Figure 1.
Four wells were used as temperature control wells (wells A1, B3, C4 and D6) and as such contained no embryos. Embryos were plated in all other wells in numbers that allowed for respiration and media acidification measurements to be made within the recommended range. Inset: Expanded view of a 30 hpf zebrafish embryo in an islet plate well prior to assay.
Figure 2.
Total basal respiration and acidification of the media by zebrafish embryos.
Total basal respiration (OCR, in pmol O2/min/embryo) and acidification of the media (PPR, in nmol H+/min/embryo) increased in a linear and reproducible fashion during zebrafish embryonic development (blastula (3 hpf), gastrula (7 hpf), segmentation (12 hpf), segmentation/pharyngula transition (24 hpf), mid-pharyngula (30 hpf), and hatching (48 hpf) periods). (A) Total basal respiration of developing zebrafish embryos (mean +/− SEM; n = 8). (B) Acid extrusion of developing zebrafish embryos (mean +/− SEM; n = 8).
Table 1.
Zebrafish embryos were staged at 28.5°C.
Table 2.
Pharmacological inhibitors used for deconvolution of total respiration, the mechanism of action, and the final concentrations used.
Figure 3.
Partitioning of total basal respiration changes with zebrafish embryonic development.
(A) The components of total respiration, per embryo. Non-mitochondrial respiration (dark grey), mitochondrial respiration (black), respiration due to ATP turnover (white) and respiration due to proton leak (light grey). Mean +/− SEM; n = 8. (B) Total basal respiration (100%) is composed of a non-mitochondrial fraction (white) and a mitochondrial fraction. The mitochondrial fraction is further composed of the respiration associated with proton leak (black) and ATP turnover (grey). The proportion of total respiration due to non-mitochondrial respiration was greatest at 3 hpf (p<0.0001) (a) and 7 hpf (p<0.02) (b) as determined by one-way ANOVA and Student-Newman-Keuls post hoc test. Non-mitochondrial respiration as a proportion of total respiration did not change between 12 and 48 hpf (p>0.05). At 3 hpf, the proportion of total respiration due to mitochondrial ATP turnover was lowest when compared with 7, 30 and 48 hpf (p<0.05) (c). Mitochondrial ATP turnover did not change much at other time-points. Proton leak as a proportion of total respiration at 3 hpf and 7 hpf was not significantly different to zero as determined by Student's t-test (p>0.05). Proton leak was significantly higher at 12 hpf (d) and 24 hpf (e) than at 3 and 7 hpf, as determined by one-way ANOVA and Student-Newman-Keul's post hoc test (p>0.05). Furthermore, proton leak was significantly higher at 24 hpf as compared to 48 hpf (p<0.05) (e).
Figure 4.
Normalization of respiration measurements by COX IV protein content (an index of mitochondrial content).
(A) Western blots of COX IV and beta actin (loading control) proteins in zebrafish embryos aged 3 to 48 hpf. (B) COX IV protein levels, an index of mitochondrial content, in zebrafish embryos aged 3 to 48 hpf (mean +/− SEM; n = 3). COX IV densitometry values were normalized to beta actin values, with the value at 3 hpf set to 1. (C) Mitochondrial respiration (white) and maximal FCCP-uncoupled rates (black) normalized to mitochondrial COX IV protein levels (mean +/− SEM; n = 7 or 8). Mitochondrial respiration was lowest at 3 hpf, as determined by one-way ANOVA with Student-Newman-Keuls post hoc test (p<0.0001) (a). Mitochondrial respiration was maximal at 24 hpf (p<0.05) (b). However, mitochondrial respiration was similar for 7, 12, 30 and 48 hpf (p>0.05). Maximal FCCP-uncoupled rates were similar for 3, 30 and 48 hpf (p>0.05). However maximal FCCP-uncoupled rates were greatest at 7 hpf (p<0.0001) (c), followed by 12 hpf (p<0.05) (d) and 24 hpf (p<0.05) (e).
Figure 5.
O2 level data for a 48 hpf embryo, as measured in the XF-24.
As the sensor cartridge is lowered into the plate, a transient microchamber is formed, thus the O2 levels in the microchamber decrease due to oxygen use by the embryo. Arrows indicate injection of FCCP into the well. Inset: Expanded view of the O2 level data before and after FCCP injection.
Figure 6.
Titration curves for each agent were performed at each time-point post-fertilization.
Shown here are the FCCP titration curves for 7 hpf (diamonds) and 24 hpf (squares), mean +/− SEM, n = 2-3. Arrows indicate the concentration that produced the maximum change in respiration without inducing death within the experimental timeframe.