The effect of bicarbonate, BSA and calcium on capacitation-related events in stallion spermatozoa
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Conditions for successful in vitro capacitation of equine spermatozoa are not established. In other species, increased plasma membrane fluidity, externalization of phosphatidylethanolamine (PE) and phosphatidylserine, cholesterol efflux and hyperactivated motility in response to bicarbonate (HCO3-) are considered to indicate capacitation and preparation of spermatozoa for the acrosome reaction. We investigated whether extracellular bicarbonate (bic), bovine serum albumin (BSA) and calcium (Ca2+) modulated membrane fluidity, PE exposure, cholesterol efflux, motility and acrosome integrity in equine spermatozoa. Semen was initially diluted in INRA96 (30x106 spermatozoa/mL). After Percoll® washing, spermatozoa were incubated at 37°C in Tyrode’s medium without (TyrControl) or with bic (TyrBic, 5% CO2 in air, 100% humidity). Simultaneous staining for viability (Hoechst 33258), acrosome integrity (PNA-FITC or PNA-AlexaFluor™647), membrane fluidity (merocyanine 540) or PE (duramycine-Cy5) was evaluated by flow cytometry after 15, 30 or 60 minute incubation. Results showed that high membrane fluidity can be induced in live acrosome intact spermatozoa in the presence of bicarbonate (LAIMFhigh). The effect of Ca2+ (no addition or 2 mM) in the presence of 1 mg/mL BSA or 0.5 mg/mL each of PVA and PVP was investigated (n=6 stallions). After 60 min in Tyrbic, the live MFhigh sperm population was reduced by both BSA and Ca2+ (p<0.05, ANOVA for repeated measures) presumably because BSA and Ca2+ both simultaneously promoted (p<0.05) development of a live, acrosome-reacted sperm population. Viable spermatozoa with exposed PE were virtually absent in all treatments, whereas nearly all dead spermatozoa were PE positive. Confocal microscopy demonstrated that PE staining on dead sperm was confined to the mid-piece. No hyperactivated motility was induced by bicarbonate, BSA and calcium. In conclusion, balanced bic, BSA and calcium concentrations can prime equine spermatozoa for a (spontaneous) acrosome reaction in vitro. PE exposure in equine spermatozoa is neither part of bicarbonate-stimulated activation in vitro nor under the control of cAMP/sAC/PKA.