Morphological markers associated with oocyte metabolism such as the motile mitochondria population and the filamentous transzonal projections (TZPs), that connect the oocyte to its attendant cumulus cells, have been traditionally studied by 2D imaging methods that miss most of the detail within the 3-dimensional oocyte.
We have developed a 3D quantification procedure using fluorescent labelling of live cultured oocytes with both Tetramethylrhodamine (TMRM) and Mitotracker Green to map active and total mitochondrial populations respectively, or with the Sir-Actin stain to visualise the TZPs. High resolution 3D modelling/visualisation of Z-stack confocal images using Imaris software allowed each fluorescent point to be tracked and analysed in a 3D space and quantitative comparison of details of fluorescent signal position and intensity. This novel method for visualising oocyte metabolic morphology was tested with a simple oocyte stress model. GV and MII stage cumulus oocyte complexes were subjected to either control culture conditions or 50uM H2O2 for 30min followed by fluorescent staining. As expected H2O2 treatment reduced the volumetric ratio of active compared to inactive mitochondria of 0.638 and 0.338 between control and H2O2 treated oocytes respectively. We analysed the spatial arrangement of ‘active’ mitochondria in relation to the meiotic spindle and each other. On average 4000 individual TMRM fluorescent points per oocyte were clustered predominantly between 10-15um from the meiotic spindle and 1.29um from the nearest fluorescent spot. For TZPs, the mean control oocyte contained 1445±647 filaments, on average 3.57um long whereas H2O2 treated oocyte had 725±396 filaments, averaging 3.06um long, though these differences were non-significant. In conclusion the short H2O2 treatment had a limited effect however this proof of concept experiment demonstrates that 3D quantification techniques can allow a new level of investigation into subcellular features providing a tool for greater understanding of conditions that impact oocyte morphology and competence.