Abstract
Cortical alveoli (CA) play a crucial role in teleost egg activation and polyspermy prevention, yet their full proteomic composition and broader biological functions remain poorly understood. Initially, this project was focused on identifying the glycoprotein component from the CA of Anguilla australis (shortfin eel; Chapter 2). However, owing to the difficulties associated with the collection of sufficiently matured oocytes and eggs from eels, the focus quickly changed to identifying the proteomic composition of zebrafish CA (Chapters 3-5). Therefore, the primary aim of this thesis was to identify several CA-associated proteins in zebrafish (Danio rerio) using an integrative approach combining proteomic identification, gene expression analysis, and intracellular protein localisation.
A novel and sensitive technique was developed for extracting perivitelline fluid (PVF) from live, water-activated zebrafish eggs. As CA are major contributors to the proteome of the PVF, it was anticipated that a significant proportion of the highly abundant proteins present within the PVF during this stage would be CA in origin. The proteomic profile of three major proteinaceous sources of the egg (the PVF, yolk/cytoplasm and the chorion) were analysed using mass-spectrometry-based shotgun proteomics. A list of 44 proteins were subsequently found to be enriched in the PVF, revealing a subset of proteins which have been widely implicated in innate immunity. Several PVF-derived proteins were selected for further analysis to explore their putative associations with zebrafish CA.
As an initial assessment, qPCR was conducted to assess the expression patterns of the selected target genes amongst various zebrafish tissues and throughout several major stages of oogenesis. All target genes exhibited significantly elevated expression in the ovary relative to all other tissues, consistent with their expected CA associations. Expression was localised to the ovarian follicle. However, no discernible pattern in expression was observed amongst the target genes throughout oogenesis. These findings mirrored previous observations of gene expression profiles for putative CA-related genes in other teleost species. Finally, intracellular protein localisation was investigated using fusion-protein overexpression, followed by western blotting and immunohistochemical co-staining analyses. These approaches provided further evidence in support of the associations between the selected target proteins and zebrafish CA, with all five overexpressed fusion-target proteins being successfully localised to CA within cortical alveolus- and early vitellogenic-staged zebrafish oocytes.
Collectively, this work represents the first identification of specific CA protein constituents in zebrafish and provides novel evidence for the inclusion of alpha-2 macroglobulin and peptide-N-glycosidase F as CA components in any species. These findings contribute to a growing understanding of the molecular composition of teleost CA and their potential functional significance beyond polyspermy prevention, particularly in relation to the provision of innate immune defence during early embryonic development.