WD-repeat containing protein-61 regulates endometrial epithelial cell adhesion indicating an important role in receptivity

Abstract

Endometrial receptivity is crucial for successful embryo implantation during early pregnancy. The human endometrium undergoes remodeling within each menstrual cycle to prepare or become receptive to an implanting blastocyst in the mid-secretory phase. However, the mechanisms behind these changes are not fully understood. Recently, using hormone-treated endometrial organoids to model receptivity, we identified that the transcriptional regulator WD-repeat-containing protein-61 (WDR61) was reduced in organoids derived from infertile women. In this study, we aimed to determine the role of WDR61 in endometrial receptivity. Here, we demonstrated that WDR61 immunolocalizes in the nuclei and cytosol of endometrial glandular epithelium, luminal epithelium, and stroma. The staining intensity of WDR61 was significantly higher during the receptive mid-secretory phase compared to the non-receptive proliferative phase in fertile women. In a functional experiment to model blastocyst adhesion to the endometrial epithelium, we found that adhesion of cytotrophoblast progenitor spheroids was blocked when siRNA was used to knockdown WDR61 in primary endometrial epithelial cells. Similarly, in Ishikawa cells (a receptive human endometrial epithelial cell line), siRNA knockdown of WDR61 significantly reduced the cell adhesive and proliferative capacities. qPCR revealed that WDR61 knockdown reduced expression of key genes involved in receptivity including HOXD10, MMP2, and CD44. Chromatin immunoprecipitation sequencing demonstrated that WDR61 directly targeted 2022 genes in Ishikawa cells, with functions including focal adhesion, intracellular signaling and epithelial-mesenchymal transition. Overall, these findings suggest that WDR61 promotes endometrial receptivity by modulating epithelial cell focal adhesions, proliferation, and epithelial-mesenchymal transition.

Keywords: Homeobox genes; WDR61; Wnt pathway; cell adhesion; chromatin immunoprecipitation sequencing; embryo implantation failure; endometrial epithelium; endometrial receptivity.

Figure 1.

Spatial and temporal localization of WDR61 in fertile human endometrial tissue. (A) Immunohistochemistry staining of WDR61 in endometrial tissue from fertile women during the mid-secretory phase (n = 7) and proliferative phase (n = 8). Sections were co-stained with hematoxylin (blue) to denote nuclear components. (B) WDR61 expression in luminal epithelium, glands and stroma was semi-quantified by giving stain frequency and intensity a score out of 4. (C) Localization of WDR61 and acetylated α-tubulin (ciliated epithelial cells) was determined by immunohistochemistry staining on a series of endometrial sections. An isotype negative control was included, consisting of the same IgG isotype and concentration with a non-immune antibody in place of the primary antibody. Data are presented as mean ± SD. *P < 0.05, ****P < 0.0001. Prolif, proliferative phase; Mid-sec, mid-secretory phase.

Figure 2.

Functional effect on endometrial epithelial cell adhesion following WDR61 knockdown. (A) Immunocytochemistry was performed on primary HEEC monolayers to determine WDR61 localization and confirm the epithelial purity of HEECs via E-cadherin staining. Cells were co-stained with hematoxylin to denote nuclear components. (B) The effect of WDR61 knockdown in primary HEECs on the adhesion of trophoblast spheroids was measured 72 h post-transfection. The percentage of trophoblast spheroids adhering to primary HEECs was significantly reduced following WDR61 knockdown (n = 5). (C–G) Ishikawa cells were used as a receptive endometrial epithelial cell model to test WDR61 functions on cell adhesion and proliferation. (C) Immunocytochemistry was performed on Ishikawa monolayers to determine WDR61 localization. (D–F) Adhesive capacity of Ishikawa cells was measured via xCELLigence and spheroid adhesion assay following treatment with WDR61 or scramble control siRNA (n = 4). (G) Proliferation of Ishikawa cells was determined by xCELLigence. (H) Levels of expression of two proliferation genes following siRNA treatment was measured at 72 h post transfection in Ishikawa cells and normalized to 18S (n = 6; qPCR). Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. HEEC, primary human endometrial epithelial cells.

Figure 3.

qPCR analysis of Homeobox genes and Wnt pathway target genes following WDR61 knockdown in Ishikawa cells. Levels of gene expression were normalized to 18S (n = 5). Data are presented as mean ± SD. *P < 0.05, ns, not significant.

Figure 4.

ChIP-Seq analysis to determine genome-wide binding of WDR61 in Ishikawa cells. (A) Gel electrophoresis of sonicated Ishikawa cells following the protocol outlined in methods. DNA fragments were dominantly between 300 and 500 bp which was adequate to continue immunoprecipitation. (B) Immunoprecipitation was coupled with qPCR using primers c-MYC and CXCL12 as positive controls for WDR61 binding. Gene expression was normalized to 18S and fold change was calculated compared to negative IgG control. Ishikawa cell replicates demonstrated enrichment of positive controls compared to negative control, thus the samples were used for Illumina sequencing. (C) Distribution of genomic regions that WDR61 bound to in Ishikawa cells (n = 3) were evaluated relative to the total gene body using fastQC. Approximately half of the peaks were intergenic, followed by intronic, promoter, exonic, and TTS regions. (D) Heatmap of genome wide occupancy of WDR61 in three Ishikawa cell ChIP-Seq replicates measured as reads per million mapped reads (RPM) per bp. TSS, transcription start site; TES, transcription end site; TTS, transcription termination site.

Figure 5.

WDR61 target gene pathway enrichment analysis. (A) Ishikawa cells treated with scramble control or WDR61 siRNA (n = 4) were analyzed by qPCR to confirm ChIP-seq data. Data are presented as mean ± SD. WDR61 was found to bind to promoter regions of CTNNA1 and PDLIM2. Both CTNNA1 and PDLIM2 exhibited significantly downregulated expression following WDR61 siRNA treatment (*P < 0.05). Visualization of WDR61 binding regions on CTNNA1 and PDLIM2 gene using Interactive Genomic Viewer. Binding peaks were calculated as significantly enriched binding of WDR61 compared to input control. (B) Top enriched KEGG pathways (adjusted P-value <0.05) for genes (Supplementary Table S2) identified to be targets for WDR61 binding at their promotor region. (C) Chord plot with identified pathways and representative genes involved in endometrial receptivity and implantation following the literature search. Genes listed in these pathways were identified as promoter binding sites in ChIP-seq data.

Figure 6.

Pathways enriched following functional analyses of WDR61 target genes in Ishikawa cells, and their role in endometrial epithelial cell adhesion. Schematic of an endometrial epithelial cell and the identified receptivity pathways enriched in WDR61 ChIP-seq targets. Wingless (Wnt), mitogen-associated protein kinase (MAPK), phosphoinositide 3-OH kinase (PI3K)/protein kinase (Akt) have complex, interrelated roles in proliferation, adhesion molecule expression, and the epithelial mesenchymal transition in preparation for embryo implantation. Stars indicate WDR61 involvement in individual gene expression or multiple genes involved in a pathway extrapolated from ChIP-seq and qPCR data.