Effect of bovine follicular fluid on reactive oxygen species and glutathione in oocytes, apoptosis and apoptosis-related gene expression of in vitro-produced blastocysts.

The reactive oxygen species (ROS) generated during the in vitro maturation of oocytes affect oocyte maturation and subsequent embryonic development. Bovine follicular fluid (bFF) has an effective antioxidant capacity. This study was conducted to investigate the effects of supplementing oocyte maturation media with bFF from different size classes (3-8 and 9-13 mm) on the glutathione (GSH) and ROS levels of oocytes. Embryonic development and apoptosis, as well as the relative abundance of INFτ, BAX, BCL2 and HSP70 transcripts in blastocysts, were also monitored. Oocytes collected from ovaries were matured in TCM-199 with FBS (control) and 10% 3-8 mm (M), 9-13 mm (L) or a mixture of 3-8 mm and 9-13 mm (M + L) bFF. Glutathione and ROS levels in oocytes after 24 h were assessed by Cell Tracker Blue CMF2HC and DCHFDA staining, respectively. Apoptosis in day-8 blastocysts was assessed by TUNEL staining. The relative abundance of BAX, BCL2, HSP70 and INFτ transcripts was assessed using quantitative real-time polymerase chain reaction (PCR). The GSH level was significantly higher in the L group compared to the other groups (p < 0.05), while the ROS levels in the M group were significantly higher than in the other groups (p < 0.05). The apoptosis levels of blastocysts in the FBS group were significantly higher than those in the M + L group (p < 0.05), although the embryonic development did not differ between the groups. The HSP70 and INFτ expression levels in group M were significantly greater than in the controls (p < 0.05). There was no significant difference in BAX expression between the groups. Supplementation with bFF from various sizes of follicles into the maturation medium was capable of supporting oocyte cytoplasmic maturation by decreasing the ROS. Moreover, bFF subsequently affected antioxidative gene expression, increasing HSP70 and INFτ expressions.