Molecular mechanisms of gamete maturation-inducing hormone synthesis

为了研究促性腺激素诱导鱼类合成配子成熟诱导激素（17α， 20β-二羟基-4-妊娠-3- 1,17 α, 20β-DP）的基因调控机制，我们分析了虹鳟鱼卵泡中分离的20β-羟基类固醇脱氢酶（20β-HSD，将17α-羟基孕酮转化为17α， 20β-DP）基因。两个密切相关的20 β- hsd cdna编码两种蛋白，只有三个氨基酸差异。在大肠杆菌中表达的重组蛋白活性分析表明，1型重组蛋白具有20β-HSD酶活性。而另一株（tyae II）不具有20β-HSD活性。Northern blot分析显示，在卵母细胞最终成熟过程中，颗粒细胞中有明显的20 β- hsd基因表达。I型基因的5'侧区（- 750bp）包含多个顺式元件基序，包括GC-rich区域、CRE、Ad4位点，并存在两个TATA-box样序列。相反，II型基因不具有这些顺式元件基序或TATA-box至少高达-750 bp。荧光素酶活性比较分析显示，启动子活性仅在I型20β-HSD基因中存在。对一系列5′缺失突变体的分析表明，I型20β-HSD基因的启动子活性与5′侧碱基-477 ~ -467（假定的TATA-box）和-50 ~ -30（具有GC-rich区域的功能性CRE）有关。Ad4一致基序未显示任何启动子活性。在本研究中，我们还首次在鱼类中克隆并鉴定了来自虹鳟和罗非鱼卵巢的两种不同的促性腺激素受体。时间和空间表达模式表明，LH受体参与了促性腺激素（LH）诱导的20 β- hsd基因在这两种硬骨鱼雌雄配子最终成熟过程中的转录。

To investigate the gene regulatory mechanisms of gonadotropin-induced synthesis of gamete maturation-inducing hormone (17α, 20β-dihydroxy-4-pregnen-3-one, 17α, 2Oβ-DP) in fish, we have analyzed the 20β-hydroxysteroid dehydrogenase (20β-HSD, the enzyme which converts 17α-hydroxyprogesterone to 17α, 20β-DP) genes isolated from rainbow trout follicles. Two closely related 2Oβ-HSD cDNAs which encode two proteins with only three amino acid differences were isolated. Analysis of activity of recombinant proteins resulting from expression in E.coli showed that one type I had 20β-HSD enzyme activitv. but the other (tyae II) did not possess 20β-HSD activity. Northern blot analysis showed a marked 2Oβ-HSD gene expression in granulosa cells during final oocyte maturation.The 5' flanking region (-750 bp) of the type I gene contained multiple cis-element motifs including GC-rich region, CRE, Ad4 sites with the presence of two TATA-box like sequences. Conversely, the type II gene did not possess either such cis-element motifs or TATA-box at least up to -750 bp. Comparative analysis of luciferase activity revealed the presence of promoter activity only in type I 20β-HSD gene. Analysis of series of 5'-deletion mutants showed that the 5'-flanking bases -477 to -467 (putative TATA-box) and -50 to -30 (a functional CRE with GC-rich region) are responsible for the promoter activity in type I 20β-HSD gene. Ad4 consensus motif did not show any promoter activity. In this study, we have also cloned and characterized, for the first time in fish, two different gonadotropin receptors from rainbow trout and tilapia ovaries. Temporal and spatial expression patterns suggest that LH receptors are involved in the gonadotropin (LH)-induced transcription of 2Oβ-HSD gene during final maturation of female and male gametes in these two teleost species.