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A monoclonal antibody against Lates calcarifer vitellogenin and a competitive ELISA to evaluate vitellogenin induction after exposure to xenoestrogen

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Journal of Environmental Sciences 2019年第1期31卷 325-333页

作者： Witchuda Prasatkaew Phochit Nanthanawat Nanthika Khongchareonporn Sutin Kingtong Environmental Science program Faculty of Science Burapha University Department of Biotechnology Faculty of Science Burapha University The Institute of Biotechnology and Genetic Engineering Chulalongkorn University Department of Biology Faculty of Science Burapha University

A monoclonal antibody specific to sea bass(Lates calcarifer) vitellogenin(VTG) was developed,for use as a tool for monitoring endocrine disrupting chemicals(EDCs). VTG was induced in sea bass by intramuscular injection of 17β-estradiol(E_2: 2 mg/kg) every three days. Blood was collected three days after the last injection. Plasma VTG was then purified by chromatography in hydroxyapatite and a sephacryl-S300 column. Characterizations of purified VTG were done by phospholipoglycoprotein staining on a native-PAGE with confirmation by mass spectrometry(LC-MS/MS). Antibody was raised in mice by injection of purified VTG. After monoclonal antibody production, the hybridoma clone No. 41(MAb-sea bass VTG 41)was selected and developed for quantification of VTG by competitive enzyme-linked immunosorbent assay(ELISA). The ELISA method was sensitive with a detection limit of VTG 40 ng/mL. MAb-sea bass VTG 41 was specific to VTG from E_2-treated sea bass and others EDCs(Nonylphenol, Benzo[a]pyrene and CdCl_2). Moreover, cross-reactivity was also found in E_2-treated coral grouper(Epinephelus corallicola). The ELISA method obtained from this work can be further applied for the assessment of EDCs in Thailand and Southeast Asia's aquatic environment.

关键词： Monoclonal antibody vitellogenin Biomarker Endocrine disrupting chemicals(EDCs) ELISA

Assessing the anti-estrogenic activity of sodium pentachlorophenol in primary cultures of juvenile goldfish (Carassius auratus) hepatocytes using vitellogenin as a biomarker

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Journal of Environmental Sciences 2006年第3期18卷 519-524页

作者： ZHAO Bing LIU Zheng-tao XU Zhang-fa China Research Academy of Environmental Sciences Beijing 100012 China Department of Environmental Science and Technology East China Normal University Shanghai 200061 China

Both pentachlorophenol and 2,3,7,8-tetrachlorodibenzo-p-dioxin （TCDD） had been studied widely because of their probable anti-estrogenic activity. Sodium pentachlorophenol （PCP-Na）, as a industrial product used in many fields, usually contains a trace of TCDD. The aim of this study was to assess the anti-estrogenic effect of PCP-Na in juvenile goldfish （Gurassius auratus） hepatocyte cultures using vitellogenin （VTG） as the biomarker. The ID50 of PCP-Na was investigated and then a series of concentrations （0.001 0.5 μg/ml） of PCP-Na were evaluated to estimate the anti-estrogenic activity. Results showed that PCP-Na was cytotoxic for hepatocytes even at very low concentration 〈1.21 μg/ml, and it could not induce VTG at any concentrations tested. Since it failed to stimulate VTG production, the possibility of its anti-estrogenic effect was tested, and a well-known anti-estrogenic compound-tamoxifen was used as positive control. PCP-Na caused a reduction in VTG synthesis in juvenile goldfish （Carassius auratus） hepatocytes at concentrations 〉0.1μg/ml when co-exposure with 1μg/ml 17β-estradiol （E2）, making its anti-estrogenic activity approximately as potent as tamoxifen. Our results indicate that PCP-Na can act as negative modulators of estrogenic function in juvenile goldfish （Carassius auratus） hepatocytes.

关键词： PCP-Na vitellogenin juvenile goldfish hepatocytes anti-estrogenic activity

Combined effect of tributyltin and benzo[a] pyrene on the levels of sex hormone and vitellogenin in female Sebastiscus marmoratu

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Journal of Environmental Sciences 2006年第2期18卷 359-363页

作者： ZHENG Rong-hui WANG Chong-gang ZUO Zheng-hong CHEN Yi-xin ZHAO Yang School of Life-Sciences Xiamen University Xiamen 361005 China State Key Laboratory of Marine Environmental Science Xiamen University Xiamen 361005 China

Tributyltin（TBT）, an organometal used as an antifouling biocide, has been reported to induce masculinization of fish. Benzo [a]pyrene （BaP）, a widespread carcinogenic polycyclic aromatic hydrocarbon, has been reported that its microsomal metabolites can produce an estrogenic response when tested in vitro. This study was therefore designed to examine the potential in vivo influence of TBT, BaP and their mixture on sex hormone levels in serum of Sebastiscus marmoratus, which were given 2 separate intraperitoneally （ip） injections（a single injection every 7 d） of TBT（0.5, 1, 5 and 10 mg/kg）, BaP（0.5, 1, 5 and 10 mg/kg）, or both in combination（0.5, 1, 5 and 10 mg/kg）; control fish received olive oil vehicle only. Six days after the 2nd injection, serum samples were collected and analyzed for sex hormone levels and alkali labile protein phosphorus （ALPP）, which is related to the yolk precursor protein vitellogenin. The pollutants at all doses significantly reduced serum testosterone, estradiol and ALPP content after 2 injections compared with the corresponding controls. The reduction of the estradiol levels should be response for the decrease of the vitellogenin levels. The results in the present study suggested that aromatase seems not the major target acted by TBT and BaP in fish. This study demonstrated that TBT or BaP exposure both inhibit the reproductive potential in female Sebastiscus marmoratus. Combined effect of TBT and BaP on the serum testosterone, estradiol and ALPP was not antagonism from the anticipation.

关键词： tributyltin benzo[a]pyrene estosterone 17β-estradiol vitellogenin Sebastiscus marmoratus

The expression characteristics of vitellogenin(VTG) in response to B(a)p exposure in polychaete Perinereis aibuhitensis

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Journal of Oceanology and Limnology 2018年第6期36卷 2297-2307页

作者： LI Wanjuan XUE Shenglun PANG Min YUE Zonghao YANG Dazuo ZHOU Yibing ZHAO Huan Key Laboratory of Marine Bio-resource Restoration and Habitat Reparation in Liaoning Province Dalian Ocean University Key Laboratory of Marine Ecology and Environmental Science and Engineering First Institute of OceanographyState Oceanic Administration

In order to investigate the endocrine toxicity of B(a)p to marine polychaete P erinereis aibuhitensis, vitellogenin(VTG) cDNA from the P. aibuhitensis was isolated, recombinated and expressed for the first time. The full length P. aibuhitensis vitellogenin gene(PaVTG) was 5 325 bp, and encoded 1 692 amino acids. It contained the vitellogenin_N domain of unknown function(DUF1943), a von Willebrand factor type D domain, as well as a conserved KALGNAG motif. The expression of VTG gene and protein were mainly up-regulated after exposed to B(a)p at transcriptional and translational levels. PaVTG gene expression did not change significantly at day 4. At day 7 PaVTG expression was up-regulated in 0.5 μg/L and 5 μg/L B(a)p group. At day 14 PaVTG was significantly up-regulated in 0.5–10 μg/L B(a)p. The protein expression of PaVTG in 0.5 μg/L and 10 μg/L B(a)p group was up-regulated with time prolonging, but the expression in 5 μg/L and 50 μg/L B(a)p group exhibited first increased and then decreased trend. With the increasing of B(a)p concentration PaVTG mRNA and protein expression both firstly increased then decreased. In contrast to B(a)p exposure, estradiol did not induce PaVTG gene and protein expression, until late times of exposure(14 d). Overall, the results in this study indicate that PaVTG could be used as a potential indicator of the effects environmental estrogenic compounds.

关键词： Perinereis aibuhitensis vitellogenin B(a)p expression profiles

Molecular cloning, expression profiling and RNA interference of a vitellogenin gene from Harmonia axyridis(Coleoptera: Coccinellidae)

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Journal of Integrative Agriculture 2019年第10期18卷 2311-2320页

作者： LIANG Chao LIU Ting-hui HAN Shi-peng HE Yun-zhuan College of Plant Protection Hebei Agricultural University

In this study, the Harmonia axyridis vitellogenin gene 2 (HmVg2) sequence was identified from transcriptomic databases of female adult H. axyridis and cloned into pMD18-T vector. The HmVg2 gene was 5 460 bp in length, and formed with an open reading frame (ORF) of 5 361 nucleotides (GenBank accession no. KY794939). The putative molecular weight of the primary HmVg2 protein was 203.459 kDa and the predicted isoelectric point (pI) was 8.59. HmVg2 contained a signal peptide, vitellogenin N-terminal (vitellogenin-N) domain, domain of unknown function 1943 (DUF1934) domain and von Willebrand factor type D (VWD) domain. The developmental expression profiling showed that HmVg2 was extremely highly expressed in female insects, but was expressed at lower levels in male insects. In female insects, HmVg2 was mainly expressed in the wing and fat body. The double-stranded RNA-HmVg2/-GFP was injected into H. axyridis, and qRT-PCR results showed that the HmVg2 gene was specifically silenced. The eggs laid during the first five days and the hatching rate of eggs was lower than controls after dsHmVg2 injection. This investigation demonstrated that the HmVg2 gene plays an important role in H. axyridis reproduction and enriches the function of the insect vitellogenin gene.

关键词： vitellogenin Harmonia axyridis expression profiling RNAi

Production of the yolk protein precursor vitellogenin is mediated by target of rapamycin (TOR) in the soft tick Ornithodoros moubata (Acari: Argasidae)

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Insect Science 2022年第5期29卷 1299-1308页

作者： Meryem Behri Haruki Teshima Keisuke Kutsuwada Shoko Nakatake Mari H.Ogihara DeMar Taylor Graduate School of Life and Environmental Sciences University of TsukubaTsukubaIbarakiJapan Animal Breeding and Reproduction Research Division Institute of Livestock and Grassland ScienceNational Agriculture and Food Research OrganizationTsukubaIbarakiJapan

Initiation of vitellogenesis by blood feeding is essential for egg maturation in *** derived from the blood meal are utilized by female ticks to synthesize the yolk protein precursor vitellogenin(Vg).Engorged Ornithodoros moubata ticks can synthesize Vg whether mated or virgin,thus *** is an excellent model for studying the relative roles of blood feeding and mating in tick *** of rapamycin into engorged *** resulted in a reduction of ovarian growth and yolk accumulation in the oocytes of mated *** expression in the midgut and fat body and protein concentrations in the hemolymph significantly decreased in mated ticks after injection with rapamycin,indicating that inhibition of the nutrient-sensing target of rapamycin(TOR)pathway disrupts egg maturation at the levels of Vg expression and *** results suggest that the TOR-signaling pathway induces vitellogenesis in response to nutritional stimulation after a blood meal in *** and is functionally independent of the mating-induced pathway.

关键词： Ornithodoros moubata target of rapamycin ticks vitellogenesis vitellogenin

Changes in Lipid Profiles and ^(32)P-Uptake Into Phosphoprotein (vitellogenin) Content of the Ovary and Liver in the Female Catfish, Clarias batrachus,Exposed to Mercury

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Biomedical and Environmental Sciences 1995年第1期8卷 35-44页

作者： R.KIRUBAGARN K.P.JOY CentreofAdvancedStudyinZoology BanarasHinduUniversityVaranasi－221005India CentreofAdvancedStudyinZo

Toxic effects of selected sublethal concentrations of mercuric chloride (HgCl2, 0.05 mg ·L-1), methylmercuric chloride (CH3HgCl, 0.04 mg·L-1) and Emisan 6 (an organic mcrcurial fungicide, 0.5 mg·L-1) on levels of lipids (total lipids-TL, phospholipids-PL, free cholesterol-FC; esterified cholesterol-EC and free fatty acids-FFA), and uptake of 32P into phosphoprotein fraction of the ovary and liver are reported in Clarias batrachus exposed for 45, 90 and 180 days from February to July (preparatory to spawning phase of the annual reproductive cycle). The TL level decreased significantly only in the ovary of 180-day Hg groups. The ovarian PL and FC levels showed a significant reduction at all durations of Hg exposure. In contrast, the hepatic PL and FC levels increased significantly at all durations except the PL content in the 45-day Emisan 6 and CH3HgCl groups. The EC level in the ovary showed a significant decline after both 90 and 180 days of Hg treatment whereas in the liver, it decreased significantly only after 180 days. The level of FFA in the ovary decreased significantly only in the 180-day HgCl2 group while in the liver, it showed a significant increase in all the 180-day Hg groups. The rate and level of incorporation of 32P into phosphoprotein fraction of the ovary and liver showed a progressive decline in the Hg-treated groups, the effect being significantly different only after 90 and 180 days. The results suggest that the decrease in ovarian lipid levels is lareely due to their immobilization from the liver and the reduction in vitellogenin content may be due to both the inhibition of its synthesis in the liver and subsequent incorporation into the ovary.

关键词： vitellogenin Content of the Ovary and Liver in the Female Catfish Hg In Clarias batrachus,Exposed to Mercury Changes in Lipid Profiles and

Advancement of Studies on vitellogenin of Crustaceans

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Animal Husbandry and Feed Science 2012年第2期4卷 86-90,92页

作者： SHUI Yan 1 ,ZHOU Xin 1,2 1. Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization,Ministry of Agriculture,Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences,Wuxi 214081,China 2. Wuxi Fishery College,Nanjing Agricultural University,Wuxi 214081,China

[Objective]The paper briefly summarizes the gene cloning and molecular research progress of vitellogenin in crustaceans belonging to Decapod. [Method]The molecular characteristics of identified Vg proteins in crustacean are reviewed from the aspects of protein synthesis,molec- ular structure,phylogenetic evolution,hormonal regulation of gene expression and biological functions. [Result]The similarity of structure and func- tion in homologous protein are pointed out; phylogenetic relationships among molecules are analyzed; the main characteristics of Vg interspecific protein in Decapod are summarized,and the special parts different from other species are raised. [Conclusion]The study can offer some help for the further study of Vg gene,and provides theoretical basis for ovary development mechanism of crabs.

关键词： Decapod vitellogenin Gene

Response of vitellogenin in Tilapia Liver to 2,2′,4,4′-Tetrabromodiphenyl Ether Stress

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Agricultural Biotechnology 2021年第3期10卷 52-54,58页

作者： Shunlong MENG Xi CHEN Zeqiu CHEN Liping QIU Chao SONG Limin FAN Yao ZHENG Jiachang CHEN Pao XU Laboratory of Quality&Safety Risk Assessment for Aquatic Products on Environmental Factors(Wuxi)of Ministry of Agriculture and Rural Affairs Key Open Laboratory of Ecological Environment and Resources of Inland Fisheries of Chinese Academy of Fishery SciencesFreshwater Fisheries Research CenterChinese Academy of Fishery SciencesWuxi 214081China Wuxi Fishery College Nanjing Agricultural UniversityWuxi 214081China

[Objectives]This study was conducted to understand the potential harm of BDE-47 to fish and aquatic ecosystems and obtain relevant toxicological data from the perspective of vitellogenin.[Methods]Adopting the semi-static water exposure method,three exposure concentrations of 5,50,and 500μg/L and five sampling time of 1,3,7,15,and 30 d were set to investigate the effect of BDE-47 on vitellogenin in tilapia liver.[Results]The low concentration of BDE-47(5μg/L)had no effect on the level of vitellogenin in the liver of *** exposed to high concentrations of BDE-47(50 and 500μg/L),the VTG content of tilapia liver showed a trend of first decreasing,then returning to normal,and then *** abnormal VTG content indicates that the endocrine system of tilapia is disturbed to a certain extent.[Conclusions]This study plays a role in promoting the formulation of relevant water quality standards and the protection of aquatic living resources.

关键词： 2,2′,4,4′-Tetrabromodiphenyl ether Tilapia Liver vitellogenin