Effect of two intracellular calcium modulators on sperm motility and heparin-induced capacitation in cryopreserved bovine spermatozoa

Abstract

Spermatozoa require a preparatory process called capacitation to fertilize mature oocytes. Two events related to capacitation of mammalian spermatozoa are an increase in intracellular Ca2+ and protein tyrosine phosphorylation. The sites that regulate intracellular Ca2+ concentration are plasma membrane and mitochondria. There are different systems for mitochondrial Ca2+ influx and efflux. Our aim was to study the involvement of mitochondrial Ca2+ cycle during heparin-induced capacitation in cryopreserved bovine spermatozoa. Samples were incubated at 38°C for 45min, in TALP medium, in the presence of: (a) heparin (H), a well known capacitation inducer; (b) H+CGP 37157, a specific inhibitor of mitochondrial Ca2+ efflux; (c) H+RU 360, a specific inhibitor of Ca2+ influx to the mitochondria and (d) H+CGP 37157+RU 360. In every treatment, capacitation (by CTC), progressive motility (by optical microscopy), viability (by the eosin/nigrosin technique) and protein tyrosine phosphorylation (by Western Immuno-blotting), were evaluated. The addition of CGP 37157 (20μM) decreased progressive motility (p<0.05), without affecting capacitation or protein tyrosine phosphorylation, indicating the importance of calcium efflux for maintaining progressive motility. RU 360 (5μM) significantly reduced capacitation without affecting progressive motility, sperm viability or protein tyrosine phosphorylation, showing that inhibition of the mitochondrial calcium uptake, negatively affect the capacitation process. The addition of both inhibitors showed the effect of RU 360. According with these results, there would exist a differential participation of the income and outcome mitochondrial calcium carriers, in the capacitation process. In conclusion, this research demonstrates the importance of normal mitochondrial calcium cycle in the achievement of sperm capacitation and the maintenance of progressive motility in cryopreserved bovine spermatozoa.