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

Estrogen sulfotransferase (EST) catalyzes the specific sulfonation and inac tivation of estrogens. A common site for EST expression in mammalian specie s is the testicular Leydig cells. In previous in vivo studies, we have show n that testicular expression of EST is under the regulation of LH. Thus, ES T expression in mouse Leydig cells was abolished by hypophysectomy, but cou ld be restored by hCG injection. In this study, we have evaluated the downs tream mechanisms by which LH exerts its regulatory effect on EST. Primary m ouse Leydig cells were isolated and purified by collagenase digestion and P ercoll density gradient centrifugation. They were cultured in serum-free me dium 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 expressio n, treatment of primary mouse Leydig cells in vitro with 100 mu M 8-bromo-d ibutyryl cAMP [(Bu)(2)cAMP] increased EST expression 3- to 5-fold. The effe ct of(Bu)(2)cAMP was attenuated by the steroidogenesis inhibitor aminoglute thimide and was mimicked by the potent androgen 5 alpha-dihydrotestosterone (5-DHT). The activity of B-DHT in stimulating EST expression was blocked b y 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 t o suppress Leydig cell steroidogenesis by downregulating P450c17 gene expre ssion. Treatment of Leydig cells with 0.2 ng/ml interleukin-1 beta inhibite d (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 expr ession is completely absent, whereas messenger RNAs of steroidogenic enzyme s such as P450c17 and 3 beta-hydroxysteroid dehydrogenase are relatively ab undant. We conclude that, by acting as an autocrine or paracrine factor, an drogen plays an essential role in the regulation of estrogen sulfotransfera se expression in Leydig cell by LH and cAMP.