The initial molecular events mediating mammalian sperm binding to the zona pellucida (ZP) of the oocyte are highly complex and still not fully elucidated. Recent advances have identified multiple candidate sperm surface proteins, often functioning as part of high-molecular-weight complexes that mediate this critical fertilization event in a species-specific and coordinated manner. To address a significant gap in the literature, we provide an in-depth overview of the functional assays employed to investigate sperm-ZP interactions, emphasizing their underlying principles, potential applications, and key methodological strengths and limitations. The techniques discussed range from classical in vitro sperm-oocyte and hemizona binding assays, including antibody-blocking and competitive strategies, to cutting-edge in vivo genetic models, each contributing unique insights into the physiological relevance of the proposed ZP receptors. Robust experimental design, including the use of appropriate controls and validation strategies, is essential for accurately interpreting the role of candidate sperm receptors. This review provides a structured overview of current methodologies to support researchers in critically evaluating and applying functional assays in future studies.

• Keywords
• antibody, antibody block, binding assay, competitive assay, hemizona binding assay, knockout model, recombinant protein, sperm receptor, sperm–ZP binding,
• Publication type
• Journal Article MeSH
• Review MeSH

Sperm-zona pellucida (ZP) interaction, involving the binding of sperm surface ligands to complementary carbohydrates of ZP, is the first direct gamete contact event crucial for subsequent gamete fusion and successful fertilization in mammals. It is a complex process mediated by the coordinated engagement of multiple ZP receptors forming high-molecular-weight (HMW) protein complexes at the acrosomal region of the sperm surface. The present article aims to review the current understanding of sperm-ZP binding in the four most studied mammalian models, i.e., murine, porcine, bovine, and human, and summarizes the candidate ZP receptors with established ZP affinity, including their origins and the mechanisms of ZP binding. Further, it compares and contrasts the ZP structure and carbohydrate composition in the aforementioned model organisms. The comprehensive understanding of sperm-ZP interaction mechanisms is critical for the diagnosis of infertility and thus becomes an integral part of assisted reproductive therapies/technologies.

• Keywords
• ZP-ligands, gamete interaction, sperm-ZP receptors, spermatozoa, zona pellucida,
• MeSH
• Humans MeSH
• Ligands MeSH
• Membrane Glycoproteins metabolism   MeSH
• Cell Communication * MeSH
• Receptors, Cell Surface metabolism   MeSH
• Mammals metabolism   MeSH
• Spermatozoa cytology   metabolism   MeSH
• Zona Pellucida metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Ligands MeSH
• Membrane Glycoproteins MeSH
• Receptors, Cell Surface MeSH