The Binding of Concanavalin A to the Surface of Intact and Denuded Sea Urchin Eggs Affects the Fertilization Process by Altering the Structural Dynamics of Actin Filaments

Sea urchin eggs are surrounded by a network of extracellular matrix, consisting of the jelly coat (JC) and vitelline layer (VL). While the voluminous JC evokes acrosomal reaction in the approaching sperm, the tight VL ensheathing the plasma membrane of the subjacent microvilli is known to be the subcellular site where 'sperm receptors' reside. In this study, we have examined the roles of JC and VL at fertilization in a combinatorial approach utilizing two different pretreatments of the eggs: (i) incubation with dithiothreitol (DTT) in alkaline seawater to remove JC and VL, (ii) masking the egg extracellular matrix with a carbohydrate-binding protein concanavalin A (Con A). Surprisingly, the results showed that the DTT-denuded eggs still engulfed sperm at fertilization, even more effectively than intact eggs, as multiple sperm entered. On the other hand, Con A appeared to interfere with sperm entry in a dose-dependent manner and to delay the onset of the Ca²⁺ wave in intact eggs after the cortical Ca²⁺ release, representing sperm-egg fusion. This prolonged time lag in triggering the Ca²⁺ wave at fertilization was associated with compromised dynamics of the subplasmalemmal actin filaments in Con A-pretreated eggs. By using Alexa Fluor 633 Con A and BPA-C8-Cy3, respectively, we also report unprecedented fluorescent labeling of the egg JC and the spontaneous 'acrosomal protrusion' on the head of <i>Paracentrotus lividus</i> sperm diluted in natural seawater. Combined with electron microscopy observations of intact and denuded eggs, our results suggest that the glycoconjugate on the egg surface contributes to the fertilization signal transduction, affecting the Ca²⁺ wave via actin cytoskeletal changes and sperm entry.