SUMMARYThere 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 Ca 2+ is necessary for hyperactivation, although it has been demonstrated that the [Ca 2+ ] i increase is indispensable to occurrence of hyperactivation. In this study, we examined necessity of extracellular Ca 2+ for the initiation and maintenance of hyperactivation and then sought possible target molecule of Ca 2+ 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 CaCl 2 to induce the cAMP-triggered events including capacitation-associated changes. Subsequently, they were incubated with CaCl 2 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 CaCl 2 in the medium. However, CaCl 2 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 Ca 2+ -chelating reagent. On the other hand, the pre-treatment with the inhibitor for Ca 2+ -dependent protease 'calpain 1 and 2' clearly suppressed the occurrence of CaCl 2 -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 fulltype hyperactivated spermatozoa exhibited extremely asymmetrical beating. On the basis of these results, we conclude that the millimolar concentration of extracellular Ca 2+ 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 Ca 2+ signal transduction leading to full-type hyperactivation.

In Japanese black cattle, AI severely subfertile males have occasionally been found. In order to solve this problem, we previously asserted the need for exact examinations of acrosomal tyrosine-phosphorylated proteins and acrosome morphology in cryopreserved spermatozoa. In the present study, we further investigated acrosomal tyrosine-phosphorylated proteins in spermatozoa before cryopreservation and examined possible relationships between these phosphoproteins and acrosome stability. Ejaculated, epididymal and cryopreserved spermatozoa were subjected to examinations of general characteristics (motility, shape and acrosome morphology) and indirect immunofluorescence of acrosomal phosphoproteins. Unlike all general characteristic parameters, the distribution of acrosomal tyrosine-phosphorylated proteins in ejaculated and cauda epididymal spermatozoa varied considerably among bulls and was linked to the maintenance of morphologically normal acrosomes in cryopreserved spermatozoa or ejaculated spermatozoa after 270min incubation. Moreover, the distribution of these phosphoproteins was arranged in the spermatozoa of the proximal epididymides. These findings indicate that acrosomal tyrosine-phosphorylated proteins are distributionally arranged during early process of sperm maturation, that their distribution of cauda epididymal and ejaculated spermatozoa are largely different among bulls, and that varied states of acrosomal phosphoproteins may result in individual differences in acrosome stability among bulls.

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