Role of endometrial estrogen and progesterone receptors on protein Disulphide isomerase 1 (PDIA1) expression on regulating endometrial receptivity

Abstract

Introduction / Background / Objectives: Human endometrium is receptive to embryo implantation at a particular time during the menstrual cycle. Differential expression of receptive proteins on the endometrial surface is a prerequisite for successful embryo attachment. Protein disulphide isomerase (PDI) family contains 21 member proteins that function as chaperones with redox activity. Our preliminary study indicated that PDIA1 is hormonally regulated, and a lower membrane protein expression level was found in receptive endometrial cell lines. Therefore, we hypothesized that steroid hormones regulate endometrial receptivity via modulating surface PDIA1 expression. Furthermore, surface expression of PDIA1 generated an oxidized microenvironment not conducive for embryo implantation. Methods: We used receptive (Ishikawa & RL95-2) and non-receptive (AN3CA & HEC1B) human endometrial epithelial cells for PDIA1 expression analysis. The expression levels of total and membrane PDIA1 proteins were determined and correlated with spheroid attachment rate onto endometrial cells. The effects of estrogen, progesterone and the inhibitors of their receptors on PDIA1 expression in Ishikawa cells were studied. The expressions levels of total and membrane PDIA1 protein in the Ishikawa cells were determined after a 24-hr treatment of estrogen (0.01-100 nM) and/or progesterone (0.01-1 µM) by western blotting and immunohistochemistry. PDIA1 small interfering RNA (siRNA) and inhibitor bacitracin, disulphide reducing agent TCEP and oxidizing agent diamide were used to treat the non-receptive (AN3CA), receptive (Ishikawa) or human primary endometrial epithelial cells collected at the receptive phase of the cycle. PDIA1 overexpression plasmid was transfected into the receptive Ishikawa cells. The spheroid attachment rate of the treated cells was determined. Furthermore, endometrial tissues were collected from the proliferative (n = 12), secretary (n = 24), and pre-receptive (n = 17) and receptive (n = 16) phases of the menstrual cycle, and PDIA1 expression was determined by immunohistochemistry. Results / Outcomes: Expression of total PDIA1 protein was high in the 4 endometrial epithelial cell lines tested; a higher membrane PDIA1 expression was found in non-receptive AN3CA cells. Ishikawa and RL95-2 cells expressed estrogen receptor alpha (ESR1), estrogen receptor beta (ESR2) and progesterone receptors (PGR). Moreover, ESR1, ESR2 and PGR showed a cyclic expression pattern during the human menstrual cycle. Estrogen at 0.1 to 100 nM up-regulated, while progesterone at 0.1 to 1 µM down-regulated PDIA1 expression. Mid-luteal estrogen and progesterone level down-regulated membrane PDIA1 but upregulated cytosolic PDIA1 expression in Ishikawa cells. PDIA1 expression is high in luminal epithelium at the proliferative phase of human endometrium compared to the secretary phase, and there was no change in the expression of PDIA1 in glandular epithelium between the two phases. PDIA1 expression in luminal epithelium at the pre-receptive phase was higher than the receptive phase. ESR1 antagonist MPP, but not ESR2 antagonist PHTPP, blocked the stimulatory effect of estrogen (10 nM) on PDIA1 expression in Ishikawa cells. Similarly, knockdown of ESR1 by siRNA suppressed the stimulatory effect of estrogen (10 nM) on PDIA1 expression. Addition of PGR antagonist mifepristone nullified the suppressive effect of progesterone (0.1 µM) on PDIA1 expression. The Jeg-3 spheroid attachment rate is significantly higher in receptive (Ishikawa & RL95-2) than nonreceptive (AN3CA & HEC1-B) cells. Inhibition of PDIA1 by bacitracin (1mM) or knockdown of PDIA1 by siRNA increased Jeg-3 spheroid attachment on non-receptive AN3CA cells, whereas forced expression of PDIA1 reduced the attachment in receptive Ishikawa cells. Furthermore, forced expression of PDIA1 in Ishikawa cells significantly down-regulated expression of cell adhesion molecule integrin β3, but not integrin αv and E cadherin. Interestingly, disulphide reducing TCEP (1 mM) treatment increased spheroid attachment in AN3CA, but not in Ishikawa cells. Conversely, oxidizing reagent diamide (0.1 mM) treatment reduced Jeg-3 spheroid attachment in Ishikawa, but not in AN3CA cells. Conclusion: Our findings suggest that estrogen, progesterone and their receptors modulate PDIA1 expression on endometrial epithelial cells and endometrial receptivity. The increased attachment was exerted partly through up-regulation of adhesion molecule integrin β3 and a reduced microenvironment for embryo attachment. Screening for membrane PDIA1 expression before embryo transfer in infertile women may open a new approach to enhance embryo implantation in clinical setting.