Abstract
Assessment of intracellular reactive oxygen species (ROS) is important for evaluating the developmental ability of cumulus-oocyte complexes (COC) and embryos. Although, fluorescence-based 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) staining method is used widely for detecting intracellular ROS in COC and embryos, it is associated with several limitations. This study aimed to develop an alternative method for detecting and quantifying intracellular ROS in oocytes, cumulus cells and embryos based on nitroblue tetrazolium (NBT) staining and bright-field microscopy. Nitroblue tetrazolium reacts with ROS and forms formazan precipitate that can be detected as dark purple/blue spots under bright-field microscope. Ovine COC were matured in vitro without (control) or with the supplementation of Interleukin-7 (IL-7; for stimulating intracellular ROS), Tempol (superoxide scavenger) or combination of IL-7 and Tempol. The matured COC were stained with NBT and the formation of intracellular formazan precipitates was assessed. Additionally, the matured COC were stained with DCFH-DA to compare the level of intracellular ROS. Further, ovine embryos (8-cell, morula, and degenerating) were generated in vitro and stained with NBT for assessing intracellular ROS. The level of intracellular ROS was expressed as the proportion (%) of the NBT stained area of oocytes, compact cumulus cell masses or embryos. The proportions of NBT stained area in the matured oocytes and cumulus cells was found significantly lesser in the control as compared to the IL-7 (1 and 5 ng/ml) treated groups. A similar trend in the intracellular ROS level was also observed in the matured COC, when assessed based on the DCFH-DA staining. Following the treatment with Tempol (100 mM), negligible NBT stained area in oocytes and cumulus cells was observed. The NBT staining patterns of the oocytes and cumulus cells following the combined treatment with IL-7 (5 ng/ml) and Tempol (10 and 25 mM) were comparable with that of the control. The proportion of NBT stained area did not differ significantly between the 8-cell embryos and morula, but was found significantly greater in the degenerating embryos. In conclusion, the developed NBT staining method was found effective for detecting and interpreting the level of intracellular ROS in oocytes, cumulus cells and embryos. This method can be used as an alternative to the DCFH-DA staining method.
Highlights
Reactive oxygen species (ROS) are the by-products of different metabolic pathways, which are produced by the incomplete reduction of oxygen
The current study aimed to develop an efficient alternative method based on NBT staining and bright-field microscopy for detecting and quantifying intracellular ROS in ovine oocytes, cumulus cells, and embryos
NBT Staining for Assessing Intracellular ROS in Oocytes and Cumulus Cells
Summary
Reactive oxygen species (ROS) are the by-products of different metabolic pathways, which are produced by the incomplete reduction of oxygen. They are essential for cellular functioning and their level in cellular system is tightly regulated by the antioxidant defense system. High level of intracellular ROS induces apoptosis in granulosa cells and oocytes (Khazaei and Aghaz, 2017). High intracellular ROS level is associated with poor oocyte maturation, embryonic block and cell death (Bain et al, 2011; Kala et al, 2017). Most studies focusing the developmental ability and cellular functions of oocytes and embryos include the level of intracellular ROS as an important parameter for better interpretation of results
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