Fig. 4

The basal level of cytosolic calcium activity and the fluorescence analysis of autophagy activity in TLR2 agonist treated bovine Day 7 blastocysts. (a) Representative images of Fluo-4 AM fluorescence (green) in Day 7 embryos treated with control (0.05% ethanol) or 100 ng/ml TLR2 agonist. Scale bar = 500 μm. (b) (Left) Quantification of the relative levels of basal cytosolic Ca²⁺ of embryos in control and TLR2 agonist embryos. (Right) Quantification of the relative levels of basal cytosolic Ca²⁺ in control and TLR2 agonist at different stages (BL, ExB, and HB) of embryonic development. The asterisk denotes a significant variance between the ‘control (n = 93)’ and ‘TLR2 agonist (n = 110)’ groups. (c) Representative images of autophagy dye (green) in Day 7 embryos treated with control (0.05% ethanol) or 100 ng/ml TLR2 agonist. Scale bar = 500 μm. (d) (Left) Quantification of the relative levels of autophagy levels in control and TLR2 agonist. (Right) Quantification of the relative levels of autophagy activity in control and TLR2 agonist at different stages (BL, ExB, and HB) of embryonic development. Examples of a quantitative assessment of autophagy (green) using ImageJ. Experiments were repeated 5 times. The data are expressed as mean ± S.E.M. and the student’s t-test was utilized for statistical analyses. The asterisk denotes a significant variance between the ‘control (n = 96)’ and ‘TLR2 agonist (n = 111)’ groups. *P < 0.05; **P < 0.01; ****P < 0.0001 vs. control group.