Glutathione Redox Cycle-Driven Recovery of Reduced Glutathione after Oxidation by Tertiary-butyl Hydroperoxide in Preimplantation Mouse Embryos

The purpose of this study was to determine whether mouse embryos at the two-cell to blastocyst stage have the capacity to reduce glutathione disulfide (GSSG) and to elucidate the mechanism that embryos utilize to recover from tertiary-butyl hydroperoxide (tBH)-induced oxidative stress. Experiments were conducted on embryos in vitro and tBH was used to induce oxidation of embryonic reduced glutathione (GSH). After derivatization of extracted embryo samples with dansyl chloride, GSH and GSSG were measured at picomole levels by fluorometric HPLC. Two-cell- and blastocyst-stage embryos were able to recover their GSH levels within 45 min after depletion of GSH by incubation in tBH for 15 min. Addition of 1,3-bis(2-chloroethyl)-1-nitrosourea to the culture medium blocked recovery of GSH and resulted in continued elevation of GSSG. Addition of buthionine sulfoximine (BSO) to the culture medium did not affect GSH levels in two-cell-stage embryos, but did reduce GSH levels in blastocysts by 1.5 h. Culture of two-cell embryos in the presence of BSO for 45 h decreased embryonic GSH content and percentage of embryos developing to the blastocyst stage. These results indicate that preimplantation mouse embryos have the capacity to reduce GSSG and suggest that under normal conditions, depletion and synthesis of GSH occur to a greater extent in the blastocyst than in the two-cell-stage embryo. A major protective role for glutathione reductase during specific stages of embryo development is indicated.