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This is an early access version, the complete PDF, HTML, and XML versions will be available soon.
Review

Functional Methods for Studying Sperm–Zona Pellucida Interaction in Mammals

by
Natalie Zelenkova
1,
Veronika Kraus
2,
Alexandra Maresova
1,
Zuzana Pilsova
1,
Aneta Pilsova
1,
Barbora Klusackova
1,
Eva Chmelikova
1,
Katerina Komrskova
2,3 and
Pavla Postlerova
1,2,*
1
Department of Veterinary Sciences, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, 165 00 Prague, Czech Republic
2
Laboratory of Reproductive Biology, Institute of Biotechnology of the Czech Academy Sciences, BIOCEV, 252 50 Vestec, Czech Republic
3
Department of Zoology, Faculty of Science, Charles University, 128 00 Prague, Czech Republic
*
Author to whom correspondence should be addressed.
Methods Protoc. 2025, 8(4), 95; https://doi.org/10.3390/mps8040095
Submission received: 30 June 2025 / Revised: 1 August 2025 / Accepted: 8 August 2025 / Published: 13 August 2025
(This article belongs to the Section Molecular and Cellular Biology)
Versions Notes

Abstract

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: sperm–ZP binding; sperm receptor; binding assay; antibody block; competitive assay; hemizona binding assay; knockout model; antibody; recombinant protein sperm–ZP binding; sperm receptor; binding assay; antibody block; competitive assay; hemizona binding assay; knockout model; antibody; recombinant protein

Share and Cite

MDPI and ACS Style

Zelenkova, N.; Kraus, V.; Maresova, A.; Pilsova, Z.; Pilsova, A.; Klusackova, B.; Chmelikova, E.; Komrskova, K.; Postlerova, P. Functional Methods for Studying Sperm–Zona Pellucida Interaction in Mammals. Methods Protoc. 2025, 8, 95. https://doi.org/10.3390/mps8040095

AMA Style

Zelenkova N, Kraus V, Maresova A, Pilsova Z, Pilsova A, Klusackova B, Chmelikova E, Komrskova K, Postlerova P. Functional Methods for Studying Sperm–Zona Pellucida Interaction in Mammals. Methods and Protocols. 2025; 8(4):95. https://doi.org/10.3390/mps8040095

Chicago/Turabian Style

Zelenkova, Natalie, Veronika Kraus, Alexandra Maresova, Zuzana Pilsova, Aneta Pilsova, Barbora Klusackova, Eva Chmelikova, Katerina Komrskova, and Pavla Postlerova. 2025. "Functional Methods for Studying Sperm–Zona Pellucida Interaction in Mammals" Methods and Protocols 8, no. 4: 95. https://doi.org/10.3390/mps8040095

APA Style

Zelenkova, N., Kraus, V., Maresova, A., Pilsova, Z., Pilsova, A., Klusackova, B., Chmelikova, E., Komrskova, K., & Postlerova, P. (2025). Functional Methods for Studying Sperm–Zona Pellucida Interaction in Mammals. Methods and Protocols, 8(4), 95. https://doi.org/10.3390/mps8040095

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