Molecular mechanisms of oocyte and sperm maturation-inducing hormone

本研究旨在探讨卵母细胞和精子成熟诱导激素(17α，20β-二羟基-4-孕烯-3-酮)在鱼性腺中的分子机制。17α，20β-二羟基-4-孕烯-3-酮是由17α-羟孕酮在类固醇生成酶20β-羟基类固醇脱氢酶(20β-HSD)的作用下生物合成的。我们首先分离了编码AYU(Plecoglossus Altivelis)和Medaka(Oryzias Latipe)20β-HSD的cDNA克隆，并以AYU 20β-HSD为探针，从虹鱼(Oncorhynchus MykISS)的卵泡中分离到两个不同的20β-HSD样基因。CDNA和基因组DNA分析表明，这两个20β-HSD编码在两个不同的基因中。Northern印迹分析和RT-PCR结果表明，这两种基因在不同组织中均有表达。这两个cDNA在大肠杆菌中的体外表达使我们能够确定两个cDNA中的一个(A型)的产物具有将17α-羟孕酮转化为17α，20β-二羟基-4-孕烯-3-酮的能力。黄体后卵泡颗粒细胞与促性腺激素孵育后，20β-HSD基因表达明显增强。此外，值得注意的是，促性腺激素刺激可以在颗粒细胞中诱导A型基因，而不是B型基因。由于20β-HSD A型具有20β-HSD活性，20β-HSD A型基因的增强表达增加了17α-羟孕酮向17α，20β-二羟基-4-孕烯-3-酮的转化。这些数据表明，20β-HSD的表达受促性腺激素的调控。促性腺激素对20β-HSD基因启动子区域的影响将有助于进一步了解类固醇和蛋白质激素对类固醇合成酶的调节作用。

This study was designed to investigate the molecular mechanism of oocyte and sperm-maturation inducing hormone (l7alpha, 20beta-dihydroxy-4-pregnen-3-one) in fish gonads. l7alpha, 20beta-Dihydroxy-4-pregnen-3-one is biosynthesized by the action of a steroidogenic enzyme, 20beta-hydroxysteroid dehydrogenase (20beta-HSD) from 17alpha-hydroxyprogesterone. We first isolated cDNA clones encoding ayu (Plecoglossus altivelis) and medaka (Oryzias latipes) 20beta-HSD.Using the ayu 20beta-HSD cDNA clone as a probe, we isolated two different 2Obeta-HSD like cDNAs from rainbow trout (Oncorhynchus mykiss) ovarian follicles. cDNA and genomic DNA analysis show that the two 20beta-HSDs are encoded in two different genes. Northern blot analysis and RT-PCR show that these two are expressed in different tissues. In vitro expression of the two cDNAs in E.colienabled us to establish that the product of one (type A) of the two cDNAs has the ability to convert 17alpha-hydroxyprogesterone to 17alpha, 2Obeta-dihydroxy-4-pregnen-3-one. 20beta-HSD gene expression was markedly enhanced when granulosa cells isolated from postvitellogenic follicles were incubated with gonadotropin. Furthermore, it is of importance to note that type A gene, but not type B gene, is inducible in granulosa cells by gonadotropin stimulation. Since 20beta-HSD type A exhibits 20beta-HSD activity, the enhanced expression of 20beta-HSD type A gene increases the conversion of 17alpha-hydroxyprogesterone to 17alpha, 2Obeta-dihydroxy-4-pregnen-3-one. These data demonstrate that the expression of 20beta-HSD is controlled by gonadotropin. The effects of gonadotropin on the promoter regions of 20beta-HSD genes will provide further understanding of steroid and protein hormone modulation of steroidogenic enzymes.