Stimulation of mitochondrial embryo metabolism by dichloroacetic acid in an aged mouse model improves embryo development and viability  Nicole O. McPherson,

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Stimulation of mitochondrial embryo metabolism by dichloroacetic acid in an aged mouse model improves embryo development and viability  Nicole O. McPherson, B.Hlth.Sc.(Hons.), Deirdre Zander-Fox, Ph.D., Michelle Lane, Ph.D.  Fertility and Sterility  Volume 101, Issue 5, Pages 1458-1466.e5 (May 2014) DOI: 10.1016/j.fertnstert.2013.12.057 Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Schematic of the template used to analyze mitochondrial parameters Confocal images from fluorescently stained two cells, showing four regions (1–4) within areas 1–3. Mitochondrial membrane potential was determined by analysis of the ratio of red to green JC-1 fluorescence. Fertility and Sterility 2014 101, 1458-1466.e5DOI: (10.1016/j.fertnstert.2013.12.057) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 2 The effect of dichloroacetic acid on embryo mitochondrial membrane potential. (A) Control 2-cell embryos stained with the fluormetric dye JC-1. (B) Dichloroacetic acid-treated 2-cell embryos stained with fluorometric dye JC-1. Green fluroescence represent the amount of JC-1 dye that has penetrated into the mitochondria. Red fluorescence represents the amount of JC-1 dye that has formed J-aggregate complexes through increased mitochondrial metabolism, and merged is a combination of both. Pictures are representative of treatment groups. Fertility and Sterility 2014 101, 1458-1466.e5DOI: (10.1016/j.fertnstert.2013.12.057) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 3 The effect of dichloroacetic acid (DCA) on embryo development and cell number. (A) Images of day 5 blastocyst development from control G1/G2 media or G1/G2 media supplemented with 1.0 mM DCA. Pictures of blastocysts are representative of each treatment group. (B) Day 6 blastocysts from each treatment group stained for cell number with Oct4 (inner cell mass [ICM]), Nanog (pluripotent cells) and 6-diamino-2-phenylindole (DAPI) (total cell number). Arrows indicate pluripotent cells located to ICM. Pictures of blastocysts are a representative of each treatment group. Fertility and Sterility 2014 101, 1458-1466.e5DOI: (10.1016/j.fertnstert.2013.12.057) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions