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The Role of the Orphan Nuclear Receptor, Liver Receptor Homologue-1, in the Regulation of Human Corpus Luteum 3ß-Hydroxysteroid Dehydrogenase Type II

Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas Texas 75390-9032

Address all correspondence and requests for reprints to: George R. Attia, M.D., Department of Obstetrics and Gynecology, University of Miami, 7007 Holtz Center, JMH East Tower, 1611 NW 12th Avenue, Miami, Florida 33136. E-mail: gattia{at}med.miami.edu.

	Abstract

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After ovulation, ovarian 3ß-hydroxysteroid dehydrogenase type II (HSD3B2) expression increases to enhance the shift of steroidogenesis toward progesterone biosynthesis. Steroidogenic factor-1 (SF-1) is a transcription factor for several genes encoding steroidogenic enzymes. However, the level of SF-1 expression decreases in the human corpus luteum (CL) after ovulation. Liver receptor homolog-1 (LRH-1) is another member of the orphan nuclear receptor family. We hypothesize that LRH-1, rather than SF-1, plays an essential role in the regulation of corpus luteum steroidogenesis. Semiquantitative RT-PCR and real-time PCR were performed to quantify the level of LRH-1 expression and correlate with HSD3B2 level. Cell transfection, mutation analysis, and EMSA were performed to examine the role of LRH-1 in the regulation of HSD3B2. LRH-1 expression was higher in CL, compared with mature ovarian follicles. Cotransfection of granulosa cells with HSD3B2 and LRH-1 resulted in a 10-fold increase of transcription. DAX-1 inhibited LRH-1-stimulated HSD3B2, which was maintained in the presence of dibutyryl cAMP. Mutation of the either of the two putative LRH-1 binding sites, which were confirmed by EMSA, in the HSD3B2 promoter decreased LRH-1 stimulation. Our findings suggest that LRH-1 is highly expressed in CL, and it plays an essential role in the regulation of HSD3B2.

	Introduction

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OVARIAN PRODUCTION OF progesterone and estrogen is determined by the cell-specific expression of a variety of steroidogenic enzymes and transcription factors (1, 2, 3, 4). Although the tropic hormones and steroidogenic enzymes responsible for ovarian steroid hormone biosynthesis have been well defined, the mechanisms controlling expression of steroid-metabolizing enzymes remain an area of active research. After ovulation, there is a shift from a predominantly estrogen-producing ovarian follicle to a predominantly progesterone-producing corpus luteum (CL). This shift in steroidogenesis is still poorly understood, but alterations in gene transcription appear to be a major mechanism controlling this transition (5, 6, 7, 8).

3ß-Hydroxysteroid dehydrogenase type 2 (HSD3B2) is a microsomal enzyme that is present in the theca interna and granulosa cells (9, 10). During the follicular phase, thecal cells express low levels of HSD3B2 for C₁₉ steroid production to occur. After ovulation, there is an increase in ovarian follicle HSD3B2 expression that enhances the shift of steroid production toward progesterone biosynthesis. HSD3B2 mRNA levels increase greatly in the corpora lutea and particularly the luteinized granulosa cells at a time when progesterone becomes the primary ovarian product (11, 12).

Orphan nuclear receptors are transcription factors for which ligands have not been identified. Two such factors, steroidogenic factor-1 (SF-1; designated NR5A1) and dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX-1; designated NR0B1) are involved in the regulation of steroidogenesis (13, 14, 15). SF-1 has been demonstrated as a regulator of the transcription of several genes encoding steroidogenic enzymes including HSD3B2 (16, 17, 18, 19, 20, 21, 22). DAX-1 tissue expression colocalizes with SF-1 and has the ability to inhibit SF-1-mediated transcription (6, 24).

Liver receptor homolog-1 (LRH-1; designated NR5A2) is another orphan nuclear receptor, which is expressed in multiple tissues (25, 26, 27, 28, 29). LRH-1 and SF-1 are members of the nuclear receptor family that recognize the canonical recognition motif for Drosophila fushi tarazu factor receptors and bind to DNA as monomers to enhance transcription of targeted genes. LRH-1 has an overall 60% amino acid similarity to SF-1 with virtually identical DNA binding domain. Initially, it was thought that LRH-1 was only expressed in tissues derived from the gut endoderm such as pancreas, liver, and intestine (27, 28, 29). More recently, LRH-1 was found in mouse, equine, and human ovaries (29, 30, 31), human testis, and at low levels in the human adrenal (32), raising the possibility that LRH-1 could play a role in regulation of steroidogenesis. However, the role of LRH-1 in the regulation of ovarian steroidogenesis is still poorly defined.

In human ovarian cycle, the LH surge is essential for ovulation and subsequent conversion of mature ovarian follicle to CL. Although SF-1 can regulate enzymes that are essential for progesterone production, the expression of SF-1 was found to be down-regulated in ovarian cells after LH surge (33, 34, 35), which raises questions regarding the role of SF-1 in corpus luteum steroidogenesis. Thus, we hypothesize that LRH-1, rather than SF-1, could play an essential role in the regulation of human corpus luteum function and expression of steroidogenic enzymes, specifically HSD3B2.

In this study we examined the differential expression of LRH-1 between human mature ovarian follicles and CL. We also examined the role of LRH-1 in the regulation of HSD3B2 promoter.

	Materials and Methods

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Cell preparation and RNA extraction

Ovarian cortex, ovarian follicle, and corpus luteum were isolated from human ovarian tissues. Human ovaries were obtained from women of reproductive age (aged 30–40 yr) at the time of hysterectomy and were determined to be normal. Human luteinized granulosa cells (HLGCs) were obtained by follicular aspiration from women of reproductive age (aged 25–35 yr) undergoing oocyte retrieval for in vitro fertilization. Briefly, women were treated with GnRH agonist before and during follicular stimulation using recombinant human gonadotropin. After follicular aspiration, HLGCs were isolated as previously described (36). The HLGCs were washed twice with DMEM and Ham’s F-12 medium (DME/F12) (GIBCO BRL, Gaithersburg, MD) and then incubated for 30 min at 37 C in DME/F-12 containing 0.1% hyaluronidase to disperse them. The dispersed cells were resuspended in 20 ml medium and transferred to 50-ml tubes containing 3.5 ml Histopaque 1077 (Sigma Chemical Co., St. Louis, MO). HLGCs were separated from red blood cells by centrifugation at 600 x g for 15 min. HLGCs formed a thin layer between the Histopaque and the medium. Cells were removed and then washed three times using DME/F-12 containing 5% fetal bovine serum; 5% horse serum; 1% ITS Plus (Collaborative Research, Waltham, MA); 2% Ultroser G (IBF Biotechnics, Sepracor, Inc., Marlborough, MA); and antibiotics.

Total RNA was extracted from ovarian cortex, mature ovarian follicles, CL, and HLGCs as previously described (37). Purity and integrity of RNA was checked spectroscopically and by gel electrophoresis before use. The use of human tissues was approved by the Institutional Review Board of the University of Texas Southwestern Medical Center at Dallas.

RT-PCR

Total RNA (1 µg) prepared from human ovarian follicles, CL, and ovarian cortex was used for reverse transcription (RT) reaction in a final volume of 20 µl. PCR was performed using equal amounts of RT product standarized to the amounts of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) present as template with human LRH-1-specific primers (32), human SF-1-specific primers (38), and G3PDH-specific primers (Accession AF261085) (Table 1). The PCR conditions were 94 C for 20 sec, 54 C for 20 sec, and 72 C for 40 sec. PCR product (10 µl) of each sample was loaded and electrophoresed on 1% agarose gel.

Four micrograms of total RNA each from human ovarian cortex, ovarian follicle, CL, and HLGCs was reverse transcribed in a final volume of 100 µl using a high-capacity cDNA archive kit (Applied Biosystem, Foster City, CA). Primers for the amplifications (Table 1) were based on published sequences for LRH-1 (GenBank accession no. NM_003822), HSD3B2 (accession no. M77144), and SF-1 (NM_004959). PCRs were performed in the ABI Prism 7000 sequence detection system (Applied Biosystems) in a total volume of 30 µl reaction mixture following the manufacturer’s recommendations using the SYBR Green Universal PCR Master Mix 2X for LRH-1 and SF-1 and Taqman 2X PCR Master-Mix for HSD3B2 (Applied Biosystems). Forward and reverse primers were added at 0.1 µM using the manufacturer’s recommended dissociation protocol. Standard curves were prepared using the human LRH-1 (in Topo pCRII), HSD3B2 (in pVL), and SF-1 (in pcDNA3) (Invitrogen, Carlsbad, CA) eukaryotic expression vector. Negative controls contained water instead of first-strand cDNA. Each sample was normalized on the basis of its 18S rRNA content. The 18S quantification was performed using the TaqMan rRNA control reagent kit (Applied Biosystems) and following the method protocol provided by the manufacturer.

Preparation of reporter constructs and expression vectors

The 5' flanking DNA from the human HSD3B2 gene(-963 bp) (22) was inserted upstream of the firefly luciferase gene in the reporter vector pGL3-Basic (Promega, Madison, WI). For all transfections, empty pGL3-Basic was used as the control vector to measure basal activity. The coding region of LRH-1 (provided by Dr. David Mangelsdorf, University of Texas Southwestern) and SF-1 (39) were inserted into pcDNA3 (Invitrogen) eukaryotic expression vector.

Cell culture and transfection assay

Human ovarian granulosa cell model comprised of granulosa cell tumor (GCT) cells isolated from patient with GCT as previously described (40). This model is able to reproduce many of the differentiated functions of granulosa cells. Specifically, they have maintained the production of progesterone and are able to convert androstenedione to estradiol. This cell model also responds to forskolin and dibutyryl cAMP (dbcAMP) by increasing production of progesterone and estradiol (data not shown). GCT cells were cultured in DME/F12 (GIBCO BRL) supplemented with 5% NU Serum (Collaborative Biomedical, Bedford, MA) and antibiotics. For transfection experiments, Fugene 6 (Roche, Indianapolis, IN) was used to transfect 0.5 µg of reporter plasmid and the indicated amounts of expression vectors. pcDNA3 empty vector was used to assure constant amounts of DNA per well for each transfection. Cells were assayed for reporter activity using the Luciferase assay system (Promega,) 18–20 h after transfection.

Preparation of deletions and mutant HSD3B2 5'-flanking constructs

Several 5'-deletion plasmids were constructed using available restriction endonuclease sites. Mutant constructs containing mutated putative LRH-1 binding sites in the HSD3B2 promoter regions were generated by using introduction of restriction endonuclease site (EcoRI) by PCR and introduction of point mutation by PCR (Table 2).

Human ovarian GCT cell and HLGC nuclear extracts (NEs) were prepared as previously described (41). The human LRH-1 was synthesized by coupled in vitro transcription/translation by T7 polymerase using the reticulocyte lysate system (Promega). Double-stranded oligonucleotides (25 pmol) containing the wild-type and mutant LRH-1 response elements (Table 2) were labeled with [-³²P] dCTP by Moloney murine leukemia virus reverse transcriptase at 37 C for 30 min. The probes (50,000 cpm) were incubated with 5 µg of LRH-1 in vitro protein (IVP LRH-1), HLGC NEs, or human ovarian GCT NEs in 20 µl reaction mixture [20 mM HEPES (pH 8.0), 80 mM KCL, 1 mM EDTA, 10% glycerol, 1 mM dithiothreitol, 0.5 mg/ml BSA, and 0.075 mg/ml poly dI-dC to block nonspecific binding]. The reactions were incubated at room temperature for 20 min. For competition analysis, reaction mixtures containing 100-fold molar excess of nonradiolabeled oligonucleotide (cold) were added simultaneously with probe. The resulting DNA/protein complexes were then separated from free probe (FP) by electrophoresis using a 4% high-ionic-strength native polyacrylamide gel with 1 x Tris-glycine running buffer. The gel was dried and visualized after autoradiography at -70 C for 24 h.

Statistical analysis

Data were analyzed by ANOVA using STATPAC software (Minneapolis, MN).

	Results

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Human ovarian follicle and corpus luteum express LRH-1 mRNA

The first objective was to determine whether human corpus luteum and ovarian follicle expressed LRH-1 and compare the level of expression to that seen for SF-1. To accomplish this goal, total RNA isolated from human ovarian follicles, corpora lutea, and ovarian cortex were used to detect transcript levels using semiquantitative RT-PCR (Fig. 1). At earlier cycles of RT-PCR, we were able to detect LRH-1 in corpus luteum but not SF-1. At 40 cycles of semiquantitative RT-PCR, we showed a lower level of SF-1 expression in human corpus luteum, compared with mature ovarian follicles. In contrast, there was a higher level of LRH-1 expression in human corpus luteum, compared with mature ovarian follicles.

View larger version (37K): [in this window] [in a new window]   FIG. 1. Expression of LRH-1 and SF-1 in human ovarian tissue. Semiquantitative RT-PCR was used to measure expression of LRH-1 and SF-1. Total RNAs were extracted from human mature ovarian follicle, corpus luteum, and ovarian cortex. PCR of G3PDH was used to standardize cDNA generated from the RT reaction. At 40 cycles, we demonstrated a lower level of SF-1 expression in human corpus luteum, compared with mature ovarian follicles. In contrast, LRH-1 expression in human corpus luteum is higher, compared with mature ovarian follicles.

View larger version (17K): [in this window] [in a new window]   FIG. 2. Quantification of LRH-1, SF-1, and HSD3B2 transcript levels in human ovarian tissues. Real-time RT-PCR was used to quantify the level of LRH-1, SF-1, and HSD3B2 mRNA in human ovarian cortex, ovarian follicle, corpora lutea, and HLGCs as described in Materials and Methods. Data represent independent RNA samples and are expressed as attomoles of mRNA per microgram of 18S rRNA. Note that the data are presented with a log scale. A, The amount of HSD3B2 mRNA expression highly correlated with the amount of LHR-1 mRNA expression in the corpora lutea and HLGCs. B, In contrast, there is no correlation between the amount of HSD3B2 mRNA and SF-1 mRNA.

To test the hypothesis that LRH-1 could play a role in progesterone biosynthesis by enhancing transcription of HSD3B2 gene, cultured GCT cells were cotransfected with HSD3B2 promoter construct alone and with expression vectors containing either LRH-1 or SF-1. Both LRH-1 (Fig. 3A) and SF-1 (Fig. 3B) were able to increase luciferase reporter driven by the HSD3B2 promoter in a concentration-dependent manner. Maximal stimulation of reporter activity was observed using 0.5 µg/well for both vectors. LRH-1 cotransfection, however, was more effective (>8-fold) in the induction of the HSD3B2 reporter construct than SF-1 (<4-fold) (Fig. 4).

View larger version (12K): [in this window] [in a new window]   FIG. 3. Concentration-dependent effects of LRH-1 or SF-1 on HSD3B2 reporter gene activity. GCT cells were transfected with luciferase reporter constructs containing the HSD3B2 promoter construct (0.5 µg/well). Cells were cotransfected with empty pcDNA3 expression vector or the indicated amounts of LRH-1 or SF-1 expression plasmid. After recovery for 24 h, cells were lysed and assayed for luciferase activity. Results represent the mean ± SE of pooled data from three to four experiments. A, 0.1 (P 0.05) and 0.5 (P 0.01) of LRH-1 were significantly different from vector alone. B, 0.1 (P 0.05), 0.3 (P 0.01), and 0.5 (P 0.01) of SF-1 were significantly different from vector alone.

View larger version (17K): [in this window] [in a new window]   FIG. 4. Effect of LRH-1 or SF-1 on HSD3B2 promoter activity. GCT cells were transfected with luciferase reporter constructs containing the HSD3B2 promoter construct (0.5 µg/well). Cells were cotransfected with empty pcDNA3 expression vector or the indicated amounts of LRH-1 (0.5 µg/well) or SF-1 (0.5 µg/well) expression plasmid. After recovery for 24 h, cells were lysed and assayed for luciferase activity. Results represent the mean ± SE of pooled data from three to four experiments. At optimal concentrations, LRH-1 has greater effect on HSD3B2 promoter than SF-1. Both LRH-1 and SF-1 were significantly different from vector alone (P 0.01). LRH-1 was significantly different from SF-1 (P 0.05).

View larger version (22K): [in this window] [in a new window]   FIG. 5. Effect of PKA agonist on LRH-1-stimulated HSD3B2 promoter activity. GCT cells were transfected with luciferase reporter constructs containing the HSD3B2 promoter construct (0.5 g/well). Cells were cotransfected with empty pcDNA expression vector or LRH-1 expression plasmid. After recovery for 22 h, cells were treated with 1 x low serum (1XLS) medium or dbcAMP (1 mM). After 6 h, cells were lysed and assayed for luciferase activity. Data were expressed as percentage of the basal. Results represent the mean ± SE of pooled data from three to four experiments. Treatment with dbcAMP was significantly different from vector alone (P 0.05). Treatment with dbcAMP plus LRH-1 was significantly different from LRH-1 alone (P 0.05).

Because DAX-1 can repress SF-1-mediated transcription, we investigated its effect on LRH-1. DAX-1 inhibited LRH-1 stimulated HSD3B2 in a dose-dependent fashion (Fig. 6). This inhibition was maintained in the presence of PKA pathway agonist (data not shown).

View larger version (22K): [in this window] [in a new window]   FIG. 6. Effect of DAX-1 on LRH-1-stimulated HSD3B2 promoter activity. GCT cells were transfected with luciferase reporter constructs containing the HSD3B2 promoter construct (0.5 µg/well). Cells were cotransfected with empty pcDNA3 expression vector, LRH-1 (0.5 µg/well), or LRH-1 plus increasing concentrations of DAX-1 expression plasmid (µg/well). After recovery for 24 h, cells were lysed and assayed for luciferase activity. Results represent the mean ± SE of pooled data from three to four experiments. Concentrations of DAX-1 greater than 0.03 µg/well were significantly different from LRH-1 alone (P 0.01).

Deletion analyses of HSD3B2 promoter were performed to determine the cis-elements used by LRH-1 to enhance its transcription (Fig. 7). Deletion analysis demonstrated a putative LRH-1 binding site between -963 and -515 and another between -345 and -210. Sequence analysis revealed two cis-elements similar to the nuclear receptor half-sites known to bind both LRH-1 and SF-1. Mutation of the first putative LRH-1 binding site from 5'-AAGGTTC-3' (-906/-900) to 5'-AgaaTTC-3' decreased LRH-1 stimulation by almost half, and mutation of the second putative LRH-1 binding site from 5'-AAGGACA-3' (-315/-309) to 5'-AtgGACA-3' completely abolished LRH-1 stimulation (Fig. 8). These inhibitions were maintained in the presence of PKA agonist (data not shown).

View larger version (12K): [in this window] [in a new window]   FIG. 7. Deletional analysis of the HSD3B2 5'-flanking DNA to determine LRH-1 sensitive regions. GCT cells were transiently transfected with luciferase reporter constructs containing serial deletions of HSD3B2 5'-flanking DNA. Transfection of reporter constructs was performed with either empty pcDNA3 expression vector (0.5 µg/well) or expression vector containing the coding sequence for LRH-1 (0.5 µg/well). After recovery for 24 h, cells were lysed, and luciferase activity was measured. Luc on the Y-axis corresponds to empty vector. Results are expressed as a percentage of the empty reporter vector and represent the mean ± SE of data from three to four independent experiments. -963 and -345 were significantly different from vector alone (P 0.05).

View larger version (18K): [in this window] [in a new window]   FIG. 8. Mutational analysis of the HSD3B2 5'-flanking DNA to determine LRH-1 sensitive regions. GCT cells were transiently transfected with luciferase reporter constructs containing mutations of the two putative LRH-1 binding sites in the HSD3B2 5'-flanking DNA. Transfection of reporter constructs was performed with either empty pcDNA3 expression vector (0.5 µg/well) or expression vector containing the coding sequence for LRH-1 (0.5 µg/well). After recovery for 24 h, cells were lysed, and luciferase activity was measured. Results are expressed as a percentage of the basal reporter activity of the -963 and represent the mean ± SE of data from three to four independent experiments. LRH-1 stimulation of mutations 1 and 2 and double mutation are significantly different from LRH-1 stimulation of wild type (P 0.01).

To confirm that LRH-1 could interact directly with the two putative response element sites, two synthetic oligonucleotides encompassing the two cis-elements were generated and used for EMSA (Fig. 9). IVP LRH-1, HLGC, and GCT cell NEs were prepared. LRH-1 bound to both sites and the protein/DNA complex was completely displaced by adding 100-fold molar excess of nonradiolabeled oligonucleotides (cold). In addition, when the two oligonucleotide probes were incubated with HLGC and GCT cell NEs, a similar protein/DNA complex was found. These were displaced by excess nonradiolabeled oligonucleotides. The second band seen with IVP LRH-1 and probe 2 may be a truncated variant of LHR-1. Radiolabeled mutated oligonucleotides did not bind to GCT NE (Fig. 9B).

View larger version (63K): [in this window] [in a new window]   FIG. 9. LRH-1 can bind to the two putative cis-elements in the HSD3B2 promoter in EMSA. The two labeled oligonucleotides (FP1 and FP2) were incubated with 5.0 µg IVP LRH-1, GCT cell NE (GCT NE), or HLGC nuclear extract (HLGC NE). A, Nonradiolabeled competitor DNA (cold) was added to a 100-fold molar excess to identify nonspecific protein/DNA interaction. B, Radiolabeled mutated DNAs (mutated probes 1 and 2) were used to identify nonspecific protein/DNA interaction. The resulting protein/DNA complexes (shown by arrows) were separated from FP by electrophoresis. Experiments were repeated twice.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The relatively high expression of LRH-1, compared with SF-1, in the human ovarian cortex, mature ovarian follicle, and corpus luteum extends a previous report demonstrating high abundance of LRH-1 transcripts in the human ovary by real-time PCR (31). It also agrees with previous studies demonstrating a high expression of LRH-1 in mouse and equine ovaries by Northern analysis (29, 30). Interestingly, in equine ovary, LRH-1 has higher expression in preovulatory follicle and corpus luteum, compared with SF-1. Furthermore, LRH-1 was much more highly expressed in granulosa cells than SF-1 (30).

In macaques undergoing controlled ovarian stimulation, HSD3B2 mRNA increased within 12 h of HCG administration (42). In human ovarian cycle, LH surge is essential for ovulation and subsequent conversion of mature ovarian follicle to corpus luteum. The expression of SF-1 was found to be down-regulated in ovarian cells after LH surge (30, 33, 34, 35), which raises the question regarding the role of SF-1 in corpus luteum steroidogenesis. LRH-1 enhances transcription of HSD3B2 in a dose-dependent manner and to a greater degree than SF-1 in human GCT cells, which is consistent with a previous report using human embryonic kidney-293 cells (31). Furthermore, using real-time PCR, we demonstrated a high correlation between the expression of HSD3B2 and LRH-1. This correlation was absent for SF-1. These findings raise the possibility that LRH-1 rather SF-1 could play an essential role in the transition of ovarian steroidogenesis from estrogen to progesterone.

Previous study suggests that stimulation of PKA pathway resulted in increased expression of HSD3B2 in ovarian thecal cells (43). Even though dbcAMP increased HSD3B2 transcription almost 3-fold, the stimulation of HSD3B2 by LRH-1 was weakly augmented in the presence of dbcAMP. This positive response of PKA pathway agonist on LRH-1 stimulated HSD3B2 expression raises the possibility that the regulation of HSD3B2 transcription in ovarian granulosa cells may involve the PKA pathway.

DAX-1 colocalizes with SF-1 during mouse development (24) and inhibits SF-1-mediated transactivation of target genes. HSD3B2 promoter was shown previously to be inhibited by DAX-1 in the presence or absence of PKA agonist (44). In the present study, DAX-1 inhibited LRH-1 stimulated HSD3B2 in a dose-dependent fashion. This inhibition may be mediated through direct interaction of LRH-1 with DAX-1 (45).

Deletion, mutation, and EMSA analyses of the HSD3B2 promoter allowed the identification of two putative LRH-1 binding sites that appear to be important regulators for HSD3B2 transcription. These two sites are different than a previously described SF-1 regulatory element in the HSD3B2 promoter after study in an adrenal cell model (22). This difference may be due to the possibility that both SF-1 and LRH-1 have slightly different DNA binding specificities or to differences in the adrenal vs. ovarian cell models. There might also be tissue-specific differences in the orphan nuclear receptor expression and their mechanisms of control and cis-elements involved in the regulation of HSD3B2 gene expression. In addition, the deletion analysis suggests that there could be one or more repressor elements between -585 to -345 in the HSD3B2 promoter. The complete regulation of HSD3B2 promoter will need to be further elucidated.

In conclusion, our findings suggest that LRH-1 is highly expressed in corpus luteum and plays an essential role in the regulation of HSD3B2. Furthermore, we believe that LRH-1 could be the major transcriptional factor responsible for the shift in human ovarian steroidogenesis from estrogen predominant to progesterone predominant milieu.

	Footnotes

 
Abbreviations: DAX-1, Dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1; dbcAMP, dibutyryl cAMP; DME/F12, DMEM and Ham’s F-12 medium; FP, free probe; GCT, granulosa cell tumor; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; HLGC, human luteinized granulosa cell; HSD3B2, 3ß-hydroxysteroid dehydrogenase type II; IVP, in vitro protein; LRH-1, liver receptor homologue-1; NE, nuclear extract; PKA, protein kinase A; RT, reverse transcription; SF-1, steroidogenic factor-1.

Received May 20, 2003.

Accepted August 26, 2003.

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