Abstract

Recent studies have shown that short-term exposure of oocytes to hydrostatic pressure, osmotic stress, thermal, stress, mechanical or oxidative stress might induce stress tolerance in mammalian embryos. The objective of the present study was to evaluate the impact of the short-term exposure of bovine matured oocytes to a nitric oxide and anion superoxide donor (3-morpholinosydnonimine, SIN-1) on subsequent in vitro embryo development, embryo quality and relative gene expression. Immature oocytes were matured in TCM-199 supplemented medium for 22-23 hours. Matured oocytes were then incubated with SIN-1 (Control, 0.1, 1, 10 and 100 μM SIN-1) for 1 hour. The sperm-oocyte incubation was carried out for 18 hours. Zygotes were cultured until Day 7. The cleavage rate at 72 hours did not show significant differences as compared to control treatment. However, the blastocyst rate on Day 7 decreased gradually doses-dependently in all groups, compared to the control group. At Day 7, embryo quality analysis in blastocyst showed no significant differences at different SIN-1 concentrations in TCN and ICM in all treatments compared to control group. Relative gene expression analysis showed eNOS downregulation and nNOS upregulation in all treatments. Downregulation was also observed in some treatments: SOD2 at 0.1 μM SIN-1; SOD1 at 0.1 y 100 μM SIN-1; PRDX5 at 0.1, 10 y 100 μM SIN-1; y NANOG at 10 y 100 μM; and an upregulation in CDX2 expression at 100 μM SIN-1. The genes OCT4, HIF1A, HSPA1A, BCL2A and iNOS did not show differences in the relative gene expression. The use of nitrosative stress inductor, SIN-1, in concentration at 0.1 and 10 μM maintains a proper embryonic development, embryo quality and increase de number of expanded blastocyst compared to the control group. The number increased of expanded blastocyst is important for used in assistant reproductive techniques as cryopreservation and embryo transfer. Acknowledgments: The authors thank “Frigorífico Temuco, Chile” for kindly providing the cow ovaries used in this study. This work was supported by a FONDECYT grant N° 1130888, CONICYT, Gobierno de Chile. Pía Loren was supported by a doctoral scholarship awarded by CONICYT, Gobierno de Chile.