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Molecular mechanisms of gamete maturation-inducing hormone synthesis

配子成熟诱导激素合成的分子机制

基本信息

批准号：
10440247
负责人：
NAGAHAMA Yoshitaka
金额：
$ 7.55万
依托单位：
Okazaki National Research Institutes, National Institute for Basic Biology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

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.

为了研究促性腺激素诱导鱼类配子成熟诱导激素（17α，20β-dihydroxy-4-diperene-3-one，17α，20 β-DP）合成的基因调控机制，我们分析了虹鳟鱼卵泡中20β-羟类固醇脱氢酶（20β-HSD，the enzyme which converts 17α-hydroxyprogesterone to 17α，20β-DP）基因。分离到两个高度相关的2 O β-HSD cDNA，它们编码两种蛋白质，只有三个氨基酸的差异。对重组蛋白的活性分析表明，Ⅰ型重组蛋白具有20种β-HSD酶活性。而另一株（tyae Ⅱ）则不具有20β-HSD活性。北方杂交结果显示，2 O β-HSD基因在卵母细胞成熟过程中有明显的表达，Ⅰ型基因5'侧翼区（~ 750 bp）含有多个顺式元件，包括GC富集区、CRE、Ad 4位点和两个TATA盒样序列。相反，II型基因不具有至少高达~ 750 bp的这种顺式元件基序或TATA盒。荧光素酶活性的比较分析表明，只有I型20β-HSD基因存在启动子活性。对一系列5 ′端缺失突变体的分析表明，Ⅰ型20β-HSD基因启动子的活性主要由5 ′端侧翼的-477 ~-467（TATA盒）和-50 ~-30（富含GC的功能性CRE）决定。Ad 4共有基序没有显示任何启动子活性。在这项研究中，我们还克隆和特点，第一次在鱼，两种不同的促性腺激素受体从虹鳟鱼和罗非鱼卵巢。LH受体的时空表达模式表明，在这两种硬骨鱼的雌、雄配子最终成熟过程中，LH受体参与了促性腺激素（LH）诱导的2 O β-HSD基因的转录。