Tsai-Morris C H, Khanum A, Tang P Z, Dufau M L
Section on Molecular Endocrinology, Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-4510, USA.
Endocrinology. 1999 Aug;140(8):3534-42. doi: 10.1210/endo.140.8.6944.
17Beta-hydroxysteroid dehydrogenase (17betaHSD), the enzyme that catalyzes the final step of testosterone biosynthesis in the testis, was cloned from a rat Leydig cell complementary DNA library to gain insights into the functional requirements, activation mechanisms, and molecular regulation. The 17betaHSD complementary DNA encoded 306 amino acids (molecular mass of 33.7 kDa) and displayed 75% and 85% amino acid sequence homology to the human and mouse 17betaHSD type III enzymes, respectively. Northern analysis revealed a single 1.4-kb messenger RNA (mRNA) species in rat Leydig cells, whereas ovarian mRNA was detected only by RT-PCR amplification. The cloned 17betaHSD expressed in mammalian cell lines specifically catalyzed the reductive reaction in androgen formation with androstenedione as the preferred substrate. This reaction was significantly reduced in the absence of glucose. Expression of the endogenous 17betaHSD gene in rat Leydig cells was inhibited by a single dose of hCG in vivo, with maximum reduction of steady state mRNA levels at 24 h and recovery at 9 days. Such agonist-induced down-regulation of 17betaHSD expression, which preceded the marked reduction of LH receptors, resulted from changes at the transcriptional level and was accompanied by loss of enzymatic activity. These studies have demonstrated a glucose requirement for optimal activity of the enzyme in vitro and for a role of gonadotropin in regulating the expression of 17betaHSD gene in vivo. Cloning of the 17betaHSD type III enzyme from rat Leydig cells will facilitate further investigation of the molecular regulation of its activity in the testis.
17β-羟基类固醇脱氢酶（17βHSD）是催化睾丸中睾酮生物合成最后一步的酶，从大鼠睾丸间质细胞互补DNA文库中克隆该酶，以深入了解其功能需求、激活机制和分子调控。17βHSD互补DNA编码306个氨基酸（分子量为33.7 kDa），与人及小鼠Ⅲ型17βHSD的氨基酸序列同源性分别为75%和85%。Northern分析显示大鼠睾丸间质细胞中有单一的1.4 kb信使核糖核酸（mRNA），而卵巢mRNA仅通过逆转录聚合酶链反应（RT-PCR）扩增检测到。在哺乳动物细胞系中表达的克隆17βHSD以雄烯二酮为首选底物，特异性催化雄激素形成中的还原反应。在无葡萄糖的情况下，该反应显著减少。体内单次注射人绒毛膜促性腺激素（hCG）可抑制大鼠睾丸间质细胞中内源性17βHSD基因的表达，在24小时时稳态mRNA水平最大程度降低，9天时恢复。这种激动剂诱导的17βHSD表达下调先于促黄体生成素（LH）受体的显著减少，是由转录水平的变化引起的，并伴有酶活性丧失。这些研究证明了体外该酶最佳活性对葡萄糖的需求以及促性腺激素在体内调节17βHSD基因表达中的作用。从大鼠睾丸间质细胞克隆Ⅲ型17βHSD酶将有助于进一步研究其在睾丸中活性的分子调控。
