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Chemiluminescent immunoassays (CLIAs) were developed for each of three subtypes of vitellogenin (VtgAa, VtgAb and VtgC) in grey mullet, primarily for use in monitoring estrogenic pollution of the environment. The working range of VtgAa-CLIA and VtgAb-CLIA was from 0.975 to 1,000 ng/ml, while that of VtgC-CLIA was from 0.487 to 1,000 ng/ml. Each CLIA appeared to be specific to the targeted Vtg subtype. Intra- and inter-assay coefficients of variation in the developed CLIAs were lower than 10%. In male serum, VtgAa, VtgAb and VtgC were detected in ranges from 0.01 to 0.38, 0.02 to 1.01, and 0.01 to 3.12 μg/ml, respectively, during various sampling periods. In vitellogenic females (October), serum VtgAb levels (1,192.05 ± 237.81 μg/ml) were significantly higher than levels of the other two Vtg subtypes (120.82 ± 30.42 and 119.23 ± 16.95 μg/ml for VtgAa and VtgC, respectively). When immature mullet were fed diets containing 17α-ethinylestradiol (EE2) at three different doses (0.4, 40 and 4,000 ng/g body weight), all Vtg subtypes were induced by 40 ng/g and 4,000 ng/g EE2. The VtgC (610.30 ± 150.18 μg/ml) was most highly expressed among the three Vtgs in fish fed 40 ng/g EE2, while VtgAb (33.25 ± 13.58 mg/ml) was highest in expression in fish fed 4,000 ng/g EE2. The present study provided practical subtype-specific Vtg assays for the first time in grey mullet, providing the necessary means to evaluate estrogenic activities in aquatic environments., (Copyright © 2018 Elsevier Inc. All rights reserved.)

We investigated the continuing effects of exposure to ethynylestradiol (EE(2)) in juvenile grey mullet after transfer to a clean environment. Eleven-month-old juvenile fish containing immature phenotype gonad were fed dry diets; the low and high EE(2)-treated groups were fed diets with 0.04 and 4 μg EE(2)/g body weight for 4 weeks, respectively. After treatment, they were transferred to clean seawater, and reared with an EE(2) free diet for 350 days. Vitellogenin (VTG) was not detected in the serum of the control group throughout the experimental period. However, in both treatment groups, abnormal values of serum VTG were detected until approximately 100 days after transfer to a clean environment. In the control group, sex differentiation was not confirmed until 206 days after transfer to a clean environment. However, some of the fish in the 0.04 μg EE(2)-treated group had ovarian cavity and oocytes at 26 days. In most of the fish in the 4 μg EE(2)-treated group, the ovarian cavity had already appeared at the end of EE(2) treatment (0 day), and oocytes were observed at 26 days, suggesting that EE(2) accelerates ovarian differentiation. These results suggest that previous exposure to EE(2) has long-term effects on VTG synthesis and gonadal development., (Copyright © 2011 Elsevier Inc. All rights reserved.)