Figure 3

Photomicrographs of frozen/thawed and vitrified/warmed mouse embryos at the morula and blastocyst stage of development as assessed for their nuclear chromatin and bioenergy/oxidative potential. MitoTracker Orange, and DCDH FDA were used to label mitochondria and ROS, respectively. Nuclear chromatin was stained with Hoechst 33258. Representative photomicrographs showing mt distribution pattern and ROS intracellular localization in a control morula (row A) and a control blastocyst (row B) with P/P mt pattern, a SF morula with SA pattern (row C), a SF blastocyst with P/P pattern (row D), a VF morula with SA pattern (row E) and a VF blastocyst with P/P pattern (row F). In embryos at the blastocyst stage, 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 cryopreservation. For each embryo, the corresponding epifluorescence images showing nuclear chromatin (line 1) and confocal images showing mt distribution pattern (line 2), ROS localization (line 3), mt/ROS merge (line 4) are shown. Scale bar represents 20 μm.