The objective of this study was to determine the mechanism by which RU 486 (mifepristone) protects sperm to undergo premature capacitation during cryopreservation. For this, semen ejaculate (n = 20) was divided into four equal fractions and diluted using egg yolk-based extender supplemented with different concentrations of RU 486 (0, 5, 10 and 20 µM) and cryopreserved. We found that RU 486 did not impair the post-thaw sperm kinetics and motility but prevented cholesterol efflux, calcium influx, and protected CatSper channels during cryopreservation. The RU 486 protected sperm from premature capacitation which was confirmed by intracellular calcium level, expression of tyrosine phosphorylated proteins (75 and 80 kDa) and CTC (chlortetracycline) assay. Furthermore, antioxidant ability of RU 486 was reflected by the ferric reducing ability, lower production of sperm malondialdehyde and intracellular reactive oxygen species. Also, we demonstrated that RU 486 treated sperm underwent normal capacitation, zona pellucida binding and zygote cleavage indicating normal fertilizing ability of sperm. In conclusion, we report a new role of RU 486 in protecting buffalo sperm from premature capacitation during cryopreservation.

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