Characterization of extracellular Ca²⁺ -dependent full-type hyperactivation in ejaculated boar spermatozoa...

Ejaculated boar spermatozoa exhibit two types of hyperactivation: full and non-full. Full-type hyperactivation is characterized by the asymmetrical bending of the entire middle piece-principal piece and a twisting/figure-eight-like trajectory, and can be induced by simple incubation with CaCl₂ after preincubation with a cAMP analog (Sp-5,6-dichloro-1-β-D-ribofuranosyl-benzimidazole-3',5'-cyclic monophosphorothioate [cBiMPS]). Here, we compared the sperm flagellar motility after treatments with elevators of [Ca²⁺ ]_i (cBiMPS/CaCl₂ , thimerosal, procaine, and 4-aminopyridine) to characterize the regulatory mechanism of extracellular Ca²⁺ -dependent, full-type hyperactivation in ejaculated boar spermatozoa, and examined the possible involvement of Transient receptor potential cation channel subfamily C member 3 (TRPC3) in this event using the specific inhibitor Pyr3. Full-type hyperactivation was induced by a 60-min incubation with CaCl₂ following a 180-min preincubation with cBiMPS but without Ca²⁺ . Thimerosal-treated spermatozoa exhibited full-type hyperactivation in a manner independent of extracellular Ca²⁺ ; conversely, this was not observed in procaine- or 4-aminopyridine-treated spermatozoa. A 20-min treatment with Pyr3 between preincubation with cBiMPS and incubation with CaCl₂ , significantly suppressed the normal phenotype. These observations indicated that mechanisms underlying full-type hyperactivation in spermatozoa incubated with CaCl₂ after preincubation with cBiMPS are different from those in the thimerosal-treated spermatozoa. Furthermore, indirect immunofluorescence localized TRPC3 in the upper segment of the middle piece, which bends asymmetrically during full-type hyperactivation but not in non-full-type hyperactivation, suggesting that TRPC3 may be involved in the extracellular Ca²⁺ -dependent full-type hyperactivation in ejaculated boar spermatozoa.