A characteristic of post-compaction embryo development is the formation of gap junctions that allow for the intercellular communication through the transport of ions, metabolites and signalling agents. However, it is not until after the 8-cell stage of development that mouse embryos develops these connections. Pre-compaction blastomere metabolic variability has been proposed as the basis of embryonic heterogeneity, and is hypothesised to be exacerbated by the absence of gap junctions. To examine blastomere heterogeneity as a consequence of cellular stress, we utilised a model of hyperglycaemic stress and analysed pathways involved in changes in metabolism and DNA damage and repair.
CBA F1 mice were stimulated with 5 IU eCG/5IU hCG, and cumulus oocyte complexes were collected 16 h post hCG and fertilised in vitro. One-cell embryos were cultured in either control (5.6mM glucose) or hyperglycaemic (30mM glucose) media for 20 hours until cleaved, before being metabolically assessed using the mitochondrial activity probe, Mitotracker Deep Red (MTDR, 200nM), a specific H2O2 fluorophore , peroxyfluor-1 (PF-1, 20μM) and a reduced GSH probe, monochlorobimane (MCB, 12.5mM). A grey-scale co-occurrence matrix (GLCM) was used for the first time on phase contrast images, to quantify its potential as an additional non-invasive method to assess embryo viability. The images were analysed for intensity and different textural features such as the degree of heterogeneity, homogeneity, smoothness and entropy using GLCM.
We found that embryos exposed to hyperglycaemic stress demonstrate a higher degree of mitochondrial activity, but not of the other metabolic measures. Phase contrast images of pre-implantation embryos revealed that hyperglycaemic embryos were more heterogeneous and were less ‘smooth’ than control embryos, supporting the hypothesis.