Effects of metformin on fertilisation of bovine oocytes and early embryo development: possible involvement of AMPK3-mediated TSC2 activation
Effects of metformin on fertilisation of bovine oocytes and early embryo development: possible involvement of AMPK3-mediated TSC2 activation
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DOI：
10.1017/s0967199413000300
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发表时间：
2013-07
期刊：
影响因子：
1.7
通讯作者：
Olympia Pikiou;A. Vasilaki;George Leondaritis;N. Vamvakopoulos;I. Messinis
中科院分区：
文献类型：
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作者：
Olympia Pikiou;A. Vasilaki;George Leondaritis;N. Vamvakopoulos;I. Messinis
对牛卵母细胞的研究表明，毫摩尔浓度的二甲双胍激活腺苷一磷酸活化蛋白激酶（AMPK）控制核成熟。脑硬化复合物2（TSC 2）已被确定为AMPK的下游靶点。本研究的目的是研究添加低浓度二甲双胍（1 nM至10 μM）对培养的卵丘-卵母细胞复合体（COC）百分比的影响，该复合体导致卵裂期胚胎和AMPK介导的TSC 2激活。在整个体外胚胎生产（IVP）过程中或仅在体外受精（IVF）过程中补充蛋氨酸。COC在体外成熟，授精，并推定受精卵培养72小时后，受精卵和早期胚胎发育产生的COC的百分比进行了评估。通过免疫细胞化学评估牛胚胎中TSC 2的存在及其可能的AMPK诱导的激活。二甲双胍对培养的COC产生胚胎的数量具有剂量依赖性影响。在IVP期间或仅在IVF期间给药均使≥8-细胞胚胎的百分比降低（1 μM，P < 0.05; 10 μM，P < 0.01; 0.1 μM，10 μM，P < 0.01），而2-细胞胚胎的百分比增加（10 μM，P < 0.01和P < 0.05）。产生合子的培养COC的百分比不受二甲双胍的影响。TSC 2在早期胚胎中表达。无论是在整个IVP期间还是仅在IVF期间，二甲双胍（10 μM）均增加AMPK诱导的PhosphoS 1387-TSC 2免疫反应性（P < 0.01），这种增加对应于细胞中表达的总TSC 2蛋白水平。我们的研究结果表明，二甲双胍对受精后卵母细胞裂解能力存在剂量依赖性的负面影响，可能通过AMPK诱导的TSC 2激活介导。
Summary Studies on bovine oocytes have revealed that the activation of adenosine monophosphate activated protein kinase (AMPK) by millimolar concentrations of metformin controls nuclear maturation. Tuberous sclerosis complex 2 (TSC2) has been identified as a downstream target of AMPK. The objective of this study was to investigate the effects of addition of low concentrations of metformin (1 nM to 10 μM) on the percentage of cultured cumulus–oocyte complexes (COC) giving rise to cleavage-stage embryos and AMPK-mediated TSC2 activation. Metformin was supplemented either throughout in vitro embryo production (IVP) or only during in vitro fertilization (IVF). COC were matured in vitro, inseminated, and presumptive zygotes cultured for a further 72 h post insemination before the percentage of COC that gave rise to zygotes and early embryo development was assessed. The presence of TSC2 in bovine embryos and its possible AMPK-induced activation were assessed by immunocytochemistry. Metformin had a dose-dependent effect on the numbers of cultured COC that gave rise to embryos. Drug treatment either throughout IVP or only during IVF decreased the percentage of ≥8-cell embryos (1 μM, P < 0.05; 10 μM, P < 0.01; and 0.1 μM, 10 μM, P < 0.01, respectively) and increased the percentage of 2-cell embryos (10 μM, P < 0.01 and P < 0.05 respectively). The percentage of cultured COC that gave rise to zygotes was not affected by metformin. TSC2 is expressed in early embryos. Metformin (10 μM) either throughout IVP or during IVF only, increased AMPK-induced PhosphoS1387-TSC2 immunoreactivity (P < 0.01) and this increase corresponded to the total TSC2 protein levels expressed in cells. Our results suggest that there is a dose-dependent negative effect of metformin on the ability of oocytes to cleave following insemination, possibly mediated through an AMPK-induced activation of TSC2.