Spermatozoa function is particularly dependent on pH variations and, consequently, Na+/H+ exchangers (NHEs) have been suggested to play significant roles in regulating the intracellular pH throughout the fertilization process. In this context, the presence of the NHE-11 exchanger in porcine spermatozoa is poorly understood. Therefore, the aim of this study was to accurately characterize the immunolocalization of the NHE-11 exchanger in porcine spermatozoa during in vitro capacitation and acrosome reaction using both fluorescence and Field Emission Scanning Electron Microscopy (FE-SEM). As a result, we recognized different fluorescence patterns according to membrane domains in the spermatozoa head. Thus, we identified NHE-11 predominantly in the apical ridge and in pre-equatorial subdomain both before capacitation and in capacitated spermatozoa with intact acrosome. However, following the acrosome reaction induction, immunofluorescence analysis identified two spermatozoa populations: one retaining NHE-11 labeling in the apical ridge and another lacking detectable fluorescence. Consistent with these observations, FE-SEM revealed persistent localization of NHE-11 in the apical ridge of acrosome-reacted spermatozoa. Quantitative immunogold analysis further demonstrated a significantly higher NHE-11 labeling density in the apical ridge of acrosome-reacted spermatozoa than in capacitated spermatozoa. In conclusion, these data demonstrate for the first time the presence and relocalization of NHE-11 immunoreactivity during capacitation and acrosome reaction in porcine spermatozoa. Overall, these findings provide new insights into the distribution of immunoreactivity attributed to NHE-11 in porcine spermatozoa and establish a basis for future studies aimed at clarifying its potential functional role during capacitation and the acrosome reaction.
