Reproduction and Early Development Research Group, Leeds Institute of Cardiovascular and Metabolic Medicine, School of Medicine, University of Leeds, Clarendon Way, Leeds, LS2 9JT, UK.
Centre for Anatomical and Human Sciences, Hull York Medical School, University of Hull, Hull, HU6 7RX, UK.
Sci Rep. 2021 Oct 28;11(1):21245. doi: 10.1038/s41598-021-99512-5.
We report a novel method to profile intrcellular oxygen concentration (icO) during in vitro mammalian oocyte and preimplantation embryo development using a commercially available multimodal phosphorescent nanosensor (MM2). Abattoir-derived bovine oocytes and embryos were incubated with MM2 in vitro. A series of inhibitors were applied during live-cell multiphoton imaging to record changes in icO associated with mitochondrial processes. The uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) uncouples mitochondrial oxygen consumption to its maximum, while antimycin inhibits complex III to ablate mitochondrial oxygen consumption. Increasing oxygen consumption was expected to reduce icO and decreasing oxygen consumption to increase icO. Use of these inhibitors quantifies how much oxygen is consumed at basal in comparison to the upper and lower limits of mitochondrial function. icO measurements were compared to mitochondrial DNA copy number analysed by qPCR. Antimycin treatment increased icO for all stages tested, suggesting significant mitochondrial oxygen consumption at basal. icO of oocytes and preimplantation embryos were unaffected by FCCP treatment. Inner cell mass icO was lower than trophectoderm, perhaps reflecting limitations of diffusion. Mitochondrial DNA copy numbers were similar between stages in the range 0.9-4 × 10 copies and did not correlate with icO. These results validate the MM2 probe as a sensitive, non-toxic probe of intracellular oxygen concentration in mammalian oocytes and preimplantation embryos.
我们报告了一种使用市售多模态磷光纳米传感器（MM2）在体外哺乳动物卵母细胞和胚胎发育过程中分析细胞内氧浓度（icO）的新方法。从屠宰场获得的牛卵母细胞和胚胎在体外与 MM2 一起孵育。在活细胞多光子成像过程中应用了一系列抑制剂，以记录与线粒体过程相关的 icO 变化。解偶联剂羰基氰化物-p-三氟甲氧基苯腙（FCCP）将线粒体氧消耗解偶联至最大值，而抗霉素抑制复合物 III 以消除线粒体氧消耗。预计增加氧消耗会降低 icO，减少氧消耗会增加 icO。使用这些抑制剂可以量化与线粒体功能的上限和下限相比，基础状态下消耗了多少氧气。icO 测量结果与通过 qPCR 分析的线粒体 DNA 拷贝数进行了比较。抗霉素处理增加了所有测试阶段的 icO，表明基础状态下线粒体氧消耗显著。FCCP 处理对卵母细胞和胚胎前体的 icO 没有影响。内细胞团的 icO 低于滋养外胚层，这可能反映了扩散的限制。线粒体 DNA 拷贝数在 0.9-4×10 拷贝之间的各个阶段相似，与 icO 不相关。这些结果验证了 MM2 探针作为哺乳动物卵母细胞和胚胎前体中细胞内氧浓度的敏感、无毒探针的有效性。
