Effect of Exogenous L-Glutathione on vitrified preimplantation murine embryos

Vitrification is an important tool for the storage of surplus embryos in assisted reproductive technology (ART). However, vitrification increases oxidative damage and leads to reduced embryo viability. Studies have shown that supplementation with L-glutathione (GSH) improves the preimplantation development of mouse embryos. Glutathione is the major non-protein sulphydryl compound in mammalian cells that provides protection against oxidative damage. However, the effect of GSH supplementation on embryonic vitrification outcomes has not yet been reported. This study aims to determine whether GSH supplementation in the culture media improves the results of in vitro culture and vitrification, as observed by embryo morphology and preimplantation development. In this study, female BALB/c mice were superovulated at 6–8 weeks of age by an intraperitoneal injection of 10 IU of pregnant mare serum gonadotropin (PMSG), followed by 10 IU of human chorionic gonadotropin (hCG) 48 hours later. The mated mice were euthanised by cervical dislocation 48 hours after hCG injection to obtain the embryos. Various concentrations of GSH ranging from 0.01, 0.5, 1.0, 1.5 and 2 mM GSH to the culture media were determined. It was found that 0.01 mM GSH notably increased embryo quality, and thus, was selected for use in this study. Two-cell embryos were randomly cultured in either Group 1 (GSH-free medium), Group 2 (GSH-free medium with vitrification), Group 3 (0.01 mM GSH￾supplemented medium) or Group 4 (0.01 mM GSH-supplemented medium with vitrification). The non-vitrified (Groups 1 and 3) and vitrified (Groups 2 and 4) embryos were examined for morphological quality and preimplantation development after 24, 48, 72 and 96 hours. In the non-vitrified groups, the number of Grade 1 blastocysts in the GSH cultures was significantly increased (p < 0.05). GSH supplementation also led to a significant increase in blastocyst formation in the vitrified groups. Exogenous GSH supplementation led to a significant increase in intracellular GSH, a release of cytochrome C from mitochondria and a parallel decrease in intracellular reactive oxygen species (ROS) in vitrified eight-cell embryos (p < 0.05). It was shown that GSH supplementation upregulated Bcl2 expression and downregulated the expression of Bax, Bid and Casp3 in the group of vitrified preimplantation embryos. The effect of exogenous GSH was accompanied by an increase in the relative abundance of Gpx1 and Sod1. The addition of GSH to the culture media was observed to reduce embryo mortality, as evidenced by a reduction in the expression of the Cirbp, Rbm3 and Hspa1b genes in Group 4 (medium enriched with 0.01 mM GSH with vitrification) compared to Group 2 (GSH-free medium with vitrification). Recent research results indicate an antagonistic relationship between intracellular GSH levels and LDH. In conclusion, our study demonstrates the novel benefit and practicality of GSH supplementation to improve embryonic cryotolerance by lowering ROS levels and inhibiting apoptotic events by improving oxidative status. Exploring the future impact of GSH on vitrification processes is crucial. Enhanced understanding of GSH’s role could significantly improve embryo vitrification outcomes. Long-term studies are essential to uncover the full potential of GSH supplementation, and promising advancements in reproductive technologies.

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