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

(1)在鳗鱼肝脏的胞浆和胞核提取液中检测到雌激素受体。细胞内雌激素受体(CER)的Kd=0.97 nM，核雌激素受体(NER)的Kd=0.85 nM。CER和NER的结合是雌激素(己烯雌酚：DES&GT；雌二醇-17β：E_2&GT；&GT；雌三醇：E_3&GT；雌酮：E_1)所特有的。ER的这些特征与血清雌激素结合组分有很大不同，[~3H]E_2与血清的结合是不饱和的，可被睾酮取代，但不能被DES、E_1或E_3取代。用每周注射鲑鱼脑下垂体匀浆20微克/克体重的鳗鱼，研究了人工成熟鳗鱼肝脏ER、循环E_2和卵黄蛋白原(VTG)水平之间的关系。血清E_2水平在卵黄发生前期到中期持续较低，而在偏头痛(MIGR…)时显著升高更具活力的核期。然而，VTG水平从卵黄发生早期到中期逐渐升高，并在迁移核期显著升高。肝脏CER水平从卵黄发生早期到中期略有升高，在游走核期显著升高。肝脏CER水平在卵黄发生早期略有升高，然后从卵黄发生中期到迁移核期显著升高。与之相反，尽管迁移核期的NER水平高于其他时期，但NER水平无明显变化。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强。与自然性成熟相比，NER水平缺乏变化可能是人工成熟的一个特征。(2)建立了一种特异而灵敏的酶联免疫吸附试验(EL ISA)，用于测定鳗鱼血清中低水平的VTG。用抗鳗鱼卵黄蛋白(a-Lipovitellin)的特异性抗血清和a-LipoVitellin的生物素标记的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。该方法可在2天内检测到低至0.8 ng/m l的VTG，一次注射E_2后9-12 h出现低水平的血清VTG的现象显著增加。VTG酶联免疫吸附试验的建立使血清VTG的定量成为可能，从而使体外分析卵黄发生的机制和过程成为可能。较少

(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