Figure 6: The involvement of FGF signaling in MVB biogenesis of activated blastocysts after delayed implantation.

(a) mRNA expression of FGFRs in normal, dormant, and activated blastocysts. Day 4 normal blastocysts were obtained from day 4 pregnant mice by uterine flushing. Dormant and activated blastocysts were obtained from day 8 delayed implanting mice. In each group, 10 embryos were pooled and subjected to RNA extraction, reverse transcription, and PCR with specific primers for mouse FGFRs (Table 1). All these receptors were expressed in day 4 normal, day 8 dormant, and day 8 activated blastocysts. (b) The experimental scheme. Ovariectomy was performed on mice on the morning of pregnancy day 4 and mice subsequently received P₄ injection from day 5 to 8. On day 8, 1 h prior to P₄ and E₂ injections, a small volume of PD173074 (50 μM in DMSO) was injected into one uterine horn. The volume was less than 5 μl. The other horn was injected with same volume of 0.25% DMSO. Hormones were injected 1 h later to ensure that blastocyst activation did not occur before PD173074 injection. Activated blastocysts were obtained 14 h post-E₂ injection and subjected to LBPA immunofluorescence staining. (c) Expression of LBPA in activated blastocysts collected from PD- or vehicle-injected horns. The experiment was performed in 8 mice. A dramatic reduction of LBPA (green) accumulation was noted in 16 embryos for each group that were collected from 5 mice. These data were included for statistical analysis as shown. Signal intensities were obtained by setting the whole blastocyst as the region of interest. The mean of values obtained for embryos collected from DMSO-injected horns for each experiment was normalized to 1. A set of embryos from one mouse is shown. For each image, five Z-series sections (1 μm) were stacked. Scale bar, 20 μm. ***p < 0.0001.