Involvement of Ca²⁺-ATPase in suppressing the appearance of bovine helically motile spermatozoa with intense force prior to cryopreservation

Abstract

In cattle, cryopreserved spermatozoa are generally used for artificial insemination (AI). Many of these specimens exhibit helical movement, although the molecular mechanisms underlying this phenomenon remain unclear. This study aimed to characterize helically motile spermatozoa, investigate the involvement of Ca 2+ -ATPase in suppressing the appearance of these spermatozoa prior to cryopreservation, and examine the potential of helical movement as an index of sperm quality. In the cryopreserved semen, approximately 50% of spermatozoa were helically motile, whereas approximately 25% were planarly motile. The helically motile samples swam significantly faster than those with planar movement, in both non-viscous medium and viscous medium containing polyvinylpyrrolidone. In contrast, in non-cryopreserved semen, planarly motile spermatozoa outnumbered those that were helically motile. Fluorescence microscopy with Fluo-3/AM and propidium iodide showed that flagellar [Ca 2+ ] i was significantly higher in cryopreserved live spermatozoa than in non-cryopreserved live ones. The percentage of non-cryopreserved helically motile spermatozoa was approximately 25% after washing, and this increased significantly to approximately 50% after treatment with an inhibitor of sarcoplasmic reticulum Ca 2+ -ATPases (SERCAs), “thapsigargin.” Immunostaining showed the presence of SERCAs in sperm necks. Additionally, the percentages of cryopreserved helically motile spermatozoa showed large inter-bull differences and a significantly positive correlation with post-AI conception rates, indicating that helical movement has the potential to serve as a predictor of the fertilizing ability of these spermatozoa. These results suggest that SERCAs in the neck suppress the cytoplasmic Ca 2+ -dependent appearance of helically motile spermatozoa with intense force in semen prior to cryopreservation.

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

“…This type of flagellar bending causes spermatozoa to exhibit circular and 2D planar movements. Moreover, the combination of extremely asymmetrical flagellar bending with steady rotation of the head yields hyperactivation with 3D helical movement [ 25 ], and both sperm behaviors are promoted by an increase in intracellular Ca 2+ in cryopreserved bovine sperm flagella [ 22 , 26 ]. In this study, Fig.…”

“…These results suggest that the origins from which adequate Ca 2+ molecules are supplied to the flagellar cytoplasm to induce hyperactivation with 3D helical movement differ between cryopreserved bovine spermatozoa and mouse epididymal spermatozoa. Although further experiments are required to reveal the factors causing the difference in flagellar Ca 2+ homeostasis between these spermatozoa, one of the causal factors may be explained by our previous observation that the cryopreservation process partially destroys SERCAs which increase the cumulative dosage of Ca 2+ in the RNE internal store of the bovine sperm neck [ 22 ].…”

“…Cytoplasmic Ca 2+ in sperm flagella was detected as described previously [ 22 ]. Briefly, the washed spermatozoa (1.0 × 10 8 cells/ml) were loaded with Fluo-3/AM (F023, Dojindo) in the presence of Pluronic F127 (0.02% v/v, R2443, Sigma-Aldrich) for 10 min in the dark and then washed.…”