Fish Physiology and Biochemistry

, Volume 41, Issue 1, pp 107–117 | Cite as

Characterization of incomplete vitellogenin (VgC) in the Indian freshwater murrel, Channa punctatus (Bloch)

  • S. Pipil
  • V. S. Rawat
  • L. Sharma
  • N. Sehgal


A novel incomplete vitellogenin (VgC) was purified from the plasma of estradiol-treated male murrel, Channa punctatus, by gel filtration chromatography. The native mass of VgC protein was 180 kDa, and it resolved as a single peptide of 100 kDa on SDS-PAGE. The peptide on subjecting to matrix-assisted laser desorption/ionization-time of flight produced a peptide mass fingerprint. On tandem mass spectrometry, some of these peptides showed mass to charge (m/z) ratio and amino acid sequence similarity with VgC peptides of other teleosts. Phylogenetic analysis revealed a similarity of murrel VgC with fish species of the order Perciformes. Semi-quantitative RT-PCR assay was developed to study expression of vgc gene at variable levels of estradiol exposure. Presence of VgC in males indicates that fish has been exposed to estrogens; hence, it can be used as a biomarker for estrogenic exposure.


Incomplete vitellogenin (VgC) MALDI-TOF–MS/MS Channa punctatus RT-PCR 



This work was supported by Research Grant from the University of Delhi, Delhi. One of us (SP) is thankful to CSIR, New Delhi, for the award of a Senior Research Fellowship.


  1. Amano H, Fujita T, Hiramatsu N, Sawaguchi S, Matsubara T, Sullivan CV, Hara A (2007) Purification of multiple vitellogenins in grey mullet (Mugil cephalus). Mar Biol 152(6):1215–1225CrossRefGoogle Scholar
  2. Amano H, Mochizuki M, Fujita T, Hiramatsu N, Todo T, Hara A (2010) Purification and characterization of a novel incomplete-type vitellogenin protein (VgC) in Sakhalin taimen (Hucho perryi). Comp Biochem Physiol A 157:41–48CrossRefGoogle Scholar
  3. Babin PJ (2008) Conservation of a vitellogenin gene cluster in oviparous vertebrates and identification of its traces in the platypus genome. Gene 413:76–82PubMedCrossRefGoogle Scholar
  4. Babin PJ, Carnevali O, Lubzens E, Schneider WJ (2007) Molecular aspects of oocyte vitellogenesis in fish. In: Babin PJ, Cerdà J, Lubzens E (eds) The fish oocyte: from basic studies to biotechnological applications. Springer, Netherlands, pp 39–76CrossRefGoogle Scholar
  5. Banoub J, Cohen A, Mansour A, Thibault P (2004) Characterization and de novo sequencing of Atlantic salmon vitellogenin protein by electrospray tandem and matrix-assisted laser desorption/ionization mass spectrometry. Eur J Mass Spectrom 10:121–134CrossRefGoogle Scholar
  6. Barton SJ, Whittaker JC (2009) Review of factors that influence the abundance of ions produced in a tandem mass spectrometer and statistical methods for discovering these factors. Mass Spectrom Rev 28(1):177–187PubMedCrossRefGoogle Scholar
  7. Bergink EW, Wallace RA (1974) Precursor-product relationship between amphibian vitellogenin and the yolk proteins, lipovitellin and phosvitin. J Biol Chem 249(9):2897–2903PubMedGoogle Scholar
  8. Cerdà J, Fabra M, Raldúa D (2007) Physiological and molecular basis of fish oocyte hydration. In: Babin PJ, Cerdà J, Lubzens E (eds) The fish oocyte: from basic studies to biotechnological applications. Springer, Dordrecht, pp 349–396CrossRefGoogle Scholar
  9. Chen JS, Sappington TW, Raikhel AS (1997) Extensive sequence conservation among insect, nematode, and vertebrate vitellogenins reveals ancient common ancestry. J Mol Evol 44:440–451PubMedCrossRefGoogle Scholar
  10. Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins DG, Thompson JD (2003) Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res 31:3497–3500PubMedCentralPubMedCrossRefGoogle Scholar
  11. Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol-chloroform extraction. Anal Biochem 162:156–159PubMedCrossRefGoogle Scholar
  12. Cohen AM, Mansour AA, Banoub JH (2005) ‘De novo’ sequencing of Atlantic cod vitellogenin tryptic peptides by matrix-assisted laser desorption/ionization quadrupole time-of-flight tandem mass spectrometry: similarities with haddock vitellogenin. Rapid Commun Mass Spectrom 19:2454–2460PubMedCrossRefGoogle Scholar
  13. Cutting JA, Roth TF (1973) Staining of phosphoproteins on acrylamide gel electropherograms. Anal Biochem 54:386–394PubMedCrossRefGoogle Scholar
  14. Ding JL, Hee PL, Lam TJ (1989) Two forms of vitellogenin in plasma and gonads of male Oreochromis aureus. Comp Biochem Physiol B 93:363–370Google Scholar
  15. Finn RN, Kristoffersen BA (2007) Vertebrate vitellogenin gene duplication in relation to the “3R Hypothesis”: correlation to the pelagic egg and the oceanic radiation of teleosts. PLoS One 2(1):e169. doi: 10.1371/journal.pone0000169 PubMedCentralPubMedCrossRefGoogle Scholar
  16. Fujiwara Y, Fukada H, Shimizu M, Hara A (2005) Purification of two lipovitellins and development of immunoassays for two forms of their precursors (vitellogenins) in medaka (Oryzias latipes). Gen Comp Endocrinol 143:267–277PubMedCrossRefGoogle Scholar
  17. Hamazaki T, Iuchi I, Yamagami K (1985) A spawning female-specific substance reactive to anti-chorion (egg-envelope) glycoprotein antibody in the teleost, Oryzias latipes. J Exp Zool 235:269–279CrossRefGoogle Scholar
  18. Hamazaki TS, Iuchi I, Yamagami K (1987) Purification and identification of vitellogenin and its immunohistochemical detection in growing oocytes of the teleost, Oryzias latipes. J Exp Zool 242:333–342CrossRefGoogle Scholar
  19. Hayes RN, Gross ML (1990) Collision induced dissociation. Methods Enzymol 193:237–263PubMedCrossRefGoogle Scholar
  20. Henzel WJ, Watanabe C, Stults JT (2003) Protein identification: the origins of peptide mass fingerprinting. J Am Soc Mass Spectrom 14:931–942PubMedCrossRefGoogle Scholar
  21. Hiramatsu N, Matsubara T, Hara A, Donato D, Hiramatsu K, Denslow N, Sullivan C (2002) Identification, purification and classification of multiple forms of vitellogenin from white perch (Morone americana). Fish Physiol Biochem 26:355–370CrossRefGoogle Scholar
  22. Hiramatsu N, Cheek AO, Sullivan CV, Matsubara T, Hara A (2005) Vitellogenesis and endocrine disruption. In: Mommsen TP, Moon TW (eds) Biochemistry and molecular biology of fishes. Environmental toxicology, 6. Elsevier, Amsterdam, pp 431–471Google Scholar
  23. Hiramatsu N, Matsubara T, Fujita T, Sullivan CV, Hara A (2006) Multiple piscine vitellogenins: biomarkers of fish exposure to estrogenic endocrine disruptors in aquatic environments. Mar Biol 149:35–47CrossRefGoogle Scholar
  24. Idler DR, Campbell CM (1980) Gonadotropin stimulation of estrogen and yolk precursor synthesis in juvenile rainbow trout. Gen Comp Endocrinol 41:384–391PubMedCrossRefGoogle Scholar
  25. Kidd KA, Blanchfield PJ, Mills H, Palace VP, Evans RE, Lazorchak JM, Flick R (2007) Collapse of a fish population following exposure to a synthetic estrogen. PNAS 104:8897–8901PubMedCentralPubMedCrossRefGoogle Scholar
  26. Kishida M, 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 12:171–182PubMedCrossRefGoogle Scholar
  27. Lattier DL, Gordon DA, Burks DJ, Toth GP (2001) Vitellogenin gene transcription: a relative quantitative exposure indicator of environmental estrogens. Environ Toxicol Chem 20:1979–1985PubMedCrossRefGoogle Scholar
  28. Matsubara T, Nagae M, Ohkubo N, Andoh T, Sawaguchi S, Hiramatsu N, Sullivan CV, Hara A (2003) Multiple vitellogenins and their unique roles in marine teleosts. Fish Physiol Biochem 28:295–299CrossRefGoogle Scholar
  29. McLuckey SA (1992) Principles of collisional activation in analytical mass spectrometry. J Am Soc Mass Spec 3:599–614Google Scholar
  30. Mommsen TP, Korsgaard B (2008) Vitellogenesis. In: Rocha MJ, Arukwe A, Kapoor BG (eds) Fish reproduction. Science Publisher, New York, pp 113–169Google Scholar
  31. Mommsen SP, Walsh KL (1988) Vitellogenesis and oocyte assembly. In: Hoar WS, Randall DJ, Donaldson EM (eds) “Fish physiology”. New York: Academic Press Vol 11A, pp 347–406Google Scholar
  32. Murata K, Yamamoto K, Iuchi I, Yasumasu I, Yamagami K (1997) Intrahepatic expression of genes encoding choriogenins: precursor proteins of the egg envelope of fish, the medaka, Oryzias latipes. Fish Physiol Biochem 17:135–142CrossRefGoogle Scholar
  33. Ohkubo N, Mochida K, Adachi S, Hara A, Hotta K, Nakamura Y, Matsubara T (2003) Development of enzyme-linked immunosorbent assays for two forms of vitellogenin in Japanese common goby (Acanthogobius flavimanus). Gen Comp Endocrinol 131:353–364PubMedCrossRefGoogle Scholar
  34. Paizs B, Suhai S (2005) Fragmentation pathways of protonated peptides. Mass Spectrom Rev 24(4):508–548PubMedCrossRefGoogle Scholar
  35. Palumbo AJ, Linares-Casenave J, Jewell W, Doroshov SI, Tjeerdema RS (2007) Induction and partial characterization of California halibut (Paralichthys californicus) vitellogenin. Comp Biochem Physiol A Mol Integr Physiol 146(2):200–207Google Scholar
  36. Papayannopoulos IA (1995) The interpretation of collision-induced dissociation tandem mass spectra of peptides. Mass Spectrom Rev 14(1):49–73CrossRefGoogle Scholar
  37. Patiño R, Sullivan CV (2002) Ovarian follicle growth, maturation, and ovulation in teleost fish. Fish Physiol Biochem 26:57–70CrossRefGoogle Scholar
  38. Rawat VS, Rani KV, Phartyal R, Sehgal N (2013a) Vitellogenin genes in fish: differential expression on exposure to estradiol. Fish Physiol Biochem 39:39–46PubMedCrossRefGoogle Scholar
  39. Rawat VS, Pipil S, Luni Sharma, Sehgal N (2013b) Purification, characterization and expression of two vitellogenins in the Indian freshwater murrel Channa punctatus. Gen Comp Endocrinol 189:119–126PubMedCrossRefGoogle Scholar
  40. Sawaguchi S, Ohkubo N, Koya Y, Matsubara T (2005) Incorporation and utilization of multiple forms of vitellogenin and their derivative yolk proteins during vitellogenesis and embryonic development in the mosquitofish, Gambusia affinis. Zool Sci 22:701–710PubMedCrossRefGoogle Scholar
  41. Sawaguchi S, Kagawa H, Ohkubo N, Hiramatsu N, Sullivan C, 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 73:719–736PubMedCrossRefGoogle Scholar
  42. Scholz S, Kordes C, Hamann J (2004) Induction of vitellogenin in vivo and in vitro in the model teleost medaka (Oryzias latipes): comparison of gene expression and protein levels. Mar Environ Res 57:235–244PubMedCrossRefGoogle Scholar
  43. Sehgal N, Goswami SV (2001) Purification of vitellogenin from the plasma of the Indian freshwater murrel, Channa punctatus (Bloch) by different methods: a comparative study. Indian J Biochem Biophys 38:263–269PubMedGoogle Scholar
  44. Sehgal N, Goswami SV (2002) Immunological identification of two female-specific proteins from the plasma of Indian freshwater murrel, Channa punctatus (Bloch). Indian J Exp Biol 40:288–295PubMedGoogle Scholar
  45. Sehgal N, Goswami SV (2005) Vitellogenin exists as charge isomers in the Indian freshwater murrel, Channa punctatus (Bloch). Gen Comp Endocrinol 141:12–21PubMedCrossRefGoogle Scholar
  46. Sharrock WJ, Rosenwasser TA, Gould J, Knott J, Hussey D, Gordon JI, Banaszak L (1992) Sequence of lamprey vitellogenin. Implications for the lipovitellin crystal structure. J Mol Biol 226:903–907PubMedCrossRefGoogle Scholar
  47. Specker JL, Sullivan CV (1994) Vitellogenesis in fishes: status and perspectives. In: Davey KG, Peter RE, Tobe SS (eds) Perspectives in comparative endocrinology. National Research Council, Ottwa, pp 304–315Google Scholar
  48. Takemura A, Kim BH (2001) Effects of estradiol-17 beta treatment on in vitro and in vivo synthesis of two distinct vitellogenins in tilapia. Comp Biochem Physiol A 129:641–651CrossRefGoogle Scholar
  49. Wahli W (1988) Evolution and expression of vitellogenin genes. Trends Genet 4:227–232PubMedCrossRefGoogle Scholar
  50. Wang H, Yan T, Tan JTT, Gong ZY (2000) A zebrafish vitellogenin gene (vg3) encodes a novel vitellogenin without a phosvitin domain and may represent a primitive vertebrate vitellogenin gene. Gene 256:303–310PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  1. 1.Department of ZoologyUniversity of DelhiDelhiIndia
  2. 2.Department of Zoology, Hindu CollegeUniversity of DelhiDelhiIndia

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