Abstract
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.
Highlights
10 mm embedded in a dense stroma [3]
Studies using freshly isolated and short and long term cultures of theca cells have demonstrated that androgen synthesis is increased in Polycystic ovary syndrome (PCOS) compared with normal theca cells, suggesting that this PCOS phenotype is an intrinsic property of the theca cell (1, 4 – 6)
PCOS Theca Cells Exhibit a Distinct Gene Expression Profile Compared with Normal Theca Cells—Previous studies indicated that excess androgen synthesis is an intrinsic property of PCOS theca cells [6, 7]
Summary
Theca Cell Culturing and RNA Isolation—Theca cells were isolated from 3–5-mm follicles from the ovaries of four normal women and five PCOS patients, and independent cultures were established using the cells that were isolated from each woman as previously described [6, 7]. For each of the 28 targets, quantitative PCRs were carried out using equivalent dilutions of each cDNA sample, the fluorescent indicator SYBR green, the empirically determined concentration of each primer, and the Applied Biosystems model 7700 sequence detector PCR machine (PerkinElmer Life Sciences) as previously described [10]. To account for differences in starting material, quantitative PCR was carried out for each cDNA sample using the Applied Biosystems human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 20ϫ primer and probe reagent (PerkinElmer Life Sciences). These quantitative PCRs defined a threshold cycle (Ct) of detection for the target or the GAPDH in each cDNA sample. The unpaired Student’s t test was used to detect statistically significant differences (p value Ͻ 0.05) between pcDNAG6 and pcDNAG3-transfected cells
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