Comparative characteristics between calyculin A-induced and thimerosal-induced hyperactivation of cryopreserved...

This study aimed to characterize calyculin A (CL-A)-induced and thimerosal-induced hyperactivation of cryopreserved bovine spermatozoa. Hyperactivation was effectively induced by treating with 10 nM CL-A for 60 min in the presence of cyclic AMP analogs, extracellular Ca²⁺, and albumin or with 12.5 µM thimerosal briefly in the absence of these capacitation-supporting factors. Majority of the spermatozoa exhibiting CL-A-induced hyperactivation were characterized by the 3-dimensional helical movement with head rotation, higher degree of flagellar curvature, and faster beating of the flagella than those exhibiting thimerosal-induced hyperactivation of the 2-dimensional planar movement without head rotation. The CL-A-induced hyperactivation was linked to the activation of cAMP/protein phosphorylation-dependent signaling cascades and to the decreased activity of glycogen synthase kinase-3α (GSK-3α). In contrast, the thimerosal-induced hyperactivation was suppressed by pretreatment with CL-A and cyclic AMP analogs in the absence of CaCl₂ to activate cAMP/protein phosphorylation-dependent signaling cascades. Additionally, the intracellular Ca²⁺ level in live sperm flagella was significantly higher in the CL-A-treated samples than in the thimerosal-treated samples. These results indicate that CL-A-induced hyperactivation of cryopreserved bovine spermatozoa is an extracellular Ca²⁺-dependent type with the 3-dimensional helical movement, which can be regulated not only by the activation of cAMP/protein phosphorylation-dependent signaling cascades, leading to a large enhancement of the intracellular Ca²⁺ level, but also by the reduction in GSK-3α activity. Considering the different characteristics of thimerosal-induced hyperactivation, our results suggest that the diversity of sperm hyperactivation arises from different combinations of flagellar bending and head rotation.