Figure 5

Effect of 460 mOsm Hyperosmotic Treatment on Ccm2 mRNA and CCM2 Immunofluorescence. Quantitative analysis of Ccm2 in blastocyst stage mouse embryos following treatment in KSOMaa + 0.2 M sorbitol (approximately 460 mOsm) and KSOMaa only (approximately 260 mOsm) (A). Data is normalized to Luciferase and relative to mRNA transcript levels detected in the KSOMaa only control group at each individual time-point of treatment. Relative mRNA levels are presented as the mean ± s.e.m. representative of three independent replicates. No significant differences in relative mRNA transcript levels were observed (P ≤ 0.05). There was a noticeable increase in the level of CCM2 immunofluorescence detected in blastocysts cultured for 15 minutes in KSOMaa + 0.2 M sorbitol (D) when compared with blastocysts cultured in KSOMaa + 1.4% glycerol (C) or KSOMaa only (B). No primary control group is shown (E). Green indicates positive staining for CCM2 proteins (when phosphorylated at Thr334), red indicates F-actin (rhodamine-phalloidin) and blue indicates nuclei (DAPI). Scale bars represent 50 μm. Scion Image analysis resulted in the quantification of FITC immunofluorescence representing detectable CCM2 polypeptides (F). Relative signal strengths are presented as the mean ± s.e.m. Bars with different letters represent significant differences in relative signal strength between treatment groups (P ≤ 0.05).