The relation between intracellular calcium concentration and reactive oxygen species production in fresh and cryopreserved-thawed human spermatozoa

Sperm cryopreservation in liquid nitrogen is a widely used method of preserving them for a long time. Many of these sperms are damaged and died or lost their motility after thawing. These cryoinjuries are caused by damage to the sperm membrane and changes in permeability to ions due to changes in osmotic pressure, the formation of ice crystals, and increased production of oxygen free radicals. Calcium ions play an important role in the physiological activities of sperm and this study was performed to investigate the effect of increased intracellular calcium on cryoinjuries and the production of oxygen free radicals in fresh and frozen-thawed sperm.

Thirty-five human sperm were washed and randomly allocated into fresh and frozen-thawed groups. The sperm of the frozen-thawed group were preserved under liquid nitrogen for one month. The effect of 10 µM calcium ionophore A23187 was evaluated on fresh and thawed sperm motility, viability, and acrosome reaction. Oxygen free radicals production and intracellular calcium content were evaluated using luminol and Flou-3/AM staining by chemiluminescence and flowcytometric method, respectively. Data were compared using the Mann–Whitney test. P<0.05 was considered statistically significant.

The percentage of live, motile and progressive sperm and the percentage of sperm with intact acrosome decreased significantly after the freezing-thawing process. The addition of A23187 to the medium increased intracellular calcium in live sperm but decreased the percentage of live and motile sperm in both groups. The production of free radicals increased per live cell in both groups, but this increase was significantly higher in the frozen-thawed group than in the fresh group. The results showed a significant positive relationship between intracellular calcium and the production of oxygen free radicals.

Calcium ions are required for normal sperm function, but increased calcium entry by A23187 into fresh or frozen-thawed sperm causes more damage and reduced motility and survival. The amount of oxygen free radicals has a direct and significant relationship with intracellular calcium. Freezing damage is exacerbated by higher levels of intracellular calcium, which may be due to increased production of oxygen free radicals.