Comparative proteomic analysis of the zebrafish (Danio rerio) perivitelline fluid; identifying putative cortical alveoli-associated proteins.

Upon fertilization, the egg must have the resources to facilitate successful fertilization and regulate zygotic growth prior to the activation of the embryonic genome. A key biomarker of egg activation is the synchronized release of cortical alveoli (CA) from the egg's cortex into the perivitelline space. Teleost CA are primarily associated with their highly conserved roles in polyspermy prevention. However, several CA-associated proteins with functions related to innate immunity have been isolated. While there is growing evidence that teleost CA have extended biological roles outside of polyspermy prevention, little work has been done toward developing an understanding of their general proteomic composition. Therefore, the aim of this study was to identify proteins which were proportionally over-represented in the zebrafish (Danio rerio) perivitelline fluid (PVF) directly following CA exocytosis to identify a candidate list of CA-associated proteins. This study utilized a novel technique for PVF extraction from water-activated eggs shortly following CA exocytosis which negated the use of fixatives, thereby increasing PVF sample integrity for downstream mass-spectrometry analysis. Moreover, this study provides the first comparative proteomic analysis of the PVF relative to the yolk and cytoplasm of the zebrafish egg. As a result, 44 proteins were identified which were proportionally over-represented in the PVF, relative to the yolk and cytosol, of the water-activated unfertilized egg. The resulting PVF proteome was comprised of proteins associated with functions in carbohydrate binding and peptidase regulation. Many of these proteins are compelling candidates for being CA-derived and have been previously implicated in innate immunity.