Abstract
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.
Highlights
In spite of advancements in sperm cryopreservation, achieving optimum conception rate with frozen buffalo semen remains a challenge
The contribution of progesterone from seminal plasma to the extender is negligible in comparison to egg yolk (Supplementary Fig. 1)
As 20% egg yolk is used in semen extender, the final progesterone concentration (438.44 ng/mL) in extender was as high as the physiological concentration of progesterone in oviduct[15]
Summary
In spite of advancements in sperm cryopreservation, achieving optimum conception rate with frozen buffalo semen remains a challenge. Cryopreservation, being a damaging phenomenon causes the loss of viability to around 50% during this process[1,2] and remaining motile sperm undergo premature capacitation (cry-capacitation)[3]. A variety of protocols, cryoprotectants and additives have been tried to protect sperm during cryopreservation with varied degree of success, but unable to prevent premature capacitation[4,5,6,7]. The loss of cholesterol during cryopreservation increases the permeability of sperm membrane by stimulating several factors that allow frozen-thawed spermatozoa to undergo precocious capacitation[8]. Higher progesterone concentration in egg yolk-based extender might play role in triggering signaling pathway leading to capacitation-like changes in already destabilized sperm membrane during cryopreservation. We report a new role of RU 486 to prevent premature capacitation of sperm during cryopreservation
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