| Budget Amount *help |
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1994: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1993: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
(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
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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
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