Polycystic ovary syndrome (PCOS) is a poorly understood syndrome of chronic hyperandrogenism and anovulation which appears to be due to dysregulation of steroidogenesis resulting in a high level of serum testosterone. Testosterone biosynthesis from androstenedione is carried out by androgenic 17b-hydroxysteroid dehydrogenase (17B-HSD) activity. The molecular basis for androgenic 17B-HSD activity within the ovary was unknown until we recently demonstrated that 17B-HSD5 is expressed in human ovary. In addition, a number of structural variants of 17B-HSD5 exist and appear to vary in activities. We propose to characterize the cell-specific expression of the 17B-HSD5 gene in the ovary, the regulation of expression of the gene in the ovary, and determine if candidate activating gene variants, which seem to be fairly common, are related hyperandrogenic states such as PCOS, all of which are unknown.
The specific aims are to test the hypotheses that: 1) ovarian 17B-HSD5 is mainly expressed in theca-interstitial-stromal (thecal) cells. 2) 17B-HSD5 in thecal cells is regulated by LH and that insulin modulates this effect. 3) activating variants of the 17B-HSD5 gene contribute to hyperandrogenism in women and that a specific phenotype is associated with these variants. The experimental design to test these hypotheses is as follows: 1) The localization of 17B-HSD5 gene expression will be determined in ovarian tissue by using in situ polymerase chain reaction (PCR) hybridization, reverse transcriptase PCR, and Northern techniques. 2) LH-regulahon of the 17B-HSD5 gene in thecal cells will be examined by analysis of constructs of the 5-untranslated region of the 17B-HSD5 gene, which will be subcloned into pGL3-reporter plasmids and transfected into thecal cells to monitor luciferase activity. 3) We will then functionally characterize candidate activating 17B-HSD gene variants by a variety of molecular and enzymatic techniques and determine the clinical significance of such these variants. The latter will be done in three ways: a) first, we will seek to identify candidate variants by comparing the sequence of the 17B-HSD5 gene in well-characterized normal women and women with diverse forms of PCOS (classic and nonclassic), b) then we will determine whether each candidate variant is associated with specific hyperandrogenic indices by comparison of its prevalence in hyperandrogenic and control populations and through use of family data, and c) finally, we will correlate genotype with phenotype using both case-control and family-based data. The overall goal of this proposal is to understand the factors regulating the production of testosterone in women, particularly in their ovaries.
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