A gene network of uterine luminal epithelium organizes mouse blastocyst implantation

Abstract

Purpose: The receptive endometrium is critical for blastocyst implantation. In mice, after blastocysts enter the uterine cavities on day 4 of pregnancy (day 1 = vaginal plug), blastocyst attachment is completed within 24 h, accompanied by dynamic interactions between the uterine luminal epithelium and the blastocysts. Any failures in this process compromise subsequent pregnancy outcomes. Here, we performed comprehensive analyses of gene expression at the luminal epithelium in the peri-implantation period.

Methods: RNA-seq combined with laser microdissection (LMD) was used to reveal unique gene expression kinetics in the epithelium.

Results: We found that the prereceptive epithelium on day 3 specifically expresses cell cycle-related genes. In addition, days 3 and 4 epithelia express glutathione pathway-related genes, which are protective against oxidative stresses. In contrast, day 5 epithelium expresses genes involved in glycolysis and the regulation of cell proliferation. The genes highly expressed on days 3 and 4 compared to day 5 are related to progesterone receptor signaling, and the genes highly expressed on day 5 compared to days 3 and 4 are associated with the ones regulated by H3K27me3.

Conclusions: These results suggest that specific gene expression patterns govern uterine functions during early pregnancy, contributing to implantation success.

Keywords: blastocyst implantation; gene expression; laser microdissection; luminal epithelium; uterus.

FIGURE 1

RNA‐seq analyses on the uterine luminal epithelium in the peri‐implantation period. (A) The analysis scheme of this study. Females on day 3 a.m., day 4 a.m. and p.m., and day 5 a.m. were used. (B) Representative pictures of the uterine tissue before and after the epithelial dissection using LMD. (C) Heatmaps depict log2 fold enrichment of DEGs during the peri‐implantation period. Numbers shown on the left indicate different gene clusters

FIGURE 2

K‐means clustering shows differential gene kinetics during the peri‐implantation period. Ten different clusters were revealed by k‐means clustering. The numbers of genes in each cluster are shown on the top of each graph

FIGURE 3

Validation of DEGs by immunostaining. The expression patterns of (A) Cox‐2 (Ptgs2 in cluster 1), (B) Ki67 (Mki67 in cluster 5), and (C) Pgr (cluster 6) were confirmed by immunostaining on uterine sections throughout day 3 to day 5. At least three different tissues were tested for each time point. CK8 (cytokeratin 8), an epithelial marker; Nu, nuclei; *, a blastocyst

FIGURE 4

Unique GO terms among genes expressed on each day of early pregnancy. GO terms were analyzed by Enrichr for day 3 (A), days 3 and 4 (B), and day 5 with blastocyst attachment (C) and day 5 without attachment (D)

FIGURE 5

Gene expression is differently governed before and after blastocyst attachment. Gene regulatory systems were predicted in Enrichr by comparing our data and publicly available datasets. Genes expressed on days 3 and 4 (A) are progesterone receptor (Pgr)‐related, while the day 5 epithelium expresses the genes regulated by PRC2 and the trimethylation of histone H3 at lysine 27 (H3K27me3) (B)