Experiments were conducted to elucidate the status of glutathione present in the oxidized (GSSG), reduced (GSH), and protein-mixed disulfide (GSSprotein) forms in preimplantation mouse embryos during development and after treatment with tertiary-butyl hydroperoxide (tBH) to cause oxidative stress. Glutathione was measured at picomolar levels by fluorimetric HPLC after derivatization of extracted embryonic samples with dansyl chloride. GSH content decreased approximately 10-fold from that in the unfertilized oocyte to 0.12 pmol/blastocyst, representing an estimated change in concentration from 7 to 0.7 mM. GSH levels were lower in embryos cultured in vitro than in embryos that developed in vivo. Addition of GSH to the culture medium improved in vitro development of mouse embryos, but surprisingly the addition of glutathione monoethyl ester did not. Addition of low levels of the oxidant tBH (13.2 microM) to culture medium decreased the percentage of two-cell and blastocyst stage embryos that exhibited further development. After 15-min exposure to 13.2 microM tBH, GSH levels were markedly decreased in the two-cell stage embryo (75%), but only slightly decreased (25%) in the blastocyst. The loss of GSH was accounted for by increases in GSSG and GSSprotein, indicating that the embryo was undergoing oxidative stress. These data indicate that preimplantation embryos are very sensitive to conditions that can cause oxidative stress and show also that their glutathione status changes dramatically during development.
We investigated the hypothesis that reduced glutathione (GSH) is present in secretions of the female reproductive tract and that this extracellular GSH may protect preimplantation mouse embryos after intracellular GSH depletion. The cleavage-stage mouse embryo cannot synthesize GSH de novo and is unable to recover from glutathione depletion in vitro. Analysis of GSH and total protein of oviduct flushings, quantified by HPLC and the Bradford method, respectively, revealed 51 nmol GSH per mg total protein. Embryos were treated with 60 microM diethyl maleate (DEM) to deplete cellular GSH. When cultured with 1 mM GSH, these embryos exhibited improved development compared to those cultured in control medium (96% vs. 87% morula [p < 0.05], 78% vs. 75% blastocyst, 58% vs. 54% expanded blastocyst, 21% vs. 17% initiating hatching blastocyst). However, intracellular GSH content of embryos was not significantly increased by the culture of DEM-treated embryos in medium containing GSH for 16, 40, or 64 h of incubation, suggesting that the embryo is not capable of taking up intact GSH. Furthermore, addition of buthionine sulfoximine (which inhibits synthesis of GSH) or acivicin (which inhibits breakdown of GSH at the membrane) to culture medium blocked the improvement in development. These data suggest that GSH in reproductive tract fluid may help protect preimplantation embryos from the adverse effects of toxicant-induced and endogenous depletion of embryonic GSH.
We investigated the hypothesis that glutathione (GSH) in reproductive tract secretions (RTS) protects the preimplantation embryo from endogenous reactive oxygen species and is important for normal development during the embryo's sensitive period when it is incapable of synthesizing GSH de novo. Mice were administered buthionine sulfoximine (BSO) to inhibit GSH synthesis and decrease GSH concentration in RTS. Embryos were then allowed to develop either in vivo or in vitro in the presence of RTS and the GSH concentration of the embryos was quantified by HPLC and embryonic development was recorded. GSH concentration in RTS did not differ over the phases of the estrous cycle, but there were significant decreases in GSH concentration on Day 2 of gestation and due to BSO treatment. Embryos allowed to develop in vivo and in vitro in RTS with decreased GSH concentration did not exhibit decreased development or GSH concentration. Oocytes exposed to BSO during maturation in vivo experienced a significant decrease in GSH concentration and an increase in percent of degenerate embryos when compared with control. These data suggest that most of the GSH in RTS does not play a critical role in normal preimplantation embryo development but that GSH stored in the oocyte during maturation has an important role in subsequent embryo development. Our studies do not exclude the possibility that GSH in RTS plays an important role in protection of the preimplantation embryo during exposure to some toxicants.
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