Liver Receptor Homolog-1 Protein Research Articles

NF-κB Regulation of LRH-1 and ABCG5/8 Potentiates Phytosterol Role in the Pathogenesis of Parenteral Nutrition-Associated Cholestasis.

Chronically administered parenteral nutrition (PN) in patients with intestinal failure carries the risk for developing PN-associated cholestasis (PNAC). We have demonstrated that farnesoid X receptor (FXR) and liver X receptor (LXR), proinflammatory interleukin-1 beta (IL-1β), and infused phytosterols are important in murine PNAC pathogenesis. In this study we examined the role of nuclear receptor liver receptor homolog 1 (LRH-1) and phytosterols in PNAC. In a C57BL/6 PNAC mouse model (dextran sulfate sodium [DSS] pretreatment followed by 14 days of PN; DSS-PN), hepatic nuclear receptor subfamily 5, group A, member 2/LRH-1 mRNA, LRH-1 protein expression, and binding of LRH-1 at the Abcg5/8 and Cyp7a1 promoter was reduced. Interleukin-1 receptor-deficient mice (Il-1r-/- /DSS-PN) were protected from PNAC and had significantly increased hepatic mRNA and protein expression of LRH-1. NF-κB activation and binding to the LRH-1 promoter were increased in DSS-PN PNAC mice and normalized in Il-1r-/- /DSS-PN mice. Knockdown of NF-κB in IL-1β-exposed HepG2 cells increased expression of LRH-1 and ABCG5. Treatment of HepG2 cells and primary mouse hepatocytes with an LRH-1 inverse agonist, ML179, significantly reduced mRNA expression of FXR targets ATP binding cassette subfamily C member 2/multidrug resistance associated protein 2 (ABCC2/MRP2), nuclear receptor subfamily 0, groupB, member 2/small heterodimer partner (NR0B2/SHP), and ATP binding cassette subfamily B member 11/bile salt export pump (ABCB11/BSEP). Co-incubation with phytosterols further reduced expression of these genes. Similar results were obtained by suppressing the LRH-1 targets ABCG5/8 by treatment with small interfering RNA, IL-1β, or LXR antagonist GSK2033. Liquid chromatography-mass spectrometry and chromatin immunoprecipitation experiments in HepG2 cells showed that ATP binding cassette subfamily G member 5/8 (ABCG5/8) suppression by GSK2033 increased the accumulation of phytosterols and reduced binding of FXR to the SHP promoter. Finally, treatment with LRH-1 agonist, dilauroyl phosphatidylcholine (DLPC) protected DSS-PN mice from PNAC. This study suggests that NF-κB regulation of LRH-1 and downstream genes may affect phytosterol-mediated antagonism of FXR signaling in the pathogenesis of PNAC. LRH-1 could be a potential therapeutic target for PNAC.

LRH1 Acts as an Oncogenic Driver in Human Osteosarcoma and Pan-Cancer.

Osteosarcoma (OS) that mainly occurs during childhood and adolescence is a devastating disease with poor prognosis presented by extreme metastases. Recent studies have revealed that liver receptor homolog 1 (LRH-1) plays a vital role in the metastasis of several human cancers, but its role is unknown in the metastasis of OS. In this study, Gene Ontology (GO) enrichment analyses based on high-throughput RNA-seq data revealed that LRH-1 acted a pivotal part in the positive regulation of cell migration, motility, and angiogenesis. Consistently, LRH-1 knockdown inhibited the migration of human OS cells, which was concurrent with the downregulation of mesenchymal markers and the upregulation of epithelial markers. In addition, short hairpin RNAs (shRNAs) targeting LRH-1 inactivated transforming growth factor beta (TGF-β) signaling pathway. LRH-1 knockdown inhibited human umbilical vein endothelial cell (HUVEC) proliferation, migration, and tube formation. Vascular endothelial growth factor A (VEGFA) expression was also downregulated after LRH-1 knockdown. Immunohistochemistry (IHC) revealed that the expression of LRH-1 protein was significantly higher in tumor tissues than in normal bone tissues. We found that high LRH-1 expression was associated with poor differentiation and advanced TNM stage in OS patients using IHC. Based on The Cancer Genome Atlas (TCGA) database, high LRH-1 expression predicts poor survival in lung squamous cell carcinoma (LUSC), kidney renal papillary cell carcinoma (KIRP), and pancreatic adenocarcinoma (PAAD). The downregulation of LRH-1 significantly hindered the migration and motility of LUSC cells. Using multi-omic bioinformatics, the positive correlation between LRH-1- and EMT-related genes was found across these three cancer types. GO analysis indicated that LRH-1 played a vital role in “blood vessel morphogenesis” or “vasculogenesis” in KIRP. Our results indicated that LRH-1 plays a tumor-promoting role in human OS, could predict the early metastatic potential, and may serve as a potential target for cancer therapy.

Regulation of liver receptor homologue-1 by DDB2 E3 ligase activity is critical for hepatic glucose metabolism

Liver receptor homologue-1 (LRH-1) plays a critical role in hepatic metabolism and disease. Here we show that LRH-1 protein stability is regulated by the cullin 4 (CUL4) E3 ubiquitin ligase complex. We found that DNA damage-binding protein 2 (DDB2) directly interacts with LRH-1 and functions as a substrate recognition component of CUL4-DDB1 to promote LRH-1 ubiquitination and proteasomal degradation. In human hepatoma (HepG2) cells, we observed that protein levels of endogenous LRH-1 are increased by insulin without a change in mRNA levels of LRH-1. However, overexpression of DDB2 impaired the insulin-stimulated increase in LRH-1 levels. In addition, DDB2 overexpression decreased LRH-1 transcriptional activation and expression of target genes, such as glucokinase, whereas knockdown of DDB2 increased the expression of glucokinase. Finally, we demonstrated that DDB2 knockdown increases glucose uptake and intracellular levels of glucose-6-phosphate in HepG2 cells. Our study reveals a novel regulatory mechanism of LRH-1 activity and suggests a role for DDB2 in hepatic glucose metabolism.

Expression of Liver Receptor Homolog-1 (LRH-1) in Villi and Decidua of Patients with Unexplained Recurrent Spontaneous Abortion.

BackgroundIn view of the important function of nuclear receptor liver receptor homolog 1 (LRH 1) in various biological processes and the physiological changes accompanying unexplained recurrent spontaneous abortion (USRA), our study was carried out to investigate the potential roles of LRH-1 in USRA.Material/MethodsThirty patients with URSA at early the early state of pregnancy were selected, and 30 patients with normal early pregnancy were also selected from Aug 2015 to Sep 2016 as a control group. The expression of LRH-1 protein in decidua and villi were detected by immunohistochemistry and Western blot analysis, and the expression of LRH-1 mRNA was detected by RT-PCR. The expression levels of CYP19 and P450scc were detected by RT-PCR and Western blot analysis at mRNA and protein levels, respectively.ResultsThe levels of LRH-1, CYP19, and P450scc mRNA and protein in villi of the patients in the URSA group were significantly lower than in the control group. There were no significant differences between the URSA group and control group in the levels of LRH-1, CYP19, and P450scc mRNA and protein in villi in decidua.ConclusionsURSA was related to the reduced expression level of LRH-1 in villous tissues but not in decidua, and expression of LRH-1 may be related to the expression of CYP19 and P450scc. We believe that the expression level of LRH-1 can be used as a marker in the early diagnosis of URSA, and the regulation of LRH-1 expression many lead to new URSA treatments.

A calreticulin-dependent nuclear export signal is involved in the regulation of liver receptor homologue-1 protein folding

As an orphan member of the nuclear receptor family, liver receptor homologue-1 (LRH-1) controls a tremendous range of transcriptional programmes that are essential for metabolism and hormone synthesis. Our previous studies have shown that nuclear localization of the LRH-1 protein is mediated by two nuclear localization signals (NLSs) that are karyopherin/importin-dependent. It is unclear whether LRH-1 can be actively exported from the nucleus to the cytoplasm. In the present study, we describe a nuclear export domain containing two leucine-rich motifs [named nuclear export signal (NES)1 and NES2] within the ligand-binding domain (LBD). Mutation of leucine residues in NES1 or NES2 abolished nuclear export, indicating that both NES1 and NES2 motifs are essential for full nuclear export activity. This NES-mediated nuclear export was insensitive to the chromosomal region maintenance 1 (CRM1) inhibitor leptomycin B (LMB) or to CRM1 knockdown. However, knockdown of calreticulin (CRT) prevented NES-mediated nuclear export. Furthermore, our data show that CRT interacts with LRH-1 and is involved in the nuclear export of LRH-1. With full-length LRH-1, mutation of NES1 led to perinuclear accumulation of the mutant protein. Immunofluorescence analysis showed that these perinuclear aggregates were co-localized with the centrosome marker, microtubule-associated protein 1 light chain 3 (LC3), ubiquitin and heat shock protein 70 (Hsp70), indicating that the mutant was misfolded and sequestered into aggresome-like structures via the autophagic clearance pathway. Our study demonstrates for the first time that LRH-1 has a CRT-dependent NES which is not only required for cytoplasmic trafficking, but also essential for correct protein folding to avoid misfolding-induced aggregation.

Oestradiol reduces Liver Receptor Homolog-1 mRNA transcript stability in breast cancer cell lines

The expression of orphan nuclear receptor Liver Receptor Homolog-1 (LRH-1) is elevated in breast cancer and promotes proliferation, migration and invasion in vitro. LRH-1 expression is regulated by oestrogen (E2), with LRH-1 mRNA transcript levels higher in oestrogen receptor α (ERα) positive (ER+) breast cancer cells compared to ER− cells. However, the presence of LRH-1 protein in ER− cells suggests discordance between mRNA transcript levels and protein expression. To understand this, we investigated the impact of mRNA and protein stability in determining LRH-1 protein levels in breast cancer cells.LRH-1 transcript levels were significantly higher in ER+ versus ER− breast cancer cells lines; however LRH-1 protein was expressed at similar levels. We found LRH-1 mRNA and protein was more stable in ER− compared to ER+ cell lines. The tumor-specific LRH-1 variant isoform, LRH-1v4, which is highly responsive to E2, showed increased mRNA stability in ER− versus ER+ cells. In addition, in MCF-7 and T47-D cell lines, LRH-1 total mRNA stability was reduced with E2 treatment, this effect mediated by ERα.Our data demonstrates that in ER− cells, increased mRNA and protein stability contribute to the abundant protein expression levels. Expression and immunolocalisation of LRH-1 in ER− cells as well as ER− tumors suggests a possible role in the development of ER− tumors. The modulation of LRH-1 bioactivity may therefore be beneficial as a treatment option in both ER− and ER+ breast cancer.

Reversible infertility in a liver receptor homologue-1 (LRH-1)-knockdown mouse model

Liver receptor homologue-1 (LRH-1) is an orphan nuclear receptor that has been implicated in steroid hormone biosynthesis and fertility. Herein we describe a transgenic inducible short hairpin (sh) RNA mouse model that was used to study the effect of transient LRH-1 knockdown in vivo. Induction of expression of the shRNA directed against LRH-1 for 2-6 weeks resulted in 80% knockdown of LRH-1 protein in the ovary and complete infertility. Gonadotropin hyperstimulation could not rescue the observed defects in ovulation and corpus luteum formation in LRH-1-knockdown mice. The infertility phenotype was fully reversible because LRH-1-knockdown females became pregnant and delivered normal size litters and healthy pups after cessation of LRH-1 shRNA expression. Timed ovarian microarray analysis showed that, in line with the observed decrease in plasma progesterone levels, key steroid biosynthesis genes, namely Star, Cyp11a1, Hsd3b and Scarb1, were downregulated in LRH-1-knockdown ovaries. In contrast with what has been described previously, no clear effect was observed on oestrogenic activity in LRH-1-knockdown mice. Only Sult1e1 and, surprisingly, Hsd17b7 expression was modulated with potentially opposite effects on oestradiol bioavailability. In conclusion, the fully reversible infertility phenotype of LRH-1-knockdown mice shows the feasibility of an LRH-1 antagonist as new contraceptive therapy with a mechanism of action that most prominently affects cholesterol availability and progesterone production.

Chronic psychosocial stress increases the risk for inflammation-related colon carcinogenesis in male mice

Patients with inflammatory bowel diseases (IBDs) have a higher risk of developing colorectal cancer (CRC) than the general population. Furthermore, chronic psychosocial stress increases the likelihood of developing IBD and multiple types of malignant neoplasms, including CRC. Here, for the first time, we investigate the effects of chronic psychosocial stress in male mice on an artificially induced CRC, by employing the chronic subordinate colony (CSC) housing paradigm in combination with the reliable azoxymethane (AOM)/dextran sodium sulfate (DSS) CRC model. Colonoscopy revealed that CSC mice showed accelerated macroscopic suspect lesions. In addition, more CSC mice developed low-grade dysplasia (LGD) and/or high-grade dysplasia (HGD) in the colonic tissue compared to the single-housed control mice (SHC). CSC mice showed an increased number of Ki67+ and a decreased number of terminal deoxynucleotidyl transferase dUTP nick end labeling epithelial cells in colonic tissue. Colonic liver receptor homolog-1 (LRH-1), cyclooxygenase II (COXII), tumor necrosis factor, forkhead box P3 (FoxP3) mRNA as well as colonic ß-catenin, COXII, and LRH-1 protein expression were also increased in CSC compared with SHC mice. Although the number of CD4+ Th cells was increased, a tendency toward a decreased colonic interferon-γ (IFN-γ) mRNA expression was observed. Furthermore, despite an increased percentage of CD3+ cells and CD3+/FoxP3+ double-positive cells within mesenteric lymph node cells of CSC mice, IFN-γ secretion from these cells was unaffected. Altogether, our results suggest that chronic psychosocial stress increases the risk for AOM/DSS-induced and, thus, inflammation-related CRC. Finally, assessment of additional time points may test whether the shift from tumor-protective Th1 cell to regulatory T-cell immunity represents a consequence of increased carcinogenesis or a causal factor involved in its development.

Nuclear receptor liver receptor homologue 1 (LRH-1) regulates pancreatic cancer cell growth and proliferation

An essential regulator of gene transcription, nuclear receptor liver receptor homologue 1 (LRH-1) controls cell differentiation in the developing pancreas and maintains cholesterol homeostasis in adults. Recent genome-wide association studies linked mutations in the LRH-1 gene and its up-stream regulatory regions to development of pancreatic cancer. In this work, we show that LRH-1 transcription is activated up to 30-fold in human pancreatic cancer cells compared to normal pancreatic ductal epithelium. This activation correlates with markedly increased LRH-1 protein expression in human pancreatic ductal adenocarcinomas in vivo. Selective blocking of LRH-1 by receptor specific siRNA significantly inhibits pancreatic cancer cell proliferation in vitro. The inhibition is tracked in part to the attenuation of the receptor's transcriptional targets controlling cell growth, proliferation, and differentiation. Previously, LRH-1 was shown to contribute to formation of intestinal tumors. This study demonstrates the critical involvement of LRH-1 in development and progression of pancreatic cancer, suggesting the LRH-1 receptor as a plausible therapeutic target for treatment of pancreatic ductal adenocarcinomas.

Abstract 4943: Regulation of human Cripto-1 gene expression by nuclear receptors and DNA promoter methylation

Abstract Human Cripto-1 (CR-1) is a membrane bound protein that belongs to the epidermal growth factor (EGF)-CFC family. CR-1 plays an important role in regulating embryonic development while also having significant implications in various forms of carcinogenesis. Previous studies have shown that germ cell nuclear factor (GCNF), a member of the nuclear receptor superfamily of transcription factors, inhibits CR-1 expression in human embryonal carcinoma NTERA-2 cells following retinoic acid-induced differentiation due to direct binding to a DR0 element within the CR-1 promoter. In the present study, we investigated the effects on CR-1 gene expression of another member of the nuclear receptor superfamily, liver receptor homologue-1 (LRH-1), which can bind to the same GCNF-binding site within the promoter of several genes. NTERA-2 cells transfected with a CR-1 promoter luciferase reporter vector showed substantially increased CR-1 promoter activity in the presence of LRH-1 and decreased CR-1 promoter activity in the presence of GCNF. Retinoic acid treatment of NTERA-2 cells induced downregulation of CR-1 and LRH-1 mRNA and protein expression. In contrast, a strong increase in GCNF was detected in NTERA-2 cells that have been treated with retinoic acid. Additionally, chromatin immunoprecipitation assay displayed direct binding of LRH-1 to the CR-1 promoter in NTERA-2 cells. Real-time PCR and Western blot analysis of NTERA-2 cells transfected with LRH-1 siRNA revealed a significant decrease in CR-1 mRNA and protein expression, suggesting that LRH-1 is required for mantaining high levels of CR-1 expression in embryonal carcinoma cells. Real time PCR analysis for GCNF and LRH-1 in human embryonal carcinoma cells and in a panel of human breast cancer cell lines, expressing very low levels of CR-1, revealed that GCNF is highly expressed in MCF7, T47D and ZR75-1 breast cancer cells as compared to NTERA-2 and NCCIT embryonal carcinoma cells. Since one of the mechanisms used by GCNF to repress gene expression is through promoter DNA methylation, we investigated the role of epigenetic modification of the CR-1 promoter in embryonal and breast carcinoma cells. While NTERA-2 and NCCIT cells showed very low levels of CR-1 promoter methylation, in human breast cancer cells CR-1 promoter was highly methylated in agreement with low levels of CR-1 expression in these cell lines. Finally, treatment of various breast cancer cell lines with the demethylating agent 5-aza-2′-deoxycytidine alone or in combination with the histone deacetylase inhibitors trichostatin A or valproic acid induced a significant increase in CR-1 expression, thus overcoming the repressive effects of GCNF. Collectively, these findings offer promising insight into the transcriptional regulation of CR-1 gene expression and its critical role in the pathogenesis of human cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4943.

Liver Receptor Homolog-1 Regulates the Transcription of Steroidogenic Enzymes and Induces the Differentiation of Mesenchymal Stem Cells into Steroidogenic Cells

Steroidogenic factor-1 (SF-1, also known as Ad4BP) has been demonstrated to be a primary transcriptional regulator of steroidogenic-related genes. However, mRNA for liver receptor homolog-1 (LRH-1), which together with SF-1, belongs to the NR5A nuclear receptor family, is expressed at much higher levels than SF-1 mRNA in the human gonad. In our previous studies, we demonstrated that SF-1 induced the differentiation of bone marrow-derived mesenchymal stem cells (MSCs) into steroidogenic cells such as Leydig or adrenocortical cells. The introduction of LRH-1 into human MSCs (hMSCs) with the aid of cAMP also induced the expression of steroidogenic enzymes, including CYP17, and their differentiation into steroid hormone-producing cells. Promoter analysis, EMSA, and chromatin immunoprecipitation assay using LRH-1-transduced hMSCs indicated that three LRH-1 binding sites were responsible for CYP17 transactivation. Immunohistochemical studies showed that LRH-1 protein was expressed in human Leydig cells. The CYP17 promoter region was highly methylated in hMSCs, whereas it was demethylated by the introduction of LRH-1 and cAMP treatment. These results indicate that LRH-1 could represent another key regulator of the steroidogenic lineage in MSCs and play a vital role in steroid hormone production in human Leydig cells.

A Key Role for Orphan Nuclear Receptor Liver Receptor Homologue-1 in Activation of Fatty Acid Synthase Promoter by Liver X Receptor

Liver X receptor (LXR) activates fatty acid synthase (FAS) gene expression through binding to a DR-4 element in the promoter. We show that a distinct nuclear receptor half-site 21 bases downstream of the DR-4 element is also critical for the response of FAS to LXR but is not involved in LXR binding to DNA. This half-site specifically binds liver receptor homologue-1 (LRH-1) in vitro and in vivo, and we show LRH-1 is required for maximal LXR responsiveness of the endogenous FAS gene as well as from promoter reporter constructs. We also demonstrate that LRH-1 stimulation of the FAS LXR response is blocked by the addition of small heterodimer partner (SHP) and that FAS mRNA is overexpressed in SHP knock-out animals, providing evidence that FAS is an in vivo target of SHP repression. Taken together, these findings identify the first direct lipogenic gene target of LRH-1/SHP repression and provide a mechanistic explanation for bile acid repression of FAS and lipogenesis recently reported by others.

The nuclear receptor liver receptor homolog-1 is an estrogen receptor target gene.

Liver receptor homolog-1 (LRH-1) is a nuclear receptor previously known to have distinct functions during mouse development and essential roles in cholesterol homeostasis. Recently, a new role for LRH-1 has been discovered in tumor progression, giving LRH-1 potential transforming functions. In order to identify critical factors stimulating LRH-1 expression leading to deregulated cellular proliferation, we studied its expression and its regulation in several breast cancer cell lines. We observed that LRH-1 expression was increased in estrogen receptor (ER) alpha expressing cell lines, whereas weak-to-no expression was found in nonexpressing ERalpha cell lines. In MCF7, LRH-1 expression was highly induced after treatment with 17beta-estradiol (E2). This transcriptional regulation was the result of a direct binding of the ER to the LRH-1 promoter, as demonstrated by gelshift and chromatin immunoprecipitation assays. Interestingly, siRNA-mediated inactivation of LRH-1 decreased the E2-dependent proliferation of MCF7 cells. Finally, LRH-1 protein expression was detected by immunohistochemistry in tumor cells of human mammary ductal carcinomas. Altogether, these data demonstrate that LRH-1 is transcriptionally regulated by the ER alpha and reinforce the hypothesis that LRH-1 could exert potential oncogenic effects during breast cancer formation.

Liver Receptor Homologue-1 Mediates Species- and Cell Line-specific Bile Acid-dependent Negative Feedback Regulation of the Apical Sodium-dependent Bile Acid Transporter

Intestinal reclamation of bile salts is mediated in large part by the apical sodium-dependent bile acid transporter (ASBT). The bile acid responsiveness of ASBT is controversial. Bile acid feeding in mice results in decreased expression of ASBT protein and mRNA. Mouse but not rat ASBT promoter activity was repressed in Caco-2, but not IEC-6, cells by chenodeoxycholic acid. A potential liver receptor homologue-1 (LRH-1) cis-acting element was identified in the bile acid-responsive region of the mouse but not rat promoter. The mouse, but not rat, promoter was activated by LRH-1, and this correlated with nuclear protein binding to the mouse but not rat LRH-1 element. The short heterodimer partner diminished the activity of the mouse promoter and could partially offset its activation by LRH-1. Interconversion of the potential LRH-1 cis-elements between the mouse and rat ASBT promoters was associated with an interconversion of LRH-1 and bile acid responsiveness. LRH-1 protein was found in Caco-2 cells and mouse ileum, but not IEC-6 cells or rat ileum. Bile acid response was mediated by the farnesoid X receptor, as shown by the fact that overexpression of a dominant-negative farnesoid X-receptor eliminated the bile acid mediated down-regulation of ASBT. In addition, ASBT expression in farnesoid X receptor null mice was unresponsive to bile acid feeding. In summary cell line- and species-specific negative feedback regulation of ASBT by bile acids is mediated by farnesoid X receptor via small heterodimer partner-dependent repression of LRH-1 activation of the ASBT promoter.

Expression and functional analysis of liver receptor homologue 1 as a potential steroidogenic factor in rat ovary.

Liver receptor homologue 1 (LRH-1) is a member of the nuclear receptor superfamily originally found in liver cells. LRH-1 participates in regulation of cholesterol metabolism and bile acid synthesis. Recent studies have shown that LRH-1 is even more highly expressed in the ovary, and LRH-1 has been implicated as a key transcriptional regulator of cytochrome P450 aromatase (P450arom) in vitro. In the present study, we investigated the spatiotemporal expression patterns of LRH-1 using in situ hybridization and immunohistochemistry in ovaries from rats with a 4-day estrous cycle, from pregnant rats, from immature rats treated with eCG to stimulate follicular development, and from eCG-treated rats that were subsequently given hCG to stimulate ovulation and luteinization. To establish a potential connection between the expression of LRH-1 and that of the steroidogenic genes in vivo, we directly compared the localization patterns of LRH-1 and P450arom transcripts in consecutive ovarian sections from these animals. LRH-1 mRNA and protein were primarily localized to granulosa cells and luteinized follicles or newly formed corpora lutea (CLs) of immature and adult rats, and the levels of expression increased during eCG-hCG-induced follicular development and ovulation. In the functional CLs of pregnant rats, a biphasic change in LRH-1 mRNA content occurred throughout the gestation process, whereas LRH-1 protein was persistently detected during the entire pregnancy. In the consecutive ovarian sections, expression of LRH-1 was approximately colocalized with that of P450arom in both tertiary and Graafian follicles and the functional CLs of pregnant rats. LRH-1 mRNA and protein expression preceded those of P450arom during early follicular development. Stage-specific expression of LRH-1 in rat granulosa and luteal cells suggests a role for LRH-1 in the regulation of ovarian function. The overlapping but distinct expression patterns of LRH-1 and P450arom circumstantially support the recent finding that LRH-1 serves as a critical upstream regulator of P450arom gene expression in ovarian cells, but LRH-1 also may be a multifunctional steroidogenic factor in ovarian physiology.