Regulation of estrogen sulfotransferase expression in Leydig cells by cyclic adenosine 3′,5′-monophosphate and androgen

Estrogen sulfotransferase (EST) catalyzes the specific sulfonation and inactivation of estrogens. A common site for EST expression in mammalian species is the testicular Leydig cells. In previous in vivo studies, we have shown that testicular expression of EST is under the regulation of LH. Thus, EST expression in mouse Leydig cells was abolished by hypophysectomy, but could be restored by hCG injection. In this study, we have evaluated the downstream mechanisms by which LH exerts its regulatory effect on EST. Primary mouse Leydig cells were isolated and purified by collagenase digestion and Percoll density gradient centrifugation. They were cultured in serum-free medium at 32 C and treated with various agents for 24 or 48 h, and levels of EST messenger RNA and enzyme activity were determined. Consistent with the in vivo data suggesting an essential role of LH in regulating EST expression, treatment of primary mouse Leydig cells in vitro with 100 mu M 8-bromo-dibutyryl cAMP [(Bu)(2)cAMP] increased EST expression 3- to 5-fold. The effect of(Bu)(2)cAMP was attenuated by the steroidogenesis inhibitor aminoglutethimide and was mimicked by the potent androgen 5 alpha-dihydrotestosterone (5-DHT). The activity of B-DHT in stimulating EST expression was blocked by the androgen receptor antagonist, hydroxyflutamide. These data suggested the involvement of androgen in (Bu)(2)cAMP-induced EST expression. Further evidence came from the study with interleukin-1 beta, another agent known to suppress Leydig cell steroidogenesis by downregulating P450c17 gene expression. Treatment of Leydig cells with 0.2 ng/ml interleukin-1 beta inhibited (Bu)(2)cAMP-induced EST expression, which was overcome by the addition of 5-DHT. Finally, in the testis-feminized mouse (Tfm) in which the androgen receptor is nonfunctional due to a frameshift mutation, testicular EST expression is completely absent, whereas messenger RNAs of steroidogenic enzymes such as P450c17 and 3 beta-hydroxysteroid dehydrogenase are relatively abundant. We conclude that, by acting as an autocrine or paracrine factor, androgen plays an essential role in the regulation of estrogen sulfotransferase expression in Leydig cell by LH and cAMP.