Formation of protein–glutathione mixed disulfides in the developing rat conceptus following diamide treatment in vitro

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Abstract
Protein‐glutathione mixed disulfide (protein‐S‐SG) formation was investigated in developing rat conceptuses during early organogenesis (gestational day 10, GD 10) using the whole embryo culture system. Low levels of protein‐S‐SG (25.0±6.6 pmoles resolved GSH/conceptus) were found in conceptuses under normal culture conditions. Incubation of the conceptuses with 75‐500 μM diamide (a thiol oxidant) resulted in rapid increases in protein‐S‐SG (to 2‐ to 16‐ fold that of control values) in a dose‐dependent manner during 30 min of the culture period. Approximately 20% of the observed cytosolic glutathione (GSH) depletion following diamide (500 μM) could be accounted for as mixed disulfides of protein sulfhydryls, when determined in whole conceptual tissues after 15 min. The most extensive S‐thiolation of protein sulfhydryls by GSH was observed in visceral yolk sac (VYS) when compared to embryo proper and ectoplacental cone. This result indicates that the most abundant, sensitive, or accessible protein sulfhydryls were found in the VYS. Inhibition of glutathione disulfide reductase activity by pretreatment of the conceptuses with 25 μM BCUN for 2 hr potentiated protein‐S‐SG formation elicited by 75 μM diamide. Reincubation of the conceptuses in fresh media, following the 15‐min treatment with 500 μM diamide, reversed both the GSH depletion and the protein‐S‐SG formation in conceptal tissues. The reduction of the protein‐S‐SG was dependent on adequate intracellular GSH levels and was inhibited when GSH was rapidly depleted by subsequent addition of N‐ethylmaleimide (NEM, 100 μM). Under the same experimental conditions, addition of 1 mM dithiothreitol (DTT) did not significantly enhance the GSH restoration rate nor the protein‐S‐SG reduction rate. The results also indicated that low levels of intracellular cysteine do not play an important role in the reduction of protein‐S‐SG. Protein‐S‐SG formation may be important for cellular regulation and in mediating the embryotoxicity elicited by diamide or other oxidative stresses.

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