Calcium influx and the progesterone-induced acrosome reaction in stallion spermatozoa
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In vitro fertilization has been successfully applied in humans, rodents and dairy cattle but remains unsuccessful in the horse. One of the main proposed reasons is that the sperm cells fail to penetrate the zona pellucida on their own, most likely due to incomplete capacitation of spermatozoa because of inadequate activation or an unsuitable fertilization media. The main components in medium to induce in vitro capacitation in mammalian spermatozoa have been identified as bicarbonate, calcium and albumin. In other mammalian species, but the horse, sperm incubated in a medium with the three main components showed capacitation-related characteristics as high membrane fluidity, protein tyrosine phosphorylation, hyperactivated motility and acrosome reaction. We investigated whether bicarbonate (HCO3-), extracellular calcium and progesterone modulate membrane fluidity and acrosome reaction in stallion 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 30 mM bicarbonate (TyrBic). The media were either calcium free, i.e. contained ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), or an increasing amount of calcium (0.1 mM - 2 mM). Simultaneous staining for viability (Hoechst 33258), acrosome integrity (PNA-AlexaFluor™647), membrane fluidity (merocyanine 540) was evaluated by flow cytometry after 15, 30, 60 and 120 minutes of incubation of the equine spermatozoa in the first experiment and after 30, 60 and 120 minutes in the second experiment. The first experiment showed that bicarbonate and calcium are necessary to induce early steps of capacitation, such as high membrane fluidity. An amount of calcium ranging from 0.1 mM to 2 mM had the same effect on the percentage of spermatozoa with high membrane fluidity. The aim of the second experiment was to determine the amount of progesterone needed to induce an acrosome reaction. However, in the current experiment it appeared that there was no significant difference between the three progesterone concentrations (10 ng, 100 ng and 1000 ng) and the control group with only ethanol (p>0.05 , ANOVA for repeated measures). In conclusion, progesterone appears not to be the relevant molecule to induce the acrosome reaction in stallion spermatozoa. Further research is needed to elucidate this finding, which is in contradiction to earlier published research.