Abstract
Mammalian sperm must undergo a functionally defined process called capacitation to be able to fertilize oocytes. They become capacitated in vivo by interacting with the female reproductive tract or in vitro in a defined capacitation medium that contains bovine serum albumin, calcium (Ca2+ ), and bicarbonate (HCO3- ). In this work, sperm were double stained with propidium iodide and the Ca2+ dye Fluo-4 AM and analyzed by flow cytometry to determine changes in intracellular Ca2+ concentration ([Ca2+ ]i ) in individual live sperm. An increase in [Ca2+ ]i was observed in a subpopulation of capacitated live sperm when compared with noncapacitated ones. Sperm exposed to the capacitating medium displayed a rapid increase in [Ca2+ ]i within 1 min of incubation, which remained sustained for 90 min. These rise in [Ca2+ ]i after 90 min of incubation in the capacitating medium was evidenced by an increase in the normalized median fluorescence intensity. This increase was dependent on the presence of extracellular Ca2+ and, at least in part, reflected the contribution of a new subpopulation of sperm with higher [Ca2+ ]i . In addition, it was determined that the capacitation-associated [Ca2+ ]i increase was dependent of CatSper channels, as sperm derived from CatSper knockout (CatSper KO) or incubated in the presence of CatSper inhibitors failed to increase [Ca2+ ]i . Surprisingly, a minimum increase in [Ca2+ ]i was also observed in CatSper KO sperm suggesting the existence of other Ca2+ transport systems. Altogether, these results indicate that a subpopulation of sperm increases [Ca2+ ]i very rapidly during capacitation mainly due to a CatSper-mediated influx of extracellular Ca2+ .