Time to revise: impact of methodology on boar sperm capacitation in vitro via phosphotyrosine patterns

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.