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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.

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.The objective of the study was to explore mechanisms by which GATA6 mRNA levels are increased in PCOS theca cells.Theca cell cDNA and genomic DNA from normal individuals and PCOS patients were subjected to quantitative RT-PCR and sequence analysis, respectively.The experiments were performed in a university laboratory.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.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.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.

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 17α-hydroxylase (CYP17) [steroid 17α-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.

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.

Polycystic ovary syndrome (PCOS) is characterized by increased ovarian androgen secretion, anovulatory infertility due to arrested folliculogenesis, and is frequently found in association with insulin resistance and obesity. Characterization of PCOS theca cells demonstrated that elevated expression of the steroidogenic enzymes 17alpha hydroxylase/17,20 lyase (CYP17) and P450 side chain cleavage enzyme (CYP11A1) play a role in increased androgen production by 3beta-hydroxysteroid dehydrogenase in the PCOS theca cell. However, the gene networks and signal transduction pathways which cause the altered expansion of the steroid enzymes remain to be determined. In order to identify these gene networks and/or signaling pathways, we carried out global gene expression profiling of normal and PCOS theca cells using subtractive suppressive hybridization and oligonucleotide microarray analysis. These analyses demonstrated that approximately 2% of genes expressed in the theca cell exhibit altered mRNA abundance in PCOS. Characterization of these genes revealed that retinoic acid synthesis and Wnt signal transduction are altered in the PCOS theca cell. In addition, the transcription factor GATA6, which regulates the promoter activity of CYP17 and CYP11A, was increased in the PCOS compared to normal theca cells. Thus, global gene expression profiling has identified potential pathways which may determine the PCOS theca cell phenotype.

Polycystic ovary syndrome (PCOS) affects 5% of reproductive aged women and is the leading cause of anovulatory infertility. A hallmark of PCOS is excessive theca cell androgen secretion, which is directly linked to the symptoms of PCOS. Our previous studies demonstrated that theca cells from PCOS ovaries maintained in long term culture persistently secrete significantly greater amounts of androgens than normal theca cells, suggesting an intrinsic abnormality. Furthermore, previous studies suggested that ovarian hyperandrogenemia is inherited as an autosomal dominant trait. However, the genes responsible for ovarian hyperandrogenemia of PCOS have not been identified. In this present study, we carried out microarray analysis to define the gene networks involved in excess androgen synthesis by the PCOS theca cells in order to identify candidate PCOS genes. Our analysis revealed that PCOS theca cells have a gene expression profile that is distinct from normal theca cells. Included in the cohort of genes with increased mRNA abundance in PCOS theca cells were aldehyde dehydrogenase 6 and retinol dehydrogenase 2, which play a role in all-trans-retinoic acid biosynthesis and the transcription factor GATA6. We demonstrated that retinoic acid and GATA6 increased the expression of 17alpha-hydroxylase, providing a functional link between altered gene expression and intrinsic abnormalities in PCOS theca cells. Thus, our analyses have 1) defined a stable molecular phenotype of PCOS theca cells, 2) suggested new mechanisms for excess androgen synthesis by PCOS theca cells, and 3) identified new candidate genes that may be involved in the genetic etiology of PCOS.

To test the hypothesis that the hyperandrogenemia associated with polycystic ovary syndrome (PCOS) results from an intrinsic abnormality in ovarian theca cell steroidogenesis, we examined steroid hormone production, steroidogenic enzyme activity, and mRNA expression in normal and PCOS theca cells propagated in long-term culture. Progesterone (P4), 17alpha-hydroxyprogesterone (17OHP4), and testosterone (T) production per cell were markedly increased in PCOS theca cell cultures. Moreover, basal and forskolin-stimulated pregnenolone, P4, and dehydroepiandrosterone metabolism were increased dramatically in PCOS theca cells. PCOS theca cells were capable of substantial metabolism of precursors into T, reflecting expression of an androgenic 17beta-hydroxysteroid dehydrogenase. Forskolin-stimulated cholesterol side chain cleavage enzyme (CYP11A) and 17alpha-hydroxylase/17,20-desmolase (CYP17) expression were augmented in PCOS theca cells compared with normal cells, whereas no differences were found in steroidogenic acute regulatory protein mRNA expression. Collectively, these observations establish that increased CYP11A and CYP17 mRNA expression, as well as increased CYP17, 3beta-hydroxysteroid dehydrogenase, and 17beta-hydroxysteroid dehydrogenase enzyme activity per theca cell, and consequently increased production of P4, 17OHP4, and T, are stable properties of PCOS theca cells. These findings are consistent with the notion that there is an intrinsic alteration in the steroidogenic activity of PCOS thecal cells that encompasses multiple steps in the biosynthetic pathway.

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.The study was performed to examine whether there are differential effects of retinol and retinoids on normal and PCOS theca cell function.We used in vitro assays.The study was conducted at the university laboratory.We studied theca interna cells isolated from normal-cycling women and women with PCOS.Theca cells were treated with all-trans-retinoic acid (atRA), 9-cis retinoic acid (9-cis RA), or the retinoic acid precursor retinol.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.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. P0.05 was considered statistically significant.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.

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.

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 μm), we observed an increase in basal and forskolin-stimulated dehydroepiandrosterone (DHEA), androstenedione, and 17α-hydroxyprogesterone production compared with control values. In contrast, low doses of VPA treatment (i.e. 30–300 μm) had no effect on basal and forskolin-stimulated progesterone production, whereas higher doses of VPA (1000–3000 μm) inhibited progesterone production. The most pronounced effect of VPA on androgen biosynthesis was observed in the dose range of 300-3000 μm, 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 17α-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 μm) treatment was observed to increase histone H3 acetylation and P450 17α-hydroxylase mRNA accumulation. The HDAC inhibitor butyric acid (500 μm) similarly increased histone H3 acetylation and DHEA biosynthesis, whereas the VPA derivative valpromide (500 μm), 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.

Ovarian theca cells propagated from patients with polycystic ovary syndrome (PCOS) convert steroid precursors into T more efficiently than normal theca cells. To identify the basis for increased T production by PCOS theca cells, we examined type I-V 17 beta-hydroxysteroid dehydrogenase (17 beta HSD) isoform expression in long-term cultures of theca and granulosa cells isolated from normal and PCOS ovaries. RT-PCR analysis demonstrated that theca cells express type V 17 beta HSD a member of the aldo-keto reductase (AKR) superfamily (17 beta HSDV, AKR1C3), whereas expression of type I, II, and IV 17 beta HSD, which are members of the short-chain dehydrogenase/reductase superfamily, was limited to granulosa cells. Type III 17 beta HSD, the testicular isoform, was not detected in either granulosa or theca cells. Northern and real-time PCR analyses demonstrated that 17 beta HSDV transcripts were not significantly increased in PCOS theca cells compared with normal theca cells. RT-PCR analysis revealed that theca cells also express another AKR, 20 alpha HSD (AKR1C1). Both basal and forskolin-stimulated 20 alpha HSD mRNA levels were increased in PCOS theca cells compared with normal theca cells. However, 17 beta HSD enzyme activity per theca cell was not significantly increased in PCOS, suggesting that neither AKR1C3 nor AKR1C1 contributes to the formation of T in this condition. In contrast, 17 alpha-hydroxylase/C17,20 lyase and 3 beta HSD enzyme activities were elevated in PCOS theca cells, driving increased production of T precursors. These findings indicate that 1) increased T production in PCOS theca cells does not result from dysregulation of "androgenic" 17 beta HSD activity or altered expression of AKRs that may express 17 beta HSD activity; and 2) increased synthesis of T precursors is the primary factor driving enhanced T secretion in PCOS.

Hyperandrogenism is characteristic of women with polycystic ovary syndrome (PCOS). Ovarian theca cells isolated from PCOS follicles and maintained in long-term culture produce elevated levels of progestins and androgens compared to normal theca cells. Augmented steroid production in PCOS theca cells is associated with changes in the expression of genes for several steroidogenic enzymes, including CYP11A1, which encodes cytochrome P450 cholesterol side-chain cleavage. Here, we further examined CYP11A1 gene expression, at both the transcriptional and post-transcriptional level in normal and PCOS theca cells propagated in long-term culture utilizing quantitative RT-PCR, functional promoter analyses, and mRNA degradation studies. The minimal element(s) that conferred increased basal and cAMP-dependent CYP11A1 promoter function were determined. CYP11A1 mRNA half-life in normal and PCOS theca cells was compared. Results of these cumulative studies showed that basal and forskolin stimulated steady state CYP11A1 mRNA abundance and CYP11A1 promoter activity were increased in PCOS theca cells. Deletion analysis of the CYP11A1 promoter demonstrated that augmented promoter function in PCOS theca cells results from increased basal regulation conferred by a minimal sequence between -160 and -90 bp of the transcriptional start site. The transcription factor, nuclear factor 1C2, was observed to regulate basal activity of this minimal CYP11A1 element. Examination of mRNA stability in normal and PCOS theca cells demonstrated that CYP11A1 mRNA half-life increased >2-fold, from approximately 9.22+/-1.62 h in normal cells, to 22.38+/-0.92 h in PCOS cells. Forskolin treatment did not prolong CYP11A1 mRNA stability in either normal or PCOS theca cells. The 5'-UTR of CYP11A1 mRNA confers increased basal mRNA stability in PCOS cells. In conclusion, these studies show that elevated steady state CYP11A1 mRNA abundance in PCOS cells results from increased transactivation of the CYP11A1 promoter and increased CYP11A1 mRNA stability.