Roles of extracellular Ca2+ in the occurrence of full-type hyperactivation in boar ejaculated spermatozoa pre-incubated to induce the cAMP-triggered events

Abstract

There are species differences in the regulatory system for sperm capacitation and subsequent hyperactivation between livestock and laboratory animals. In livestock spermatozoa, it is poorly understood when and how extracellular Ca2+ is necessary for hyperactivation, although it has been demonstrated that the [Ca2+]i increase is indispensable to occurrence of hyperactivation. In this study, we examined necessity of extracellular Ca2+ for the initiation and maintenance of hyperactivation and then sought possible target molecule of Ca2+ that was involved in hyperactivation of boar spermatozoa. Boar ejaculated spermatozoa were pre-incubated with a cell-permeable cyclic adenosine monophosphate (cAMP) analog 'cBiMPS' and without CaCl2 to induce the cAMP-triggered events including capacitation-associated changes. Subsequently, they were incubated with CaCl2 to induce hyperactivation and then used for motility assessment. Many of the spermatozoa after the incubation exhibited full-type hyperactivation which was characterized by high-amplitude and extremely asymmetrical beating of whole middle piece and principal piece. The initiation of full-type hyperactivation required the millimolar concentration of CaCl2 in the medium. However, CaCl2 of the medium was less necessary for maintenance than initiation of full-type hyperactivation, as hyperactivated spermatozoa were barely affected by the incubation with the Ca2+-chelating reagent. On the other hand, the pre-treatment with the inhibitor for Ca2+-dependent protease 'calpain 1 and 2' clearly suppressed the occurrence of CaCl2-induced hyperactivation without influences on the percentages of motile spermatozoa. Western blotting and indirect immunofluorescence showed distribution of calpain 2 in the middle and principal pieces in which full-type hyperactivated spermatozoa exhibited extremely asymmetrical beating. On the basis of these results, we conclude that the millimolar concentration of extracellular Ca2+ is necessary for the initiation, but not for the maintenance of full-type hyperactivation in boar spermatozoa that beforehand undergo the cAMP-triggered events including capacitation-associated changes. Moreover, we suggest possible involvement of calpain 2 in the intracellular Ca2+ signal transduction leading to full-type hyperactivation.