Study of gonad-stimulating substance (GSS) and 1-methyladenine which are related to starfish oocyte maturation

1. 糖蛋白激素受体家族的cDNA克隆利用从哺乳动物糖蛋白激素（LH， FSH和TSH）受体的一致序列衍生的退化引物，从海星Asterina pectinifera的卵巢中克隆了cDNA （apGPHR）。然而，部分纯化的性腺刺激物质（GSS）未能增加表达apGPHR的COS-7细胞中的环AMP水平。这表明除了GSS外，还有一种新的神经肽是海星apGPHR的配体。1-甲基腺苷的生物合成及其调控先前的体外研究表明，1-甲基腺苷（1-MeA）是由ATP和s -腺苷蛋氨酸（SAM）合成的。在使用缓冲溶液均质的滤泡细胞的无细胞系统中，在环状AMP存在的情况下，ATP和SAM也以浓度依赖的方式产生1-MeA。可能是一种独特的甲基转移酶被环AMP激活产生1-MeA，尽管尚不清楚ATP是被sam直接甲基化还是间接甲基化。新合成了10个n1取代腺嘌呤作为1-MeA的类似物，用于分析卵母细胞表面1-MeA与受体的相互作用。这表明腺嘌呤n1位点的区域特异性结构对于与1-MeA受体相互作用很重要。此外，在分子结构上，腺嘌呤n1位点的负电荷是受体结合所必需的。其他：卵泡细胞表面存在高活性的外atp二磷酸水解酶（atpase）。外腺atpase不太可能直接参与1-MeA的产生，但外腺atpase产生的腺苷和AMP负责预防卵母细胞成熟阶段前的早熟。

1. cDNA cloning of glycoprotein-hormone receptor familyUsing degenerate primers derived from consensus sequences of the memmalian glycoprotein hormone (LH, FSH and TSH) receptors, we cloned cDNAs (apGPHR) from ovaries of the starfish Asterina pectinifera. However, a partially purified gonad-stimulating substance (GSS) failed to increase cyclic AMP levels in COS-7 cells expressing apGPHR.This suggests that a new neural petide other than GSS is a ligand for the apGPHR in starfish.2. 1-Methyladenine biosynthesis and its regulationPrevious in vitro studies have shown that 1-methyladenine (1-MeA) is produced from ATP and S-adenosylmethionine (SAM). In a cell-free system using follicle cells homogenized with a buffer solution, 1-MeA was also produced from ATP and SAM in the presence of cyclic AMP in a concentration dependent manner. It is possible that a unique methyltransferase is activated by cyclic AMP to produce 1-MeA, although it is unclear whether ATP is methylated directly or indirectly by SAM.3. 1-Methyladenine receptorTen N1-substituted adenines as 1-MeA analogues were newly synthesized and used for analysis of the interaction between 1-MeA and its receptors on the surface of oocytes. It was suggested that regional specific structures of the N1-site of adenine are important to interact with 1-MeA receptors. In addition, on the molecular structure a negative charge in N1-site of adenine is required for the binding of the receptors.Others : A high activity of ecto-ATP diphosphohydrolase (ATPDase) was present on the surface of ovarian follicle cells. It is unlikely that ecto-ATPDase is directly involved in 1-MeA production, but adenosine and AMP produced by ecto-ATPDase is responsible for prevention of precocity before oocyte maturation stage.