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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.

Intracellular oxygen metabolism during bovine oocyte and preimplantation embryo development