Role of endometrial protein disulphide isomerase (PDIA1 and PDIA6) on regulating endometrial receptivity

Abstract

Abstract of thesis entitled ROLE OF ENDOMETRIAL PROTEIN DISULPHIDE ISOMERASE (PDIA1 AND PDIA6) ON REGULATING ENDOMETRIAL RECEPTIVITY

Submitted by Thevarathanthrige Sudini Ranshaya Fernando For the degree of Doctor of Philosophy at the University of Hong Kong In June 2021 Proteins on the endometrial surface regulate embryo implantation process. However, the underlying mechanism on how embryo-endometrium interacts and subsequent successful implantation remains largely unknown. Accumulating evidence suggest steroid hormones transform the non-receptive endometrium to receptive endometrium during the mid-luteal phase of menstrual cycle through expression of surface protein markers including mucins and integrins. By using mass spectrometric analysis, we identified protein Disulphide isomerase as one of the surface marker differentially expressed in receptive and non-receptive endometrial epithelial cells. Protein Disulphide Isomerase (PDI) family consists of 21 members exhibiting various functions including redox reactivity and chaperone activity. PDI proteins mainly located in endoplasmic reticulum and other locations include cytosol, nucleus, cell membrane and extracellular matrix. In this study, the expression of PDI transcripts (PDIA1-6) and the functional role of PDI proteins (PDIA1 and PDIA6) in endometrial epithelial cells on embryo implantation were studied. In human endometrial epithelial Ishikawa cells, PDIA1 and PDIA6 transcripts were upregulated, and PDIA4 and PDIA5 transcripts were downregulated by estrogen; whereas the expression of PDIA2 and PDIA3 remained unchanged. PDIA1-6 transcripts were downregulated progesterone. PDIA1 and PDIA6 proteins were highly expressed in receptive (Ishikawa and RL95-2) and non-receptive (HEC1-B and AN3CA) endometrial epithelial cells. Higher PDIA1 and PDIA6 proteins were found in the membrane protein fraction of non-receptive cells. However, only receptive endometrial (Ishikawa and RL95-2) cells express estrogen receptor alpha (ESR1), estrogen receptor beta (ESR2) and progesterone receptor (PR) transcripts. In Ishikawa cells, MPP (ER antagonist) and ESR1 siRNA, but not PHTPP (ER antagonist) reversed estrogen induced total and membrane PDIA1 and PDIA6 expression; while mifepristone (PR antagonist) revered progesterone reduced total and membrane PDIA1 and PDIA6 expression. In non-receptive AN3CA cells, addition of PDI inhibitors (bacitracin, 16F16 and LOC14) or PDIA1 siRNA increase attachment of Jeg-3 spheroids; whereas overexpression of PDIA1 in receptive Ishikawa cell decreased spheroid attachment and this is associated with reduced expression of Integrin β3 in treated Ishikawa cells. Addition of TCEP (disulphide reducing agent) increased spheroid attachment in non-receptive AN3CA, but not Ishikawa cells. Conversely, addition of diamide (oxidizing reagent) reduced spheroid attachment in Ishikawa cells. In human endometrial samples taken from pregnant and non-pregnant women, PDIA1 and PDIA6 proteins are highly expressed at the luminal epithelium of pre-receptive (LH+2 days) than receptive (LH+7 days) phase when the expression of integrin β3 protein is low. Interestingly, higher luminal expression of PDIA1 and PDIA6, and lower integrin 3 proteins are associated with non-pregnant women in IVF setting. Taken together, PDIA1 and PDIA6 are expressed in endometrial epithelial cells and regulated by estrogen and progesterone. Higher membrane expression of PDIA1 and PDIA6 proteins are associated with low spheroid attachment and embryo implantation. Decreased membrane PDIA1 and PDIA6 protein during mid-luteal phase hormone levels provide a reduced micro-environment conducive for embryo implantation. PDI proteins could be used as marker for pregnancy prediction in clinical setting (472 words).