Identification of isoforms of calyculin A-sensitive protein phosphatases which suppress full-type hyperactivation in...

In bull spermatozoa, extracellular Ca²⁺-dependent full-type hyperactivation, which is characterized by the asymmetrical beating in whole parts of the middle/principal pieces, is suppressed by calyculin A-sensitive protein phosphatases. The aim of this study was to identify isoforms of these protein phosphatases. Ejaculated spermatozoa were used for the investigation on effects of protein phosphatase inhibitors (calyculin A with high specificity for both of protein phosphatases 1 and 2A, and okadaic acid with relatively higher specificity for protein phosphatase 2A than protein phosphatase 1) on the induction of extracellular Ca²⁺-dependent full-type hyperactivation by incubation with CaCl₂ and cAMP analog (cBiMPS). They were also used for the immunodetection of protein phosphatases 1α, 1β, 1γ, 2Aα and 2Aβ. Percentages of full-type hyperactivated spermatozoa significantly increased after incubation with calyculin A (10 nM) in a concentration-dependent manner of CaCl₂ (0-3.42 mM), though only minor increases in the percentages of full-type hyperactivated spermatozoa were observed after incubation with okadaic acid (10 nM). Moreover, the immunodetection of protein phosphatase isoforms showed sperm connecting piece and flagellum included protein phosphatases 1α and 1γ, but did not do the other isoforms. These results suggest that calyculin A-sensitive and okadaic acid-less sensitive protein phosphatases (1α and 1γ) are suppressors for the extracellular Ca²⁺-dependent full-type hyperactivation in bull ejaculated spermatozoa.