STATUS OF GLUTATHIONE DURING OXIDANT-INDUCED OXIDATIVE STRESS IN THE PREIMPLANTATION MOUSE EMBRYO
STATUS OF GLUTATHIONE DURING OXIDANT-INDUCED OXIDATIVE STRESS IN THE PREIMPLANTATION MOUSE EMBRYO
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DOI：
10.1095/biolreprod51.6.1307
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发表时间：
1994-12-01
影响因子：
3.6
通讯作者：
REED, DJ
中科院分区：
文献类型：
--
作者：
GARDINER, CS;REED, DJ
本实验旨在研究小鼠胚胎在发育过程中和用叔丁基过氧化氢（tBH）处理后谷胱甘肽的氧化型（GSSG）、还原型（GSH）和蛋白质混合二硫化物（GSSprotein）的状态。在用丹磺酰氯衍生提取的胚胎样品后，通过荧光HPLC测定皮摩尔水平的谷胱甘肽。GSH含量下降约10倍，在未受精的卵母细胞的0.12 pmol/囊胚，代表浓度从7至0.7 mM的估计变化。GSH水平较低，在体外培养的胚胎比在体内发育的胚胎。在培养基中添加谷胱甘肽可以改善小鼠胚胎的体外发育，但令人惊讶的是，添加谷胱甘肽单乙酯却没有。在培养基中加入低水平的氧化剂tBH（13.2 μ M），可降低进一步发育的两细胞和囊胚期胚胎的百分比。暴露于13.2 μ M tBH 15分钟后，GSH水平显着下降，在两细胞期胚胎（75%），但只有轻微下降（25%）的囊胚。GSH的损失是由于GSSG和GSS蛋白的增加造成的，表明胚胎正在经历氧化应激。这些数据表明，植入前胚胎对可能导致氧化应激的条件非常敏感，并且还表明它们的谷胱甘肽状态在发育过程中发生了显着变化。
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 mu M) to culture medium decreased the percentage of two-cell and blastocyst stage embryos that exhibited further development. After 15-min exposure to 13.2 mu M 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.