Non-invasive imaging of mouse embryo metabolism in response to induced hypoxia

Abstract

Purpose: This study used noninvasive, fluorescence lifetime imaging microscopy (FLIM)-based imaging of NADH and FAD to characterize the metabolic response of mouse embryos to short-term oxygen deprivation. We investigated the response to hypoxia at various preimplantation stages.

Methods: Mouse oocytes and embryos were exposed to transient hypoxia by dropping the oxygen concentration in media from 5-0% over the course of ~1.5 h, then 5% O₂ was restored. During this time, FLIM-based metabolic imaging measurements of oocyte/embryo cohorts were taken every 3 minutes. Experiments were performed in triplicate for oocytes and embryos at the 1- to 8-cell, morula, and blastocyst stages. Maximum hypoxia response for each of eight measured quantitative FLIM parameters was taken from the time points immediately before oxygen restoration.

Results: Metabolic profiles showed significant changes in response to hypoxia for all stages of embryo development. The response of the eight measured FLIM parameters to hypoxia was highly stage-dependent. Of the eight FLIM parameters measured, NADH and FAD intensity showed the most dramatic metabolic responses in early developmental stages. At later stages, however, other parameters, such as NADH fraction engaged and FAD lifetimes, showed greater changes. Metabolic parameter values generally returned to baseline with the restoration of 5% oxygen.

Conclusions: Quantitative FLIM-based metabolic imaging was highly sensitive to metabolic changes induced by hypoxia. Metabolic response profiles to oxygen deprivation were distinct at different stages, reflecting differences in metabolic plasticity as preimplantation embryos develop.

Keywords: Embryo metabolism; FLIM; Hypoxia; Metabolic imaging; Mitochondria; Noninvasive assessment.

Fig. 1

(a) A schematic of the on-stage, enclosed incubation system, which allows for exchange of pre-mixed gases with 0 or 5% O2. Oocytes and embryos were imaged continuously in a microwell dish with an oil overlay of fixed thickness to produce consistent O2 exchange rates with the media. (b) An NADH fluorescence intensity image of embryos at the 1-cell stage generated from a FLIM measurement. 50 μm scale bar. (c) Representative images of the six stages of development, for which oxygen deprivation experiments were performed. 50 μm scale bar

Fig. 2

(a) FLIM NADH imaging of a 1-cell embryo under experimental hypoxic conditions. Estimated oxygen concentration percentage in media shown below the corresponding embryo images. NADH intensity (pseudocolored) increases during hypoxia, and recovers when 5% oxygen is restored. 50 μm scale bar. (b) 1-cell control embryo with no oxygen deprivation undergoes no significant change in brightness. 50 μm scale bar. (c) Corresponding quantitative metabolic parameters for individual embryos displayed in A (left plots) and B (right plots). Four (4) metabolic parameters displayed: NADH intensity and fraction engaged (blue) and FAD intensity and fraction engaged (orange). The oxygen deprivation samples show significant changes in all four (4) parameters in response to hypoxia. Here, the vertical dashed red line indicates the time when the oxygen drop was initiated, and the later green line indicates the oxygen restoration time. Control samples (at 5% oxygen throughout) show no significant metabolic change. Consistent data ranges displayed for comparison with standard error bars

Fig. 3

(a) Changes in absolute values of metabolic parameters in response to oxygen deprivation for embryos at the 1-cell stage. Time courses for individual embryos for all eight parameters shown using light traces. Each trace represents an individual embryo. Average metabolic parameter values are shown in black with standard error bars displayed. Time is represented in scaled units where oxygen drop begins at t = 0 and is restored at t = 1. (b) Average changes in metabolic response to oxygen deprivation of all embryos, arranged by stage of development. Standard error bars are displayed. Time is represented in scaled units where oxygen drop begins at t = 0 and is restored at t = 1

Fig. 4

Bar plots show the maximum response values of all eight FLIM parameters for the following developmental preimplantation stages: unfertilized oocytes, 1 cell embryos, 2 cell embryos, 3-8 cell embryos, compaction or morula stage embryos and blastocysts. Bar plots projecting upwards from zero showed positive relative change and those projecting downwards showed negative change. Standard error bars are displayed, with asterisks indicating the following p-values: (*:p < 0.05) and (**:p < 10-8)