Rab2A and Lactadherin: Potential Players in Sperm-Zona Pellucida Interaction in Pigs?

Natalie Zelenkova¹; Veronika Kraus²; Barbora Klusackova¹; Lukas Ded²; Ondrej Simonik²; Eva Chmelikova¹;  Tereza Krejčova¹; Michal Zigo³; Peter Sutovsky^3,4; Katerina Komrskova^2,5; Pavla Postlerova^1,2

1. Department of Veterinary Sciences, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Czech Republic

2. Laboratory of Reproductive Biology, Institute of Biotechnology of the Czech Academy   Sciences, v.v.i., BIOCEV, Vestec, Czech Republic

3. Division of Animal Sciences, College of Agriculture, Food and Natural Resources, University of Missouri, USA

4. Department of Obstetrics, Gynaecology & Women’s Health, University of Missouri, Columbia, USA

5. Department of Zoology, Faculty of Science, Charles University, Prague, Czech Republic

Abstract Text:

The interaction of sperm cell with the zona pellucida (ZP) of the oocyte is one of the key events in mammalian fertilization. The initial interaction of the gametes, also known as the primary binding of sperm to the ZP, is facilitated by sperm surface molecules that are localized either peripherally or integrally in the plasma membrane (PM). Protein receptors may become exposed or activated on the sperm surface during capacitation. In recent years, great progress has been made in identifying sperm receptor proteins that are capable of recognizing and binding to ZP glycoproteins in a variety of mammals; however, the current paradigm states that this interaction is a multimolecular event and many receptors likely remain unknown. The aim of this study is to validate two proteins present on the boar sperm surface with previously demonstrated affinity for ZP glycoproteins as novel potential candidates for the sperm-ZP binding. These proteins are targeted by our in-house prepared antibodies that were obtained by immunizing mice with isolated boar sperm surface proteome. Mass spectrometry analysis revealed the targeted proteins to be Rab-2A protein, a small GTPase of the Ras superfamily that is involved in the regulation of intracellular membrane trafficking, and lactadherin (P47/SED1), membrane-associated protein previously described as a ZP receptor in mouse spermatozoa. To gain deeper insights into the role of these proteins and their function in boar sperm, immunodetection and fluorescence and confocal microscopy were applied, mapping the origin and detailed changes in the localization of these proteins in spermatozoa throughout their development and maturation. Importantly, in vitro antibody block assays were carried out to decipher the role these proteins play in the sperm binding to zona pellucida-intact porcine oocytes. To confirm the results of the antibody block binding assays, competitive binding assays were performed using corresponding recombinant proteins. To explore potential cross-species reactivity, we investigated the reactivity of the anti-Rab2A antibody on human sperm and successfully localized the protein for the first time. Additionally, the commercial anti-Rab2 antibody was employed to test the accuracy of the in-house made monoclonal antibody. While characterizing the roles of Rab2A and lactadherin in spermatozoa and sperm-ZP interaction, this work also aims to highlight the inherent challenges and limitations of in vitro sperm-ZP binding assays and the need for a deep understanding of the sperm protein structure, epitope accessibility, and experimental conditions when conducting such assays. These findings contribute to the broader understanding of the intricacy of identifying potential sperm receptors for the zona pellucida/oocyte interaction. This work is supported by the Internal Grant Agency of the Czech University of Life Sciences in Prague (SV24-22-21230) and by the Ministry of Education, Youth and Sports of the Czech Republic under the INTER-EXCELLENCE II program, subprogram INTER-ACTION (LUAUS25072), and the institutional support of Institute of Biotechnology (RVO: 86652036).