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Sperm motility, a feature essential for in vivo fertilization, is influenced by intracellular pH (pH_i) homeostasis. Several mechanisms are involved in pH_i regulation, among which sodium–hydrogen exchangers (NHEs), a family of integral transmembrane proteins that catalyze the exchange of Na⁺ for H⁺ across lipid bilayers. A preliminary characterization of NHE activity and kinetic parameters, followed by analysis of the expression and localization of the protein in ram spermatozoa was performed. NHE activity showed an apparent K _m for external Na⁺ of 17.61 mM. Immunoblotting revealed a molecular mass of 85 kDa. Immunolocalization pattern showed some species-specific aspects, such as positive labeling at the equatorial region of the sperm head. Cariporide, a selective NHE1 inhibitor, significantly reduced pH_i recovery (85%). Similarly, exposure to cariporide significantly inhibited different motility parameters, including those related to sperm capacitation. In vitro fertilization (IVF) was not affected by cariporide, possibly due to the non-dramatic, although significant, drop in motility and velocity parameters or due to prolonged exposure during IVF, which may have caused progressive loss of its inhibitory effect. In conclusion, this is the first study documenting, in a large animal model (sheep) of well-known translational relevance, a direct functional role of NHE on sperm pH_i and motility. The postulated specificity of cariporide toward isoform 1 of the Na⁺/H⁺ exchanger seems to suggest that NHE1 may contribute to the observed effects on sperm cell functionality.

The development of fertilizing ability in sperm cells is associated with changes in the plasma membrane. However, to date the exact nature of sequentially activated primary receptors and channels and the signal transduction pathways derived from these remains elusive. We analyzed the expression and localization of the μ-opioid receptossr in equine spermatozoa. A transcript corresponding to the third extracellular loop that selectively binds μ agonists was amplified, sequenced and compared with the known sequences in humans, rats and cattle. The amplification product showed a high degree of nucleotide conservation. By immunofluorescence, μ-opioid receptor labeling was found on the sperm head and on the tail and disappeared in the acrosomal region of acrosome-reacted sperm cells. Immunoblotting revealed two bands of 50 and 65 kDa. Effects of the opioid antagonist naloxone on motility and on viability and capacitation/acrosome reaction were investigated by computer-assisted sperm analysis and Hoechst 33258/chlortetracycline (H258/CTC) staining. Progressive motility was significantly reduced after 3 h incubation in 10⁻³ M naloxone (P < 0.05), whereas it increased significantly after 5 h in 10⁻⁸ M naloxone (P < 0.05). Sperm velocity at 5 h was significantly reduced by the addition of 10⁻³ M naloxone (P < 0.05), but increased significantly in the presence of 10⁻⁸ M (P < 0.001). Curvilinear velocity and amplitude of lateral head displacement in spermatozoa incubated in the presence of naloxone were not indicative of hyperactivation. H258/CTC staining showed that 10⁻⁸ M naloxone significantly stimulated capacitation (P < 0.01) after 3 h. However, it had no effect on sperm cell viability and acrosomal status. Overall, this study provides the first evidence that the μ-opioid receptor is expressed in equine spermatozoa and that naloxone significantly affects motility and capacitation.