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J. Biol. Chem., Vol. 283, Issue 18, 12438-12445, May 2, 2008

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Recombinant Mouse Sperm ZP3-binding Protein (ZP3R/sp56) Forms a High Order Oligomer That Binds Eggs and Inhibits Mouse Fertilization in Vitro^*

From the Department of Obstetrics and Gynecology, Center for Research on Reproduction and Women's Health, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

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.

Received for publication, August 3, 2007 , and in revised form, March 3, 2008.

^* This work was supported by Grant HD-41552 (to G. L. G.) and the Fogarty International Center Fellowship Program 5D43TW000671 (to M. G. B.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Current address: Abramson Research Center, Rm. 415H, 3516 Civic Center Blvd., Children's Hospital of Philadelphia, Philadelphia, PA 19104-4318.

² To whom correspondence should be addressed: Center for Research on Reproduction and Women's Health, Department of Obstetrics and Gynecology, University of Pennsylvania School of Medicine, 421 Curie Blvd., 1311 BRB II/III, Philadelphia, PA 19104-6160. Tel.: 215-573-4781; Fax: 215-573-7627; E-mail: gerton{at}mail.med.upenn.edu.

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