Expression of testicular 3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase: regulation by luteinizing hormone and forskolin in Leydig cells of adult rats.
LH is required to maintain the activity of 3 beta-hydroxysteriod 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-3H]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-[35S]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 microM) 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 microM forskolin, 1 ng/ml cholera toxin, and 1 mM (Bu)2cAMP.(ABSTRACT TRUNCATED AT 400 WORDS)
Keeney
D S
DS
Cecil H. and Ida Green Center for Reproductive Biology Sciences, Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235-9051.
Mason
J I
JI
D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology*; D007985:Leydig Cells / Q000187:drug effects / Q000201:enzymology*; D007986:Luteinizing Hormone / Q000494:pharmacology*; D008297:Male; D009097:Multienzyme Complexes / Q000032:analysis* / Q000235:genetics; D011375:Progesterone Reductase / Q000032:analysis* / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011919:Rats, Inbred Strains; D013251:Steroid Isomerases / Q000032:analysis* / Q000235:genetics
C021425:3 beta-hydroxysteroid oxidoreductase-delta(5) 3-ketosteroid isomerase; D009097:Multienzyme Complexes; D012333:RNA, Messenger; D005576:Colforsin; D003994:Bucladesine; D007986:Luteinizing Hormone; D011375:Progesterone Reductase; D013251:Steroid Isomerases