• Korean Journal of Ichthyology
• /
• v.12 no.3
• /
• pp.180-185
• /
• 2000

The effects of bisphenol-A(BPA), a monomer of plastics used in many consumer products, on vitellogenin(VTG) synthesis were examined in primary hepatocyte culture of olive flounder, Paralichthys olivaceus. The hepatocytes were precultured for 2 days, and then estradiol-$17\beta(10^{-6}M)$ and BPA were simultaneously added to the incubation medium. The hepatocytes were cultured for 6 more days and then spent medium was analyzed by SDS-PAGE. BPA increased the rate of VTG to total protein concentrations in a concentration-dependent way, and a significant difference was obtained at concentrations of $10^{-6}M$ and $10^{-5}M$ (P<0.05). In particular, the rate of VTG to total protein concentrations was 26.36% at $10^{-5}M$ of BPA, and its level did not differ from the control level with $E_2$ alone. $E_2$ and/or BPA-primed VTG synthesis was markedly inhibited to about 80% of the control(with $E_2$) by the addition of tamoxifen($10^{-6}M$) to the incubation medium. Furthermore, In vivo $E_2$-primed VTG synthesis was significantly inhibited by in vitro $E_2$-free incubation of hepatocyte to about 22% of the control (with $E_2$) on Day 6. The effect of reducing was delayed in a BPA concentration-dependent way. These results suggest that BPA induce VTG synthesis by estrogenic activity through estrogen receptor mediated response and prolong VTG synthesis on vitellogenesis in olive flounder.

• Fisheries and Aquatic Sciences
• /
• v.8 no.4
• /
• pp.213-219
• /
• 2005

Vitellogenin (VTG) was purified from the blood plasma of estradiol-17$\beta$ ($E_2$)-treated male marbled sole (Limanda yokohamae) using gel filtration and anion exchange chromatography. The purity of the marbled sole VTG (msVTG) was confirmed by polyacrylamide gel electrophoresis (SDS-PAGE) and N-terminal amino acid sequencing. The purified msVTG was used to produce monoclonal and polyclonal antibodies in mice and rabbits, respectively, and the specificity of the polyclonal antisera for msVTG was confirmed by Western blot analysis. The antibodies cross­reacted with a protein of molecular mass approximately 160 kDa in the plasma samples of mature female marbled sole. No cross-reactivity was observed with the plasma of male fish. A direct non-competitive sandwich enzyme-linked immunosorbent assay (ELISA) was developed using the monoclonal anti-msVTG and polyclonal anti-msVTG antibodies, with purified msVTG as the standard protein. The values of the intra- and inter-assay variations were within the ranges of $8.l-9.8\%$ and $8.5-12.2\%$, respectively. The sensitivity was about 0.3 ng/mL. Serial dilutions of plasma from mature female sole reacted with the msVTG-antibodies in the sandwich ELISA, whereas the plasma from male fish did not. The results indicate that the maturation status of female marbled sole can be identified using a sandwich ELISA for msVTG.

• Fisheries and Aquatic Sciences
• /
• v.1 no.1
• /
• pp.19-23
• /
• 1998

Effects of pituitary and thyroid hormones on estradiol-induced vitellogenin (VTG) induction were electrophoretically examined in primary hepatocyte cultures of rainbow trout. Hepatocytes were precultured for 2 days and then estradiol-17 $\beta$ $(E_2,\;2 \times 10^{-6}M)$>, triiodothyronine $(T_3,\;10^{-8}-10^{-6}M)$, bovine growth hormone (bGH, 10-100 ng/ml), ovine prolactin (oPRL, 100-500 ng/ml), and pituitary extract (PE) of rainbow trout (0.75PE/dish) were added to the incubation medium. The hepatocytes were cultured for 7 more days. The addition of oPRL to the incubation medium was not effective in increasing VTG production at any concentrations. The addition of PE to the incubation medium with $E_2$ was not effective in increasing VTG production. The addition of bGH to the incubation medium with $E_2$ was not effective in increasing the rate of VTG production at concentrations of 10-50 ng/ml. However, a higher concentration of bGH, 100 ng/ml, increased VTG production. The various concentrations of $T_3$ were ineffective in stimulating VTG production. These results suggest that GH could be one of stimulus factors for VTG production in rainbow trout.

• Korean Journal of Fisheries and Aquatic Sciences
• /
• v.28 no.6
• /
• pp.753-760
• /
• 1995

This study was conducted to determine the serum vitellogenin (VTG) concentration changes during the ovulation period in elkhorn sculpin, Alcichthys alcicornis. The results of sepacryl S-300 showed that the molecular weight of VTG could be 380,000. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis may indicate that the purified VTG consists of three subunits with molecular weights of 180,000, 118,000 and 85,000, respectively. Yolk protein purified from the egg extracts was eluted on an equilibrated sephacryl S-300 column, and its molecular weight was estimated 250,000. The precipitation lines of the female serum against the antiserum of the egg extracts were fused completely by immunoelectrophoresis and immunodiffusion analysis. VTG was detected in the serum, and hepatocytes from males injected with $17\beta-estradiol\;(E_2).$ Furthermore, VTG was immunochemically similar to yolk proteins. The concentration of VTG was high before ovulation $(9.80\pm0.81-11.02\pm0.09 mg/ml),$ and then decreased rapidly after ovulation $(less\;than\;6.19\pm0.59 mg/ml).$ This study suggested that VTG was synthesized in the liver by the action of $E_2$ and released to blood, and then incorporated into oocytes.

• Journal of Aquaculture
• /
• v.11 no.3
• /
• pp.311-317
• /
• 1998

Effects of Cu and Zn on estradiol-17$\beta$-Induced vitellogenin (VTG) production were electro-phoretically examined in hepatocyte cultures of rainbow trout. Hepatocytes were precultured for 2 days and then Cu ($10^{-5}$ ~$10^{-4}$M) and Zn ($10^{-5}$~$10^{-3}$M) were added to the incubation medium with estradiol-17${\beta}$ ($2{\times}10^{-6}$M). The hepatocytes were cultured for 5 more days. The relative VTG production rate was expressed as the percentage of VTG to total proteins including the VTG. The addition of CU and Zn to the incubation medium had no appreciable toxin effect on the viability of hepatocytes in the culture. However, Cu markedly reduced VTG production at any concentration used. Zn also specifically reduced VTG production by hepatocytes in a concentration dependent way and there was a significnt reduction at Zn concentrations of $10^{-3}$M. The reduction recovered by removing Zn from the media, but Cu did not. Additionally, enriched Ca concentrations (1.8 to 2.5 or 5.0 mM) in the incubation medium had no protective effect on the reduction of VTG production by Cu $10^{-4}$ M. These results suggest that the production of VTG is more susceptible to Cu and Zn than are other hepatocyte-derived proteins.

• Journal of fish pathology
• /
• v.26 no.3
• /
• pp.241-254
• /
• 2013

In order to establish bio-marker systems for the screening of endocrine-disrupting chemicals contaminated in various environment, Vitellogenin(Vtg) bio-marker have been developed to detect Scorpion fish's(Sebastiscus marmoratus) Vtg. Vtg has been induced by administration of estradiol into S. marmoratus, and purified by gel filtration and ion-exchange chromatography from serum of the fish. After immunization of the purified Vtg into BALB/c mouse, hybridomas secreting anti-Vtg antibodies have been produced. The size of induced Vtg in the serum was about 440 kDa by gel filtration using Sepharose CL-6B. By SDS-PAGE analysis, the main band of Vtg, however, was at 175 kDa, and several minor bands have been detected with the main band. Eight different monoclonal antibodies have been produced from established hybridomas and the antibodies did not cross-react with sera from different species of fishes tested in this study except with that of Sebastes hubbsi. These results suggested that the monoclonal antibody of S28 and S15 can used as capture and tracer antibodies for ELISA and ICG assays. The detection systems developed in this study can be used as Bio-marker assays to check endocrine disrupting activity of various chemicals as well as to detect known endocrine disrupting chemicals contaminated in environment.

• Journal of fish pathology
• /
• v.17 no.3
• /
• pp.191-198
• /
• 2004

Temporal changes of plasma vitellogenin (VTG), alkaline-labile protein phosphorus (ALPP), calcium (Ca), glutamate pyruvate transaminase (GPT) and hepatosomatic index (HSI) were examined in the $estradiol-17\beta$ ${E_2}$-administered immature rockfish, Sebastes schlegeli. Fish were intraperitoneally injected with ${E_2}$ (5 ㎎/kg B.W.) in 70% ethanol and then plasma were extracted at 0, 1, 3, 6, 9, 12 and 15 days. VTG band was detected at a molecular weight position of about 170 kDa on Day 3 in SDS-PAGE. This band became more distinct at 6 days but its was gradually thinned with time-course, and not detected at 15 days. Plasma ALPP and Ca increased suddenly at 1 day and the highest concentrations were detected at 6 days and then these concentrations decreased gradually with time-course. ALPP and Ca concentrations at 15 days after E2 administration were very similar to that before E2 administration. GPT was increased at 1 day and higher GPT was detected at 3 days. However, GPT was gradually decreased with time-course. GPT and HSI at 15 days after E2 administration were also very similar to that before E2 administration. HSI was also increased at 1 day and the highest value was detected at 3 days and then gradually decreased with time-course. These results suggest that plasma ALPP, Ca, GPT and HSI could be utilized as a biomarker of exogenous E2 exposure in coastal ecosystem, because the changes of ALPP, Ca, GPT and HSI after E2 administration are very similar to that of VTG.

• Korean Journal of Ichthyology
• /
• v.12 no.3
• /
• pp.173-179
• /
• 2000

Effects of Estradiol-$17\beta(E_2)$ and 2, 4-dichlorophenoxy acetic acid (2, 4-D) on vitellogenin(VTG) production were investigated in primary hepatocyte culture of olive flounder, Paralichthys olivaceus. Highest survival rate of hepatocyte were observed at $27^{\circ}C$, which markedly declined equal to 50% of those of $15^{\circ}C$. Vitellogenin production peaked at the concentration of $10^{-6}M\;E_2$. No effect was observed on VTG production at various concentrations of 2, 4-D. However, a low concentration of 2, 4-D (ie, $10^{-8}M$) only appeared increased VTG production. $E_2$ or $10^{-8}M$ 2, 4-D-primed VTG production was markedly inhibited by the addition of $10^{-6}M$ tamoxifen to the culture medium(P<0.01). Inhibition was not affected by combinational treatment with $10^{-6}M$ $E_2$ and $10^{-6}M$ 2, 4-D. These results from the current investigation suggest that 2, 4-D mimics $E_2$, but the mechanism of reaction in inducing the $E_2$ receptor are different in VTG production in oliver flounder hepatocytes.

• Animal cells and systems
• /
• v.7 no.3
• /
• pp.227-235
• /
• 2003

Bisphenol A (BPA), nonylphenol (NP), and 4-tert-octylphenol (OP) are known endocrine disrupting chemicals (EDCs) with estrogenic activity in fish. This study compared the effects of BPA, NP and OP on in vitro vitellogenin (VTG) synthesis in primary cultures of hepatocytes of the Chinese minnow Phoxinus oxycephalus. The VTG secreted into the culture medium was measured using enzyme-linked immunosorbent assay (ELISA), which we developed in this study using an antibody prepared from homogenates of Chinese minnow egg. VTG synthesis was induced by estradiol-17$\beta$ ($E_2$) and phenols (BPA, NP and OP) treatment. $E_2$ at concentrations of 10$^{-6}$ M or higher increased VTG levels significantly (P < 0.05). Exposure to 10^5\;M\;BPA\;or\;10^-4$M NP and OPinduced in vitro VTG synthesis (P < 0.01). However, $10^-3$ M BPA, NP or OP did not induce VTG synthesis. These results suggest that SPA has the highest estrogenic potential in Chinese minnow hepatocytes. Tamoxifen, an anti-estrogen, drastically blocked the production of VTG by phenols (BPA, NP and OP) suggesting that phenols (BPA, NP and OP) may act via binding to estrogen receptor (ER) in Chinese minnow hepatocytes.

• Korean Journal of Veterinary Research
• /
• v.46 no.1
• /
• pp.35-41
• /
• 2006

Vitellogenin (Vtg), a phospholipoglycoprotein precursor of egg yolk is synthesized and secreted from the liver in response to estrogens in female fish. Vtg is normally undetectable in the blood of male fish, but can be induced by exposure to chemicals possessing estrogenic activity. Thus, the presence of Vtg in blood of male fish can serve as a useful biomarker for assessing previous exposure to estrogenic compounds. In the present study, Vtg was abnormally expressed in Rhynchocypris oxycephalus using estradiol benzoate ($E_2$). As the result, it was found that the level of Vtg in blood from R. oxycephalus was increased by treated quantity of $E_2$ with dose-effect manner. Monoclonal antibodies were generated against Vtg of R. oxycephalus. The hybridoma were screened with an enzyme immunoassay for the production of specific anti-Vtg antibodies. Five positive cell lines with a high specificity were selected. Monoclonal antibodies against vtg of R. oxycephalus that was developed in this study, may be a useful bio-indicator for the detection of estrogenic contamination in the aquatic ecosystem.