Distinct segment‐specific functions of calyculin A‐sensitive protein phosphatases in the regulation of cAMP‐triggered events in ejaculated bull spermatozoa

Abstract

Livestock spermatozoa possess more tenacious suppressors of cAMP-triggered events-including capacitation-associated changes-than laboratory animal spermatozoa, leading to flagellar hyperactivation. In order to identify the suppressors, we examined effects of an inhibitor of serine/threonine protein phosphatases (calyculin A) on cAMP-triggered changes in the protein phosphorylation state, and subsequent occurrence of hyperactivation and acrosome reaction in ejaculated bull spermatozoa. Ejaculated spermatozoa were incubated in cAMP-supplemented medium, then assessed for motility, acrosome morphology, and phosphorylated protein localization. The addition of calyculin A greatly enhanced cAMP-triggered protein phosphorylation at serine/threonine and tyrosine residues in the connecting piece and induction of flagellar hyperactivation. Most hyperactivated spermatozoa exhibited extremely asymmetrical bends at the middle piece, which produced intensive twisting or figure-eight movements. In the sperm head, however, cAMP-triggered dephosphorylation of serine/threonine-phosphorylated proteins and subsequent acrosome reaction were abolished by the addition of calyculin A. Based on these results, we suggest that calyculin A-sensitive protein phosphatases in the connecting piece are suppressors of cAMP-triggered events leading to hyperactivation. By contrast, similar protein phosphatases in the sperm head accelerate cAMP-triggered events leading to the acrosome reaction. These findings are consistent with the indication that calyculin A-sensitive protein phosphatases have distinct functions in the regulation of cAMP-triggered events in different regions of ejaculated bull spermatozoa.

Translocation of Tektin 3 to the equatorial segment of heads in bull spermatozoa exposed to dibutyryl cAMP and calyculin A

“…Enhanced immunolabeling at both the annulus of flagella and the post‐acrosomal region was clearly observed at higher magnification (Fig. I), which is similar to the pattern reported previously for activated spermatozoa (Mizuno et al, ). Thus, spermatozoa exposed to dbcAMP and calyculin A almost achieve a hyperactivation status.…”

“…In vitro incubation of ejaculated porcine spermatozoa or frozen‐stored bovine spermatozoa (Japanese black bulls) with a cell‐permeable cyclic AMP (cAMP) analog induced hyperactivation, which might be mediated by cAMP‐induced protein phosphorylation in flagella (Harayama and Miyake, ; Harayama and Nakamura, ; Pons‐Rejraji et al, ; Murase et al, ). Calyculin A, a protein phosphatase inhibitor, also greatly enhanced cAMP‐triggered protein phosphorylation at serine/threonine and tyrosine residues in the connecting piece and also induced full hyperactivation of flagella, which is characterized by asymmetrical bending of the middle piece with a large amplitude (Mizuno et al, ).…”

“…Several molecules, including protein kinase A (PKA) substrates, were reported to relocate in sperm heads during capacitation or hyperactivation (Toshimori, ; Mizuno et al, ). Given the importance of protein phosphorylation during sperm behavior, we examined the effects of cAMP‐triggered activation on the distribution of TEKT3 in bull spermatozoa.…”

“…Calyculin A‐sensitive protein phosphatases are known to dephosphorylate serine/threonine‐phosphorylated protein substrates of PKA. Indeed, calyculin A‐sensitive protein phosphatases suppressed capacitation‐associated protein phosphorylation at serine/threonine and tyrosine residues in mouse spermatozoa (Krapf et al, ), and greatly enhanced cAMP‐triggered protein phosphorylation at serine/threonine and tyrosine residues in the connecting piece while also inducing flagellar hyperactivation in bull spermatozoa (Mizuno et al, ). Calyculin A‐dependent hyperactivation was also reported for spermatozoa from mouse (Goto and Harayama, ) and hamster (Si and Okuno, ).…”

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

“…Interestingly, this anti‐hook‐type hyperactivation is predominately observed in the oviduct of mated female mice (Chang & Suarez, ), suggesting that spermatozoa hyperactivated under a physiologically capacitation‐supporting conditions in vivo exhibit high‐amplitude and asymmetrical flagellar bending in the entire middle piece‐principal pieces. Analogous to mouse spermatozoa, gametes from livestock—for example, bulls (Marquez & Suarez, ; Mizuno et al, ) or boars (Kojima et al, )—exhibit non‐full‐type and full‐type hyperactivation, which includes asymmetrical flagellar bending in the distal middle piece‐principal piece and along the entire middle piece‐principal piece; we consider the pro‐hook and anti‐hook types of murine sperm hyperactivation equivalent to non‐full and full types of sperm hyperactivation of livestock, respectively, based on which flagellar regions exhibit asymmetrical flagellar bending. Full‐type hyperactivation of boar spermatozoa is characterized by a larger curvature of the proximal (head‐side) segment of middle piece than that of bull spermatozoa.…”