Molecular mechanisms of gamete maturation-inducing hormone synthesis

配子成熟诱导激素合成的分子机制

• 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
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10440247/
• 关键词: rainbow trout; gamete maturation-inducing hormone; oocyte maturation; 17α, 20β-dihydroxy-4-pregnen-3-one; ovary; cDNA; 20β-HSD; gonadotropin; 17α、20β-ジヒドロキ-4-プレグネン]-3-オン; 生殖腺刺激ホルモン; 17α,20β-ジヒドロキシ-4-プレクネン-3-オン

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.

为了研究鱼类中促性腺激素诱导配子成熟诱导激素（17α，20β-二羟基-4-pregnen-3-one，17α，2Oβ-DP）合成的基因调控机制，我们分析了20β-羟基类固醇脱氢酶（20β-HSD，该酶将 从虹鳟鱼卵泡中分离出 17α-羟基孕酮至 17α, 20β-DP) 基因。分离出两个密切相关的 2Oβ-HSD cDNA，它们编码两种仅具有三个氨基酸差异的蛋白质。对大肠杆菌中表达的重组蛋白的活性分析表明，一种 I 型蛋白具有 20β-HSD 酶活性。但另一个（tyae II）不具有 20β-HSD 活性。 Northern印迹分析显示，在最终卵母细胞成熟过程中，颗粒细胞中有显着的2Oβ-HSD基因表达。I型基因的5'侧翼区域（-750 bp）包含多个顺式元件基序，包括富含GC的区域、CRE、Ad4位点，并存在两个TATA盒样序列。相反，II型基因至少不具有-750bp的顺式元件基序或TATA盒。荧光素酶活性的比较分析揭示仅在I型20β-HSD基因中存在启动子活性。对一系列 5'-缺失突变体的分析表明，5'-侧翼碱基 -477 至 -467（推定的 TATA-box）和 -50 至 -30（具有 GC 丰富区域的功能性 CRE）负责 I 型 20β-HSD 基因中的启动子活性。 Ad4共有基序没有显示任何启动子活性。在这项研究中，我们还首次在鱼类中克隆并鉴定了来自虹鳟鱼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，在这两种硬骨鱼雌性和雄性配子的最终成熟过程中，LH 受体参与促性腺激素 (LH) 诱导的 2Oβ-HSD 基因转录。

项目成果

Guan, G. et al.: "Isoleucine-15 rainbow trout carbonyl reductase-like 20β-hydmxysteroid dehydrogenase is crucial for co-enzyme"Proceedings of Nation Academy of Science, USA. (印刷中). (2000)

guan, G. 等人：“异亮氨酸 15 虹鳟鱼羰基还原酶样 20β-羟基类固醇脱氢酶对于辅酶至关重要”，美国国家科学院院刊（2000 年出版）。

Katsu, Y., Yamashita, M.and Nagahama, Y.: "Translational regulation of cyclin B mRNA during 17α, 20β-dihydroxy-4-pregnen-3-one (maturation-inducing hormone)-induced oocyte maturation in goldfish, Carassius auratus."Mol. Cell. Endocrinol.. 158. 79-85 (1999

Katsu, Y.、Yamashita, M. 和 Nagahama, Y.：“17α、20β-二羟基-4-pregnen-3-one（成熟诱导激素）诱导金鱼、鲫鱼卵母细胞成熟过程中细胞周期蛋白 B mRNA 的翻译调节auratus.“分子细胞内分泌学.. 158. 79-85 (1999

Kajiura-Kobayashi, H., Yoshida, N., Sagata, N., Yamashita, M.and Nagahama, Y.: "The Mos/MAPK pathway is involved in metaphase II arresst as a cytostatic factor but is neither necessary nor sufficient for initiating oocyte maturation in goldfish."Dev. Gene

Kajiura-Kobayashi, H.、Yoshida, N.、Sagata, N.、Yamashita, M. 和 Nagahama, Y.：“Mos/MAPK 通路作为细胞抑制因子参与中期 II 阻滞，但对于

Oba, Y., Hirai, T., Yoshiura, Y., Kobayashi, T.and Nagahama, Y.: "Cloning, functional characterization, and expression of thyrotropin receptors in the thyroid of amago salmon (Oncorhynchus rhodurus)."Biochem. Biophys. Res. Comm.. 276. 258-263 (2000)

Oba, Y.、Hirai, T.、Yoshiura, Y.、Kobayashi, T. 和 Nagahama, Y.：“Amago 鲑鱼 (Oncorhynchus rhodurus) 甲状腺中促甲状腺激素受体的克隆、功能表征和表达。”Biochem。

Kajiura-Kobayashi,H. et al.: "The MOS/MAPK pathway is involved in metaphase II arrest as a cytostatic factor but is neither necessary nor sufficient for initiating oocyte maturation in goldfish"Development, Genes and Evolution. 210. 416-425 (2000)

梶浦小林，H.