Effects of Cryopreservation on Ca2+ Signals Induced by Membrane Depolarization, Caffeine, Thapsigargin and Progesterone in Boar Spermatozoa

Abstract

Although the fertilizing ability of spermatozoa is greatly reduced after freezing, complete understanding of alterations induced by cryopreservation has not been elucidated. The present study evaluates the effects of cryopreservation on the Ca2+ handling of boar spermatozoa using several sperm activators. Intracellular Ca2+ signals from single spermatozoa were measured using confocal Ca2+ imaging of unfrozen samples and of other spermatozoa after having been frozen. Elevation of the external K+ concentration elicited a three times larger Ca2+ increase in fresh spermatozoa than in cryopreserved spermatozoa. Caffeine elicited Ca2+ transients with some oscillations in the fresh spermatozoa, but not in the thawed spermatozoa. Depletion of the Ca2+ store with thapsigargin induced a rapid rise in Ca2+ in the control but generated a smaller increase of Ca2+ after thawing. Exposure to progesterone induced a biphasic rise of the Ca2+ level in the fresh spermatozoa only. Sperm viability was reduced by cryopreservation. Resting Ca2+ levels in fresh and cryopreserved spermatozoa were similar. Longer incubation (2.5 h) of thawed spermatozoa partly recovered the Ca2+ response to the interventions. These results suggest that cryopreservation reduces the responsiveness of spermatozoa to depolarization, modulators of the internal Ca2+ store and progesterone in terms of the Ca2+ signal, thus providing a possible mechanism for reduced fertility observed in cryopreserved boar spermatozoa.