Fig. 6: Ex vivo uterine system suggests a possible signaling that mediates uterine COX-2 and embryonic AKT.

A Bulk RNA-seq of embryos cultured solely and co-cultured with ex vivo uterus for 24 h. The number of DEGs increased in the co-cultured embryos attached to the endometria, suggesting the effect of celecoxib predominantly on the uterus and the signals from the uterus to embryos. DESeq2 Wald test with an adjusted P value cutoff 0.05 using the Benjamini and Hochberg method for multiple testing was performed. The plots were automatically generated by RaNA-seq, and the vertical axis shows the P value before adjustment. B A bobble plot of enrichment analysis on the 244 downregulated DEGs (A) by celecoxib exhibits the suppression of embryonic AKT signaling in ex vivo co-cultured embryos. See also Supplementary Table 1A and B. Accumulative hypergeometric test was used, and all the results involving KEGG Pathway, Reactome Gene Sets, and WikiPathways showing log₁₀ P value less than -2 were plotted. C Representative pictures of immunofluorescence for AKT and CK8 in sequential sections. D Representative pictures of immunofluorescence for pAKT. Asterisks indicate the location of embryos and three independent experiments with different animals were performed (C, D). E Representative slices from three-dimensional images of whole-mount immunofluorescence for AKT and pAKT with celecoxib treatment. Yellow dotted lines indicate the outline of the embryos. The white arrow indicates the activation of AKT in invading trophoblasts. Three-dimensional mean fluorescent intensity values within the surface were computed by Imaris. Each value for AKT (F), pAKT (G), and pAKT/AKT (H) in DMSO group were normalized to 1. Mean ± SEM. I Attachment at 24 h/initially loaded. J. Shedding between 24 h and 48 h/attached at 24 h. K. Sustention on the endometria at 48 h/initially loaded. Mean ± SEM (I–K). L Representative slices from three-dimensional images. M. Quantitative data of the invading volumes. The box plot is presented as minimum, lower quartile, median, upper quartile, and maximum. + represents the mean value. LE, luminal epithelium; Str, stroma. Two-tailed unpaired Student’s t-test (F, G, H, M) and ordinary one-way ANOVA with post hoc Tukey’s multiple comparisons test (I, J, K) were used (n = 4 vs 3 (F–H), 4 vs 3 vs 3 (I–K), 6 vs 7 (M) biologically independent samples). *, P < 0.05; **, P < 0.01; ****, P < 0.0001; ns, not significant. tdTomato indicates the cell membrane of all embryonic/extraembryonic cell lineages. White and orange dotted lines represent the surface of LE and the boundary of LE and Str, respectively. Scale bars: 50 μm. Each plot represents the result per a recipient mouse (I–K) or the value for an embryo (F–H, M). Scale bars: 50 μm. A. created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en).