Calcium ionophore a23187 effect on acquisition of fertilizing ability in equine cryopreserved spermatozoa

Abstract

Spermatozoa must undergo three events to fertilize anoocyte in vivo: capacitation, hyperactivation and acrosome reaction. Therefore,it is necessary to induce these physiological events in vitro in themasculine gamete during assisted reproductive techniques. Even though the invitro conditions under which many species acquire their fertilizing abilityare well known, to this day, there is no standard protocol to induce theseevents in equine cryopreserved spermatozoa. Thus, there is no available protocolfor embryo production through in vitro fertilization (IVF) with these samples.It has been proven that brief exposure of murine andbovine spermatozoa to calcium ionophore A23187 and the consequent calcium influxto the cell cytoplasm increases their fertilizing ability in vitro andembryo production due to the induction of capacitation and hyperactivation. Moreover,A23187 increases events related to capacitation without inducing the classicalcapacitation signaling pathway such as PKA-activation (pPKA) or proteintyrosine phosphorylation (pTyr). Given the current difficulties in equine embryoproduction through IVF with cryopreserved spermatozoa and the previously showneffects of the calcium ionophore in spermatozoa from other species, this workaimed to study the potential fertilizing ability of equine cryopreserved spermatozoathat were previously exposed to A23187.First, we studied the effect of A23187 (1 µM) on spermatozoa.After 10 minutes of exposure, the ionophore decreased the spermatozoa motility(CASA, p<0,05) without affecting their viability (HOS test, p>0,05) or acrosomestatus (PSA-FITC, IF, p>0,05). After removing the ionophore, the spermatozoawere incubated under non-capacitating conditions for 20 minutes and motilitywas reassessed, and their fertilizing ability was studied. The previousincubation with A23187 increased the hyperactivated sperm population (CASA,p<0,05) and the induction of capacitation-associated events such as progesterone-inducedacrosome reaction (IF, p<0,05) and the sperm ability to bind to bovineoocyte zona pellucida (p<0,001) without activating the pTyr and pPKA pathways(IF, p>0,05). Our results suggest that brief exposure of cryopreservedequine spermatozoa to calcium ionophore A23187 could be incorporated into the assistedreproductive techniques to increase spermatozoa fertilizing ability in vitrodue to its effect on hyperactivation and capacitation in this species.