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

(1)在日本鳗鲡肝脏的胞浆和核提取物中鉴定了雌激素受体。发现了一个高亲和力结合位点的单一类，细胞溶质雌激素受体（cER）的Kd=0.97 nM，核雌激素受体（nER）的Kd=0.85 nM。cER和nER对雌激素的结合是特异性的（己烯雌酚：DES>雌二醇-17 β：E_2>>雌三醇：E_3>雌酮：E_1）。雌激素受体的这些特性与血清雌激素结合组分的特性有很大的不同，[3 H]E_2与血清的结合是不饱和的，它被睾酮取代，而不被DES、E_1或E_3取代。用20 μ g/g体重的鲑鱼脑垂体匀浆每周注射一次，研究了人工成熟过程中鳗鱼肝脏雌激素受体（ER）、循环E_2和卵黄蛋白原（VTG）水平的关系。血清E_2水平在卵黄发生前至中期持续偏低，而在移行期显著升高。 关于我们 裂核期然而，VTG水平逐渐增加，从早期到midvitellogenesis，并大大升高，在迁移核阶段。肝cER水平略有逐渐增加，从早期到midvitellogenesis，并大大升高，在迁移核阶段。肝cER水平在卵黄发生早期略有上升，然后在卵黄发生中期至移行核期显著上升。与此相反，nER水平没有显着变化，虽然nER水平在迁移核阶段高于其他阶段。cER水平的变化代表了人工成熟过程中肝脏对雌激素刺激的反应性增加。与自然界中的性成熟相比，nER水平缺乏变化可能是人工成熟的一个特征。(2)建立了一种特异、灵敏的日本鳗鲡血清VTG的酶联免疫吸附试验（ELISA）。用抗卵黄脂磷蛋白（α-lipovitellin）的特异性抗血清和生物素化的α-lipovitellin的F（ab '）_2片段，用夹心法进行非竞争性的亲和素-生物素相互作用。该测定可在2天内运行，并常规检测低至0.8 ng/ml的浓度的VTG。幼鱼注射E_2后9-12 h，血清VTG低水平的出现显著增加。建立了VTG的ELISA检测方法，可用于血清VTG的定量检测，从而在体外分析卵黄发生的发生和发展的机制。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