Abstract
The vitelline layer (VL) of a sea urchin egg is an intricate meshwork of glycoproteins that intimately ensheathes the plasma membrane. During fertilization, the VL plays important roles. Firstly, the receptors for sperm reside on the VL. Secondly, following cortical granule exocytosis, the VL is elevated and transformed into the fertilization envelope (FE), owing to the assembly and crosslinking of the extruded materials. As these two crucial stages involve the VL, its alteration was expected to affect the fertilization process. In the present study, we addressed this question by mildly treating the eggs with a reducing agent, dithiothreitol (DTT). A brief pretreatment with DTT resulted in partial disruption of the VL, as judged by electron microscopy and by a novel fluorescent polyamine probe that selectively labelled the VL. The DTT-pretreated eggs did not elevate the FE but were mostly monospermic at fertilization. These eggs also manifested certain anomalies at fertilization: (i) compromised Ca2+ signaling, (ii) blocked translocation of cortical actin filaments, and (iii) impaired cleavage. Some of these phenotypic changes were reversed by restoring the DTT-exposed eggs in normal seawater prior to fertilization. Our findings suggest that the FE is not the decisive factor preventing polyspermy and that the integrity of the VL is nonetheless crucial to the egg’s fertilization response.
Highlights
For a cell like the sea urchin egg, the presence of a well-developed extracellular matrix is of particular importance
Spawning was induced by intracoelomic injection of 0.5 M KCl, and the resulting eggs were collected in natural seawater (NSW) filtered with a Millipore membrane of 0.2 μm pore size (Nalgene vacuum filtration system, Thermo Fisher Scientific, Rochester, NY, USA)
When the DTTpreincubated eggs were rinsed and restored in NSW before fertilization, the latent period for the Ca2+ response in these eggs was nearly doubled (11.3 ± 7.6 s, n = 15, p < 0.01) in comparison with both CTL and DTT. These results suggest that the alteration of the Ca2+ -release systems induced by DTT pretreatment is reversed to some extent by the restoration of the eggs in NSW, but the recovery path may not be the same as the path of modification induced by DTT
Summary
For a cell like the sea urchin egg, the presence of a well-developed extracellular matrix is of particular importance. Sperm-activating peptides released from the jelly coat of sea urchin eggs serve as a chemoattractant for the sperm [1,2,3]. The vitelline layer (VL) of a sea urchin egg, which intimately covers the plasma membrane, has long been recognized as the primary subcellular site of sperm attachment during fertilization [4,5,6]. Bindin, a protein isolated from the acrosomal process of sea urchin sperm, was shown to mediate species-specific attachment to the egg’s VL, and the egg’s receptor for sperm or bindin has been isolated from the VL [7,8,9,10,11,12,13,14]. It has been generally assumed that such a practice does not affect the fertilization process and embryonic development, while its effect on the cytological and biochemical changes characterizing egg activation has not been addressed sufficiently
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