Figure 2

Photomicrographs of fresh and vitrified/warmed mouse embryos at early (4/16-cell), morula and blastocyst stages of development as assessed for their nuclear chromatin and bioenergy/oxidative potential. For each embryo, corresponding bright-field (phase contrast; lane 1), UV light (lane 2) and confocal images showing mt distribution pattern (lane 3), ROS localization (lane 4) and mt/ROS merge (lane 5), are shown. Representative photomicrographs of control non-vitrified and vitrified/warmed embryos at the 4- (row A) and 8-cell (row B), morula (rows C and D) and blastocyst stages (rows E and F), are shown. Nuclear chromatin was stained with Hoechst 33258.MitoTracker Orange CMTM Rosand DCDH FDA were used to label mitochondria and ROS, respectively. In control fresh early embryos and in morulae, in all blastomeres, there were detectable MitoTracker signals in the form of continuous rings around the nuclei and clusters of mitochondria at the cortex, namely perinuclear/pericorticalmt pattern (heterogeneous, healthy P/P mt pattern) (A3, C3). Vitrified early embryos(B3) showed a uniform/diffused (homogeneous) mt distribution pattern throughout the blastomere cytoplasm. In embryos at the morula and blastocyst stage, the mt pattern was apparently not affected by vitrification (D3 vs C3 and F3 vs E3). A higher number of red fluorescent spots is evident on the trophoectoderma (white arrows) compared with the inner cell mass, indicating differences in mt number/cell between these two embryo lineages and higher mt/number and aggregate formation in the trophoectoderma compared with ICM. This feature can be observed in embryos of both groups, thus it was not influenced by vitrification. ROS intracellular localization (lane 4) corresponded to the distribution pattern of mitochondria. In fresh control embryos, apart areas/sites of mt/ROS overlapping (merge, lane 5), intracellular ROS appeared diffused throughout the cytoplasm (A4, C4, E4). In vitrified (B4, D4 and F4) embryos, diffused MitoTracker and DCDH FDA labelling were evident throughout the cytoplasm. Scale bar represents 20 μm.