title: Relationship between cyclic AMP-dependent protein tyrosine phosphorylation and extracellular calcium during hyperactivation of boar spermatozoa.
abstract: In mammalian spermatozoa, the state of protein tyrosine phosphorylation is modulated by protein tyrosine kinases and protein tyrosine phosphatases that are controlled via cyclic AMP (cAMP)-protein kinase A (PKA) signaling cascades. The aims of this study were to examine the involvement of cAMP-induced protein tyrosine phosphorylation in response to extracellular calcium and to characterize effects of pharmacological modulation of the cAMP-induced protein phosphorylation state and calmodulin activity during hyperactivation in boar spermatozoa. Ejaculated spermatozoa were incubated with cBiMPS (a cell-permeable cAMP analog) and CaCl(2) at 38.5°C to induce hyperactivation, and then used for Western blotting and indirect immunofluorescence of phosphorylated proteins and for the assessment of motility. Both cBiMPS and CaCl(2) were necessary for hyperactivation. The increase in hyperactivated spermatozoa exhibited a dependence on the state of cBiMPS-induced protein tyrosine phosphorylation in the connecting and principal pieces. The addition of calyculin A (an inhibitor for protein phosphatases 1/2A (PP1/PP2A), 50-100 nM) coincidently promoted hyperactivation and cAMP-induced protein tyrosine phosphorylation in the presence of cBiMPS and CaCl(2). Moreover, the addition of W-7 (a calmodulin antagonist, 2-4 µM) enhanced the percentages of hyperactivated spermatozoa after incubation with cBiMPS and CaCl(2), independently of protein tyrosine phosphorylation. These findings indicate that cAMP-induced protein tyrosine phosphorylation in the connecting and principal pieces is involved in hyperactivation in response to extracellular calcium, and that calmodulin may suppress hyperactivation via the signaling cascades that are independent of cAMP-induced protein tyrosine phosphorylation.
authors.affiliation: Laboratory of Reproductive Biology, Graduate School of Agricultural Science, Kobe University, Kobe 657-8501, Japan. harayama@kobe-u.ac.jp
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Harayama
authors.forename: Taichi
authors.initials: T
authors.lastname: Noda
authors.forename: Shou
authors.initials: S
authors.lastname: Ishikawa
authors.forename: Osamu
authors.initials: O
authors.lastname: Shidara
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002122:Calcium Chloride; D002147:Calmodulin / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism*; D004004:Dichlororibofuranosylbenzimidazole / Q000031:analogs & derivatives / Q000494:pharmacology; D005110:Extracellular Space / Q000378:metabolism*; D005407:Flagella / Q000378:metabolism; D008297:Male; D008387:Marine Toxins; D010080:Oxazoles / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D013094:Spermatozoa / Q000378:metabolism*; D034421:Sus scrofa; D013873:Thionucleotides / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D002147:Calmodulin; D008387:Marine Toxins; D010080:Oxazoles; D013873:Thionucleotides; C071644:5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-3',5'-monophosphorothioate; D014443:Tyrosine; D004004:Dichlororibofuranosylbenzimidazole; C059041:calyculin A; D000242:Cyclic AMP; D011505:Protein-Tyrosine Kinases; D002122:Calcium Chloride; D002118:Calcium