Abstract
We report a novel method to profile intrcellular oxygen concentration (icO(2)) 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(2) 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(2) and decreasing oxygen consumption to increase icO(2). Use of these inhibitors quantifies how much oxygen is consumed at basal in comparison to the upper and lower limits of mitochondrial function. icO(2) measurements were compared to mitochondrial DNA copy number analysed by qPCR. Antimycin treatment increased icO(2) for all stages tested, suggesting significant mitochondrial oxygen consumption at basal. icO(2) of oocytes and preimplantation embryos were unaffected by FCCP treatment. Inner cell mass icO(2) was lower than trophectoderm, perhaps reflecting limitations of diffusion. Mitochondrial DNA copy numbers were similar between stages in the range 0.9-4 × 10(6) copies and did not correlate with icO(2). These results validate the MM2 probe as a sensitive, non-toxic probe of intracellular oxygen concentration in mammalian oocytes and preimplantation embryos.