Abstract
Many candidates have been proposed as zona pellucida-binding proteins. Without precluding a role for any of those candidates, we focused on mouse sperm protein ZP3R/sp56, which is localized in the acrosomal matrix. The objective of this study was to analyze the role of ZP3R/sp56 in mouse fertilization. We expressed recombinant ZP3R/sp56 as a secreted protein in HEK293 cells and purified it from serum-free, conditioned medium. In the presence of reducing agents, the recombinant ZP3R/sp56 exhibited a molecular weight similar to that observed for the native ZP3R/sp56. Reminiscent of the native protein, recombinant ZP3R/sp56 formed a high molecular weight, disulfide cross-linked oligomer consisting of six or more monomers under non-reducing conditions. Recombinant ZP3R/sp56 bound to the zona pellucida of unfertilized eggs but not to 2-cell embryos, indicating that the changes that take place in the zona pellucida at fertilization affected the interaction of this protein with the zona pellucida. The extent of in vitro fertilization was reduced in a dose-dependent manner when unfertilized eggs were preincubated with recombinant ZP3R/sp56 (74% drop at the maximum concentrations assayed). Eggs incubated with the recombinant protein showed an absence of or very few sperm in the perivitelline space, suggesting that the reduction in the fertilization rate is caused by the inhibition of sperm binding and/or penetration through the zona pellucida. These results indicate that sperm ZP3R/sp56 is important for sperm-zona interactions during fertilization and support the concept that the acrosomal matrix plays an essential role in mediating the binding of sperm to the zona pellucida.
Highlights
Analysis of the conditioned culture medium by immunoblotting revealed that recombinant ZP3R/sp56 exhibited molecular weights similar to that observed for native ZP3R/sp56 under reducing and non-reducing conditions (Fig. 1)
Our studies showing that the mouse sperm zona pellucidabinding protein ZP3R/sp56 is intra-acrosomal, and not on the plasma membrane, have led us to reexamine the events of acrosomal exocytosis and the role of the sperm acrosomal matrix in the fertilization process [14, 16]
ZP3R/sp56 was expressed in Human embryonic kidney 293 cells (HEK293) cells because there is no spermatogenic cell system for the expression of recombinant proteins
Summary
Some researchers favor a two-step process whereby primary binding with the ZP occurs through a plasma membrane receptor(s) of sperm with intact acrosomes, and secondary binding utilizes intraacrosomal proteins that are exposed during the course of or following acrosomal exocytosis (“Acrosome Reaction Model”) [1, 2]. It is possible that during sperm capacitation subtle changes occur to the acrosomal region that initiate the acrosomal exocytotic process, thereby exposing acrosomal proteins involved in binding to the ZP (Acrosomal Exocytosis Model) [1, 3]. ZP3R/sp was initially discovered by photoaffinity crosslinking with ZP3 [9] and later characterized as having specific affinity for mouse ZP3 oligosaccharides involved in sperm binding [10]. ZP3-binding Protein (ZP3R/sp56) and Fertilization electron microscopy that ZP3R/sp is an intra-acrosomal protein and is, part of a stable acrosomal matrix [14, 16]
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