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

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... 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 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 mim- Materials and Methods Animals and reagents Eight-week-old mature CD-1 male mice [Cr1:CD-1(ICR)BR] were obtained from Charles River Laboratories, Inc. (Wilmington, MA). These mice were used for Leydig cell isolation, usually within a week of being received. The androgen-insensitive testis-feminized mice (Tfm) were