MAML1: a coregulator that alters endometrial epithelial cell adhesive capacity

Abstract

Background: Abnormalities in endometrial receptivity has been identified as a major barrier to successful embryo implantation. Endometrial receptivity refers to the conformational and biochemical changes occurring in the endometrial epithelial layer which make it adhesive and receptive to blastocyst attachment. This takes place during the mid-secretory phase of woman's menstrual cycle and is a result of a delicate interplay between numerous hormones, cytokines and other factors. Outside of this window, the endometrium is refractory to an implanting blastocyst. It has been shown that Notch ligands and receptors are dysregulated in the endometrium of infertile women. Mastermind Like Transcriptional Coactivator 1 (MAML1) is a known coactivator of the Notch signaling pathway. This study aimed to determine the role of MAML1 in regulating endometrial receptivity.

Methods: The expression and localization of MAML1 in the fertile human endometrium (non-receptive proliferative phase versus receptive mid-secretory phase) were determined by immunohistochemistry. Ishikawa cells were used as an endometrial epithelial model to investigate the functional consequences of MAML1 knockdown on endometrial adhesive capacity to HTR8/SVneo (trophoblast cell line) spheroids. After MAML1 knockdown in Ishikawa cells, the expression of endometrial receptivity markers and Notch dependent and independent pathway members were assessed by qPCR. Two-tailed unpaired or paired student's t-test were used for statistical analysis with a significance threshold of P < 0.05.

Results: MAML1 was localized in the luminal epithelium, glandular epithelium and stroma of human endometrium and the increased expression identified in the mid-secretory phase was restricted only to the luminal epithelium (P < 0.05). Functional analysis using Ishikawa cells demonstrated that knockdown of MAML1 significantly reduced epithelial adhesive capacity (P < 0.01) to HTR8/SVneo (trophoblast cell line) spheroids compared to control. MAML1 knockdown significantly affected the expression of classical receptivity markers (SPP1, DPP4) and this response was not directly via hormone receptors. The expression level of Hippo pathway target Ankyrin repeat domain-containing protein 1 (ANKRD1) was also affected after MAML1 knockdown in Ishikawa cells.

Conclusion: Our data strongly suggest that MAML1 is involved in regulating the endometrial adhesive capacity and may facilitate embryo attachment, either directly or indirectly through the Notch signaling pathway.

Keywords: Embryo implantation; Endometrial adhesion; Endometrial epithelial cell; Hippo pathway; MAML1; Notch pathway; Trophoblast cell.

Fig. 1

Comparison of MAML1 expression in fertile proliferative phase and mid-secretory phase endometrium. a Immunohistochemistry detection of MAML1 in luminal epithelium (L) glandular epithelium (G) and stroma (S) of human endometrium. A nuclear labeling was recorded in all cell types. The specificity of MAML1 labeling was confirmed through the inclusion of an isotype control in which the non-immune antibody of the same isotype was substituted for the MAML1 antibody at the same concertation. Sections were counterstained with hemotoxylin to highlight the cell nuclei (blue). b Staining intensity of MAML1 was semi-quantitated by scoring staining in tissues blinded to cycle stage. Data were presented as mean ± SEM. (n = 4). *P < 0.05

Fig. 2

Examination of the effect of MAML1 knockdown on Ishikawa cell adhesive capacity. Ishikawa cells were transfected with either MAML1 siRNA (20 nM) or scrambled control (20 nM) before HTR8/SVneo spheroid adhesion assay or other analysis. a MAML1 knockdown was confirmed by qPCR. Expression levels were normalized to 18S (n = 9). b MAML1 knockdown was confirmed by immunoblotting and densitometry, normalized against a loading control GAPDH (n = 4). c MAML1 knockdown significantly compromised the spheroid adhesion compared to scrambled control (n = 5). d Representative images are presented to show attached spheroids on the Ishikawa cell monolayer after adhesion assay. Data were presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

Qualitative qPCR examination of the effect of MAML1 knockdown on common Notch pathway members. HEY1 and NRARP expression were significantly decreased after MAML1 knockdown in Ishikawa cells compared to scrambled control. Expression levels were normalized to 18S (n = 7). Data were presented as mean ± SEM. *P < 0.05, **P < 0.01, ns: no significant difference

Fig. 4

Effect of MAML1 knockdown on the expression of endometrial receptivity markers and hormone receptors. Expression levels were normalized to 18S (n = 7). Data were presented as mean ± SEM. *P < 0.05, ns: no significant difference

Fig. 5

Examination of the effect of MAML1 knockdown on the expression of Hippo pathway downstream effectors. Expression levels were normalized to 18S (n = 7). Data were presented as mean ± SEM. ***P < 0.001, ns: no significant difference