Involvement of ROS in FBS-associated phenotypes during early IVC phase of PA embryos.

<p>(A) Fluorescence microscopy of 4- to 8-cell embryos treated with CM-H₂DCFDA after treatment with FBS for 48 h (left panel) and quantification of ROS levels (right panel) in the indicated groups. Bar = 100 μm. (B) Representative photographs of the blastocysts (left panel; white asterisks) and quantification of the blastocyst developmental rate (center panel) and total cell number (right panel) within the indicated groups. Bar = 100 μm. (C) Fluorescence microscopy of 1-cell embryos treated with CM-H₂DCFDA after 6 h of cultivation in the presence or absence of either FBS and/or 0.5 mM hydrogen peroxide (left panel) and quantification of ROS levels (right panel) within the indicated groups. Bar = 100 μm. (D) Representative photographs of the developed blastocysts (left panel; white asterisks) and quantification of the blastocyst developmental rate within the indicated groups. Bar = 100 μm. (E–G) Immunocytochemical analysis of Cdx2/DAPI using blastocysts developed under the indicated IVC conditions for 0–2 days of IVC (E) and quantification of the total cell number (F) and ICM/TE proportions (G) in the indicated groups. Merged images (light green) between Cdx2 (green) and DAPI (blue) signals are shown (E). Bar = 100 μm (E). The data are from three independent experiments, and values represent the means ± SE (^*<i>P</i> < 0.05; F and G). (H and I) Apoptosis detection analysis using blastocysts developed under the indicated IVC conditions for 0–2 days (H), and quantification of the number and proportion of apoptotic cells in the indicated groups (I). Merged images (light green) between TUNEL (green, white arrow) and DAPI (blue) signals are shown (H). Bar = 50 μm (H). The data are from three independent experiments, and values represent the means ± SE (^*<i>P</i> < 0.05; I).