Fisheries Science

, Volume 85, Issue 1, pp 127–135 | Cite as

Purification and biochemical characterization of a vitellogenin-like protein from sea urchin

  • Osamu NishimiyaEmail author
  • Yoshihiko Teraoka
  • Takahiro Gotoh
  • Tomoharu Yuhi
  • Ichiro Higuchi
  • Kazuhiro Ura
  • Yasuaki Takagi
Original Article Biology


The major yolk protein in sea urchins is a transferrin-like protein. In this report, a new component was detected in gonad extracts of Strongylocentrotus intermedius and Mesocentrotus nudus, which cross-reacts with antiserum against egg yolk proteins. We tentatively named them egg yolk-related proteins siYRP and mnYRP. The siYRP was purified from testis of S. intermedius by ammonium sulfate fractionation, anion exchange chromatography, affinity chromatography and gel filtration. Purified siYRP was > 900 kDa in size. The siYRP purified on SDS-PAGE under reducing conditions gave seven bands, corresponding to 93, 213 and > 250 kDa. Purified mnYRP displayed similar structural characteristics as siYRP. Purified siYRP and mnYRP were identified by tandem mass spectrometry and renamed as siVitellogenin (Vtg)-like and mnVtg-like proteins, respectively. Both Vtg-like proteins were confirmed to be lipoglycoproteins by staining with Sudan black and periodic acid-Schiff. A specific antiserum against the siVtg-like protein was raised in rabbit. Antiserum against the siVtg-like protein immunostained siVtg-like and mnVtg-like proteins. Immunochemical methods using the antiserum revealed that siVtg-like and mnVtg-like proteins were present in the ovary, testis and unfertilized eggs of both species. These results indicated that Vtg-like proteins have important physiological functions for gonadal growth and gametogenesis in sea urchins.


Strongylocentrotus intermedius Mesocentrotus nudus Egg Yolk protein Large lipid transfer protein superfamily 



We thank Dr. Hiroyuki Munehara from the Usujiri Fisheries Station, Field Science Center for the Northern Biosphere, Hokkaido University, for providing samples and helpful advice. This work was supported by the Science and Technology Research Promotion program for Agriculture, Forestry, Fisheries and Food industry, and The Hokusui Society Foundation. We thank the Edanz Group ( for editing a draft of this manuscript.


  1. Alqaisi KM, Lamare MD, Grattan DR, Damsteegt EL, Schneider WJ, Lokman PM (2016) A comparative study of vitellogenesis in Echinodermata: lessons from the sea star. Comp Biochem Physiol A Mol Integr Physiol 198:72–86CrossRefGoogle Scholar
  2. Babin PJ (2008) Conservation of a vitellogenin gene cluster in oviparous vertebrates and identification of its traces in the platypus genome. Gene 413:76–82CrossRefGoogle Scholar
  3. Babin PJ, Bogerd J, Kooiman FP, Van Marrewijk WJ, Van der Horst DJ (1999) Apolipophorin II/I, apolipoprotein B, vitellogenin, and microsomal triglyceride transfer protein genes are derived from a common ancestor. J Mol Evol 49:150–160CrossRefGoogle Scholar
  4. Brooks JM, Wessel GM (2002) The major yolk protein in sea urchins is a transferrin-like, iron binding protein. Dev Biol 245:1–12CrossRefGoogle Scholar
  5. Byrne BM, Gruber MABG, Ab G (1989) The evolution of egg yolk proteins. Prog Biophys Mol Biol 53:33–69CrossRefGoogle Scholar
  6. Damsteegt EL, Mizuta H, Hiramatsu N, Lokman PM (2015) How do eggs get fat? Insights into ovarian fatty acid accumulation in the shortfinned eel, Anguilla australis. Gen Comp Endocrinol 221:94–100CrossRefGoogle Scholar
  7. Davis B (1964) Disc electrophoresis-II. Method and application to human serum proteins. Ann NY Acad Sci 121:404–427CrossRefGoogle Scholar
  8. Evans AJ, Burley RW (1987) Proteolysis of apoprotein B during the transfer of very low density lipoprotein from hens’ blood to egg yolk. J Bio Chem 262:501–504Google Scholar
  9. 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:e169CrossRefGoogle Scholar
  10. Grabar P, Williams CA (1953) Méthode permettant l’étude conjuguée des propriétés électrophorétiques et immunochimiques d’un mélange de protéines. Biochem Biophys Acta 10:193–194CrossRefGoogle Scholar
  11. Hara A (1987) Studies on female-specific serum proteins (vitellogenin) and egg yolk proteins in teleosts: immunochemical, physicochemical and structural studies. Mem Fac Fish Hokkaido Univ 34:1–59Google Scholar
  12. Hara A, Hiramatsu N, Fujita T (2016) Vitellogenesis and choriogenesis in fishes. Fish Sci 82:187–202CrossRefGoogle Scholar
  13. Harrington FE, Easton DP (1982) A putative precursor to the major yolk protein of the sea urchin. Dev Biol 94(2):505–508CrossRefGoogle Scholar
  14. Hayley M, Sun M, Merschrod S, Erika F, Davis PJ, Robinson JJ (2008) Biochemical analysis of the interaction of calcium with toposome: a major protein component of the sea urchin egg and embryo. J Cell Biochem 103:1464–1471CrossRefGoogle Scholar
  15. Hiramatsu N, Hara A (1996) Relationship between vitellogenin and its related egg yolk proteins in Sakhalin taimen (Hucho perryi). Comp Biochem Physiol 115A:243–251CrossRefGoogle Scholar
  16. Hiramatsu N, Matsubara T, Weber GM, Sullivan CV, Hara A (2002) Vitellogenesis in aquatic animals. Fish Sci 68:694–699CrossRefGoogle Scholar
  17. 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. In: Environmental toxicology, vol 6. Elsevier, Amsterdam, pp 431–471Google Scholar
  18. Hiramatsu N, Todo T, Sullivan CV, Schilling J, Reading BJ, Matsubara T, Ryu Y, Mizuta H, Luo W, Nishimiya O, Wu M, Mushirobira Y, Yilmaz O, Hara A (2015) Ovarian yolk formation in fishes: molecular mechanisms underlying formation of lipid droplets and vitellogenin-derived yolk proteins. Gen Comp Endocrinol 221:9–15CrossRefGoogle Scholar
  19. Ii I, Deguchi K, Kawasima S, Endo S, Ueta N (1978) Water-soluble lipoprotein from yolk granules in sea urchin egg. I. Isolation and general properties. J Biol Chem 84:737–749Google Scholar
  20. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefGoogle Scholar
  21. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275Google Scholar
  22. Nishimiya O, Kunihiro Y, Hiramatsu N, Inagawa H, Todo T, Hara A (2014) Biochemical and immunochemical characterization of two discrete vitellogenin proteins and their derived lipovitellins in the inshore hagfish (Eptatretus burgeri). Zoolog Sci 31:251–257CrossRefGoogle Scholar
  23. Ouchterlony Ö (1953) Antigen-antibody reactions in gels. APMIS 32:231–240Google Scholar
  24. Perkins ND, Pappin DJ, Creasy DM, Cottrell JS (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20:3551–3567CrossRefGoogle Scholar
  25. Prat JP, Lamy JN, Weill JD (1969) Coloration des lipoprotein après electrophorese en gel de polyacrylamid. Bull Soc Chim Biol 51:1367Google Scholar
  26. Prowse TA, Byrne M (2012) Evolution of yolk protein genes in the Echinodermata. Evol Dev 14:139–151CrossRefGoogle Scholar
  27. Sammar M, Levi L, Hurvitz A, Lubzens E (2005) Studies on retinol-binding protein during vitellogenesis in the rainbow trout (Oncorhynchus mykiss). Gen Comp Endocrinol 141:141–151CrossRefGoogle Scholar
  28. Smolenaars MM, Madsen O, Rodenburg KW, Van der Horst DJ (2007) Molecular diversity and evolution of the large lipid transfer protein superfamily. J Lipid Res 48:489–502CrossRefGoogle Scholar
  29. Song JL, Wong JL, Wessel GM (2006) Oogenesis: single cell development and differentiation. Dev Biol 300:385–405CrossRefGoogle Scholar
  30. Specker JL, Sullivan CV (1994) Vitellogenesis in fishes: status and perspectives. In: Davey RE et al (eds) Perspectives in comparative endocrinology. National Research Council, Ottawa, pp 304–315Google Scholar
  31. Unuma T, Konishi K, Furuita H, Yamamoto T, Akiyama T (1996) Seasonal changes in gonads of cultured and wild red sea urchin, Pseudocentrotus depressus. Aquac Sci 44:169–175Google Scholar
  32. Unuma T, Suzuki T, Yamamoto T, Akiyama T (1998) A protein identical to the yolk protein is stored in the testis in male red sea urchin, Pseudocentrotus depressus. Biol Bull 194:92–97CrossRefGoogle Scholar
  33. Unuma T, Yamamoto T, Akiyama T, Shiraishi M, Ohta H (2003) Quantitative changes in yolk protein and other components in the ovary and testis of the sea urchin Pseudocentrotus depressus. J Exp Biol 206:365–372CrossRefGoogle Scholar
  34. Unuma T, Ikeda K, Yamano K, Moriyama A, Ohta H (2007) Zinc-binding property of the major yolk protein in the sea urchin—implications of its role as a zinc transporter for gametogenesis. FEBS J 274:4985–4998CrossRefGoogle Scholar
  35. Unuma T, Nakamura A, Yamano K, Yokota Y (2010) The sea urchin major yolk protein is synthesized mainly in the gut inner epithelium and the gonadal nutritive phagocytes before and during gametogenesis. Mol Reprod Dev 77(1):59–68CrossRefGoogle Scholar
  36. Ura K, Wang H, Hori T, Aizawa S, Tsue S, Satoh M, Takei N, Hoshino K, Higuchi I, Sanuki S, Yuhi T, Nishimiya O, Takagi Y (2017) The reproductive cycle and transcription level changes in the major yolk protein of wild northern sea urchin, Mesocentrotus nudus, in southern Hokkaido. Aqua Sci 65:231–237Google Scholar
  37. Wahli W (1988) Evolution and expression of vitellogenin genes. Trends Genet 4:227–232CrossRefGoogle Scholar
  38. Walker CW, Unuma T, McGinn NA, Harrington LM, Lesser MP (2001) Reproduction of sea urchins. In: Lawrence JM (ed) Edible sea urchins: biology and ecology. Elsevier, Amsterdam, pp 5–26CrossRefGoogle Scholar
  39. Wang H, Ura K, Takagi Y (2015) The major yolk protein in sea urchin egg yolk granules is a glycoprotein complex. Fish Sci 81:1127–1134CrossRefGoogle Scholar
  40. Yamane K, Yagai T, Nishimiya O, Sugawara R, Amano H, Fujita T, Hiramatsu N, Todo T, Matsubara T, Hara A (2013) Characterization of vitellogenin and its derived yolk proteins in cloudy catshark (Scyliorhinus torazame). Fish Physiol Biochem 39:373–390Google Scholar
  41. Yokota Y, Unuma T, Moriyama A (2004) Echinoferrin: a newly proposed name for a precursor to yolk protein in the sea urchin. In: Echinoderms: München: Proceedings of the 11th international echinoderm conference, 6–10 October 2003. CRC Press, Boca Raton, p 79Google Scholar
  42. Zacharius RM, Zell TE, Morrison JH, Woodlock JJ (1969) Glycoprotein staining following electrophoresis on acrylamide gels. Anal Biochem 30:148–152CrossRefGoogle Scholar

Copyright information

© Japanese Society of Fisheries Science 2018

Authors and Affiliations

  1. 1.Laboratory of Comparative Physiology, Division of Marine Life Sciences, Faculty of Fisheries SciencesHokkaido UniversityHakodateJapan
  2. 2.South Ehime Fisheries Research CenterEhime UniversityAinanJapan

Personalised recommendations