Isolation of Zebrafish Balbiani Bodies for Proteomic Analysis

  • Allison Jamieson-Lucy
  • Mary C. MullinsEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1920)


Proteomic characterization of isolated organelles can provide insight into the functional components of the structure and novel targets for further testing. Germplasm in developing oocytes is difficult to isolate for protein identification because not all types of germplasm are stable outside of the cytoplasm. In zebrafish, the Balbiani body forms a proteinaceous aggregate that contains the germplasm and we found is stable outside of the oocyte. Here we present a manual isolation protocol that collects intact Balbiani bodies from stage I zebrafish oocytes. We lysed oocytes by passing them through a syringe, and then used a fine injection needle to wick up Balbiani bodies by capillary action with minimal buffer solution. Using this protocol we collected sufficient material for proteomic analysis of the zebrafish Balbiani body.

Key words

Balbiani body Germplasm Proteomics Isolation Oocytes 


  1. 1.
    Bilinski SM, Jaglarz MK, Tworzydlo W (2017) The pole (germ) plasm in insect oocytes. In: Kloc M (ed) Oocytes, results and problems in cell differentiation. Springer International Publishing, New York, pp 103–126Google Scholar
  2. 2.
    Aguero T, Kassmer S, Alberio R, Johnson A, King ML (2017) Mechanisms of vertebrate germ cell determination. In: Pelegri F (ed) Vertebrate development, advances in experimental medicine and biology. Springer International Publishing, New York, pp 383–440Google Scholar
  3. 3.
    Lehman R (2016) Germ plasm biogenesis—an Oskar-centric perspective. Curr Top Dev Biol 116:679–707CrossRefGoogle Scholar
  4. 4.
    Hertig AT (1968) The primary human oocyte: some observation on the fine structure of Balbiani’s vitelline body and the origin of annulate lamellae. Am J Anat 122:107–138CrossRefGoogle Scholar
  5. 5.
    Bontems F, Stein A, Marlow F, Lyuatey J, Gupta T, Mullins MC (2009) Bucky ball organizes germ plasm assembly in zebrafish. Curr Biol 19:414–422CrossRefGoogle Scholar
  6. 6.
    Marlow F, Mullins MC (2008) Bucky ball functions in Balbiani body assembly and animal-vegetal polarity in the oocyte and follicle cell layer in zebrafish. Dev Biol 321:40–50CrossRefGoogle Scholar
  7. 7.
    Escobar-Aguirre M, Elkouby YM, Mullins MC (2017) Localization in oogenesis of maternal regulators of embryonic development. Adv Exp Med Biol 953:173–207CrossRefGoogle Scholar
  8. 8.
    Dosch R, Wagner DS, Mintzer KA, Runke G, Weimelt AP, Mullins MC (2004) Maternal control of vertebrate development before the midblastula transition: mutants from the zebrafish I. Dev Cell 6:771–780CrossRefGoogle Scholar
  9. 9.
    Rostagno A, Ghiso J (2010) Isolation and biochemical characterization of amyloid plaques and paired helical filaments. Curr Protoc Cell BiolGoogle Scholar
  10. 10.
    Lino L, Cheng D, Wang J, Duong DM, Losik TG, Gearing M et al (2004) Proteomic characterization of postmortem amyloid plaques isolated by laser capture microdissection. J Biol Chem 279:37061–37068CrossRefGoogle Scholar
  11. 11.
    Roher AE, Palmer KC, Chau V, Ball MJ (1988) Isolation and chemical characterization of Alzheimer’s disease paired helical filament cytoskeletons: differentiation from amyloid plaque core protein. J Cell Biol 107:2703–2716CrossRefGoogle Scholar
  12. 12.
    Iqbal K, Zaidi T, Thompson CH, Perz PA, Wisniewski HM (1984) Alzheimer paired helical filaments: bulk isolation, solubility and protein composition. Acta Neuropathol 62:167–177CrossRefGoogle Scholar
  13. 13.
    Jain S, Wheeler JR, Walters RW, Agrawal A, Barsic A, Parker R (2016) ATPase-modulated stress granules contain a diverse proteome and substructure. Cell 164:487–498CrossRefGoogle Scholar
  14. 14.
    Khong A, Jain S, Matheny T, Wheeler JR, Parker R (2018) Isolation of mammalian stress granule cores for RNA-Seq analysis. Methods 137:49–54CrossRefGoogle Scholar
  15. 15.
    Chang P, Torres J, Lewis RA, Mowry KL, Houliston E, King ML (2004) Localization of RNAs to the mitochondrial cloud in Xenopus oocytes through entrapment and association with endoplasmic reticulum. Mol Biol Cell 15:4669–4681CrossRefGoogle Scholar
  16. 16.
    Escobar-Aguirre M, Zhang H, Jamieson-Lucy A, Mullins MC (2017) Microtubule-actin crosslinking factor 1 (Macf1) domain function in Balbiani body dissociation and nuclear positioning. PLoS Genet 13:1–29CrossRefGoogle Scholar
  17. 17.
    Butler AM, Aguero T, Newman KM, King ML (2017) Primordial germ cell isolation from Xenopus laevis embryos. In: Buszczak M (ed) Germline stem cells, methods in molecular biology. Springer, New York, pp 115–124CrossRefGoogle Scholar
  18. 18.
    Elkouby Y, Mullins M (2017) Methods for the analysis of early oogenesis in Zebrafish. Dev Biol 430:310–324CrossRefGoogle Scholar
  19. 19.
    Wang XG, Bartfai R, Sleptzova-Freidrich I, Orban L (2007) The timing and extent of “juvenile ovary” phase are highly variable during zebrafish testis differentiation. J Fish Biol 70:33–44CrossRefGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Cell and Developmental BiologyPerelman School of Medicine, University of PennsylvaniaPhiladelphiaUSA

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