Keeney D S, Mason J I
Cecil H. and Ida Green Center for Reproductive Biology Sciences, Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235-9051.
Endocrinology. 1992 Apr;130(4):2007-15. doi: 10.1210/endo.130.4.1312436.
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)
黄体生成素（LH）是维持睾丸间质细胞中3β-羟基类固醇脱氢酶/δ5-δ4异构酶（3βHSD）活性所必需的。本研究的目的是确定LH以及通过细胞内cAMP信号转导途径起作用的效应物，如福斯可林，是否能在体外调节大鼠间质细胞中3βHSD的表达。从成年大鼠睾丸制备间质细胞原代培养物，并用促黄体生成素（oLH）、福斯可林、二丁酰环磷腺苷（Bu）2cAMP或霍乱毒素进行处理。以[3α-3H]脱氢表雄酮为底物，测定处理对3βHSD活性的影响。通过变性十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和用抗3βHSD的多克隆抗血清进行免疫印迹，对免疫反应性3βHSD进行定量。在用L-[35S]甲硫氨酸进行放射性标记的间质细胞免疫沉淀细胞裂解物的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳后，对3βHSD的合成进行定量。通过Northern分析和与编码睾丸3βHSD（大鼠I型）的cDNA杂交，对3βHSD mRNA水平进行定量。使用无细胞蛋白质合成系统来测试3βHSD mRNA翻译成免疫反应性3βHSD的能力。与对照培养物相比，用福斯可林（1μM）和（Bu）2cAMP（1 mM）处理的间质细胞培养物中，3βHSD活性分别增加了3.5倍和5.0倍。48 - 72小时后达到最大活性，并在处理120小时内维持。3βHSD活性的增加可以通过3βHSD稳态水平和合成速率的增加来定量解释。在用福斯可林和（Bu）2cAMP处理的间质细胞中，免疫反应性3βHSD的细胞水平分别增加了4.0倍和7.6倍。此外，这两种效应物在处理24 - 72小时后，使新合成的3βHSD水平增加了6 - 8倍。绵羊LH、福斯可林、霍乱毒素和（Bu）2cAMP以剂量依赖性方式增加了3βHSD mRNA的细胞水平。处理12小时后，与对照培养物相比，增加幅度在2至42倍之间。最大反应由1 ng/ml绵羊LH、1μM福斯可林、1 ng/ml霍乱毒素和1 mM（Bu）2cAMP引起。（摘要截断于400字）
