Development of Fluorescent Assays for Mitochondrial Function during Drosophila Oogenesis

Abstract

The successful development of Drosophila melanogaster egg chambers during oogenesis partly depends upon mitochondrial health and ATP production. Due to functional differences between the oocyte and nurse cells, assays are necessary to observe and quantify these differences in their respective mitochondrial populations, especially with regards to mitochondrial membrane potential, reactive oxygen species (ROS) levels, and pH. Fluorescent dyes were utilized in conjunction with live imaging techniques in order to determine and employ the most effective methods for assessing mitochondrial function. Staining with TMRM (a membrane potential probe) showed that relatively healthy, polarized mitochondria have comparable membrane potentials in the oocyte and nurse cells, reflected by a ratio of oocyte to nurse cell TMRM fluorescent intensity of 0.9 ±0.1. This correlation does not change significantly as oogenesis progresses and indicates that mitophagy may serve as a mechanism for controlling membrane potential. Nurse cells stained with H2DCFDA (a probe for ROS levels) and TMRM suggested that increases in ATP production and mitochondrial activity may be reflected by positively correlated increases in both membrane potential and ROS production. However, mitochondria in poor health exhibit a negative correlation of significant depolarization with excessively high levels of ROS, indicating inactivation of mitophagy and execution of cell death. In healthy developing egg chambers, ROS levels in nurse cell mitochondria become increasingly higher than those in oocyte mitochondria as oogenesis progresses. This result shows that nurse cell mitochondria have greater respiratory status and ROS production, and some mechanism may be in place to prevent the transfer of ROS from nurse cells to the oocyte. Carboxy SNARF-1 (a pH probe) localized non-specifically in cells, and the addition of MitoTracker Green as a mitochondrial marker resulted in significant spectral bleed-through. In addition to technical difficulties with image processing and analysis, the ratiometric images did not show any considerable variations in accordance with pH. These factors thus render this assay ineffective. On the other hand, CFDA – a different non-ratiometric pH probe – localized specifically in mitochondria, but images of eggs co-stained with CFDA and TMRM were evaluated on a case-by-case basis due to complications from TMRM quenching and expulsion of CFDA from hyperpolarized mitochondria. From these functional assays, conclusions cannot yet be drawn connecting membrane potential and ROS levels to pH and respiratory activity over the course of oogenesis, but the groundwork for doing so has been established and should be further explored.