Measurement of mitochondrial distribution and membrane potential in oocytes
Usama Alzubaidi, Deepak Adhikari, Jun Liu, Rebecca Robker, John Carroll
Department of Anatomy and Developmental Biology and Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Monash University; Australia.
Mitochondrial ATP production plays a critical role during oocyte maturation and early embryo development. ATP production is generated through oxidative phosphorylation, a process that is critically dependent on the mitochondrial membrane potential (∆Ψm). In oocytes and embryos, it has been reported that ∆Ψm varies according to stage of oocyte maturation and mitochondrial location within the cell. However, these measurements are complicated by the properties of fluorescent indicators, JC1 and tetramethylrhodamine, methyl ester, (TMRM), and the changing distribution and concentration of mitochondria in different sub-cellular compartments during meiosis and mitosis. Here we have set out to characterize and establish new reliable methods to measure ∆Ψm that allows accurate comparisons across different cells and regions of an individual cell.
We confirm that the commonly used ratiometric ∆Ψm indicator, JC-1, typically demonstrates a cortical elevation of ∆Ψm that is not evident with the single wave-length potentiometric indicator, TMRM. We show that J-aggregates are subject to concentration and time-dependent distribution, thereby explaining apparent sub-cellular differences in ∆Ψm. By ratioing fluorescence images or values from TMRM with those from a mitochondria-specific, ∆Ψm-insensitive fluorescent probe (such as mito-GFP or MitoTracker-Green), we show that ∆Ψm is relatively homogeneous across the oocyte and early embryo. Furthermore, the ratiometric approach allows comparison of ∆Ψm across different cells independent of time and concentration of mitochondria.