Enzymatic activities of P450c17 stably expressed in fibroblasts from patients with the polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder affecting approximately 5-10% of women of reproductive age. The clinical features of PCOS include oligo/anovulation, hyperandrogenemia, and hyperinsulinemia. Because P450c17 is the single enzyme catalyzing both 17alpha-hydroxylase and 17,20-lyase activities in the ovary and adrenal, some have suggested that defects in P450c17 may cause the hyperandrogenism of PCOS. Previous studies have shown that serine hyperphosphorylation of P450c17 increases the enzyme's 17,20-lyase activity, thereby favoring androgen production, and that serine phosphorylation of the insulin receptor beta-chain (IR-beta) inhibits IR-beta tyrosine phosphorylation, causing insulin resistance in vitro. We previously suggested that a gain of function mutation in a single serine kinase might cause the hyperandrogenism and insulin resistance observed in PCOS patients by excessive phosphorylation of both P450c17 and IR-beta. To test this hypothesis, we obtained fibroblasts from nine previously studied patients: three controls, three PCOS patients with normal levels of IR-beta serine phosphorylation, and three PCOS patients with increased levels of IR-beta serine phosphorylation. Initial studies showed that such skin fibroblasts could not be transfected effectively by calcium phosphate, diethylaminoethyl-dextran, lipofection or adenovirus procedures. Therefore, we employed a retroviral infection system to stably express human P450c17 in the primary cultures of fibroblast cells from the PCOS patients and controls and measured the resulting 17alpha-hydroxylase and 17,20-lyase activity. The cells were analyzed in a blinded fashion until the study was complete. The 17alpha-hydroxylase and 17,20-lyase activities in each cell line correlated well with the amount of P450c17 protein expressed, but there was no correlation between either enzymatic activity (or their ratio) with the clinical phenotype of the cells' donors even when results were corrected for the number of P450c17 complementary DNA inserts per cell line. Overnight incubation with 1 micromol/L insulin also did not affect enzymatic activity. Thus, we were unable to find evidence for the hypothesis that in PCOS a single abnormal kinase hyperphosphorylates both IR-beta, causing insulin resistance, and P450c17, causing hyperandrogenism. However, because fibroblasts do not normally express either P450c17 or the accessory proteins needed for its optimal activity, these results cannot exclude a role for serine phosphorylation in the hyperandrogenism and insulin resistance of PCOS.

Chronic high-dosage androgen administration to ovulatory women does not alter adrenocortical steroidogenesis.

OBJECTIVE: To evaluate the role of chronic long-term exogenous androgen administration to normal ovulatory women on adrenal steroidogenesis. DESIGN: Prospective study of four consecutive female-to-male transsexuals before and during chronic testosterone (T) therapy. SETTING: Clinical Research Center of the Mount Sinai Medical Center. PATIENTS: Four female-to-male transsexuals were studied before and during 6 to 12 months of chronic T enanthate therapy for desired virilization. All four subjects were ovulatory before treatment. Adrenocorticotropic hormone (ACTH) testing was performed before, and 6 and 12 months of androgen therapy and various adrenal androgens as well as precursor:product pairs were evaluated as an index of specific adrenocortical biosynthetic defects. RESULTS: Baseline and 1 hour after 0.25 mg ACTH intravenously, adrenal androgen levels as well as adrenal precursor/product pairs demonstrated no difference before and during chronic T treatment. Studies included determinations of plasma 17-hydroxypregnenolone, 17-hydroxyprogesterone, dehydroepiandrosterone, androstenedione, 11-deoxycortisol, and cortisol. CONCLUSION: It is concluded that chronic hypertestosteronemia does not alter adrenal steroidogenesis.

The effects of the aromatase inhibitor delta 1-testolactone on gonadotropin release and steroid metabolism in polycystic ovarian disease.

This study was designed to examine the importance of aromatization in the gonadotropin secretory dynamics of polycystic ovarian disease (PCOD) by using the aromatase inhibitor delta 1 testolactone (TL) as a probe and to determine the effects of TL on steroid metabolism in vivo and in vitro. The pulsatile patterns of gonadotropin secretion and peripheral steroid levels were studied in eight women with PCOD before and during TL administration. There was a significant fall in peripheral estrone (E1) levels, a rise in peripheral androstenedione levels, and an increase in the androstenedione/E1 ratio during TL administration in these women. Isotopic determinations of androgen and estrogen production and metabolism before and during TL administration in two women confirmed a 90-95% decrease in the overall rate of aromatization. One patient also had an increase in the production and clearance rates of estradiol and E1 during TL administration, suggesting resistance to TL of the ovarian aromatase enzyme system. There were significant increases in both mean LH pulse amplitude [1.2 +/- 0.3 (SE) mIU/ml LER-907 before vs. 1.7 +/- 0.3 mIU/ml LER-907 during TL, P less than 0.05, paired t test] and frequency per 6 h (median: 3 before vs. 4 during TL, P less than 0.05, Wilcoxon signed rank test). Mean levels of LH and FSH did not, however, change significantly during TL administration. TL maximally inhibited neonatal rat hypothalamic aromatase in vitro at concentrations of 200 microM, a level theoretically obtainable during pharmacological therapy. These data suggest that: 1) in humans TL is a potent inhibitor of peripheral but not ovarian aromatase, and of hypothalamic aromatase in rats; 2) TL administration increases LH pulse amplitude and frequency in PCOD, either directly via hypothalamic aromatase inhibition, or indirectly by alterations in gonadal steroid metabolism; and 3) because of the multiple potential actions of TL, its usefulness as a probe in studies of gonadotropin secretion in PCOD is limited.