Development to the blastocyst stage, the oxidative state, and the quality of early developmental stage of porcine embryos cultured in alteration of glucose concentrations in vitro under different oxygen tensions
Development to the blastocyst stage, the oxidative state, and the quality of early developmental stage of porcine embryos cultured in alteration of glucose concentrations in vitro under different oxygen tensions
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DOI：
10.1186/1477-7827-4-54
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发表时间：
2006-11-06
影响因子：
4.4
通讯作者：
Nagai, Takashi
中科院分区：
文献类型：
--
作者：
Karja, Ni Wayan Kurniani;Kikuchi, Kazuhiro;Nagai, Takashi
背景：最近的研究表明，葡萄糖可能通过自氧化产生的自由基或抑制次黄嘌呤磷酸核糖基转移酶的作用诱导细胞损伤。研究了猪胚胎体外培养初期葡萄糖对其发育能力、活性氧（ROS）、谷胱甘肽（GSH）含量及囊胚质量的影响。方法：体外成熟和受精的猪卵母细胞在NCSU37中分别添加1.5 ~ 20 mM葡萄糖（glucu -1.5 ~ -20组）或丙酮酸和乳酸（Pyr-Lac组），培养IVC前2天（Day 0 =受精第1天）。各组胚胎在添加5.5 mM葡萄糖的NCSU-37培养基中培养至第6天。在第1天和第2天测定ROS和GSH水平。第6天，用tunel染色法观察胚泡dna片段化细胞核和总细胞数。结果：5%氧下葡萄糖组囊胚率和囊胚总细胞数均显著低于Pyr-Lac组。20%氧组（不含葡萄糖-10组）囊胚率差异无统计学意义，但各组囊胚细胞总数差异无统计学意义。在两种氧压下，所有葡萄糖组第1天胚胎的H2O2水平均显著高于Pyr-Lac组，只有葡萄糖-1.5组第2天胚胎的H2O2水平显著高于Pyr-Lac组。5%氧条件下第1天和第2天胚胎的谷胱甘肽含量各组间基本一致。只有1.5 mM组第2天胚胎含量显著低于20%氧浓度下Pyr-Lac组。除葡萄糖-20组外，20%氧培养的囊胚细胞总数明显低于5%氧培养的囊胚细胞总数。只有5%氧条件下发育的葡萄糖-20囊胚的DNA断裂率显著高于Pyr-Lac囊胚。结论：用葡萄糖代替丙酮酸和乳酸培养的胚胎受精后发育能力下降可能与第1天胚胎ROS生成增加有关。此外，本研究结果表明，第1 -2天胚胎可以使用的培养基中的葡萄糖浓度限制在3.5 mM，暴露于更高的葡萄糖浓度并不能促进胚胎发育。
Background: Recent work has shown that glucose may induce cell injury through the action of free radicals generated by autooxidation or through hypoxanthine phosphoribosyltransferase inhibition. The effect of glucose during early in vitro culture (IVC) period of porcine embryos on their developmental competence, contents of reactive oxygen species (ROS) and glutathione (GSH), and the quality of the blastocysts yielded was examined.Methods: In vitro matured and fertilized porcine oocytes were cultured for the first 2 days (Day 0 = day of fertilization) of IVC in NCSU37 added with 1.5 to 20 mM glucose (Gluc-1.5 to -20 groups) or pyruvate and lactate (Pyr-Lac group). The embryos in all groups were cultured subsequently until Day 6 in NCSU-37 with 5.5 mM added glucose. The ROS and GSH level were measured at Day 1 and 2. DNA-fragmented nuclei and the total cell numbers in blastocyst were evaluated by TUNEL-staining at Day 6.Results: Under 5% oxygen the blastocyst rates and total cell numbers in the blastocysts in all glucose groups were significantly lower than that in the Pyr-Lac group. Similar result in blastocyst rate was found under 20% oxygen (excluding the Gluc-10 group), but total cell numbers in the blastocysts was similar among the groups. At both oxygen tensions, the H2O2 levels of Day 1 embryos in all glucose groups were significantly higher than that in the Pyr-Lac group, while only the Gluc-1.5 group of Day 2 embryos showed a significantly higher H2O2 level than that in the Pyr-Lac group. The GSH contents of either Day 1 or Day 2 embryos developed under 5% oxygen were similar among the groups. Only the content of Day 2 embryos in 1.5 mM group was significantly lower than the embryos in the Pyr-Lac group under 20% oxygen. Total cell numbers in the blastocysts (except in the Gluc-20 group) were significantly lower in the embryos cultured under 20% oxygen than 5% oxygen. Only the Gluc-20 blastocysts developed under 5% oxygen showed significantly higher DNA fragmentation rate than those of Pyr-Lac blastocysts.Conclusion: These results show that a decrease in developmental ability of embryos cultured by use of glucose instead of pyruvate and lactate after the ferilization may be due to the rise in ROS generation in Day 1 embryos. Moreover, results from this study suggest that the concentration of glucose in the medium that can be used by the Day 1 -2 embryos is limited to 3.5 mM and exposure to higher glucose concentrations does not improve embryo development.