Molecular cloning of a membrane receptor of maturation-inducing steroid hormone from fish oocytes

鱼卵母细胞成熟诱导类固醇激素膜受体的分子克隆

• 批准号: 09640798
• 负责人: YOSHIKUNI Michiyasu
• 金额: $ 2.18万
• 依托单位: Okazaki National Research Institutes
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09640798/
• 关键词: oocyte maturation; steroid hormone; hormone receptor; GTP-binding protein; Oryzias latipes; goldfish; G蛋白質

Fish oocyte maturation is induced by maturation-inducing hormone, 17alpha, 20beta-dihydroxy-4-pregnen-3-one (l7alpha, 2Obeta-DP) acting upon an oocyte surface receptor.We evidenced that a pertussis toxin-sensitive inhibitory GTP-binding protein (Gi) was involved in the signal transduction pathway of l7alpha, 2Obeta-DP in rainbow trout oocytes.We cloned two Gialpha subunit cDNAs, Gialpha1 and Gialpha2, from Medaka (Oryzias latipes) oocyte cDNA library. Three rabbit polyclonal antibodies, A2, C4, and M1, were generated against deduced internal amino acid sequences of Gialpha1 and Gialpha2, and a common C-terminal sequence, respectively.Ml antibody successfully immunoprecipitated Gialphal and Gialpha2 subunit proteins.However, l7alpha, 2Obeta-DP-binding acitivity was almost not detected in the immunoprecipitate.l7alpha, 2Obeta-DP receptor activity was solubilized from membrane preparations of goldfish oocytes by sucrose monolaurate. Solubilized activity was serially separated by following four-step chromatography ; gel-filtration, anion-exchange, hydrophobic interaction, and lectin affinity chromatography.Major seven proteineous bands on SDS-PAGE were detected by silver staining.In medaka (Oryzias latipes) oocytes, the amount of l7alpha, 2Obeta-DP binding to the oocyte cortices was shown to increase with maturational competence of oocytes and reach a maximum one day before spawning.When oocyte maturation was triggered with l7alpha, 2Obeta-DP, 17alpha, 2Obeta-DP binding and Gi decreased quickly, suggesting a close relation between Gi and the l7alpha, 2Obeta-DP receptor.

鱼卵母细胞成熟是由成熟诱导激素 17α, 20β-二羟基-4-pregnen-3-one (l7α, 2Obeta-DP) 作用于卵母细胞表面受体诱导的。我们证明百日咳毒素敏感的抑制性 GTP 结合Gialpha蛋白（Gi）参与虹鳟鱼卵母细胞l7α、2Obeta-DP的信号转导通路。我们克隆了两个Gialpha亚基cDNA、Gialpha1 和 Gialpha2，来自青鳉 (Oryzias latipes) 卵母细胞 cDNA 文库。分别针对推导的 Gialpha1 和 Gialpha2 的内部氨基酸序列以及共同的 C 端序列生成三种兔多克隆抗体 A2、C4 和 M1。M1 抗体成功地免疫沉淀 Gialpha1 和 Gialpha2 亚基蛋白。然而，l7alpha、2Obeta -在免疫沉淀物中几乎未检测到DP结合活性。17α、2Oβ-DP受体活性从膜制剂中溶解蔗糖单月桂酸酯对金鱼卵母细胞的影响。通过四步色谱法连续分离溶解的活性物质；凝胶过滤、阴离子交换、疏水作用和凝集素亲和层析。银染法检测 SDS-PAGE 上主要 7 个蛋白条带。在青鳉 (Oryzias latipes) 卵母细胞中，l7α、2Obeta-DP 与卵母细胞结合的量研究表明，皮质随着卵母细胞的成熟能力而增加，并在产卵前一天达到最大值。当用l7α、2Obeta-DP触发卵母细胞成熟时， 17α、2Oβ-DP结合和Gi迅速下降，表明Gi与17α、2Oβ-DP受体之间存在密切关系。

项目成果

Yoshikuni M.: "Apertussis toxin sensitive GTP-binding protein is involved in the signal truns duction pathway of the maturation-inducinghormone(17α,20β-dihydroxy-4-pregnene-3-one)in fist oocytes." Steroids. (印刷中). (1999)

Yoshikuni M.：“百日咳毒素敏感的 GTP 结合蛋白参与拳头类固醇中成熟诱导激素 (17α,20β-二羟基-4-孕烯-3-一) 的信号传导途径。”按））（1999）。

Yuichi Oba: "Inhibitory guanine-nucleotide-binding-regulatory protein α subunits in Medaka oocytes. cDNAcloning and decreased expression of proteinscturing oocyte maturation." European Journal of Biochemistry. 249. 846-853 (1997)

Yuichi Oba：“青鳉卵母细胞中的抑制性鸟嘌呤核苷酸结合调节蛋白 α 亚基。cDNA 克隆和卵母细胞成熟蛋白表达的降低。欧洲生物化学杂志 249。846-853 (1997)。

Yoshikuni M.: "A PERTUSSIS TOXIN SENSITIVE GTP-BINDING PROTEIN IS INVOLVED IN THE SIGNAL TRANSDUCTION PATHWAY OF THE MATURATION-INDUCING HORMONE (17α,20β-DIHYDROXY-4-PREGNEN-3-ONE)IN FISH OOCYTES" Steroids. (印刷中). (1999)

Yoshikuni M.：“一种百日咳毒素敏感的 GTP 结合蛋白参与鱼卵母细胞中成熟诱导激素 (17α,20β-二羟基-4-PREGNEN-3-ONE) 的信号转导途径”（类固醇）。 1999）

Yuichi Oba: "Inhibitory guanine-nucleotide-binding-regulortory protein α subunits in Medaka oocytes.cDNA cloning and decreased expression of proteins during oocyte maturation." European Journal of Biochemistry. 249. 846-853 (1997)

Yuichi Oba：“青鳉卵母细胞中的抑制性鸟嘌呤核苷酸结合调节蛋白 α 亚基。卵母细胞成熟过程中的 cDNA 克隆和蛋白质表达降低。” 249. 846-853 (1997)。