Molecular mechanism of vitellogenin synthesis in the Japanese eel, Anguilla Japonica

(1)在日本鳗鲡（Anguilla japonica）肝脏细胞质和细胞核提取物中鉴定出雌激素受体。发现单类高亲和力结合位点，胞质雌激素受体（cER） Kd=0.97 nM，核雌激素受体（nER） Kd=0.85 nM。cER和nER的结合对雌激素（二雌酚：DES b>雌二醇-17 β: E_2 b> >雌三醇：E_3>雌酮：E_1）具有特异性。雌激素受体的这些特征与血清雌激素结合成分有很大不同；[3H]E_2与血清结合不饱和，被睾酮取代，而不被DES、E_1和E_3取代。采用每周注射20 ug/g体重的鲑鱼垂体匀浆的方法，研究了人工成熟过程中鳗鱼肝脏er、循环E_2和卵黄蛋白原（VTG）水平的关系。血清E_2水平在卵黄形成前期至中期持续较低，在移行核期显著升高。然而，从卵黄形成早期到中期，VTG水平逐渐升高，并在迁移核阶段大幅升高。从卵黄形成早期到中期，肝脏cER水平逐渐升高，在迁移核期显著升高。肝细胞癌水平在卵黄形成早期略有升高，在卵黄形成中期至迁移核期显著升高。而迁移核阶段的nER含量高于其他阶段，但变化不明显。在人工成熟过程中，cER水平的变化表明肝脏对雌激素刺激的反应性增加。与自然界的性成熟相比，人工成熟缺乏内胆素水平的变化可能是人工成熟的特征。(2)建立了一种特异、灵敏的酶联免疫吸附测定法（ELISA），用于测定日本鳗鲡低水平血清VTG。利用特异性抗血清对黄鳝卵黄蛋白（a-lipovitellin）和a-lipovitellin的生物素化F (ab')_2片段的特异性，采用夹心法进行亲和素与生物素的非竞争性相互作用。该试验可在2天内运行，常规检测浓度低至0.8 ng/ml的VTG。未成熟鱼单次注射E_2后9-12 h，血清VTG水平明显降低。ELISA法的开发使血清VTG的定量分析成为可能，从而在体外分析卵黄形成的发生和过程的机制。少

(1) Estrogen receptors were identified in cytosolic and nuclear extracts of livers of Japanese eel, Anguilla japonica. A single clases of high Affinity binding sites was found, with a Kd=0.97 nM for the cytosolic estrogen receptor (cER) and Kd=0.85 nM for the nuclear estrogen receptor (nER). Binding of both the cER and nER was specific for estrogens (diethystibestrol : DES>estradiol-17beta : E_2>>estriol : E_3>estrone : E_1). These characteristics of ERs were quite different from those of the serum estrogen-binding component ; [3H]E_2 binding to serum was not saturable, and was displaced by testosterone but not by DES,E_1 or E_3. The relashionship between the levels of hepatic ERs, circulating E_2 and vitellogenin (VTG) during artificial maturation of cultivated eels were examined, using eels injected weekly with chum salmon pituitary homogenate at a dose of 20 ug/g-body weight. Serum E_2 levels were constantly low during pre- to midvitellogenesis, and dramatically increasedin the migr … More atory nucleus stage. However, VTG levels gradually increased from early to midvitellogenesis, and were greatly elevated in the migratory nucleus stage. Hepatic cER levels slightly gradually increased from early to midvitellogenesis, and were greatly elevated in the migratory nucleus stage. Hepatic cER levels slightly increased in early vitellogenesis, and then increased significantly from midvitellogenesis to the migratory nucleus stage. In contrast, nER levels did not change significantly, although nER levels in the migratory nucleus stage were higher than those at other stages. The changes in cER levels represent increased hepatic responsiveness to estrogenic stimuli during artificial maturation. Lack of change in nER levels may be a feature of artificial maturation compared to sexual maturation in nature.(2) A specific and sensitive enzyme-linked immunosorbent assay (ELISA) was developed for the measurement of low levels of serum VTG of the Japanese eel. The non-competive avidin-biotin interactionwas performed by a sandwich method using the specific of the specific antiserum to eel yolk protein (a-lipovitellin) and biotinylated F (ab')_2 fragment of a-lipovitellin. The assay could be run in 2 days and routinely detect VTG in a concentration as low as 0.8 ng/ml. The appearance of low levels of serum VTG significantly increased 9-12 h after a single injection of E_2 into immature fish. The development of an ELISA for VTG made possible the quantification of serum VTG snd thereby in vitro analysis of the mechanism of the onset and course of vitellogenesis. Less