Mechanisms of Action for an Androgen-Mediated Autoregulatory Process in Rat Thecal-Interstitial Cells

BIOLOGY OF REPRODUCTION 49, 1190-1201 (1993) Mechanisms of Action for an Androgen-Mediated Autoregulatory Process in Rat Thecal-lnterstitial Cells1 DEBORAH A. SIMONE, LYNN P. CHORICH, and VIRENDRA B. MAHESH 2 Department of Physiology and Endocrinology, Medical College of Georgia, Augusta, Georgia 30912-3000 ABSTRACT INTRODUCTION Recent investigations in our laboratory have revealed the existence of an androgen-mediated autoregulatory process for androgen production in rat ovarian thecal-interstitial cells (TIC) [1]. The synthetic androgen 17-hydroxy-7(x, 17a-dimethyl-4-estren-3-one (mibolerone) was able to antagonize both hCG- and hCG/insulin-like growth factor I (IGF-I)stimulated TIC androgen synthesis by 32% and 40%, respectively. Furthermore, it was demonstrated that mibolerone inhibits hCG action by interfering with the cAMP second messenger system. Because of the potential physiological importance of the androgen-mediated autoregulatory process for androgen production, a study of the mechanisms involved is of considerable interest. The presence of androgen receptors has been demonstrated in the ovary by several investigators [2-4], and recently androgen receptors have been demonstrated in thecal cells of the primate ovary [5]. Therefore, the autoregulatory effect of androgens may be mediated through androgen receptors. The activation of protein kinase C (PKC) by phorbol esters has been demonstrated to decrease LH-stimulated Accepted August 6, 1993. Received May 19, 1993. 'This work was supported by grant HD-244488 from the National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD. 'Correspondence. FAX: (706) 721-7299. and 8-bromo-cAMP-stimulated androgen synthesis in TIC [6]. GnRH has been found to be inhibitory to ovarian steroidogenesis, and, because it brings about PKC activation, it has been suggested that its inhibitory effect on LH-induced ovarian androgen production may be mediated through PKC activation [6, 7]. There is also considerable evidence that LH has the ability to modify phosphoinositide metabolism in granulosa cells and corpus luteal cells, leading to increases in inositol triphosphate, cAMP, and free Ca2 + [8-10]. Ca 2+ acting together with diacylglycerol has the ability to activate PKC. Ca2+ can also activate phosphodiestrases to lower cAMP levels and inhibit or stimulate adenylate cyclase depending on the cell type and the concentration of Ca2+ [11-13]. Similar to the effects of Ca 2+ on cAMP, stimulatory and inhibitory effects have also been observed on gonadal steroidogenesis [12-15]. Mibolerone has been shown to increase CaZ+ flux through L-type channels in human prostatic cancer cells [16]. Therefore, in order to characterize the mechanisms of the autoregulatory effect of mibolerone, this investigation had the following four objectives: 1) to determine whether the inhibitory action of mibolerone on androgen synthesis is mediated via an androgen receptor-mediated process, 2) to determine whether changes in PKC activity bring about a change in mibolerone's inhibitory effect on androgen synthesis, 3) to determine the effect of changes in Ca 2+ on the action of mibolerone on inhibiting androgen synthesis, 1190 In rat thecal-interstitial cells (TIC), treatment with the synthetic androgen mibolerone has led to the documentation of an autoregulatory process for androgen production. In the present study, accumulated evidence has provided insight into the mechanisms of mibolerone action that control this process. Investigations using the nonsteroidal antiandrogen hydroxyflutamide were conducted to characterize mibolerone's mode of action. Hydroxyflutamide had differential effects on hCG action, the 1-M dose stimulating hCG-induced androsterone synthesis by 27% and the 10-,M concentration decreasing the androgen levels by 84%. In addition, treatment with 1 LM hydroxyflutamide was effective in partially reversing the inhibitory action of mibolerone on hCG-stimulated androsterone production. Thus, the data indicated that mibolerone's mode of action may be mediated, at least in part, via the androgen receptor. The possibility that mibolerone had multiple sites of action prompted studies on the effectiveness of this androgen to alter various signaling pathways. Treatment with increasing concentrations (0.01-100 nM) of the phorbol ester 12-0-tetradecanoylphorbol 13-acetate (TPA), which activates protein kinase C, resulted in a 75% decrease in hCGstimulated androgen production at a dose of 100 nM TPA. Treatment with mibolerone (100 nM) was unable to alter the action 2+ of TPA on androgen synthesis when doses of 1 and 10 nM TPA were employed. It was also found that Ca can serve as a 2+ mediator of mibolerone action. Treatment with a 0.01-IM dose of A23187, a Ca ionophore known to increase intracellular Ca 2+ , was ineffective in altering hCG-stimulated androsterone synthesis. The concurrent treatment of mibolerone (100 nM) and A23187 (0.01 M) resulted in the potentiation of mibolerone's inhibitory effects on hCG-stimulated androgen production, thereby suggesting that mibolerone can stimulate Ca2+ influx. Additional studies revealed that the administration of a l-piM dose of the 2+ L-type Ca channel blocker verapamil to TIC cultures was able to partially block the inhibitory effect of mibolerone on androgen synthesis. Evidence for an additional site of mibolerone action was revealed through an analysis of the mRNA levels of P450,, and P450,,7 enzymes. Although hCG and insulin-like growth factor I treatment resulted in 20- and 32-fold increases in the amount of P450,, and P450,17 mRNA, respectively, the addition of mibolerone (100 nM) reduced only P450,, mRNA levels by 91%. Overall, the evidence indicates that mibolerone has multiple sites of action in exerting its regulatory effect on androgen synthesis. MECHANISMS FOR AUTOREGULATION IN THECAL-INTERSTITIAL CELLS and 4) to determine whether the action of mibolerone in inhibiting androgen synthesis in TIC is exerted by its effects on key rate-limiting steroidogenic enzymes, the cholesterol side-chain cleavage (P450scc) and the 17ot-hydroxylase/ C,17 201yase (P4501 7a) systems. MATERIALS AND METHODS Animals Immature female Sprague-Dawley rats hypophysectomized at 21 days of age were obtained from the Harlan Company (Madison, WI). The rats were maintained in temperature-controlled rooms on a 14L: 10OD cycle and provided with physiological saline and rat chow ad libitum. At four to five days posthypophysectomy, the rats were killed by decapitation, and the ovaries were removed and placed in Medium 199 (Sigma). The animal procedures used were approved by the institutional CAURE (Committee for Animal Use in Research and Education). Cell Dispersaland Enrichment of TIC Ovarian dispersates and enriched TIC were prepared according to the procedures described by Magoffin and Erickson [17,18]. Briefly, the ovaries were cleaned of nonovarian tissues and the surroundi (...truncated)