This study will investigate the regulation of human cytochrome P450 17-alpha- hydroxylase (CYP17) gene expression in theca interna cells from ovaries of normal cycling women and from ovaries of women with polycystic ovarian syndrome (PCOS). The hypothesis to be tested is that increased androgen production results from an intrinsic abnormality of steroid production in PCOS theca cells. The goal is to understand how LH and growth factors regulate CYP17 expression and androgen synthesis in normal cells, and how dysregulation of these processes results in increased androgen production in PCOS. Conditions have been developed to propagate functional cultures of normal and PCOS theca cells that express 17a-hydroxylase activity in response to cAMP. In normal, cultured theca cells, cAMP stimulates CYP17 mRNA, whereas epidermal growth factor (EGF), fibroblast growth factor (FGF), and transforming growth factor B (TGFB) inhibit cAMP-stimulation of CYP17 mRNA. The study will characterize the mechanisms by which CYP17 gene expression is stimulated by LH, and inhibited by growth factors in theca cells.
Specific Aim 1 will characterize the regulation of CYP17 enzyme activity, protein, and mRNA content in theca cultures, and determine whether the LH-dependent induction of CYP17 mRNA requires ongoing protein synthesis. The investigator will examine the time-and dose-dependent effects of LH and growth factors on CYP17 mRNA levels, and determine whether changes in mRNA stability also contribute to the effects of cAMP and growth factors on steady state CYP17 mRNA. Moreover, the investigator will determine whether these regulatory processes are altered in PCOS.
Specific Aim 2 will identify the cis- regulatory elements involved in the tissue-specific regulation of CYP17 gene by LH, the transcription factor steroidogenic factor-1 (SF-1), and growth factors. Should growth factor modulation of CYP17 expression in PCOs theca cells be altered, studies will begin to identify the cis-regulatory elements involved in the regulation of CYP 17 expression by the specific growth factor involved.
In Specific Aim 3, studies of steroid metabolism will be performed with fresh explant cultures and long-term cultures of theca cells from normal and PCOS patients to examine whether the abnormalities in the steroidogenic pathway that cause increased androgen production in PCOS are extrinsic or intrinsic. Experiments are planned to determine whether increased androgen production results from changes in the regulation by LH and growth factors of expression of P450 scc (CYP11A), 3B -HSD, CYP17, or some combination of these in PCOS theca cells. Information derived from these studies will provide a better understanding of the molecular basis of steroid synthesis and androgen excess in PCOS.
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