| Summary: | 碩士 === 國立海洋大學 === 水產養殖學系 === 83 ===
The objectives of the studies were to investigate the concentrations characteristics of plasma 11-ketotestosterone (11-KT) and aromatase activity in pituitaries and gonads of grey mullets (Mugil cephalus) during the period of sex differentiation. The physiological and biochemical characteristics of vitellogenin and aromatase were also studied.
Seven-month-old grey mullest were divided into three groups, and feed them with pelleted diets mixed individually ethynylestradiol (EE2, 20 mg/kg diet), 17 α-methyl-testosterone (17α-MT, 20 mg/kg diet) and control died for four months. After two and three months treatment, 100% of female and male were detected in the EE2 and 17α-MT groups, respectively; but only 30% of grey mullets had differentiated in the control group. There were no significant profiles of plasma 11-KT during the period of experimental treatment, although the control and EE2 groups had the highest and lowest levels of plasma 11-KT, respectively. Following one month treatment, the aromatase activities in pituitary increased in the EE2 group. Pituitary aromatase activities were significantly higher in EE2 and 17α-MT groups than in the control group after two months treatment, and EE2 group also had higher aromatase activities than 17α-MT group. After the sex differentiation, the aromatase activities in pituitary weresignificantly lower than that during the period of sex differentiation. The gonad aromatase activities were higher in EE2 group than in the 17α-MT and control grouups during the experimental period and there was no significant diference between 17α-MT and control groups. These results indicated that aromatase activities in pituitary and gonad of grey mullets had close relationships with sex differentiation by treatment with sex steroids.
A female-specific plasma protein (vitellogenin, Vg) was induced from grey mullet after estradiol-17β (E2) treatment. Vg, the phospholipoglycoprotein, was purified by gel filtration, hydroxylapatite column and gel electrophoresis. There were three circulating forms of Vg; the estimated molecular weights were 531 KDa (Vg I), 367 KDa (Vg II) and 295 KDa (Vg III). The purified Vg had four major subunits (49 KDa, 91 KDa, 132 KDa, 149 KDa), and contained about 35.1% non-polar amino acid. Enzyme-linked immunosorbent assay for Vg in grey mullet was set up. The highest levels of plasma Vg of E2-induced grey mullets were 657.3±160.1 mg/ml. Plasma Vg levels were 2.6±0.6 mg/ml in the cultured grey mullets with gonadosomatic indexes of 11.9±2.9%.
For preparation of aromatase in the brain of grey mullet, buffer IV (10 mM Hepes, 1 mM EDTA, 10% glycerol, 10 mM dithiothreitol, 3 mM chaps, pH 7.2) was the best buffer. The data showed that 100 mM KCl wasn't good for long-term storage of aromatase, but dithiothreitol and glycerol were better than sucrose in protecting the activity of aromatase. Brain aromatase was purified by gel filtration (Sepharose CL-2B), hydroxylapatite column and high performance liquid chromatography. The first and second fractions after gel filtration contained aromatase activity. Aromatase was further eluted with a 0.3 M potassium phosphate buffer on a hydroxylapatite column. The fraction with the retention time of 19.4 minutes contained aromatase activity after separation with high performance liquid chromatography. The data indicated that the structure of brain aromatase was very special and maybe it comprised five kinds of different molecular weight protein (130 KDa, 95 KDa, 67 KDa, 51 KDa, 43 KDa). There was no immunological cross-reactivity between grey mullet and human, catfish aromatase on the basis of Western blotting.
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