丙酮酸脱氢酶在母源糖尿病对卵子质量影响过程中的作用

Women with poorly controlled diabetes often suffer from reproductive problems, such as miscarriage and offspring with congenital malformations. Emerging evidence suggest that these effects are associated with compromised oocyte quality. Our previous work has shown that oocytes from diabetic mice displays the defects in mitochondrial metabolism and meiosis, including reduced levels of ATP and TCA cycle metabolites, increased reactive oxygen species and disorganized spindle and chromosomes. Of note, the similar phenotypes were also observed in Pdha1 (Pyruvate dehydrogenase E1 α gene) deficient oocytes from conditional knockout mice. Meanwhile, our critical preliminary data demonstrate the increased expression of phosphorylated Pdha1-ser293, indicating the down-regulated activity of pyruvate dehydrogenase (PDH). Moreover, Vitamin B1 (thiamine, an irreplaceable cofactor of PDH) supplementation in vivo significantly decreased the apoptosis of blastocyst from diabetic mice. Taken together, we hypothesize that PDH in mitochondria is an important target mediating the effects of maternal diabetes on oocyte quality. To test this hypothesis, here, we will use Akita genetic diabetic mice and oocyte Pdha1-specific knockout mice as models, to perform genetic, cellular and biochemical analysis. Our research findings could provide new theoretical basis and molecular target for oocyte quality improvement and birth defects prevention.

糖尿病女性常常会经历早产流产和出生缺陷等生殖问题。越来越多的证据表明这些异常与卵母细胞质量受损有关。我们的前期工作显示，糖尿病小鼠卵母细胞表现出线粒体代谢和减数分裂方面的缺陷，包括：ATP和三羧酸循环代谢物水平降低、活性氧含量增加、纺锤体组装和染色体排列异常等。值得注意的是，特异性敲除Pdha1基因（丙酮酸脱氢酶E1α亚基）的卵母细胞也表现出了与此极其相似的表型。同时，我们的关键基础数据证实，糖尿病小鼠卵母细胞内Pdha1磷酸化水平显著升高，暗示丙酮酸脱氢酶（PDH）活性下调。而且，体内补充维生素B1（一种不可替代的PDH辅助因子）明显降低了糖尿病小鼠囊胚凋亡率。这些结果提示，线粒体内的PDH可能是母源糖尿病对卵子质量产生影响的关键作用靶点。本研究将以Akita糖尿病小鼠和Pdha1条件性敲除小鼠为模型，应用遗传学和生物化学等手段，对上述设想予以验证，为预防出生缺陷提供理论基础和分子靶标。

线粒体内丙酮酸的氧化代谢在卵母细胞的发育过程中发挥着至关重要的作用。我们推测母源健康可能诱导了卵母细胞内PDH活性下调，而这是致使其线粒体氧化代谢能力紊乱及减数分裂异常的一个重要因素。在本项目的支持下，我们对卵母细胞中PDH活性调节以及其在母源糖尿病对卵子质量影响过程中的作用进行了研究。首先评估了卵母细胞中PDH的三个关键丝氨酸位点（Ser232、Ser293及Ser300）的磷酸化水平，发现它们在卵母细胞中呈现出阶段特异性的表达方式。为了进一步研究它们的磷酸化状态在卵母细胞中如何调控，我们应用Morpholino注射的方法，依次敲减PDH的四种上游激酶（PDKI、PDK2、PDK3和PDK4）。结果证明PDK1/2是通过调控PDH-Ser232的磷酸化水平来影响卵母细胞减数分裂活动，而PDK3则主要是通过调控PDH-Ser293的磷酸化水平来影响卵母细胞的代谢活动。在此基础上，我们评估了糖尿病小鼠卵母细胞中PDH三个位点的磷酸化水平以及四种PDKs的表达变化。发现，PDK1的蛋白水平在糖尿病小鼠卵母细胞中显著下降，与之相应，PDH-Ser232的磷酸化水平也明显降低。最后利用分子生物学技术，通过构建了一系列磷酸化突变体，经过显微操作和体外成熟系统，阐明了PDK1蛋白表达水平减少诱导的PDH活性改变可能是造成糖尿病小鼠卵母细胞质量受损的一个重要原因。我们的发现不仅揭示了PDK-PDH调控途径在正常卵母细胞成熟过程中的全新作用，而且阐明了糖尿病小鼠卵母细胞质量受损的潜在机制，对于预防生殖问题和出生缺陷具有重要意义。