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 dehyd rogenase/DELTA(5 --> 4)-isomerase (3-beta-HSD) in testicular Leydig ce lls. The objective of the present study was to determine whether LH an d effectors such as forskolin, which act via the intracellular cAMP si gnal transduction pathway, can regulate the expression of 3-beta-HSD i n rat Leydig cells in vitro. Primary cultures of Leydig cells were pre pared from testes of adult rats and treated with oLH, forskolin, (Bu)2 cAMP, or cholera toxin. The effects of treatment on 3-beta-HSD activit y were measured using [3-alpha-H-3]dehydroepiandrosterone as substrate . Immunoreactive 3-beta-HSD was quantified by denaturing sodium dodecy l 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 electro phoresis of immunoprecipitated cellular lysates of Leydig cells radiol abeled with L-[S-35]methionine. The levels of 3-beta-HSD mRNA were qua ntified by Northern analysis and hybridization with a cDNA encoding te sticular 3-beta-HSD (rat type I). A cell-free protein-synthesizing sys tem was used to test the ability of 3-beta-HSD mRNA to be translated i nto 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 t reatment. The increase in 3-beta-HSD activity could be accounted for q uantitatively 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 forskoli n and (Bu)2cAMP, respectively. Moreover, both of these effectors incre ased 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)2cAM P 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, com pared 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 l evels of 3-beta-HSD mRNA after 12 h, and these levels were still eleva ted 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-bet a-HSD, and 3-beta-HSD mRNA in response to effectors that act through t he intracellular cAMP signal transduction pathway demonstrate that thi s 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-HS D, which could be readily translated into immunoreactive 3-beta-HSD in vitro, implies that the expression of 3-beta-HSD is regulated at leas t in part at the level of gene transcription via a cAMP-dependent sign alling pathway.