(2) Comparative Analysis of Common Techniques to Detect Capacitation Status in Boar Spermatozoa

Barbora Klusackova¹; Aneta Pilsova¹; Eva Chmelikova¹; 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 of Sciences, BIOCEV, Vestec, Czech Republic

Abstract Text:

Capacitation is a crucial step in sperm maturation, which in vivo occurs in the female reproductive tract. During this process, sperm undergoes a series of physiological and biochemical changes that enable it to undergo the acrosomal reaction and fertilize the oocyte. These changes include membrane remodeling as cholesterol redistributes from a uniform distribution to the apical region of the sperm head leading to cholesterol efflux. Another critical change during capacitation is the efflux of Zn²⁺, which is essential for the influx of Ca²⁺ into the sperm cell. The changes in plasma membrane composition and intracellular ion concentrations are related to the signaling cascades in the sperm. There are several pathways resulting in tyrosine phosphorylation of several sperm proteins. Capacitation also leads to the activation of acrosomal enzymes, such as acrosin, which are crucial for penetrating the zona pellucida. Given the complexity of capacitation, various detection methods have been developed, each focusing on different functional aspects. However, the reliability and specificity of these methods can vary, making it essential to critically assess their implementation. This study aims to critically evaluate and compare commonly used capacitation detection techniques in boar sperm. Eight markers of capacitation were analyzed: (i) cholesterol efflux using BODIPY-cholesterol, (ii) Zn²⁺efflux using imaging flow cytometry with the fluorescent Zn²⁺ probe FluoZin™-3 AM, (iii) changes in the intracellular Ca²⁺ concentration using chlortetracycline (CTC) fluorescent assay, (iv) tyrosine phosphorylation via indirect immunofluorescence with the 4G10 antibody, (v) glycosylation using UEA lectin, (vi) detection of acrosin active form using indirect immunofluorescence with the ACR.2 antibody, (vii) acrosomal cap remodeling assessed with PNA lectin, and (viii) kinematic parameters using computer-assisted sperm analysis (CASA). Preliminary analysis revealed differences in detection of capacitation status via different markers. While some methods such as CTC assay and acrosin detection provided clear and reproducible results, others, including tyrosine phosphorylation pattern assessment, showed variability depending on experimental conditions. Differences in acrosome remodeling and motility parameters further highlighted the complexity of capacitation assessment. Additionally, the sensitivity of each marker in detecting capacitation-associated changes varied, indicating that a combination of multiple markers may provide a more comprehensive evaluation. This comparative analysis underscores the importance of selecting appropriate markers and methodologies for reliable capacitation evaluation in boar sperm. 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).