The spatio-temporal dynamics of mitochondrial membrane potential during oocyte maturation

Abstract

Mitochondria are highly dynamic organelles and their distribution, structure and activity affect a wide range of cellular functions. Mitochondrial membrane potential (∆Ψm) is an indicator of mitochondrial activity and plays a major role in ATP production, redox balance, signaling and metabolism. Despite the absolute reliance of oocyte and early embryo development on mitochondrial function, there is little known about the spatial and temporal aspects of ΔΨm during oocyte maturation. The one exception is that previous findings using a ΔΨm indicator, JC-1, report that mitochondria in the cortex show a preferentially increased ΔΨm, relative to the rest of the cytoplasm. Using live-cell imaging and a new ratiometric approach for measuring ΔΨm in mouse oocytes, we find that ΔΨm increases through the time course of oocyte maturation and that mitochondria in the vicinity of the first meiotic spindle show an increase in ΔΨm, compared to other regions of the cytoplasm. We find no evidence for an elevated ΔΨm in the oocyte cortex. These findings suggest that mitochondrial activity is adaptive and responsive to the events of oocyte maturation at both a global and local level. In conclusion, we have provided a new approach to reliably measure ΔΨm that has shed new light onto the spatio-temporal regulation of mitochondrial function in oocytes and early embryos.

Keywords: JC-1; TMRM; membrane potential; mitochondria; oocyte maturation.

Figure 1

Distribution of JC-1 and TMRM. (A) J-aggregate (i) and monomer (ii) distributions after loading with JC-1 (1 μg/ml). (B) TMRM (25 nM) fluorescence distribution in GV oocyte. (C) Ratio of the fluorescence intensities in cortex and cytoplasm. (D) J-aggregate and (E) TMRM-labeled 2-cell embryos. (F) Ratios of cortex/cytoplasm fluorescence intensities of J-aggregates and TMRM. (G) J-aggregate and (H) TMRM labeled 4-cell embryos. (I) Ratios of cortex/cytoplasm fluorescence intensities of J-aggregates and TMRM. Results are presented from three replicate experiments for each stage. ^*P ≤ 0.05, compared to TMRM; ^****P ≤ 0.0001, compared to TMRM. Error bars show SE of the mean. n = Total number of oocyte/embryos analyzed.

Figure 2

Effect of JC-1 concentration and time on spatial distribution of J-aggregates in oocytes. (A) Quantification of spatial distribution of J-aggregates after incubation with various JC-1 concentrations for the same period of time (30 minutes). Oocytes were also cultured in TMRM (25 nM) for 30 minutes. ANOVA was used to compare between groups. A statistical comparison was shown as letters, a versus b represents P ≤ 0.01; c versus a or b represents P ≤ 0.0001. (B) Representative images of oocytes used for quantification in A. (C) Quantification of outside/inside ratios of J-aggregates after incubation for different periods of time in the same concentration of JC-1 (1 μg/ml). For comparison, oocytes were also cultured in TMRM (25 nM) for 30 minutes. A statistical comparison was shown as letters after using ANOVA, all differences between groups were P ≤ 0.0001 except that comparison between 90 and 120 minutes (c versus d) was P ≤ 0.001. (D) Representative images of oocytes used for quantification in C. Results were combined from 20 oocytes from three replicate experiments. The ratio on the Y-axis was obtained by dividing the fluorescence intensity of the region of interest (ROI) in cortex by that in the cytoplasm. Error bars show SE of the mean.

Figure 3

Combination of probes for ratiometric measurement of ∆Ψm in oocytes. (A) After loading with TMRM and MTG for 30 minutes, loss in fluorescence intensities were monitored. (B) Representative images for MTG and TMRM fluorescence. (C) TMRM/MTG fluorescence ratio plotted after carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) addition. (D) Comparison of TMRM/MTG fluorescence over time after addition of different concentrations of FCCP. The experiments were repeated three times each and the values before FCCP addition were normalized to 1. Error bars show SE of the mean.

Figure 4

Increase in ∆Ψm during oocyte maturation. GV oocytes were incubated with TMRM and MTG for 30 minutes and then matured in vitro in medium containing low TMRM concentration (5 nM). (A) Increase in TMRM fluorescence between 1.5 and 7 hours but with constant levels of MTG fluorescence. (B) Increase in TMRM/MTG ratio during oocyte maturation. (C) Oocyte meiotic maturation was blocked by adding 200 μM 3-isobutyl-1-methylxanthine (IBMX) to culture medium. This prevented the increase in TMRM fluorescence and the increase in TMRM/MTG fluorescence ratio (D). Each experiment was repeated at least three times. Error bars show SE of the mean.

Figure 5

Spatial distribution of ∆Ψm during oocyte maturation. Mito-Dendra expressing oocytes from PhAM^loxP/loxP; Gdf9Cre mice at GV, metaphase I and II (MI and MII) were loaded with TMRM. High-resolution confocal images were obtained at both wavelengths to calculate TMRM/Dendra ratios across various regions of the oocytes. (A–D) Mitochondria around the GV have higher membrane potential than the mitochondria not surrounding the GV. n = 28. ^****, P < 0.01. (E–H) GV oocytes were cultured in vitro for 8 hours to obtain MI oocytes. MI oocytes show higher ∆Ψm in mitochondria associated with spindle than in those away from the spindle. n = 32. ^****, P < 0.0001. (I–L) Ovulated MII oocytes were collected 14 hours after hCG injection. Mitochondria around the MII spindle (spindle pole) have higher ∆Ψm than those at opposite pole. n = 16. ^**, P < 0.01. Data shown as mean ± SEM.