Capacitation is a crucial sperm maturation process occurring in vivo in the female reproductive tract, enabling spermatozoa to fertilize the oocyte. In vitro, capacitation can be induced using defined capacitation media (CM), with further assessment of protein tyrosine phosphorylation (PTyr) patterns widely used as a marker to evaluate sperm capacitation. This review critically examines the factors influencing PTyr detection in boar spermatozoa variability introduced by different methodological approaches. Discrepancies in PTyr patterns may be a result of different sperm handling, including preservation methods, selection techniques, and capacitation protocols. Semen extenders, which may contain unknown components, can affect the variability in capacitation status. Selection techniques commonly employed to isolate viable spermatozoa may initiate different capacitation regulatory pathways, resulting in variability in analyzed sperm subpopulations and inconsistencies in PTyr detection. Similarly, the lack of standardization in CM composition significantly impacts capacitation outcomes. Fixation protocols further increase variability in PTyr pattern detection, as aldehydic fixatives potentially alter protein structures, while alcohol-based fixatives cause protein aggregation and plasma membrane disruption. While PTyr immunofluorescence remains a valuable tool for capacitation assessment, its reliability is limited by methodological variability. Mimicking in vivo conditions is crucial, and even minor modifications in the sperm capacitation process may provide inconsistent results in PTyr patterns across studies. This review offers valuable insights into often-disregarded methodological details and highlights the need for improved for better standardization of capacitation protocols. The uniform methodological approach improves reproducibility and reliability in capacitation studies and stimulates further investigation leading to the discovery of alternative additional markers to determine the capacitation status in mammalian spermatozoa.

• Klíčová slova
• Capacitation media, Immunofluorescence, Protein phosphorylation, Reproduction, Signaling pathway, Sperm fixation,
• MeSH
• analýza spermatu veterinární   MeSH
• fosforylace MeSH
• fosfotyrosin * metabolismus   MeSH
• kapacitace spermií * fyziologie   MeSH
• prasata fyziologie   MeSH
• spermie * fyziologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• fosfotyrosin * MeSH

BACKGROUND: Capacitation involves physiological changes that spermatozoa must undergo in the female reproductive tract or in vitro to obtain the ability to bind, penetrate and fertilize the egg. Up to date, several methods have been developed to characterize this complex biological process. The goal of the presented study is to mutually compare several fluorescent techniques, check their ability to detect changes in molecular processes during the capacitation progress and determine their ability to predict the percentage of acrosome reacted (AR) sperm after the exposure to solubilized zona pellucida (ZP). The capacitation process was analyzed using four fluorescent techniques: 1. chlortetracycline (CTC) staining, 2. anti-acrosin antibody (ACR.2) assay, 3. anti-phosphotyrosine (pY) antibody assay, 4. fluorescein isothiocyanate-conjugated phalloidin (FITC-phall) assay. All these methods were tested using fluorescent microscopy and flow cytometry. RESULTS: All selected methods are capable to detect the capacitation progress of boar sperm in vitro, but there are significant differences in their outcome when using fluorescent microscopy or flow cytometry experimental arrangements and subsequent statistical analysis (KW-ANOVA). Also, the ability to predict the absolute numbers of sperm which will undergo ZP-induced AR differ significantly (CTC and ACR.2 gave the best predictions). CONCLUSIONS: Our study compared four largely used methods used to characterize capacitation process, highlighted their differences and showed that all are able to detect capacitation progress, CTC and ACR.2 are furthermore able to accurately predict the percentage of AR sperm after ZP-induced AR.

• Klíčová slova
• Acrosin staining, Acrosome reaction, Chlortetracycline assay, Flow cytometry, Fluorescent microscopy, Phalloidin staining, Tyrosine phosphorylation,
• MeSH
• akrozomální reakce fyziologie   MeSH
• faloidin MeSH
• fluorescein-5-isothiokyanát MeSH
• fluorescenční barviva * MeSH
• fluorescenční mikroskopie * metody   MeSH
• fluorescenční protilátková technika MeSH
• kapacitace spermií fyziologie   MeSH
• průtoková cytometrie * metody   MeSH
• spermie fyziologie   MeSH
• Sus scrofa fyziologie   MeSH
• vápník analýza   MeSH
• zona pellucida fyziologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• faloidin MeSH
• fluorescein-5-isothiokyanát MeSH
• fluorescenční barviva * MeSH
• vápník MeSH