:5000) at room temperature for 2 h, followed by incubation with horseradish peroxidase-conjugated donkey antirabbit IgG (Amersham Pharmacia Biotech) (1:12,500) at room temperature for 1 h. Immunoreactive proteins were detected using enhanced chemiluminescence (ECL kit, Amersham Pharmacia Biotech).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
FOXL2 mRNA expression in developing mouse gonads
FOXL2 expression at multiple stages of ovarian development in the mouse was initially assessed using RT-PCR. Total RNA was extracted from ovaries of mice at 17.5 dpc, and at various postnatal stages. As shown in Fig. 1Go, RT-PCR revealed FOXL2 transcripts in the ovaries of fetal mice at 17.5 dpc, at 13 and 23 d of age, and in the adult. Unlike the ovary, FOXL2 was absent in testes at 17.5 dpc, at 13 and 23 d of age, and in the adult.



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  		FIG. 1. RT-PCR analysis of FOXL2 expression from mouse ovaries and testes at different ages of development. Gonads were obtained from fetal mice at 17.5 dpc or neonatal and adult animals. mRNA was extracted and reverse transcribed before PCR analysis and fractionation using electrophoresis. The 241-bp PCR product of FOXL2 is present in the ovaries at all ages of development. Unlike the ovary, FOXL2 is absent in testes during similar ages. glyceraldehyde-3-phosphate dehydrogenase serves as a control to indicate the presence of cDNA in each sample. D, Day.

 
In situ hybridization was performed to localize the expression of FOXL2 in the developing mouse ovary. In ovaries from 17.5 dpc until birth, the ovary is filled with somatic cells and oocytes without defined follicle development (52). In situ hybridization analysis revealed the expression of FOXL2 in somatic cells, cells that have the potential to become granulosa, theca, or stromal cells. In contrast, the FOXL2 transcript was absent in oocytes (Fig. 2Go, A–C). By d 13 of life, the immature ovary is filled with small, primordial (type 2), primary (type 3a), and medium (type 3b, 4, and 5a) follicles, with small follicles arranged in the outer cortex and medium follicles in the inner cortex (52) (Fig. 2DGo). FOXL2 was expressed in the granulosa cells of all follicles at this age. Similar to earlier stages, the oocyte was devoid of the FOXL2 transcript (Fig. 2Go, E and F).



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  		FIG. 2. In situ hybridization analysis of FOXL2 in ovaries at different stages of development. A–C, In ovaries from animals at 17.5 dpc, FOXL2 is expressed in the multipotential stromal cells (SC) and is absent in the oocytes (O). D–F, In ovaries from mice at 13 d of age, FOXL2 is expressed in granulosa cells (GC) of small follicles, types 2 and 3a (arrows), and medium follicles, types 3b, 4, and 5a (arrowheads). G and H, In ovaries from mice at 23 d of age, FOXL2 is expressed in granulosa cells of small (arrows), medium (arrowhead), type 5B, and occasionally type 6 follicles. I and J, In the adult ovary, the FOXL2 transcript is expressed in the granulosa cells of small (arrows) and medium follicles (arrowhead) but absent in the antral follicles (types 7 and 8) (AF) and corpus luteum (CL) (A, C, D, F, G, and I, bright-field; B, E, H, and J, dark-field).

 
By the third week of life the ovary contains small and medium follicles. In addition, some large (type 5b) follicles and an occasional type 6 follicle, characterized by the presence of a small antral cavity, are present (52) (Fig. 2GGo). FOXL2 transcript was expressed in the granulosa cells of the small and medium follicles. FOXL2 was also expressed in a small number of the large type 6 follicles (Fig. 2HGo).

In the adult, FOXL2 expression was confined to the granulosa cells of the small and medium follicles. FOXL2 expression was absent in the granulosa cells of antral follicles (type 7 and 8) and the corpus luteum (Fig. 2Go, I and J).

FOXL2 expression in gonadotropin-treated ovaries
To study gonadotropin regulation of FOXL2 expression, immature mice at 21 d of age were treated with PMSG to stimulate the formation of preovulatory follicles. After 48 h, the animals were treated with an ovulatory dose of hCG to induce ovulation and subsequent luteinization.

Expression of FOXL2 mRNA was maintained in the granulosa cells of small and medium follicles throughout hormonal stimulation (Fig. 3Go). After PMSG treatment, FOXL2 expression was limited to less differentiated cumulus granulosa cells in large antral (type 7) follicles and was minimal in mural granulosa cells (Fig. 3Go, C and D). This pattern of expression was retained in the preovulatory (type 8) follicles 6 h after hCG treatment (Fig. 3Go, E and F). Sixteen hours after hCG treatment, FOXL2 expression remained in the small and medium follicles but was absent in the newly formed corpus luteum (Fig. 3Go, G and H).



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  		FIG. 3. In situ hybridization analysis of ovarian expression of FOXL2 in immature mice before and after gonadotropin stimulation. A and B, In ovaries from mice at 23 d of age, FOXL2 is expressed in granulosa cells of small (arrows), medium (arrowhead), type 5B, and a few type 6 follicles. C and D, At 48 h after PMSG treatment, FOXL2 is expressed in the granulosa cells of small (arrows) and medium growing follicles (arrowheads). FOXL2 is also expressed in cumulus cells (CC) of type 8 preovulatory follicles but low in mural granulosa cells (MG). E and F, At 6 h after hCG treatment, FOXL2 expression remains in the granulosa cells of small (arrows) and medium growing follicles (arrowheads) and in the cumulus cells (CC) of the type 8 follicles but is lower in mural granulosa cells. G and H, At 16 h after hCG treatment, FOXL2 is expressed in the small (arrows) and medium (arrowheads) follicles and is absent in the corpus luteum (CL) (A, C, E, and G, bright-field; B, D, F, and H, dark-field).

 
Wild-type FOXL2 is a repressor of the StAR promoter
FOXL2 is a member of the FKH/HNF3 family of transcription factors known to regulate the promoters of different genes with a unique recognition motif, 5' [(G/A) (T/C) (C/A) A A (C/T) A] 3' (19, 25, 26, 27, 28). Based on the degeneracy of the this motif (53), the core of this motif 5' [A A X A] 3' was used to screen promoter sequences of genes known to be involved in ovarian functions. In the –885-bp promoter region of the human StAR gene, 15 potential FKH/HNF3 recognition motifs were found, and this gene was selected as a target gene for testing transcriptional regulation by wild-type and mutant FOXL2.

To demonstrate whether the recombinant human FOXL2 and mutant proteins are indeed expressed, expression vectors encoding wild-type FOXL2 or mutant FOXL2 (a.a. 1–218) were transfected into CHO cells. Cell lysates were analyzed using a FOXL2 antibody that recognizes human FOXL2 at the amino terminus. As shown in Fig. 4AGo, when wild-type FOXL2 was transfected, a 45-kDa protein was detected, corresponding to the predicted size. When mutant FOXL2 (a.a. 1–218) was transfected, a truncated protein at 30 kDa could be detected (Fig. 4AGo).



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  		FIG. 4. Overexpression of wild-type, but not mutant, FOXL2 represses StAR promoter activity. A, Expression of wild-type and mutant FOXL2 after transfection of increasing amounts of expression plasmids encoding these proteins. A 45-kDa protein is detected when the wild-type FOXL2 plasmid is transfected, whereas a 30-kDa protein is detected when the mutant FOXL2 plasmid is transfected. B, Dose-dependent suppression of StAR promoter activity by FOXL2. Wild-type FOXL2 represses basal StAR promoter activity in a dose-dependent manner as reflected by luciferase activities. In contrast, there is no change in luciferase activity in the presence of mutant FOXL2.

 
To determine whether the human StAR promoter is regulated by human FOXL2, wild-type FOXL2 was cotransfected with the –885-bp human StAR promoter-luciferase reporter plasmid into CHO cells. Twenty-four hours after transfection, StAR promoter activity was determined by measuring luciferase activity in cell lysates. Wild-type FOXL2 repressed basal StAR promoter activity in a dose-dependent manner as reflected by decreases in luciferase activity (Fig. 4BGo). FOXL2 repressor activity is sufficiently robust, that in the presence of undetectable protein, repression of the StAR promoter occurs. In contrast, cotransfection of the StAR promoter luciferase reporter with a mutant FOXL2 did not suppress luciferase activity (Fig. 4BGo).

Minimal StAR promoter region responsive to FOXL2 repression
Because the –885-bp human StAR promoter contains multiple forkhead-responsive consensus sites (Fig. 5AGo), the StAR promoter was truncated to determine the minimal region that is important for transcriptional regulation by FOXL2. The –235-bp StAR promoter has four putative forkhead consensus sites and the –95-bp StAR promoter contains three putative forkhead consensus sites (at –67 bp, –24 bp, and –15 bp). These three promoters of different length showed similar basal transcriptional activity. Of interest, overexpression of wild-type FOXL2 repressed the promoter activities of the two shorter promoters (–235 bp, 40%; –95 bp, 47%) similar to that found for the larger construct (–885 bp, 47%) (Fig. 5BGo). Similar results were obtained with increasing amounts of transfected FOXL2 plasmids. In the presence of 100 ng of FOXL2 plasmid, the repression of the two shorter promoters (–235 bp, 48%; –95 bp, 58%) were similar to that found for the larger construct (–885 bp, 57%). These results suggest that the first 95 bp upstream of the transcriptional start site are sufficient for transcriptional repression by FOXL2.



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  		FIG. 5. Regions of the StAR promoter important for FOXL2 repression. A, Putative forkhead consensus sites in different StAR promoter constructs. The –885-bp StAR promoter contains 15 putative forkhead DNA consensus sites. The –235-bp StAR promoter contains four putative sites and the –95-bp StAR promoter contains three putative sites. B, Wild-type FOXL2 represses StAR promoters of different lengths. In contrast, mutant FOXL2 does not repress the luciferase activity of any StAR promoters.

 
FOXL2 binding to the StAR promoter based on EMSAs
To further confirm that the FOXL2 protein interacts directly with the StAR promoter, EMSAs were performed using nuclear extracts of CHO cells transfected with the FOXL2 cDNA. As shown in Fig. 6AGo, nuclear extracts from CHO cells containing FOXL2 bind to the StAR promoter probe (–95/+39), as demonstrated by a shift of the labeled probe to a prominent high-molecular-weight region (arrow) and a second diffuse complex of slower mobility. In contrast, no shift was evident when the StAR promoter probe was incubated with nuclear extracts from cells transfected with the empty vector. Furthermore, boiling of the nuclear extracts containing FOXL2 prevented interactions with the labeled probe. In the presence of excess unlabeled probe, FOXL2 protein-DNA complex formation was completely inhibited. Although several minor complexes also could be found, these highmolecular-weight bands could not be competed by excess unlabeled probes. To further demonstrate FOXL2 binding to the StAR promoter, EMSAs were performed using in vitro-translated FOXL2 proteins. In the presence of FOXL2, a shift of the labeled probe to a high-molecular-weight region was again identified (Fig. 6BGo, arrow) together with the second complex. Similarly, cell lysates without FOXL2 or with boiled FOXL2 did not interact with the labeled probe. In addition, binding of recombinant FOXL2 to the StAR promoter could be progressively competed by the addition of increasing amounts of unlabeled probe showing complete inhibition by a 200-fold molar excess of the unlabeled probe.



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  		FIG. 6. EMSAs showing binding of FOXL2 protein to the StAR promoter. A, Radiolabeled StAR promoter probe was incubated with or without nuclear extracts from CHO cells transfected with either FOXL2 cDNA or an empty vector. B, Radiolabeled StAR promoter probe was incubated with in vitro-translated recombinant FOXL2 protein. Some nuclear extracts and in vitro-translated proteins were boiled to denature the FOXL2 protein. Excess unlabeled probe was used to demonstrate specific binding between FOXL2 and the StAR promoter. The positions of the FOXL2 StAR promoter complexes are shown with arrows.

 
The alanine/proline-rich carboxyl terminus of FOXL2 is important for transcriptional repression
In human FOXL2, the DNA-binding domain (Forkhead domain) is located at the amino terminus, followed by an alanine/proline-rich region with two polyalanine tracts and a polyproline tract (Fig. 7AGo). In a number of Forkhead family proteins, these regions have been associated with transcriptional repression activity (22, 23, 24). To determine the particular region of the carboxyl terminus of FOXL2 responsible for its repressor activity, we sequentially truncated the carboxyl terminus of FOXL2, eliminating portions of the alanine/proline-rich regions (Fig. 7AGo). We deleted the carboxyl terminus alanine/proline-rich region but retained the polyalanine and polyproline tracts to produce FOXL2 (a.a 1–326). We also deleted the carboxyl terminal polyalanine and polyproline tract to generate FOXL2 (a.a. 1–271). Lastly, the entire carboxyl terminus was deleted, including the largest polyalanine tract, FOXL2 (a.a 1–218), similar to the predicted truncated proteins present in patients with BPES type 1. Compared with 48% repression of the StAR promoter by wild-type FOXL2 (a.a 1–376), loss of repressor activity occurred with further deletions of the carboxyl terminus in a stepwise manner (Fig. 7BGo). The StAR promoter was repressed by 30 and 19% with FOXL2 (a.a 1–326) and FOXL2 (a.a. 1–271), respectively. Similar results were obtained with increasing amounts of transfected FOXL2 plasmids. In the presence of 100 ng of different FOXL2 plasmids, the StAR promoter was repressed by 37 and 26% with FOXL2 (a.a 1–326) and FOXL2 (a.a. 1–271), respectively, compared with 66% repression by wild-type FOXL2 (a.a 1–376).



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  		FIG. 7. Regions of the FOXL2 protein responsible for its repressor function. A, Domain arrangement of FOXL2 and construction of FOXL2 mutants. In FOXL2, the DNA-binding domain (forkhead domain or FH) is located at the amino terminus followed by an alanine/proline-rich region from amino acids 221–348, with two polyalanine tracts (AAA) and a polyproline tract (PP). FOXL2 constructs with sequential deletions of the carboxyl terminus and two naturally occurring mutant FOXL2 constructs (1–218 and 1–93) were generated. B, The entire carboxyl terminus of FOXL2 is important for its repressor activity. Deletions of the carboxyl terminus result in a loss of repressor activity in a stepwise manner. Mutant FOXL2 (1–218 a.a. and 1–93 a.a.) show a complete loss of repressor activity. C, Dominant-negative effect of mutant FOXL2 (1–218 a.a.). Repressor activity of wild-type FOXL2 is lost in a dose-dependent manner when coexpressed with increasing concentrations of mutant FOXL2.

 
Nonsense mutations found in BPES type 1 produce truncated proteins lacking the region rich in alanine and proline residues (6, 7, 8, 9). These truncated proteins may be as long as 218 amino acids or as short as 93 amino acids, depending on the type of mutation in families with BPES type 1. We found that the longest FOXL2 mutants (a.a. 218) result in complete loss of repressor activity and could account for the ovarian failure phenotype. To determine whether this loss of repressor activity is universal among the other nonsense mutations, we tested whether the shortest truncated FOXL2 fragment (a.a. 1–93) known to be associated with premature ovarian failure also shows a similar loss-of-function phenotype. Mutant FOXL2 (a.a. 1–93) was cotransfected with the StAR promoter reporter plasmid into CHO cells. Similar to a longer mutant FOXL2 (a.a. 1–218), complete loss of repressor activity occurred with the shorter mutant FOXL2 (a.a. 1–93) (Fig. 7BGo). Similar results were obtained with increasing amounts of different transfected FOXL2 plasmids. Complete loss of repressor activity occurred in the presence of 100 ng of the two nonsense mutations.

Mutant FOXL2 exerts dominant-negative effects on the activity mediated by wild-type FOXL2
In BPES type I, the clinical phenotype of ovarian failure has been attributed to either haploinsuffiency, resulting from a decreased amount or activity of the protein, or a dominant-negative effect, in which the mutant protein interferes with the action of the wild-type allele (6, 7, 54). Mutant human FOXL2 (a.a. 1–218) was cotransfected with wild-type human FOXL2 to determine whether there is a dominant-negative effect by the mutant protein. As seen in Fig. 7CGo, increasing concentrations of mutant FOXL2 antagonized the repressor activity of FOXL2 on the StAR gene in a dose-dependent manner. At a 10-fold higher concentration, the mutant plasmid completely blocked the repressor effect of wild-type FOXL2.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have demonstrated that FOXL2 mRNA is expressed in mouse ovaries at all stages of development, from the fetal to the juvenile ovary, as well as in the adult. In situ analysis revealed that FOXL2 is expressed in undifferentiated granulosa cells, in either small or medium follicles or the cumulus cells of preovulatory follicles after gonadotropin stimulation. In contrast, FOXL2 expression is lower in the mural granulosa cells of preovulatory follicles and luteal cells, both being highly differentiated. Promoter reporter studies further revealed that FOXL2 is a transcriptional repressor of the StAR gene, a marker of granulosa cell differentiation. In addition, EMSA reveals that FOXL2 protein directly interacts with the first 95 bp upstream of the start site of the StAR promoter. Important for the repressor activity of FOXL2 is the entire alanine/proline-rich carboxyl terminus found to be lacking in mutant FOXL2 proteins from patients with premature ovarian failure. In addition to the loss of repressor activity, the FOXL2 mutants exhibit a dominant-negative effect by blocking the repressor activity of wild-type FOXL2. Mutant FOXL2 in patients with premature ovarian failure may be associated with accelerated differentiation of granulosa cells and secondary increases in the recruitment of dormant follicles leading to premature exhaustion of the primordial follicle pool.

The observed expression of FOXL2 transcripts in fetal and juvenile ovaries is consistent with a recent report (55) showing the expression of FOXL2 in the developing fetal gonad of the mouse, whereas the expression of FOXL2 in the adult ovary is consistent with findings by Crisponi et al. (6). In fetal ovaries, FOXL2 is expressed in the undifferentiated multipotential stromal cells including those destined to become granulosa cells. During the infant and juvenile period, FOXL2 is expressed in granulosa cells of small, medium, and a few type 6 follicles. In larger follicles, FOXL2 is expressed in cumulus cells but has lower expression in mural granulosa, consistent with the observed ability of FOXL2 to repress the follicle differentiation gene, StAR. Indeed, earlier studies on the LH receptor and steroidogenic enzymes indicated that cumulus cells are less differentiated than their mural counterparts (56, 57, 58, 59, 60).

StAR is expressed in multiple steroidogenic tissues and is responsible for a rate-limiting step in steroid hormone synthesis. It facilitates the transport of cholesterol from the outer mitochondrial membrane into the inner mitochondrial membrane before conversion to pregnenolone (29, 31). StAR is a marker of granulosa cell differentiation, and multiple forkhead consensus sites were identified by computer search within the promoter of the StAR gene. This presents StAR as a candidate target gene for FOXL2 suppression. In the adult, ovarian expression of StAR is predominantly in the theca cell layer, but it has also been localized to granulosa cells (61, 62, 63). In humans, StAR immunoreactivity is present in the granulosa cell layer of large preovulatory and luteinized follicles but absent in immature follicles (61). Similarly, in rodents, StAR protein expression is present in mural granulosa cells after PMSG treatment and is undetectable in granulosa cells of the secondary follicles of infant rats (62). Furthermore, StAR expression is confined to the granulosa cells of periovulatory follicles, whereas nonovulating follicles are devoid of StAR in the granulosa cells (63). Thus, StAR activity is present in the granulosa cell compartment during follicular differentiation, signaling early functional maturation of the rat ovarian antral follicles. The differential expression pattern between FOXL2 and StAR is consistent with the observed repressor function of FOXL2 on StAR promoter activity. During follicle development, the StAR transcript seems to be low in granulosa cells until FOXL2 expression declines. Thus, StAR is a marker of granulosa cell differentiation and was selected in our transfection studies as a potential target for repression by FOXL2.

FOXL2 is likely a repressor of StAR, a granulosa cell differentiation gene, based on our findings using StAR promoter reporter constructs and the EMSA protein-DNA binding assay. A number of forkhead transcription factors are transcriptional repressors (15, 16, 64, 65). Many of the forkhead transcriptional repressors function as determinants of tissue differentiation in a spatiotemporal manner (15, 16, 64, 65), and FOXL2 also may function as a determinant of tissue differentiation by inhibiting premature differentiation of granulosa cells. With the inhibition of granulosa cell differentiation, FOXL2 may control the number of primordial follicles that remain dormant and prevent the premature depletion of ovarian follicles. This is characteristic of another forkhead transcriptional repressor, Foxg1, whose role in neuroepithelial differentiation prevents early depletion of the progenitor pool (66, 67).

Human FOXL2 is highly homologous to mouse FOXL2 with 90% sequence identity and 95% similarity at the protein level. Within the DNA binding domain of FOXL2, there is 100% similarity at the protein level, suggesting similar promoter recognition between the two species. Comparison between human and mouse StAR promoters indicated the presence of putative steroidogenic factor 1 binding sites and repetitive motifs (35). Within the first 95 bp upstream of the human StAR promoter, found by us to be sufficient for transcriptional repression and direct binding by human FOXL2, 80% sequence similarity was evident. In addition, we identified three putative forkhead recognition sites (–67, –24, and –15 bp) within this region of the human promoter with one (–67 bp) being conserved in the mouse promoter (–70 bp). This conserved site may be involved in forkhead recognition.

Trophic hormones acutely stimulate StAR expression by activating the cAMP-mediated protein kinase A pathway in steroidogenic cells (29, 32, 33, 39) and StAR promoter activity increases in the presence of cAMP in these cells. However, in nonsteroidogenic cells, cAMP does not affect StAR promoter activity (35, 36, 38, 68). In the present CHO cell model, treatment with forskolin did not increase StAR promoter activity at 1, 3, or 6 h of incubation, despite persistent repression of the StAR promoter activity by FOXL2 (data not shown).

BPES type I is an autosomal dominant disorder in which a mutant gene encodes truncated proteins lacking the carboxyl terminus alanine/proline-rich domain (6, 7, 8, 9). The clinical phenotype of ovarian failure has been attributed to either haploinsufficiency, resulting from a decreased amount or activity of the protein, or a dominant-negative effect, in which the mutant protein interferes with the action of the wild-type allele (6, 7, 54). Overexpression of truncated FOXL2 does not lead to repression of the StAR promoter, despite the production of a recombinant protein. Furthermore, cotransfection of mutant FOXL2 with wild-type FOXL2 results in a loss of repressor activity of the wild-type protein, consistent with the dominant-negative effect of the truncated protein. Of interest, artificially produced dominant negatives (15, 69, 70) of FOXO1 also show dominant-negative effects. In particular, one dominant-negative mutant of FOXO1 (15) restores adipocyte differentiation, similar to the putative facilitation of granulosa cell differentiation by the mutant FOXL2.

The entire alanine/proline-rich region of FOXL2 is necessary for maximal transcriptional repression. Although alanine/proline-rich domains are traditionally characteristic of the transcriptional repressors Kruppel, even-skipped, and engrailed (71), a number of forkhead transcription factors also contain these domains (17, 23, 24, 66, 67, 72). Similar to FOXL2, these other forkhead transcription factors function in repressing cellular differentiation, such as neuroepithelial cells (22, 66, 67), neuroectoderm (23), and neural crest cells (17, 24, 72).

Several other forkhead transcription factors are expressed in the ovary. Richards et al. (73) characterized the expression and regulation of the O class of forkhead transcription factors, FKHR (forkhead in rhabdomyosarcoma), AFX (ALL1 fused gene from chromosome X), and FKHRL1 (FKHR-like 1). Similar to FOXL2, FKHR expression is confined to the small and medium growing follicles, and is low in the preovulatory follicles and corpus luteum. AFX and FKHRL1 are more diffusely expressed in the ovary with distinct expression in the corpus luteum. Within the O class of transcription factors, transactivation domains are located in the carboxyl terminus (74, 75, 76, 77, 78, 79). These regions are rich in acidic and serine/threonine residues (74, 75, 76, 77, 78, 79, 80) with phosphorylation of specific serine and threonine residues playing important roles in their transactivation activity (81, 82, 83). FOXL2, designated as a forkhead family member based on its winged helix DNA binding domain, has little similarity to the O class of the forkhead family members. Its DNA binding domain does not have significant homology to the O class and the presence of an alanine/proline-rich region, associated with transcriptional repression (22, 23, 24), is consistent with the present findings.

Recently mice null for Foxl2 (84) and Foxo 3a (FKHRL1) (85) were generated and found to exhibit unique ovarian phenotypes. The Foxl2 null mice showed follicles arrest between the primordial and primary stages, followed by follicle degeneration. They failed to undergo sexual maturation and hence underwent primary ovarian failure. In contrast, Foxo 3a (FKHRL1) null mice underwent normal sexual maturation but had accelerated recruitment of early follicles, followed by depletion of the primordial follicle pool and subsequent premature ovarian failure (84). Unlike the complete loss of Foxl2 alleles in null mice, patients with the heterozygous FOXL2 mutations do not show primordial follicle arrest. They exhibited a complete sequence of follicle development to the preovulatory stage with early depletion of the follicle pool and premature ovarian failure (5), much like the Foxo 3a null mice (85). Because FOXL2 is expressed in primordial, primary, and larger secondary follicles, it may play important roles during separate stages of follicle development in addition to primordial follicle arrest. Our data suggest that FOXL2 likely functions as a suppressor of ovarian follicle progression in small and medium follicles by the prevention of premature differentiation of granulosa cells. In patients with BPES type 1, mutations of FOXL2 with the resultant loss of repressor activity could lead to accelerated differentiation of granulosa cells and accelerated follicle recruitment. With an accelerated initial recruitment of follicles, the finite number of follicles that are present in the ovary may become depleted prematurely, leading to premature ovarian failure in patients with BPES type 1.


   Acknowledgments
 
We thank J. F. Strauss III for the StAR promoter constructs and Caren Spencer for editorial assistance.


   Footnotes
 
This work was supported by the National Institute of Child Health and Human Development through Cooperative Agreement U54 HD31398 as part of the Specialized Cooperative Centers Program in Reproduction Research (to A.J.W.H.) and the National Institutes of Health Women’s Reproductive Health Research Career Development Program K12 HD01249 (to M.D.P.).

Abbreviations: BPES, Blepharophimosis-ptosis-epicanthus inversus syndrome; CHO, Chinese hamster ovary; dpc, days post conception; FKH, forkhead; FOXL2, Forkhead L2; hCG, human chorionic gonadotropin; HNF3, hepatocyte nuclear factor 3; PMSG, pregnant mare serum gonadotropin; StAR, steroidogenic acute regulatory.

Received September 2, 2003.

Accepted for publication March 22, 2004.


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 Introduction
 Materials and Methods
 Results
 Discussion
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