Figure 4

Immunofluorescence Detection and Quantitation of Phosphorylated MAPKAPK2 Following Culture in 1800 mOsm Medium for 10 Minutes and 30 Minutes. Phosphorylated MAPKAPK2 was detected in the cytoplasm and the nucleus of all embryos observed following 10 minutes and 30 minutes of treatment. There was a noticeable increase in the level of phosphorylated MAPKAPK2 protein detected in blastocysts cultured in KSOMaa + 1.4 M sucrose (C, G) when compared with blastocysts cultured in KSOMaa + 10% glycerol (B, F) or KSOMaa only (A, E) at both 10 minutes and 30 minutes of treatment. No primary controls are shown (D, H). All confocal micrographs are representative images from pools of blastocysts in each treatment group. Green indicates positive staining for MAPKAPK2 proteins (when phosphorylated at Thr334), red indicates F-actin (rhodamine-phalloidin) and blue indicates nuclei (DAPI). Scale bars represent 50 μm. Scion Image analysis applied to FITC immunofluorescence images representing detectable phosphorylated MAPKAPK2 at 10 minutes (I) and 30 minutes of treatment (J). Relative signal strengths are presented as the mean ± s.e.m. representative of three independent replicates. Bars with different letters represent significant differences in relative signal strength between treatment groups (P ≤ 0.05). Immunofluorescence Detection and Quantitation of Phosphorylated MAPKAPK2 Following Culture in 460 mOsm Medium for 15 Minutes and 30 Minutes. Phosphorylated MAPKAPK2 was detected in the cytoplasm and the nucleus of all embryos observed following 15 minutes and 30 minutes of treatment. There was a noticeable increase in the level of phosphorylated MAPKAPK2 protein detected in blastocysts cultured in KSOMaa + 0.2 M sorbitol (M, Q) when compared with blastocysts cultured in KSOMaa + 1.4% glycerol (L, P) or KSOMaa only (K, O) at both 15 minutes and 30 minutes of treatment. No primary controls are shown (N, R). All confocal micrographs are representative images from pools of blastocysts in each treatment group. Green indicates positive staining for MAPKAPK2 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 phosphorylated MAPKAPK2 polypeptides at 15 minutes (S) and 30 minutes of treatment (T). Relative signal strengths are presented as the mean ± s.e.m. representative of three independent replicates. Bars with different letters represent significant differences in relative signal strength between treatment groups (P ≤ 0.05).