Flow cytometry analysis revealed a rapid increase in intracellular calcium in a subpopulation of mouse sperm during capacitation.

TOMAS DALOTTO-MORENO; LIS C PUGA MOLINA; SCHIAVI-EHRENHAUS, LIZA; PABLO E. VISCONTI; HIDALGO, DAVID; ANA ROMAROWSKI; NICOLAS GILIO; MARIANO G. BUFFONE; GUILLERMINA M LUQUE; RITAGLIATI, CARLA; PAULA A BALESTRINI; DARIO KRAPF

Conferencia; ASA 43rd Annual Conference; 2018

Ejaculated mammalian sperm do not have the ability to fertilize oocytes. They must undergo a functionally defined process called capacitation. Sperm become capacitated in vivo by interacting with the female reproductive tract or in vitro in a defined capacitation media that contains bovine serum albumin (BSA), calcium (Ca2+) and bicarbonate (HCO3-). In this work, flow cytometry was used to analyze changes in intracellular Ca2+ concentration ([Ca2+]i). For this purpose, sperm were double stained with propidium iodide (PI) and the Ca2+ dye Fluo-4 AM to analyze these changes in individual live sperm. An increase in [Ca2+]i was observed in a subpopulation of capacitated live sperm when compared with non-capacitated ones. Sperm exposed to capacitating medium displayed a very 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 capacitating medium was evidenced by an increase in the normalized median fluorescence intensity (MFI). 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 at 90 min 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. Altogether, these results indicate that a subpopulation of sperm increases [Ca2+]i very rapidly during capacitation due to a CatSper-mediated influx of extracellular Ca2+.