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Updated: Jul 7, 2026
Mouse Round Spermatid Injection
Published on: January 26, 2024
Recombinant mouse sperm ZP3-binding protein (ZP3R/sp56) forms a high order oligomer that binds eggs and inhibits
Mariano G Buffone1, Tiangang Zhuang, Teri S Ord
1Department of Obstetrics and Gynecology, Center for Research on Reproduction and Women's Health, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
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.
Insights
Mouse sperm protein ZP3R/sp56 is crucial for fertilization. This study shows recombinant ZP3R/sp56 inhibits sperm binding to eggs, highlighting its role in sperm-zona interactions.
Area of Science:
- Reproductive Biology
- Molecular Biology
- Cell Biology
Background:
- The zona pellucida (ZP) is a key structure in mammalian fertilization, and identifying sperm-egg binding proteins is critical.
- Several candidates for ZP-binding proteins exist, but the specific role of mouse sperm protein ZP3R/sp56, located in the acrosomal matrix, requires further investigation.
Purpose of the Study:
- To investigate the function of mouse sperm protein ZP3R/sp56 in the process of fertilization.
- To determine if ZP3R/sp56 mediates sperm binding to the zona pellucida.
Main Methods:
- Recombinant ZP3R/sp56 was expressed in HEK293 cells and purified.
- The binding properties of recombinant ZP3R/sp56 to unfertilized eggs and 2-cell embryos were assessed.
- In vitro fertilization assays were performed using eggs preincubated with recombinant ZP3R/sp56 to evaluate its effect on fertilization rates.
Main Results:
- Recombinant ZP3R/sp56 exhibited similar molecular weight and oligomeric properties to native ZP3R/sp56.
- Recombinant ZP3R/sp56 successfully bound to the zona pellucida of unfertilized eggs but not to 2-cell embryos.
- Preincubation with recombinant ZP3R/sp56 significantly reduced in vitro fertilization rates in a dose-dependent manner, with a 74% drop observed at maximum concentrations.
Conclusions:
- Mouse sperm ZP3R/sp56 plays a significant role in mediating sperm-zona pellucida interactions during fertilization.
- The findings support the importance of the acrosomal matrix in facilitating sperm binding to the egg's zona pellucida.
