Characterization of extracellular Ca²⁺‐dependent full‐type hyperactivation in ejaculated boar spermatozoa preincubated with a cAMP analog

Abstract

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 CaCl 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 [Ca ] (cBiMPS/CaCl , thimerosal, procaine, and 4-aminopyridine) to characterize the regulatory mechanism of extracellular Ca -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 CaCl following a 180-min preincubation with cBiMPS but without Ca . Thimerosal-treated spermatozoa exhibited full-type hyperactivation in a manner independent of extracellular Ca ; 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 CaCl , significantly suppressed the normal phenotype. These observations indicated that mechanisms underlying full-type hyperactivation in spermatozoa incubated with CaCl 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 Ca -dependent full-type hyperactivation in ejaculated boar spermatozoa.

Flagellar hyperactivation of bull and boar spermatozoa

“…Suarez and her colleagues also confirmed that Ca 2+ can antisymmetrize flagellar bending by increasing the curvatures of the principal bend in detergent‐demembranated bull spermatozoa . Moreover, the treatment with the elevators of the intracellular Ca 2+ level can induce hyperactivation rapidly and highly in the uncapacitated spermatozoa in vitro . These results have established a current theory that the most important initiator of hyperactivation is a rapid increase of cytoplasmic Ca 2+ that is induced by the influx from extracellular space and recruitment from the internal store …”

“…This suggests existence of strong suppressors (eg a calyculin‐sensitive protein phosphatase) for hyperactivation in the spermatozoa from livestock . Instead, hyperactivation can be induced highly in the uncapacitated spermatozoa in vitro by the treatment with the elevators of the intracellular Ca 2+ level [eg A23187 (extracellular Ca 2+ ‐dependent hyperactivation) and thimerosal (extracellular Ca 2+ ‐independent hyperactivation)], although the activation of the intracellular cAMP‐PKA signaling cascades is perhaps skipped in these spermatozoa . In our laboratory, extracellular Ca 2+ ‐dependent hyperactivation can be induced highly in bull and boar ejaculated spermatozoa by the treatment with the cell‐permeable cAMP analog to stimulate the intracellular cAMP‐PKA signaling cascades.…”

“…Instead, hyperactivation can be induced highly in the uncapacitated spermatozoa in vitro by the treatment with the elevators of the intracellular Ca 2+ level [eg A23187 (extracellular Ca 2+ ‐dependent hyperactivation) and thimerosal (extracellular Ca 2+ ‐independent hyperactivation)], although the activation of the intracellular cAMP‐PKA signaling cascades is perhaps skipped in these spermatozoa . In our laboratory, extracellular Ca 2+ ‐dependent hyperactivation can be induced highly in bull and boar ejaculated spermatozoa by the treatment with the cell‐permeable cAMP analog to stimulate the intracellular cAMP‐PKA signaling cascades. Specifically, replacement of NaHCO 3 (an activator of ADCY10) with a cell‐permeable, phosphodiesterase‐resistant cAMP analog (Sp‐5,6‐dichloro‐1‐β‐D‐ribofuranosyl‐benzimidazole‐3′,5′‐cyclic monophosphorothioate, cBiMPS), and supplementation of an inhibitor for protein phosphatase 1 and protein phosphatase 2A (calyculin A) dramatically improved the capacity of the capacitation‐supporting medium to induce hyperactivation in bull and boar ejaculated spermatozoa .…”

“…In bulls, boars, and mice, hyperactivation can be induced immediately in uncapacitated spermatozoa by a short treatment with putative stimulator for IP 3 R “thimerosal.” The thimerosal‐induced hyperactivation is full‐type (bulls and boars)/anti‐hock (mice) . Moreover, indirect immunofluorescence and ultrastructural observations indicate existence of the internal store with IP 3 R in the redundant nuclear envelope (RNE) of the connecting piece .…”

“…In human spermatozoa, ryanodine receptor (RyR) is likely to mediate the recruitment of cytoplasmic Ca 2+ from RNE in a manner of calcium‐induced calcium release (CICR) and potentially regulates the occurrence of hyperactivation, although existence and functions of RyR still remain to be revealed in bull and boar ejaculated spermatozoa. Furthermore, we recently reported that the other Ca 2+ channel “transient receptor potential cation channel subfamily C member 3, TRPC3” is present in the connecting and middle pieces of boar ejaculated spermatozoa and involved in the extracellular Ca 2+ ‐dependent occurrence of full‐type hyperactivation after preincubation with cAMP analog . At least in boar ejaculated spermatozoa, TRPC3 channel may mediate the cAMP signaling‐dependent influx of extracellular Ca 2+ leading to full‐type hyperactivation.…”