Vitellogenin of the Chilean flounder Paralichthys adspersus as a biomarker of endocrine disruption along the marine coast of the South Pacific. Part I: induction, purification, and identification (original) (raw)
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Archives of Environmental Contamination and Toxicology, 2012
This study evaluated the condition factor, gonadosomatic, and hepatosomatic indexes, occurrence of plasmatic vitellogenin (Vg), and frequency of spermatogenic maturity stages in male Chilean flounders, Paralichthys adspersus, caught at three different coastal sites off the Bio-Bio region, central Chile, during 1 year. The Vg was detected by polyacrylamide gel electrophoresis with sodium dodecyl sulfate and Western blot analyses using an antibody against Chilean flounder Vg. The spermatogenic maturity stages were analyzed by histological gonadic diagnostic. The prevalence of plasmatic Vg induction in male fish differed significantly among sites. The flounders sampled from the Itata area were the most affected. Evaluations of biometric data, plasmatic Vg induction, and spermatogenic maturity stages of the flounder showed the following: (1) lower gonadosomatic index, (2) greater hepatosomatic index, (3) greater prevalence of plasmatic Vg, and (4) delayed development of the gonad. The results suggest that estrogenic endocrine-disruption compounds are introduced into the marine environment, negatively affecting the fish studied. The relevance of this report is discussed in relation to estrogenic compounds introduced by industrial and municipal wastewater effluents in the areas studied.
Marine environmental …, 2006
An ELISA for cod vitellogenin (VTG) has been set up using cod lipovitellin for plate coating and standardisation. The assay has been applied to plasma samples collected from male and female cod caught in three distinct areas around the UK, three areas off the Norwegian coast and also to cod reared initially at an aquaculture site and subsequently maintained at a research station. The aim of the study was to determine whether there were any signs of oestrogenic endocrine disruption in a fish species living offshore. VTG induction was found in male cod caught in the North Sea, the Shetland Box area, in Oslofjord and also in cultivated fish. There was a strong relationship between concentrations of VTG and fish size. There was no evidence that the presence of VTG in the plasma of males is a natural part of their life cycle. On the other hand, the size of fish at which these elevated VTG concentrations appear (ca. 5 kg) is about the size that cod change from feeding primarily on benthic invertebrates to mainly other fish, both benthic and pelagic. The possibility is suggested that large cod pick up oestrogenic endocrine disrupters through the food chain. Crown
2006
Several pollutants have the potential to disrupt the endocrine system in aquatic organisms, and synthesis of vitellogenin (VTG) in male fish is a well-recognized effect of estrogenic xenobiotics. In this respect both the presence of the protein in plasma and the analysis of VTG gene induction may represent valuable biomarkers. The present article describes primers specifically designed for a RT-PCR assay of VTG mRNA in various Mediterranean fish species. All the species analyzed have great potential as bioindicators in the Mediterranean: the red mullet (Mullus barbatus) and the striped mullet (Mugil cephalus) are commonly found in coastal and estuarine waters, the black goby (Gobius niger) is an important species in harbors, the European eel (Anguilla anguilla) is more typical of brackish environments and lagoon ecosystems, and the tuna fish (Thunnus thynnus) has commercial value and, being a top predator in marine food webs, is particularly exposed to bioaccumulated halogenated hydrocarbons with possible estrogenic activity. The analysis of VTG mRNA has been standardized in feral fish, and basal expression of VTG was demonstrated in female specimens of the species analyzed. Only sexually immature specimens were analyzed for A. anguilla, and exposure to 17b-estradiol clearly induced the synthesis of VTG mRNA, confirming their responsiveness to estrogenic exposure and the specificity of the designed primers. VTG mRNA was detected in adult males of T. thynnus (4100 kg), supporting estrogenic exposure of older specimens. In this species two different VTGs were identified, and the sequences obtained in the various species were compared with available sequences.
Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 1996
Vitellogenins (VTGs) were isolated from three marine teleosts, the ocean pout (Macrozoarces americanus), lumpfish (Cyclopterus lumpus) and Atlantic cod (Gadus morhua), for the analysis of amino-acid composition and other partial biochemical characterizations. All three VTGs contained high levels of essential amino acids, arginine (5.08-6.54%), lysine (8.44-8.74%), isoleucine (5.60-6.82%), leucine (9.61-11.39%) and valine (6.76-6.99%). While histidine (2.27-2.67%) levels were low, the levels of methionine (1.51-3.38%), threonine (4.69-6.09%) and phenylalanine (3.38-4.41%) varied between species. In the category of non-essential amino acids, aspartic acid (8.60-9.68%), serine (6.09-7.68%), glutamic acid (11.45-11.97%) and alanine (6.49-7.79%) were predominant. Similarity was also noted in the VTG content of protein-bound phosphorus (0.62-0.72%) as well as total lipid (17.92-21.34%) among the three species. However, the molecular weights (485-630 KD), total phosphorus (2.15-3.56%) and the lipid-bound phosphorus contents (1.51-2.86%) of the VTGs varied. VTGs from the Atlantic cod and lumpfish did not cross-react with the antibodies made against ocean pout VTG, suggesting that VTG is species-specific. COMe BIOCHEM PHYSIOL l13B, 247-253, 1996.
Isolation and partial characterization of Asian sea bass (Lates calcarifer) Vitellogenin
Fish Physiology and Biochemistry, 2012
A study was conducted to isolate, partial characterize Asian sea bass (Lates calcarifer) vitellogenin (vtg). Two-year-old juvenile L. calcarifer (n = 10) were given three intraperitoneal injections of 17-β estradiol (E 2) at a dose of 2 mg/kg body weight to induce vitellogenesis. Blood was collected 3 days after the last injection, and plasma was purified through gel filtration chromatography. A broad single symmetrical peak consisting of vtg molecule was produced. Protein concentration was 0.059 mg/ml as determined by Bradfrod assay using bovine serum albumin as a standard. The protein appeared as one circulating form in Native PAGE considering the dimeric form of putative vtg with molecular weight of 545 kDa. In SDS-PAGE under reducing conditions, two major bands appeared at 232.86 and 118.80 kDa and minor bands at 100.60, 85.80 and 39.92 kDa, respectively. The purified vtg was used to generate a polyclonal antibody, and the specificity of antibody was assessed by Western blot analysis. Two major bands were immunoreacted, but no cross-reactivity was observed with plasma from non-induced males. The protein was characterized as phosphoglycolipoprotein as it positively stained for the presence of lipid, phosphorus and carbohydrate using Sudan Black B, methyl green and periodic acid/Schiff reagent solution, respectively. The amino acid composition was analyzed by high sensitivity amino acid analysis that showed high percentage of non-polar amino acids (~48 %). The results suggest the potential utilization of vtg as a basis tool to further study about reproductive physiology of this important economical species.
Comparative Biochemistry and Physiology Part C: Pharmacology, Toxicology and Endocrinology, 1999
The egg yolk precursor protein, vitellogenin (VTG), was purified from blood plasma of 17b-estradiol (E 2 )-treated male fathead minnows (Pimephales promelas) by anion-exchange chromatography on DEAE-agarose. A rabbit antiserum was raised against their blood plasma and then adsorbed with plasma from untreated (control) males to render the antiserum specific to VTG. The adsorbed antiserum was used to detect fathead minnow VTG (fVTG) in Western and dot blotting experiments and in an enzyme-linked immunosorbent assay (ELISA). The antiserum recognised fVTG as a 156 kDa protein in plasma from vitellogenic females and E 2 -injected males but not untreated males. Its identity was confirmed by analysis of: (1) amino acid composition; (2) an internal amino acid sequence; (3) reactivity to the homologous antiserum; and (4) recognition by monoclonal antibodies prepared against the VTG from common carp (Cyprinus carpio) and brown bullhead (Ameiurus nebulosus). Specificity of the homologous antiserum to fVTG was confirmed by Western blotting of serially diluted plasma from vitellogenic females. Utility of the antiserum and purified fVTG for detecting exposure of male fathead minnows to estrogenic compounds was verified using a dot blotting immunoassay of fVTG and detected by chemiluminescence. Adult male fish were exposed to various concentrations of E 2 (10 − 8 , 10 − 9 and 10 − 10 M) in their rearing water and plasma assayed for the presence of VTG at different time points (2, 7, 14 and 21 days). A competitive, antibody-capture, quantitative ELISA was then developed based on the purified fVTG and its respective antiserum. The ELISA was validated by demonstrating parallel binding slopes of dilution curves prepared with plasma from E 2 -injected males, vitellogenic females, and aqueous egg extracts as compared with purified fVTG standard. Plasma concentrations of VTG as low as 3 ng ml − 1 were detected in the ELISA, for which inter-and intra-assay coefficients of variation were both less than 5%. Furthermore, plasma from control males was unreactive with the fVTG antiserum. The VTG ELISA could be useful for the detection of estrogenic properties associated with certain compounds and could be easily incorporated into standard laboratory toxicity assays using this species. (C.V. Sullivan) 0742-8413/99/$ -see front matter © 1999 Elsevier Science Inc. All rights reserved. PII: S 0 7 4 2 -8 4 1 3 ( 9 9 ) 0 0 0 1 0 -9
Environmental Toxicology and Chemistry, 2007
Vitellogenin (VTG) is the major protein present in the plasma of females undergoing oogenesis. In males, the VTG gene normally is suppressed; however, synthesis of VTG can be induced by exposure to xenoestrogenic compounds. In the present study, an enzyme-linked immunosorbent assay was developed and validated to evaluate VTG levels in the California halibut (Paralichthys cali+ornicu.s). Vitellogenin and lipovitellin (LV) were identified in the plasma of 17B-estradiol-induced females and in the ovaries of wild females, to our knowledge for the first time. Purified VTG from the plasma of induced females was obtained, and polyclonal antibodies against the LV of mature female ovaries was prepared and their specificity assessed by Western blot analysis. At Bahía Magdalena, Baja California Sur, México, quantitative measurements of VTG in the plasma of female specimens were made during one reproductive cycle.