Single-Cell RNA-Seq Identifies Pathways and Genes Contributing to the Hyperandrogenemia Associated with Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder characterized by hyperandrogenemia of ovarian thecal cell origin, resulting in anovulation/oligo-ovulation and infertility. Our previous studies established that ovarian theca cells isolated and propagated from ovaries of normal ovulatory women and women with PCOS have distinctive molecular and cellular signatures that underlie the increased androgen biosynthesis in PCOS. To evaluate differences between gene expression in single-cells from passaged cultures of theca cells from ovaries of normal ovulatory women and women with PCOS, we performed single-cell RNA sequencing (scRNA-seq). Results from these studies revealed differentially expressed pathways and genes involved in the acquisition of cholesterol, the precursor of steroid hormones, and steroidogenesis. Bulk RNA-seq and microarray studies confirmed the theca cell differential gene expression profiles. The expression profiles appear to be directed largely by increased levels or activity of the transcription factors SREBF1, which regulates genes involved in cholesterol acquisition (LDLR, LIPA, NPC1, CYP11A1, FDX1, and FDXR), and GATA6, which regulates expression of genes encoding steroidogenic enzymes (CYP17A1) in concert with other differentially expressed transcription factors (SP1, NR5A2). This study provides insights into the molecular mechanisms underlying the hyperandrogenemia associated with PCOS and highlights potential targets for molecular diagnosis and therapeutic intervention.

Human DENND1A.V2 Drives Cyp17a1 Expression and Androgen Production in Mouse Ovaries and Adrenals.

The DENND1A locus is associated with polycystic ovary syndrome (PCOS), a disorder characterized by androgen excess. Theca cells from ovaries of PCOS women have elevated levels of a DENND1A splice variant (DENND1A.V2). Forced expression of this variant in normal theca cells increases androgen biosynthesis and CYP17A1 expression, whereas knockdown of the transcript in PCOS theca cells reduced androgen production and CYP17A1 mRNA. We attempted to create a murine model of PCOS by expressing hDENND1A.V2 using standard transgenic approaches. There is no DENND1A.V2 protein equivalent in mice, and the murine Dennd1a gene is essential for viability since Dennd1a knockout mice are embryonically lethal, suggesting that Dennd1a is developmentally critical. Three different hDENND1A.V2 transgenic mice lines were created using CMV, Lhcgr, and TetOn promoters. The hDENND1A.V2 mice expressed hDENND1A.V2 transcripts. While hDENND1A.V2 protein was not detectable by Western blot analyses, appropriate hDENND1A.V2 immunohistochemical staining was observed. Corresponding Cyp17a1 mRNA levels were elevated in ovaries and adrenals of CMV transgenic mice, as were plasma steroid production by theca interstitial cells isolated from transgenic ovaries. Even though the impact of robust hDENND1A.V2 expression could not be characterized, our findings are consistent with the notion that elevated hDENND1A.V2 has a role in the hyperandrogenemia of PCOS.

Overexpression of a DENND1A isoform produces a polycystic ovary syndrome theca phenotype.

Polycystic ovary syndrome (PCOS), characterized by increased ovarian androgen biosynthesis, anovulation, and infertility, affects 5-7% of reproductive-age women. Genome-wide association studies identified PCOS candidate loci that were replicated in subsequent reports, including DENND1A, which encodes a protein associated with clathrin-coated pits where cell-surface receptors reside. However, these studies provided no information about functional roles for DENND1A in the pathogenesis of PCOS. DENND1A protein was located in the cytoplasm as well as nuclei of theca cells, suggesting a possible role in gene regulation. DENND1A immunostaining was more intense in the theca of PCOS ovaries. Using theca cells isolated and propagated from normal cycling and PCOS women, we found that DENND1A variant 2 (DENND1A.V2) protein and mRNA levels are increased in PCOS theca cells. Exosomal DENND1A.V2 RNA was significantly elevated in urine from PCOS women compared with normal cycling women. Forced overexpression of DENND1A.V2 in normal theca cells resulted in a PCOS phenotype of augmented CYP17A1 and CYP11A1 gene transcription, mRNA abundance, and androgen biosynthesis. Knock-down of DENND1A.V2 in PCOS theca cells reduced androgen biosynthesis and CYP17A1 and CYP11A1 gene transcription. An IgG specific to DENND1A.V2 also reduced androgen biosynthesis and CYP17 and CYP11A1 mRNA when added to the medium of cultured PCOS theca cells. We conclude that the PCOS candidate gene, DENND1A, plays a key role in the hyperandrogenemia associated with PCOS. These observations have both diagnostic and therapeutic implications for this common disorder.

Loci on chromosome 12q13.2 encompassing ERBB3, PA2G4 and RAB5B are associated with polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is characterized by hyperandrogenemia of ovarian theca cell origin. We report significant association of androgen production with 15 single nucleotide variants (SNVs) identified by exome sequencing of theca cells from women with PCOS and normal ovulatory women. Ten SNVs are located within a 150 kbp region on 12q13.2 which encompasses loci identified in PCOS genome-wide association studies (GWAS) and contains PCOS candidate genes ERBB3 and RAB5B. The region also contains PA2G4 which encodes a transcriptional corepressor of androgen receptor and androgen receptor-regulated genes. PA2G4 has not previously been recognized as related to PCOS in published GWAS studies. Two of the SNVs are predicted to have functional consequences (ERBB3 missense SNV, PA2G4 promoter SNV). PA2G4 interacts with the ERBB3 cytoplasmic domain containing the missense variant, suggesting a potential signaling pathway disruption that could lead to the PCOS ovarian phenotype. Single cell RNA sequencing of theca cells showed significantly less expression of PA2G4 after forskolin treatment in PCOS cells compared to normal cells (padj = 3.82E-30) and in cells heterozygous for the PA2G4 promoter SNV compared to those without the SNV (padj = 2.16E-11). This is consistent with a functional effect of the PA2G4 promoter SNV. No individual SNV was significantly associated with PCOS in an independent family cohort, but a haplotype with minor alleles of three SNVs was found preferentially in women with PCOS. These findings suggest a functional role for 12q13.2 variants in PCOS and implicate variants in ERBB3 and PA2G4 in the pathophysiology of PCOS.

The PCOS GWAS Candidate Gene ZNF217 Influences Theca Cell Expression of DENND1A.V2, CYP17A1, and Androgen Production.

Polycystic ovary syndrome (PCOS), a common endocrine disorder of women, is characterized by increased ovarian androgen production and anovulatory infertility. Genome-wide association studies (GWAS) have identified more than 20 PCOS candidate loci. One GWAS candidate locus encompasses ZNF217, a zinc finger transcription factor. Immunohistochemical staining of ovarian tissue demonstrated significantly lower staining intensity for ZNF217 protein in PCOS theca interna compared to ovarian tissue from normal ovulatory women. Immunofluorescence staining of normal and PCOS theca cells demonstrated nuclear localization of ZNF217, with lower intensity in PCOS cells. Western blotting showed reduced ZNF217 protein in PCOS theca cells compared to normal theca cells, and that treatment with forskolin, which mimics the action of luteinizing hormone (LH), reduces ZNF217 expression. Lower ZNF217 expression in PCOS theca cells was confirmed by quantitative reverse transcription polymerase chain reaction. Notably, there was an inverse relationship between ZNF217 messenger RNA (mRNA) levels and theca cell androgen (dehydroepiandrosterone; DHEA) synthesis. The abundance of mRNA encoding a splice variant of DENND1A (DENND1A.V2), a PCOS candidate gene that positively regulates androgen biosynthesis, was also inversely related to ZNF217 mRNA levels. This relationship may be driven by increased miR-130b-3p, which targets DENND1A.V2 transcripts and is directly correlated with ZNF217 expression. Forced expression of ZNF217 in PCOS theca cells reduced androgen production, CYP17A1 and DENND1A.V2 mRNA, while increasing mIR-130b-3p. Conversely, knockdown of ZNF217 in normal theca cells with short hairpin RNA-expressing lentivirus particles increased DENND1A.V2 and CYP17A1 mRNA. These observations suggest that ZNF217 is part of a network of PCOS candidate genes regulating thecal cell androgen production involving DENND1A.V2 and miR-130b-3p.

PDE8A genetic variation, polycystic ovary syndrome and androgen levels in women.

Polycystic ovary syndrome (PCOS) is characterized by excessive theca cell androgen secretion, dependent upon LH, which acts through the intermediacy of 3',5'-cyclic adenosine monophosphate (cAMP). cAMP signaling pathways are controlled through regulation of its synthesis by adenylyl cyclases, and cAMP degradation by phosphodiesterases (PDEs). PDE8A, a high-affinity cAMP-specific PDE is expressed in the ovary and testis. Leydig cells from mice with a targeted mutation in the Pde8a gene are sensitized to the action of LH in terms of testosterone production. These observations led us to evaluate the human PDE8A gene as a PCOS candidate gene, and the hypothesis that reduced PDE8A activity or expression would contribute to excessive ovarian androgen production. We identified a rare variant (R136Q; NM_002605.2 c.407G > A) and studied another known single nucleotide polymorphism (SNP) (rs62019510, N401S) in the PDE8A coding sequence causing non-synonymous amino acid substitutions, and a new SNP in the promoter region (NT_010274.16:g.490155G > A). Although PDE8A kinetics were consistent with reduced activity in theca cell lysates, study of the expressed variants did not confirm reduced activity in cell-free assays. Sub-cellular localization of the enzyme was also not different among the coding sequence variants. The PDE8A promoter SNP and a previously described promoter SNP did not affect promoter activity in in vitro assays. The more common coding sequence SNP (N401S), and the promoter SNPs were not associated with PCOS in our transmission/disequilibrium test-based analysis, nor where they associated with total testosterone or dehydroepiandrosterone sulfate levels. These findings exclude a significant role for PDE8A as a PCOS candidate gene, and as a Las major determinant of androgen levels in women.

Colocalization of Polycystic Ovary Syndrome Candidate Gene Products in Theca Cells Suggests Novel Signaling Pathways.

Genome-wide association studies identified loci associated with polycystic ovary syndrome (PCOS), including those near the LH receptor gene (LHCGR), a clathrin-binding protein (DENND1A) that functions as a guanine nucleotide exchange factor, and the gene encoding RAB5B, a GTPase involved in vesicular trafficking. We proposed that these three PCOS loci could be assembled into a functional network that contributes to altered gene expression in theca cells, resulting in increased androgen synthesis. The functional significance of this network was supported by our discovery that a truncated protein splice variant of the DENND1A gene, termed DENND1A.V2, is elevated in PCOS theca cells, and that forced expression of DENND1A.V2 in normal theca cells increased CYP11A1 and CYP17A1 expression and androgen synthesis, a hallmark of PCOS. In this study, we demonstrate the colocalization of LHCGR, DENND1AV.2, and RAB5B proteins in various cellular compartments in normal and PCOS theca cells by immunofluorescence. Human chorionic gonadotropin and forskolin stimulation was shown to affect the cytoplasmic distribution of LHCGR, DENND1A.V2, and RAB5B. DENND1A.V2 accumulated in the nuclei of the theca cells. Moreover, PCOS theca cells, following forskolin treatment, had a significantly greater relative abundance of nuclear DENND1A.V2. RAB5B also accumulated in the nuclei of PCOS theca cells treated with forskolin. In contrast, LHCGR did not enter the nucleus. This cytological evidence, and the previously reported increase in androgen biosynthesis with forced expression of DENND1A.V2 in normal theca cells, raises the possibility that DENND1A.V2 and RAB5B participate in increasing transcription of genes involved in androgen synthesis.

miRNA Profiling Reveals miRNA-130b-3p Mediates DENND1A Variant 2 Expression and Androgen Biosynthesis.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder of reproductive-age women involving overproduction of ovarian androgens and, in some cases, from the adrenal cortex. Family studies have established that PCOS is a complex heritable disorder with genetic and epigenetic components. Several small, noncoding RNAs (miRNAs) have been shown to be differentially expressed in ovarian cells and follicular fluid and in the circulation of women with PCOS. However, there are no reports of global miRNA expression and target gene analyses in ovarian theca cells isolated from normal cycling women and women with PCOS, which are key to the elucidation of the basis for the hyperandrogenemia characteristic of PCOS. With the use of small RNA deep sequencing (miR-seq), we identified 18 differentially expressed miRNAs in PCOS theca cells; of these, miR-130b-3p was predicted to target one of the PCOS genome-wide association study candidates, differentially expressed in neoplastic vs normal cells domain containing 1A (DENND1A). We previously reported that DENND1A variant 2 (DENND1A.V2), a truncated isoform of DENND1A, is upregulated in PCOS theca cells and mediates augmented androgen biosynthesis in PCOS theca cells. The comparison of miR-130b-3p in normal and PCOS theca cells demonstrated decreased miR-130b-3p expression in PCOS theca cells, which was correlated with increased DENND1A.V2, cytochrome P450 17α-hydroxylase (CYP17A1) mRNA and androgen biosynthesis. miR-130b-3p mimic studies established that increased miR130b-3p is correlated with decreased DENND1A.V2 and CYP17A1 expression. Thus, in addition to genetic factors, post-transcriptional regulatory mechanisms via miR-130b-3p underly androgen excess in PCOS. Ingenuity® Pathway Analysis Core Pathway and Network Analyses suggest a network by which miR-130b-3p, DENND1A, the luteinizing hormone/choriogonadotropin receptor, Ras-related protein 5B, and signaling pathways that they potentially target may mediate hyperandrogenism in PCOS.

VCAM1 Is Induced in Ovarian Theca and Stromal Cells in a Mouse Model of Androgen Excess.

Ovarian theca androgen production is regulated by the pituitary LH and intrafollicular factors. Enhanced androgen biosynthesis by theca cells contributes to polycystic ovary syndrome (PCOS) in women, but the ovarian consequences of elevated androgens are not completely understood. Our study documents the molecular events that are altered in the theca and stromal cells of mice exposed to high androgen levels, using the nonaromatizable androgen DHT. Changes in ovarian morphology and function were observed not only in follicles, but also in the stromal compartment. Genome-wide microarray analyses revealed marked changes in the ovarian transcriptome of DHT-treated females within 1 week. Particularly striking was the increased expression of vascular cell adhesion molecule 1 (Vcam1) specifically in the NR2F2/COUPTF-II lineage theca cells, not granulosa cells, of growing follicles and throughout the stroma of the androgen-treated mice. This response was mediated by androgen receptors (ARs) present in theca and stromal cells. Human theca-derived cultures expressed both ARs and NR2F2 that were nuclear. VCAM1 mRNA and protein were higher in PCOS-derived theca cells compared with control theca and reduced markedly by the AR antagonist flutamide. In the DHT-treated mice, VCAM1 was transiently induced by equine chorionic gonadotropin, when androgen and estrogen biosynthesis peak in preovulatory follicles, and was potently suppressed by a superovulatory dose of human chorionic gonadotropin. High levels of VCAM1 in the theca and interstitial cells of DHT-treated mice and in adult Leydig cells indicate that there may be novel functions for VCAM1 in reproductive tissues, including the gonads.

Human ovarian theca cells in culture.

Elucidating the regulation of androgen biosynthesis in ovarian theca cells is not only important for determining the mechanisms of regulation of estrogen biosynthesis throughout the menstrual cycle, but is also essential for understanding the pathogenesis of excess androgen biosynthesis and polycystic ovary syndrome (PCOS). Human theca cells in primary and long-term culture have provided model systems for examining theca cell differentiation as well as the mechanisms underlying basal and cAMP-regulated steroid biosynthesis at both the transcriptional and post-transcriptional level in normal and PCOS ovaries. Results of these studies are expected to lead to the identification of novel targets for clinical treatment of infertility and PCOS.

Alterations in mitogen-activated protein kinase kinase and extracellular regulated kinase signaling in theca cells contribute to excessive androgen production in polycystic ovary syndrome.

We have investigated the involvement of the MAPK signaling pathway in increased androgen biosynthesis and CYP17 gene expression in women with polycystic ovary syndrome (PCOS). A comparison of MAPK kinase (MEK1/2) and ERK1/2 phosphorylation in propagated normal and PCOS theca cells, revealed that MEK1/2 phosphorylation was decreased more than 70%, and ERK1/2 phosphorylation was reduced 50% in PCOS cells as compared with normal cells. Infection with dominant-negative MEK1 increased CYP17 mRNA and dehydroepiandrosterone (DHEA) abundance, whereas constitutively active MEK1 reduced DHEA production and CYP17 mRNA abundance. Similarly, the MEK inhibitor, PD98059, increased CYP17 mRNA accumulation and CYP17 promoter activity to levels observed in PCOS cells. Remarkably, in theca cells maintained in the complete absence of insulin, ERK1/2 phosphorylation was decreased in PCOS theca cells as compared with normal theca cells, and CYP17 mRNA and DHEA synthesis were increased in PCOS theca cells. These studies demonstrate that in PCOS cells reduced levels of activated MEK1/2 and ERK1/2 are correlated with increased androgen production, irrespective of the insulin concentration. These findings implicate alterations in the MAPK pathway in the pathogenesis of excessive ovarian androgen production in PCOS.

Valproate-induced alterations in human theca cell gene expression: clues to the association between valproate use and metabolic side effects.

Valproic acid (VPA) is an anti-epileptic drug that has been associated with polycystic ovary syndrome (PCOS)-like symptoms, including increased ovarian androgen production. The hyperandrogenemia likely reflects the stimulatory action of VPA on theca cell androgen synthesis and has been correlated to its activity as a histone deacteylase inhibitor in these cells. To determine whether VPA induces a PCOS-like genomic phenotype, we compared the gene expression profiles of untreated (UNT) normal, VPA-treated normal, and UNT PCOS theca cells. Hierarchal cluster analysis demonstrated similarities in the gene expression profiles of VPA-treated normal and PCOS theca cells. Statistical analysis identified 1,050 transcripts that have significantly altered mRNA abundance in both VPA-treated normal and UNT PCOS theca cells compared with normal UNT theca cells. Among these 1,050 transcripts were cAMP-GEFII and TRB3, which have increased and decreased mRNA abundance, respectively. The altered abundance of these two mRNAs was correlated to increased basal and insulin-induced phosphorylation of protein kinase B (Akt/PKB). Thus these studies indicate that VPA- and PCOS-induced changes in gene expression enhance Akt/PKB signal transduction in human theca cells. Furthermore, common changes in gene expression in PCOS and VPA-treated normal theca cells suggest a possible mechanism for the development of PCOS-like symptoms, including increased steroid synthesis and arrested follicle development in women receiving chronic VPA therapy.

Dysregulation of cytochrome P450 17alpha-hydroxylase messenger ribonucleic acid stability in theca cells isolated from women with polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a common reproductive endocrine disorder characterized by ovarian hyperandrogenism. Theca interna cells isolated from the ovaries of women with PCOS are characterized by increased expression of cytochrome P450 17alpha-hydroxylase (CYP17) [steroid 17alpha-hydroxylase/17,20 lyase (P450c17)], a steroidogenic enzyme obligatory for the biosynthesis of androgens. Augmented expression of the gene encoding P450c17 (CYP17) in PCOS theca has been attributed, in part, to differential transcriptional regulation of the CYP17 promoter in normal and PCOS cells. The present studies examine whether CYP17 gene expression is also posttranscriptionally regulated at the level of mRNA stability in normal and PCOS theca cells maintained in long-term culture. Determination of endogenous CYP17 mRNA half-life by pharmacological inhibition of transcription demonstrated that the half-life of CYP17 mRNA increased 2-fold in PCOS theca cells, compared with normal theca cells. Forskolin treatment also prolonged CYP17 mRNA half-life in both normal and PCOS theca cells. In vitro mRNA degradation studies demonstrated that the 5'-untranslated region confers increased stability to CYP17 mRNA in PCOS theca cells and showed that the 5'-untranslated region of CYP17 also confers forskolin-stimulated stabilization of CYP17 mRNA. These studies indicate that a slower rate of CYP17 mRNA decay contributes to increased steady-state mRNA accumulation and augmented CYP17 gene expression in PCOS theca cells.

Retinoids and retinol differentially regulate steroid biosynthesis in ovarian theca cells isolated from normal cycling women and women with polycystic ovary syndrome.

CONTEXT: Polycystic ovary syndrome (PCOS) is characterized by ovarian androgen excess and infertility. Recent experiments have suggested that several genes involved in retinoic acid synthesis may be differentially expressed in PCOS theca cells and may contribute to excessive theca-derived androgen production. OBJECTIVE: The study was performed to examine whether there are differential effects of retinol and retinoids on normal and PCOS theca cell function. DESIGN: We used in vitro assays. SETTING: The study was conducted at the university laboratory. PATIENTS: We studied theca interna cells isolated from normal-cycling women and women with PCOS. INTERVENTIONS: Theca cells were treated with all-trans-retinoic acid (atRA), 9-cis retinoic acid (9-cis RA), or the retinoic acid precursor retinol. MAIN OUTCOME MEASURE(S): We measured dehydroepiandrosterone, testosterone, and progesterone biosynthesis as well as cytochrome P450 17alpha-hydroxylase (CYP17), cytochrome P450 cholesterol side-chain cleavage, and steroidogenic acute regulatory protein mRNA abundance and promoter function. RESULTS: Dehydroepiandrosterone production was increased by atRA and 9-cis RA in normal cells and by atRA, 9-cis RA, and retinol in PCOS. Testosterone production was increased by atRA in normal and by atRA, 9-cis RA, and retinol in PCOS. Progesterone production was not altered by retinoid treatment. Retinoids stimulated mRNA abundance and promoter function for CYP17 and steroidogenic acute regulatory protein in both cell types and cytochrome P450 cholesterol side-chain cleavage in normal cells. Retinol stimulated CYP17 mRNA accumulation and promoter function in PCOS but not normal theca cells. P < 0.05 was considered statistically significant. CONCLUSIONS: Differential responses to retinol and retinoids in normal and PCOS theca suggest that altered retinoic acid synthesis and action may be involved in augmented CYP17 gene expression and androgen production in PCOS.

Increased transcription and increased messenger ribonucleic acid (mRNA) stability contribute to increased GATA6 mRNA abundance in polycystic ovary syndrome theca cells.

CONTEXT: Polycystic ovary syndrome (PCOS) theca cells secrete increased levels of androgens. The mRNA and protein levels of the transcription factor GATA6, which regulates expression of several steroidogenic enzymes, are increased in PCOS theca cells. Thus, GATA6 is a PCOS candidate gene. OBJECTIVE: The objective of the study was to explore mechanisms by which GATA6 mRNA levels are increased in PCOS theca cells. DESIGN: Theca cell cDNA and genomic DNA from normal individuals and PCOS patients were subjected to quantitative RT-PCR and sequence analysis, respectively. SETTING: The experiments were performed in a university laboratory. PARTICIPANTS: Four hundred sixty-nine families that contain at least one PCOS patient were ascertained for genetic studies. Theca cells were obtained from four normal individuals and four PCOS patients. RESULTS: Nascent GATA6 transcript levels, which reflect GATA6 gene transcription, were significantly increased in PCOS theca cells. In normal theca cells, GATA6 mRNA has a short half-life, which was attributed to an AU-rich 3'-untranslated region sequence. The half-life of GATA6 transcripts was also significantly longer in the PCOS theca cells. However, no sequence variations in the GATA6 gene locus were associated with PCOS. CONCLUSIONS: In PCOS theca cells, GATA6 gene transcription and the stability of the GATA6 mRNA are increased. Because there is no sequence variation in the GATA6 gene locus, which is associated with PCOS, it is likely that the increased gene transcription and mRNA stability are due to intrinsic differences in PCOS theca cells.

Increased cytochrome P450 17alpha-hydroxylase promoter function in theca cells isolated from patients with polycystic ovary syndrome involves nuclear factor-1.

Cytochrome P450 17alpha-hydroxylase (CYP17) gene expression and androgen biosynthesis are persistently elevated in theca cells isolated from ovaries of women with polycystic ovary syndrome (PCOS). We previously reported that -235 to -109 bp of the CYP17 promoter confers increased CYP17 promoter function in PCOS theca cells. In this report, additional deletion and mutational analyses of the CYP17 promoter were performed to identify the sequences that contribute to increased CYP17 promoter function in PCOS theca cells. Results of these analyses established that augmented promoter function in PCOS theca cells results from preferentially increased basal regulation conferred by sequences between -188 and -147 bp of the CYP17 promoter. Scanning mutant analysis demonstrated that mutations within a 16-bp sequence, spanning -174 to -158 bp of the promoter, ablated increased basal CYP17 promoter function in PCOS theca cells. EMSA analysis demonstrated that the NF-1 family member, NF-1C, bound this sequence. Cotransfection of several NF-1C isoforms expressed in normal and PCOS cells repressed CYP17 promoter function. NF-1C protein and DNA binding were reduced in PCOS theca cell nuclear extracts, as compared with normal. Another NF-1C site between -102 and -90 bp of the promoter was also identified. However, mutation of this site had no effect on differential promoter function in PCOS theca cells. These studies demonstrate that 1) augmented CYP17 promoter function in PCOS theca cells results from increased basal regulation, and 2) diminished NF-1C-dependent repression may be one mechanism underlying increased basal CYP17 promoter activity and altered gene expression in PCOS theca cells.

Functional genomics of PCOS: from GWAS to molecular mechanisms.

Polycystic ovary syndrome (PCOS) is a common endocrinopathy characterized by increased ovarian androgen biosynthesis, anovulation, and infertility. PCOS has a strong heritable component based on familial clustering and twin studies. Genome-wide association studies (GWAS) identified several PCOS candidate loci including LHCGR, FSHR, ZNF217, YAP1, INSR, RAB5B, and C9orf3. We review the functional roles of strong PCOS candidate loci focusing on FSHR, LHCGR, INSR, and DENND1A. We propose that these candidates comprise a hierarchical signaling network by which DENND1A, LHCGR, INSR, RAB5B, adapter proteins, and associated downstream signaling cascades converge to regulate theca cell androgen biosynthesis. Future elucidation of the functional gene networks predicted by the PCOS GWAS will result in new diagnostic and therapeutic approaches for women with PCOS.

Human skin is a steroidogenic tissue: steroidogenic enzymes and cofactors are expressed in epidermis, normal sebocytes, and an immortalized sebocyte cell line (SEB-1).

Although the human sebaceous gland can synthesize cholesterol from acetate and can further metabolize steroids such as dehydroepiandrosterone into potent androgens, the de novo production of steroids from cholesterol has not been demonstrated in human skin. The goal of this study was to delineate the steroidogenic pathway upstream from dehydroepiandrosterone by documenting the presence of members of the P450 side chain cleavage system (P450scc). This system catalyzes the initial step in steroid hormone synthesis following translocation of cholesterol to the inner mitochondrial membrane. In concert with its cofactors, adrenodoxin and adrenodoxin reductase, and the transcription factor steroidogenic factor 1, P450scc converts cholesterol to pregnenolone. An SV40 immortalized human sebaceous gland cell line (SEB-1) was established in order to facilitate investigation of the P450scc system. The sebaceous phenotype of SEB-1 sebocytes was confirmed using immunohistochemistry, Oil Red O staining, and gene array expression analysis. Presence of P450scc, adrenodoxin reductase, cytochrome P450 17-hydroxylase (P450c17), and steroidogenic factor 1 was documented in human facial skin, human sebocytes, and SEB-1 sebocytes. Using immunohistochemistry, antibodies to the above proteins localized to epidermis, hair follicles, sebaceous ducts, and sebaceous glands in sections of facial skin. Results of immunohistochemistry were confirmed with Western blotting. Biochemical activity of cytochrome P450scc and P450c17 was demonstrated in SEB-1 sebocytes using radioimmunoassay. The relative abundance of mRNA for P450scc, P450c17, and steroidogenic factor 1 in SEB-1 sebocytes and sebaceous glands was compared to mRNA levels in ovarian theca and granulosa cells using real-time quantitative polymerase chain reaction. Gene array expression analysis and quantitative polymerase chain reaction indicated that mRNA for P450scc is more abundant than mRNA for both P450c17 and steroidogenic factor 1 in sebaceous glands and SEB-1 cells. These data demonstrate that the skin is in fact a steroidogenic tissue. The clinical significance of this finding in mediating androgenic skin disorders such as acne, hirsutism, or androgenetic alopecia remains to be established.

Valproate potentiates androgen biosynthesis in human ovarian theca cells.

In patients with epilepsy, treatment with valproate (VPA) has been reported to be associated with polycystic ovary syndrome-like symptoms including weight gain, hyperandrogenemia, and hyperinsulinemia. We examined the effect of VPA on androgen biosynthesis in ovarian theca cells isolated from follicles of normal cycling women to determine whether the hyperandrogenemia reported with VPA treatment could be a result of direct effects of VPA on the ovary. In long-term cultures of theca cells treated for 72 h with sodium valproate (30-3000 microm), we observed an increase in basal and forskolin-stimulated dehydroepiandrosterone (DHEA), androstenedione, and 17alpha-hydroxyprogesterone production compared with control values. In contrast, low doses of VPA treatment (i.e. 30-300 microm) had no effect on basal and forskolin-stimulated progesterone production, whereas higher doses of VPA (1000-3000 microm) inhibited progesterone production. The most pronounced effect of VPA on androgen biosynthesis was observed in the dose range of 300-3000 microm, which represent therapeutic levels in the treatment of epilepsy and bipolar disorder. Western analyses demonstrated that VPA treatment increased both basal and forskolin-stimulated P450c17 and P450scc protein levels, whereas the amount of steroidogenic acute regulatory protein was unaffected. In transient transfection studies, VPA was found to increase P450 17alpha-hydroxylase and P450 cholesterol side-chain cleavage promoter activity, whereas steroidogenic acute regulatory protein promoter activity was unaffected. Consistent with the ability of VPA to act as a histone deacetylase (HDAC) inhibitor in other cell systems, VPA (500 microm) treatment was observed to increase histone H3 acetylation and P450 17alpha-hydroxylase mRNA accumulation. The HDAC inhibitor butyric acid (500 microm) similarly increased histone H3 acetylation and DHEA biosynthesis, whereas the VPA derivative valpromide (500 microm), which lacks HDAC inhibitory activity, had no effect on histone acetylation or DHEA biosynthesis. These data suggest that VPA-induced ovarian androgen biosynthesis results from changes in chromatin modifications (histone acetylation) that augment transcription of steroidogenic genes. These studies provide the first biochemical evidence to support a role for VPA in the genesis of polycystic ovary syndrome-like symptoms, and establish a direct link between VPA treatment and increased ovarian androgen biosynthesis.

Growth differentiation factor-9 inhibits 3'5'-adenosine monophosphate-stimulated steroidogenesis in human granulosa and theca cells.

Growth differentiation factor-9 (GDF-9), a member of the transforming growth factor superfamily, modulates the development and function of granulosa and theca cells. Targeted deletion of GDF-9 in the mouse revealed that GDF-9 was essential for the establishment of the thecal cell layer during early folliculogenesis. During later stages of follicular development, the roles of GDF-9 are less well understood, but it has been postulated that oocyte-derived GDF-9 may prevent premature luteinization of follicular cells, based on its ability to modulate steroidogenesis by rodent ovarian cells. In the rodent, GDF-9 is expressed solely by the oocyte from the early primary follicular stage through ovulation. Recent studies in the rhesus monkey demonstrated that granulosa cells express GDF-9, suggesting a broader role for this protein in ovarian function in primates. We examined the effect of recombinant GDF-9 on proliferating human granulosa and thecal cell steroidogenesis and the expression of steroidogenic acute regulatory protein (StAR), P450 side-chain cleavage, and P450 aromatase. We also examined granulosa cell GDF-9 expression by quantitative RT-PCR and by Western analysis. GDF-9 inhibited 8-Br-cAMP-stimulated granulosa progesterone synthesis by approximately 40%, but did not affect basal progesterone production. Concordant with reduced steroid production, 8-Br-cAMP-stimulated StAR protein expression was reduced approximately 40% in granulosa cells, as were expression of StAR mRNA and StAR promoter activity. Additionally, GDF-9 inhibited 8-Br-cAMP-stimulated expression of P450 side-chain cleavage and P450 aromatase. Human granulosa cells expressed GDF-9, as determined by RT-PCR and Western analysis. Treatment of human thecal cells with GDF-9 blocked forskolin-stimulated progesterone, 17alpha-hydroxyprogesterone, and dehydroepiandrosterone synthesis. Thecal cells exhibited greater sensitivity to GDF-9, suggesting that this cell may be a primary target of GDF-9. Moreover, GDF-9 increased thecal cell numbers during culture, but had no effect on granulosa cell growth. Our findings implicate GDF-9 in the modulation of follicular steroidogenesis, especially theca cell function. Because GDF-9 mRNA and protein are detectable in granulosa-lutein cells after the LH surge, the concept of GDF-9 as a solely oocyte-derived luteinization inhibitor needs to be reevaluated.