A New Model for Understanding the Egg Cell Surface at Fertilization
Authors: Wiseman Emily , Carroll David 
Published: June 2021

The cell signaling pathway initiating fertilization has not been fully elucidated in any species. Several downstream components in the pathway leading to an internal Ca2+ release have been found; however, not much is known regarding the participation of proteins on the surface of the sperm and egg. The starfish Patiria miniata is an excellent model organism to study this process because they produce a significant quantity of large synchronized oocytes that are nearly transparent and rigid enough to hold their spherical shape despite the removal of extracellular layers. Recently, lipid rafts have been found on the surface of a variety of eggs, but their role at fertilization is unknown. Lipid rafts are small dynamic regions enriched with cholesterol and sphingolipids, which hold clusters of membrane bound proteins. Lipid rafts are found in a variety of cells and they are critical in certain signaling processes, such as the activation of T cells. This study develops the starfish egg as a model system for the study of lipid rafts and for the discovery of cell surface proteins necessary for fertilization. To visualize egg surface proteins in living cells, oocytes were labeled with a membrane impermeant form of biotin and imaged during fertilization by laser scanning confocal microscopy. Evidence of an increase in protein at the fertilization cone, which marks the site of sperm‐egg fusion, was seen. To further understand how these surface proteins were behaving at fertilization, samples prepared from the biotinylated eggs were subjected to gel electrophoresis followed by affinity interaction with streptavidin‐HRP to visualize the labeled proteins. This showed the presence of multiple egg surface proteins, ranging in size from 75 to 250 kDa in great quantity, as well as a few proteins ranging from 30 to 75kDa. Interestingly, biotinylated proteins were also recovered from the surrounding seawater following fertilization, suggesting the action of a protease that cleaves egg cell surface proteins. Experiments with protease inhibitors suggest that this protease activity is necessary for fertilization, leading to the idea that the protease may be involved in sperm‐egg fusion and/or signaling. To examine the idea that signaling may be initiated at the moment of sperm‐egg interaction, and to determine if lipid rafts could be involved, a detergent‐based lipid raft isolation assay was developed for the starfish egg. The eggs are separated into cytosolic, soluble membrane, and insoluble membrane (lipid rafts) fractions. The fractions were subjected to western blotting for tyrosine‐phosphorylated proteins, which were found almost exclusively in the insoluble fraction of the unfertilized egg. Therefore, the lipid rafts contain as of yet unidentified signaling molecules that could play a role in the initiation of fertilization. Understanding this mechanism at the molecular level in starfish can to eventually lead to understanding infertility issues as well as offer new possible non‐hormonal prophylactics in humans.Support or Funding InformationThis research was funded by the NSF and Florida Institute of TechnologyThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.