EXPRESSION OF TESTICULAR 3-BETA-HYDROXYSTEROID DEHYDROGENASE DELTA-S-]4-ISOMERASE - REGULATION BY LUTEINIZING-HORMONE AND FORSKOLIN IN LEYDIG-CELLS OF ADULT-RATS

LH is required to maintain the activity of 3-beta-hydroxysteroid dehydrogenase/DELTA(5 --> 4)-isomerase (3-beta-HSD) in testicular Leydig cells. The objective of the present study was to determine whether LH and effectors such as forskolin, which act via the intracellular cAMP signal transduction pathway, can regulate the expression of 3-beta-HSD in rat Leydig cells in vitro. Primary cultures of Leydig cells were prepared from testes of adult rats and treated with oLH, forskolin, (Bu)2cAMP, or cholera toxin. The effects of treatment on 3-beta-HSD activity were measured using [3-alpha-H-3]dehydroepiandrosterone as substrate. Immunoreactive 3-beta-HSD was quantified by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with a polyclonal antiserum against 3-beta-HSD. The synthesis of 3-beta-HSD was quantified after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitated cellular lysates of Leydig cells radiolabeled with L-[S-35]methionine. The levels of 3-beta-HSD mRNA were quantified by Northern analysis and hybridization with a cDNA encoding testicular 3-beta-HSD (rat type I). A cell-free protein-synthesizing system was used to test the ability of 3-beta-HSD mRNA to be translated into immunoreactive 3-beta-HSD. 3-beta-HSD activity increased 3.5- and 5.0-fold in Leydig cell cultures treated with forskolin (1-mu-M) and (Bu)2cAMP (1 mM), respectively, compared with control cultures. Maximal activity was attained after 48-72 h and maintained through 120 h of treatment. The increase in 3-beta-HSD activity could be accounted for quantitatively by increases in the steady state levels and the rates of synthesis of 3-beta-HSD. The cellular levels of immunoreactive 3-beta-HSD increased 4.0- and 7.6-fold in Leydig cells treated with forskolin and (Bu)2cAMP, respectively. Moreover, both of these effectors increased by 6- to 8-fold the levels of newly synthesized 3-beta-HSD after 24-72 h of treatment. Ovine LH, forskolin, cholera toxin, and (Bu)2cAMP increased the cellular levels of 3-beta-HSD mRNA in a dose-dependent manner. The magnitude of the increases ranged from 2- to 42-fold, compared with that in control cultures, after 12 h of treatment. Maximal responses were effected by 1 ng/ml ovine LH, 1-mu-M forskolin, 1 ng/ml cholera toxin, and 1 mM (Bu)2cAMP. Forskolin increased (5-fold) the levels of 3-beta-HSD mRNA after 12 h, and these levels were still elevated after 24 h (4-fold) and 40 h (5-fold) of treatment. When RNA from these forskolin-treated cells was translated in vitro, a 2- to 3-fold increase in immunoreactive 3-beta-HSD was observed. The increases in 3-beta-HSD activity, immunoreactive 3-beta-HSD, newly synthesized 3-beta-HSD, and 3-beta-HSD mRNA in response to effectors that act through the intracellular cAMP signal transduction pathway demonstrate that this pathway can regulate the expression of 3-beta-HSD in Leydig cells of adult rats in vitro. The increase in cellular mRNA encoding 3-beta-HSD, which could be readily translated into immunoreactive 3-beta-HSD in vitro, implies that the expression of 3-beta-HSD is regulated at least in part at the level of gene transcription via a cAMP-dependent signalling pathway.