Oocyte proteomics: Localisation of mouse zona pellucida protein 3 to the plasma membrane of ovulated mouse eggs

S. A. Coonrod, M. E. Calvert, P. P. Reddi, E. N. Kasper, L. C. Digilio, J. C. Herr

Research output: Contribution to journalArticle

Abstract

In order to gain a deeper understanding of the molecular underpinnings of sperm-egg interaction and early development, we have used two-dimensional (2D) electrophoresis, avidin blotting and tandem mass spectrometry to identify, clone and characterise abundant molecules from the mouse egg proteome. Two-dimensional avidin blots of biotinylated zona-free eggs revealed an abundant approximately 75-kDa surface-labelled heterogeneous protein possessing a staining pattern similar to that of the zona pellucida glycoprotein, mouse ZP3 (mZP3). In light of this observation, we investigated whether mZP3 specifically localises to the plasma membrane of mature eggs. Zona pellucidae of immature mouse oocytes and mature eggs were removed using acid Tyrode's solution, chymotrypsin or mechanical shearing. Indirect immunofluorescence using the mZP3 monoclonal antibody (mAb) IE-10 demonstrated strong continuous staining over the entire surface of immature oocytes and weak microvillar staining on ovulated eggs, regardless of the method of zona removal. Interestingly, in mature eggs, increased fluorescence intensity was observed following artificial activation and fertilisation, whereas little to no fluorescence was observed in degenerated eggs. The surface localisation of ZP3 on mature eggs was supported by the finding that the IE-10 mAb immunoprecipitated an approximate 75-kDa protein from lysates of biotinylated zona-free eggs. To further investigate the specificity of the localisation of mZP3 to the oolemma, indirect immunofluorescence was performed using the IE-10 mAb on both CV-1 and CHO cells transfected with full-length recombinant mZP3 (re-mZP3). Plasma membrane targeting of the expressed re-mZP3 protein was observed in both cell lines. The membrane association of re-mZP3 was confirmed by the finding that biotinylated re-mZP3 (approximately 75 kDa) is immunoprecipitated from the hydrophobic phase of Triton X-114 extracts of transfected cells following phase partitioning. Immunoprecipitation assays also demonstrated that surface re-mZP3 was released from transfected CV-1 in a time-dependent manner. These results demonstrate that ZP3 is specifically associated with the surface of mature eggs and its subsequent release from the cell surface may represent one mechanism by which ZP3 is secreted. Furthermore, the increase in ZP3 surface expression following fertilisation suggests that ZP3 may have a functional role during sperm-oolemma binding and fusion. These results also validate the usefulness of using the 2D proteomic approach to identify and characterise egg-surface proteins.

Original languageEnglish (US)
Pages (from-to)69-78
Number of pages10
JournalReproduction, Fertility and Development
Volume16
Issue number1-2
DOIs
StatePublished - 2004
Externally publishedYes

Keywords

  • Fertilisation
  • Mouse ovum
  • Oolemma

ASJC Scopus subject areas

  • Biotechnology
  • Reproductive Medicine
  • Animal Science and Zoology
  • Molecular Biology
  • Genetics
  • Endocrinology
  • Developmental Biology

Fingerprint Dive into the research topics of 'Oocyte proteomics: Localisation of mouse zona pellucida protein 3 to the plasma membrane of ovulated mouse eggs'. Together they form a unique fingerprint.

  • Cite this