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Identification, purification, and immunoassay of stone flounder (Kareius bicolouratus) vitellogenin

Journal of the Korean Society for Applied Biological Chemistry volume 55, pages 219–227 (2012)Cite this article

Abstract

Vitellogenin (Vtg), the precursor of egg yolk proteins, serves as nutrients for the developing embryos. Vtg production in male fish has become an excellent biomarker for environmental estrogens. Thus, Vtg synthesis was induced by injection of 17β-estradiol (E2) in stone flounder (Kareius bicolouratus). Plasma from uninduced and E2-treated fish was subjected to native polyacrylamide gel electrophoresis (PAGE) and the gels were stained with Coomassie brilliant blue R250 for proteins, methyl green for phosphates, Sudan black B for lipids, and periodic acid-Schiff’s reagent for carbohydrates. These analyses verified the presence of two phospholipoglycoproteins in the plasma from E2-treated stone flounder, which were tentatively identified as Vtg. Vtg was then isolated by two purification procedures: gel filtration followed by anion exchange chromatography, or selective precipitation combined with anion exchange chromatography. The latter procedure was more effective at isolating Vtg with a high degree of purity. In native PAGE, the apparent molecular masses of Vtgs were determined to be 520 and 550 kDa, respectively. Two major bands with approximate molecular masses of 165 and 106 kDa together with two additional faint bands of lower molecular weights were observed after sodium dodecyl sulfate- PAGE under reducing conditions. These bands exhibited differential affinity for polyclonal antisera against red drum (Sciaenops ocellatus) Vtg in Western blot. The present study established the purification procedure and characterized the electrophoretic and immunological properties of stone flounder Vtg.

References

  • Byrne BM, Gruber M, and Ab G (1989) The evolution of egg yolk proteins. Prog Biophys Molec Biol 53, 33–69.

    Article  CAS  Google Scholar 

  • Chen JS, Sappington TW, and Raikhel AS (1997) Extensive sequence conservation among insect, nematode, and vertebrate vitellogenins reveals ancient common ancestry. J Mol Evol 44, 440–451.

    Article  CAS  Google Scholar 

  • Copeland PA, Sumpter JP, Walker TK, and Croft M (1986) Vitellogenin levels in male and female rainbow trout (Salmo gairdneri Richardson) at various stages of the reproductive cycle. Comp Biochem Physiol B Biochem Mol Biol 83, 487–493.

    Article  CAS  Google Scholar 

  • Covens M, Covens L, Ollevier F, and Loof AD (1987) A comparative study of some properties of vitellogenin (Vg) and yolk proteins in a number of freshwater and marine teleost fishes. Comp Biochem Physiol B Biochem Mol Biol 88, 75–80.

    Article  CAS  Google Scholar 

  • Cutting JA and Roth TF (1973) Staining of phospho-proteins on acrylamide gel electropherograms. Anal Biochem 54, 386–394.

    Article  CAS  Google Scholar 

  • Finn RN (2007a) The maturational disassembly and differential proteolysis of paralogous vitellogenins in a marine pelagophil teleost: A conserved mechanism of oocyte hydration. Biol Reprod 76, 936–948.

    Article  CAS  Google Scholar 

  • Finn RN (2007b) Vertebrate yolk complexes and the functional implications of phosvitins and other subdomains in vitellogenins. Biol Reprod 76, 926–935.

    Article  CAS  Google Scholar 

  • Fukada H, Haga A, Fujita T, Hiramatsu N, Sullivan CV, and Hara A (2001) Development and validation of chemiluminescent immunoassay for vitellogenin in five salmonid species. Comp Biochem Physiol A Mol Integr Physiol 130, 163–170.

    Article  CAS  Google Scholar 

  • Hartling RC, Pereira JJ, and Kunkel JG (1997) Characterization of a heatstable fraction of lipovitellin and development of an immunoassay for vitellogenin and yolk protein in winter flounder (Pleuronectes americanus). J Exp Zool 278, 156–166.

    Article  CAS  Google Scholar 

  • Heppell SA, Denslow ND, Folmar LC, and Sullivan CV (1995) Universal assay of vitellogenin as a biomarker for environmental estrogens. Environ Health Perspect 103, 9–15.

    CAS  Google Scholar 

  • Hiramatsu N, Chapman RW, Lindzey JK, Haynes MR, and Sullivan CV (2004) Molecular characterization and expression of vitellogenin receptor from white perch (Morone americana). Biol Reprod 70, 1720–1730.

    Article  CAS  Google Scholar 

  • Hiramatsu N, Cheek AO, Sullivan CV, Matsubara T, and Hara A (2005) Vitellogenesis and endocrine disruption. In Biochemistry and Molecular Biology of Fishes, Mommsen TP, and Moon T, pp. 431–471, Elsevier Science Press, Amsterdam, Netherlands.

    Google Scholar 

  • Hiramatsu N, Matsubara T, Fujita T, Sullivan CV, and Hara A (2006) Multiple piscine vitellogenins: Biomarkers of fish exposure to estrogenic endocrine disruptors in aquatic environments. Mar Biol 149, 35–47.

    Article  CAS  Google Scholar 

  • Hiramatsu N, Matsubara T, Hara A, Donato DM, Hiramatsu K, Denslow ND et al. (2002a) Identification, purification and classification of multiple forms of vitellogenin from white perch (Morone americana). Fish Physiol Biochem 26, 355–370.

    Article  CAS  Google Scholar 

  • Hiramatsu N, Matsubara T, Weber GM, Sullivan CV, and Hara A (2002b) Vitellogenesis in aquatic animals. Fish Sci 68, 694–699.

    Article  Google Scholar 

  • Inaba K, Buerano CC, Natividad FF, and Morisawa M (1997) Degradation of vitellogenins by 170 kDa trypsin-like protease in the plasma of the tilapia, Oreochromis niloticus. Comp Biochem Physiol B Biochem Mol Biol 118, 85–90.

    Article  CAS  Google Scholar 

  • Kanungo J, Petrino TR, and Wallace RA (1990) Oogenesis in Fundulus heteroclitus. VI. Establishment and verification of conditions for vitellogenin incorporation by oocytes in vitro. J Exp Zool 254, 313–321.

    Article  CAS  Google Scholar 

  • Kishida M, Anderson TR, and Specker JL (1992) Induction by â-estradiol of vitellogenin in striped bass (Morone saxatilis): Characterization and quantification in plasma and mucus. Gen Comp Endocrinol 88, 29–39.

    Article  CAS  Google Scholar 

  • Kishida M and Specker JL (1993) Vitellogenin in tilapia (Oreochromis mossambicus): induction of two forms by estradiol, quantification in plasma and characterization in oocyte extract. Fish Physiol Biochem 12, 171–182.

    Article  CAS  Google Scholar 

  • Komatsu M, Matsunoto W, and Hayashi S (1996) Protease activity appeared after trypsin treatment of the purified vitellogenin from eel Anguilla japonica. Comp Biochem Physiol B Biochem Mol Biol 113, 561–571.

    Article  Google Scholar 

  • Korsgaard B and Pedersen KL (1998) Vitellogenin in Zoarces viviparus: purification, quantification by ELISA and induction by estradiol-17â and 4-nonylphenol. Comp Biochem Physiol C Toxicol Pharmacol 120, 159–166.

    Article  CAS  Google Scholar 

  • Koya Y, Matsubara T, Ikeuchi T, Adachi S, and Yamauchi K (1997) Annual changes in serum vitellogenin concentrations in viviparous eelpout, Zoarces elongatus. Comp Biochem Physiol A Mol Integr Physiol 118, 1217–1223.

    CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277, 680–687.

    Article  Google Scholar 

  • Lee FY, Shih TW, and Chang CF (1997) Isolation and characterization of the female-specific protein (vitellogenin) in mature female hemolymph of the freshwater prawn, Macrobrachium rosenbergii: Comparison with ovarian vitellin. Gen Comp Endocrinol 108, 406–415.

    Article  CAS  Google Scholar 

  • Magalhães I, Ledrich ML, Pihan JC, and Falla J (2004) One-step, nondenaturing purification method of carp (Cyprinus carpio) vitellogenin. J Chromatogr B Analyt Technol Biomed Life Sci 799, 87–93.

    Article  Google Scholar 

  • Maltais D and Roy RL (2009) Purification and partial characterization of vitellogenin from shorthead redhorse (Moxostoma macrolepidotum) and copper redhorse (Moxostoma hubbsi) and detection in plasma and mucus with a heterologous antibody. Fish Physiol Biochem 35, 241–254.

    Article  CAS  Google Scholar 

  • Marin GM and Matozzo V (2004) Vitellogenin induction as a biomarker of exposure to estorogenic compounds in aquatic environment. Mar Pollut Bull 48, 835–839.

    Article  CAS  Google Scholar 

  • Marx A, Sherry J, Hassen PD, and Hock B (2001) A new monoclonal antibody against vitellogenin from rainbow trout (Oncorhynchus mykiss). Chemosphere 44, 393–399.

    Article  CAS  Google Scholar 

  • Matsubara T, Nagae M, Ohkubo N, Andoh T, Sawaguchi S, Hiramatsu N et al. (2003) Multiple vitellogenins and their unique roles in marine teleosts. Fish Physiol Biochem 28, 295–299.

    Article  CAS  Google Scholar 

  • Matsubara T, Ohkubo N, Andoh T, Sullivan CV, and Hara A (1999) Two forms of vitellogenin, yielding two distinct lipovitellins, play different roles during oocyte maturation and early development of barfin flounder, Verasper moseri, a marine teleost that spawns pelagic eggs. Dev Biol 213, 18–32.

    Article  CAS  Google Scholar 

  • Matsubara T and Sawano K (1995) Proteolytic cleavage of vitellogenin and yolk proteins during vitellogenin uptake and oocyte maturation in barfin flounder (Verasper moseri). J Exp Zool 272, 34–45.

    Article  CAS  Google Scholar 

  • Meucci V and Arukwe A (2005) Detection of vitellogenin and zona radiata protein expressions in surface mucus of immature juvenile Atlantic salmon (Salmo salar) exposed to waterborne nonylphenol. Aquatic Toxicol 73, 1–10.

    Article  CAS  Google Scholar 

  • Mommsen TP and Walsh PJ (1988) Vitellogenesis and oocyte assembly. In Fish Physiology, Hoar WS, and Randall DJ, pp. 347–406, Academic Press, New York, NY.

    Google Scholar 

  • Nilsen BM, Berg K, Arukwe A, and Goksøyr A (1998) Monoclonal and polyclonal antibodies against fish vitellogenin for use in pollution monitoring. Mar Environ Res 46, 153–157.

    Article  CAS  Google Scholar 

  • Norberg B (1995) Atlantic halibut (Hippoglossus hippoglossus) vitellogenin: induction, isolation and partial characterization. Fish Physiol Biochem 14, 1–13.

    Article  CAS  Google Scholar 

  • Norberg B and Haux C (1988) An homologous radioimmunoassay for brown trout (Salmo trutta) vitellogenin. Fish Physiol Biochem 5, 59–68.

    Article  CAS  Google Scholar 

  • Norberg B and Haux C (1985) Induction, isolation and a characterization of the lipid content of plasma vitellogenin from two Salmo species: Rainbow trout (Salmo gairdneri) and sea trout (Salmo trutta). Comp Biochem Physiol B Biochem Mol Biol 81, 869–876.

    Article  CAS  Google Scholar 

  • Nunez-Rodriguez J, Kah O, Geffard M, and Le Menn F (1989) Enzyme linked immunosorbent assay (ELISA) for sole (Solea vulgaris) vitellogenin. Comp Biochem Physiol B Biochem Mol Biol 92, 741–746.

    Article  Google Scholar 

  • Ohkubo N, Mochida K, Adachi S, Hara A, Hotta K, Nakamura Y et al. (2003) Development of enzyme-linked immunosorbent assays for two forms of vitellogenin in Japanese common goby (Acanthogobius flavimanus). Gen Comp Endocriol 131, 353–364.

    Article  CAS  Google Scholar 

  • Pan ZB (2011) Preparation of polyclonal antisera against vitellogenin from Sciaenops ocellatus and its application to screening pesticides with estrogenic activities. MS Thesis, Ocean University of China, Qingdao, China.

    Google Scholar 

  • Parks LG, Cheek AO, Denslow ND, Heppell SA, McLachlan JA, LeBlanc GA et al. (1999) Fathead minnow (Pimephales promelas) vitellogenin: purification, characterization and quantitative immunoassay for the detection of estrogenic compounds. Comp Biochem Physiol C Toxicol Pharmacol 123, 113–125.

    Article  CAS  Google Scholar 

  • Prat JP, Lamy JN, and Weill JD (1969) Coloration des lipoprotéines après électrophorèse en gel de polyacrylamide. Bull Soc Chim Biol 51, 1367.

    CAS  Google Scholar 

  • Reith M, Munholland J, Kelly J, and Finn RN (2001) Lipovitellins derived from two forms of vitellogenin are differentially processed during oocyte maturation in haddock (Melanogrammus aeglefinus). J Exp Zool 291, 58–67.

    Article  CAS  Google Scholar 

  • Roubal WT, Lomax DP, Willis ML, and Johnson LL (1997) Purification and partial characterization of English sole (Pleuronectes vetulus) vitellogenin. Comp Biochem Physiol B Biochem Mol Biol 118, 613–622.

    Article  CAS  Google Scholar 

  • Roy RL, Morin Y, Courtenay SC, and Robichaud P (2004) Purification of vitellogenin from smooth flounder (Pleuronectes putnami) and measurement in plasma by homologous ELISA. Comp Biochem Physiol B Biochem Mol Biol 139, 235–244.

    Article  Google Scholar 

  • Sawaguchi S, Kagawa H, Ohkubo N, Hiramatsu N, Sullivan CV, and Matsubara T (2006) Molecular characterization of three forms of vitellogenin and their yolk protein products during oocyte growth and maturation in red seabream (Pagrus major), a marine teleost spawning pelagic eggs. Mol Reprod Dev 73, 719–736.

    Article  CAS  Google Scholar 

  • Sawaguchi S, Koya Y, Yoshizaki N, Ohkubo N, Andoh T, Hiramatsu N et al. (2005) Multiple vitellogenins (Vgs) in mosquitofish (Gambusia affinis): identification and characterization of three functional Vg genes and their circulating and yolk protein products. Biol Reprod 72, 1045–1060.

    Article  CAS  Google Scholar 

  • Sehgal N and Goswami SV (2005) Vitellogenin exists as charge isomers in the Indian freshwater murrel, Channa punctatus (Bloch). Gen Comp Endocrinol 141, 12–21.

    Article  CAS  Google Scholar 

  • Selman K and Wallace RA (1983) Oogenesis in Fundulus heteroclitus. III. Vitellogenesis. J Exp Zool 226, 441–457.

    Article  CAS  Google Scholar 

  • Silversand C and Haux C (1989) Isolation of turbot (Scophthalmus maximus) vitellogenin by high performance chromatography. J Chromatogr A 478, 387–397.

    CAS  Google Scholar 

  • Silversand C, Hyllner SJ, and Haux C (1993) Isolation, immunochemical detection and observations of the instability of vitellogenin from four teleosts. J Exp Zool 267, 587–597.

    Article  CAS  Google Scholar 

  • Specker JL and Sullivan CV (1994) Vitellogenesis in fishes: status and perspectives. In Perspectives in Comparative Endocrinology, Davey KG, Peter RE, and Tobe SS, pp. 304–315, National Research Council, Ottawa, Canada.

    Google Scholar 

  • Stifani S, Le Menn F, Rodriguez JN, and Schneider WJ (1990) Regulation of oogenesis: the piscine receptor for vitellogenin. Biochem Biophys Acta 3, 271–279.

    Google Scholar 

  • Sumpter JP and Jobling S (1995) Vitellogenesis as a biomarker for estrogenic contaminants of the aquatic environment. Environ Health Perspect. 103(Suppl 7), 173–178.

    CAS  Google Scholar 

  • Susca V, Corriero A, Bridges CR, and de Metrio G (2001) Study of the sexual maturity of female bluefin tuna: Purification and partial characterization of vitellogenin and its use in an enzyme-linked immunosorbent assay. J Fish Biol 58, 815–831.

    Article  CAS  Google Scholar 

  • Tolar JF, Mehollin AR, Watson RD, and Angus RA (2001) Mosquitofish (Gambusia affinis) vitellogenin: Identification, purification, and immunoassay. Comp Biochem Physiol C Toxicol Pharmacol 128, 237–245.

    Article  CAS  Google Scholar 

  • Tyler CR and Sumpter JP (1990) The purification and partial characterization of carp, Cyprinus carpio, vitellogenin. Fish Physiol Biochem 8, 111–120.

    Article  CAS  Google Scholar 

  • Tyler CR, van Aerle R, Hutchinson TH, Maddix S, and Trip H (1999) An in vivo testing system for endocrine disruptors in fish early life stages using induction of vitellogenin. Environ Toxicol Chemistry 18, 337–347.

    Article  CAS  Google Scholar 

  • Wallace RA (1985) Vitellogenesis and oocyte growth in nonmammalian vertebrates. In Oogenesis, Browder LW, pp. 127–177, Plenum Press, New York, NY.

    Chapter  Google Scholar 

  • Wang H, Yan T, Tan JTT, and Gong Z (2000) A zebrafish vitellogenin gene (Vg3) encodes a novel vitellogenin without a phosvitin domain and may represent a primitive vertebrate vitellogenin gene. Gene 256, 303–310.

    Article  CAS  Google Scholar 

  • Wiley HS, Opresko L, and Wallace RA (1979) New methods for the purification of vertrebrate vitellogenin. Analyt Biochem 97, 145–152.

    Article  CAS  Google Scholar 

  • Wray W, Bonlikas T, Wray VP, and Hancock R (1981) Silver staining of proteins in polyacrylamide gels. Anal Biochem 118, 197–203.

    Article  CAS  Google Scholar 

  • Zacharius RM, Zell IL, Merrison JH, and Nordlock JJ (1969) Glycoprotein staining following electrophoresis on acrylamide gels. Anal Biochem 30, 148–152.

    Article  CAS  Google Scholar 

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  1. College of Marine Life Sciences, Ocean University of China, Qingdao, 266003, China

    Zongbao Pan, Hua Tian, Wei Wang, Jun Wang & Shaoguo Ru

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  1. Zongbao Pan
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  2. Hua Tian
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  3. Wei Wang
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  4. Jun Wang
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  5. Shaoguo Ru
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Correspondence to Shaoguo Ru.

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Z. Pan and H. Tian contributed equally

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Pan, Z., Tian, H., Wang, W. et al. Identification, purification, and immunoassay of stone flounder (Kareius bicolouratus) vitellogenin. J Korean Soc Appl Biol Chem 55, 219–227 (2012). https://doi.org/10.1007/s13765-012-1014-z

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