Gonadotropin-induced receptor regulation and steroidogenic lesions in cultured Leydig cells. Induction of specific protein synthesis by chorionic gonadotropin and estradiol.

Abstract

Cultured Leydig cells were employed for in vitro studies on gonadotropin-induced regulation of luteinizing hormone (LH) receptors and desensitization of steroidogenic responses, and to analyze the role of estradiol in the development of enzymatic lesions that impair the conversion of progesterone to androgen. After 24 h in culture, adult rat Leydig cells retained 60% of their LH receptors and showed no change in maximal testosterone response to human chorionic gonadotropin (hCG). Such acutely cultured cells were incubated for 24 h with human chorionic gonadotropin or ovine LH (oLH), and then analyzed for LH binding sites and steroidogenic responses. LH receptors were increased by 1 ng of hCG or oLH to 153% and 131% of control, and were reduced at higher doses to 57% and 2% (by 10 and 100 ng of hCG) or to 70% and 44% (by 100 and 1000 ng of oLH). No change in LH receptors was seen in cells stimulated with dibutyryl cyclic AMP. The “Iate” enzyme lesion in 17a-hydroxylase and 17,220-desmolase activities was manifested at 1 to 10 ng of hCG and 10 to 1000 ng of oLH by increases in progesterone and 17u-hydroxyprogesterone, and was more marked after hCG treatment. The highest hCG dose (100 ng) also caused an “early” steroidogenic lesion prior to pregnenolone, previously observed in vivo and found to be unrelated to estrogen. This lesion was also observed after treatment of cultured cells with 0.5 to 1 mM dibutyryl cyclic AMP. The late steroidogenic lesion in cultured cells was prevented by Tamoxifen, added 20 min before hCG and present throughout the incubation. Incubation with 1 to 100 ng of estradiol for 24 h caused a dose-dependent decrease in testosterone production, with concomitant accumulation of 17a-hydroxyprogesterone and progesterone. These estrogen-induced changes, like those produced by chorionic gonadotropin, were prevented by addition of Tamoxifen. Exposure of Leydig cells to estradiol for 6 to 12 h was required to cause the steroidogenic lesions. The estrogen-dependent enzymatic defect was preceded by synthesis of a specific protein of M, 27,000, which was induced by estradiol or hCG, and was inhibited by Tamoxifen. These studies have defined an in vitro system suitable for the investigation of gonadotropin-dependent regulatory mechanisms. The increase and decrease in LH receptors produced by low and high doses * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Present address, Research Laboratories of Kakenyaku, Yamashha-ku, Kyoto, Japan. 8 To whom requests for reprints should be addressed. of gonadotropin correspond to the early up-regulation and subsequent down-regulation of testicular sites observed in vivo after treatment with LH and hCG. Modulation of the late steroidogenic enzymes by endogenous estradiol contributes to the regulation of androgen production, and could be exerted through synthesis of estrogen-dependent proteins of the Leydig cell.