Figure 5: MVB formation is associated autophagic activation in activated blastocysts.
To inhibit autophagy, 3-MA (5 mM in 100 μl PBS) injections were given daily at 9 AM to delayed implanting mice from the day of OVX to the day of E2 injection. (a) Immunofluorescence staining of LBPA or ubiquitin in day 8 activated blastocysts from PBS- or 3-MA-injected delayed implanting mice. Immunofluorescence staining was performed in indicated numbers of blastocysts for each group. For LBPA staining, four independent experiments were performed and three sets showing similar results were included in statistical analysis. For ubiquitin staining, seven independent experiments were performed and four sets showing similar results were included in statistical analysis. Signal intensities were obtained by setting the whole blastocyst as the region of interest. The mean of values obtained for blastocysts from PBS-injected mice in each experiment was normalized to 1. For each image, five Z-series sections of 1 μm (LBPA) or four sections of 2 μm (ubiquitin) were stacked. Scale bar, 20 μm. **p < 0.001; ***p < 0.0001. (b) Di-I confocal live imaging of day 18 activated blastocysts obtained from PBS- or 3-MA-injected mice. DiI-positive puncta were observed in the trophectoderm of activated blastocysts. 3-MA injection to block autophagy during delayed implantation led to a reduced number of DiI-positive puncta. Enlarged images of yellow-boxed areas are shown in the lower panel. This experiment was repeated 6 times (16 activated blastocysts from PBS-injected mice [n = 6] and 19 activated blastocysts from 3-MA-injected mice [n = 6]). (c) LBPA immunofluorescence staining in Atg5+/+, Atg5+/−, and Atg5−/− activated blastocysts. Atg5+/− female mice were bred with Atg5+/− male mice and ovariectomy was performed on day 4 of pregnancy. On day 8 of delayed implantation, mice received an E2 injection to activate implantation and all embryos were collected 14 h later and subjected to LBPA immunofluorescence staining. See Methods section for detailed information. Scale bar, 20 μm. *p < 0.01; ns, not significant.
