Molecular Mechanism Of Human Endometrial S100A11in Regulating Embryo Implantation

Embryo implantation includes the process of blastocysts adhesion to endometrium ind subsequent invasion. It is a complicated process accompaning by a complicated series of genetic, molecular and cellular interactions. Although, the overwhelming genes lave been confirmed as involved in embryo implantation, the exact mechanism is still unclear. Our previous finding showed that the expression of S100A11in the villous tissues of early pregnancy loss patients was down-regulated, suggesting the involvement of S100A11in the early embryo-implantation. S100A11is a member of S100proteins. SI00is a multigenic family of Ca2+-binding proteins of EF-hand type with both intracellular and extracellular roles.In this study, we investigate the expression, location of S100A11, and its involvement in embryo implantation, via clinical investigation, observations from in vivo animal model and in vitro cell line. Our present data indicates that,(1) deciduous S100A11in recurrent miscarriage patients,endometrial S100A11levels in tubal infertility patients with unsuccessful pregnancy, and in unexplained infertility patients,were found to be lower;(2) the deficiency of endometrial S100A11in mice result in the decreased trophoblast implantation rate;(3) The knockdown of S100A11in endometrial cells had adverse effects on the endometrium receptivity and immunotolerance;(4) Epidermal growth factor (EGF), but not human chorionic gonadotropin (hCG), estradiol or/and progesterone promoted S100A11expression;(5) S100A11sub-localized in endoplasmic reticulum, and promoted the EGF-induced intracellular Ca2+influx via activating the Ca2+release, uptake of calcium store. It provides an undocumented molecular mechanism of S100A11in embryo implantation, implicating S100A11levels may be a candidate for predicting the outcome of pregnancy in clinic.Part One:The expression and location of S100A11in human endometriumObjective:To investigate the location of S100A11in human endometrium, and analysis the levels of deciduous S100A11in recurrent miscarriage patients, endometrial S100A11in failed pregnancy outcome of women undertaking IVF-ET treatment, and in unexplained infertility patients, respectively.Methods:Immunohistochemical staining was performed to localize S100A11in human endometrium, and exhibited the levels of deciduous S100A11in recurrent miscarriage patients.qRT-PCR and Western blot assay were performed to investigate the levels of S100A11in human endometrium derived from tubal infertile patients, and unexplained infertile patients, respectively.Results:1. Immunohistochemical staining localized S100A11in both luminal and glandular epithelial cells of human mid-secretory endometrium, and weak staining was also observed in the stromal endometrium.2. Deciduous S100A11of immunohistochemical staining in recurrent miscarriage patients showed weak than that in normal patients.3. In three patients of unexplained infertility, the endometrial S100A11mRNA levels were decreased obviously, compared with the total infertile patients.4. Western blotting analysis shows the levels of endometrial S100A11of failed pregnancy outcome in women undertaking IVF-ET treatment was lower significantly than that those of successful pregnancy outcome.Conclusion:S100A11in human luminal and glandular epithelial cell is crucial for the embryo implantation and early pregnancy maintenance. Part Two:Effects of endometrial S100A11on embryo implantationObjective:To investigate the effect of S100A11on the attachment of the floating blastocyst to the receptive endometrial epithelium, and to explore the contriutions of S100A11in endometrium receptivity and immunotolerance.Methods:1. ICR mice in endometrium treated by specific S100A11siRNA were administrated to explore the outcome of embryos implantation rate in vivo.2. Specific S100A11siRNA were administrated to knock down the levels of S100A11in endometrial cells (Ishikawa cells) in vitro.3. In vitro attachment model were performed to observe the adhesion of trophablasts on endometrium.4. qRT-PCR and Western blot assay were performed to investigation the alteration of genes associated with endometrium receptivity and immunotolerance.Results:1. Specific S100A11siRNA interference to mouse endometrial cells for48hours (day4.5.p.c) resulted in significant reduction of S100A11protein expression in endometrium (Figure.2a), and on day6.5p.c, consequently, the embryo implantation number were significantly reduced. 2. Attachment rate analysis demonstrated that the knockdown of S100A11results in the significant decreased JAr speroids attachment rate (p<0.05).3. qRT-PCR assay showed expression levels of genes associated endometrium receptivity, indcluding LIF, Integrin β3, EGFR, claudin4and olfactomedin,were significantly altered, and also, expression of genes associated immunotolerance, including IL-4, IL-15, IL-12a, IL-16, altered significantly.Conclusion:S100A11plays multiple roles in embryo implantation including affecting the adhesion of trophoblast, receptivity of endometrium and the banlace of Th1/Th2. Part Three:Molecular mechanism of embryo implantation mediated by endometrial S100A11Objective:To confirm the molecular mechanism of embryo implantation mediated by endometrial S100A11Methods:1.Estrogen, progesterone,human chorionic gonadotropin (hCG), and EGF were administrated to treat endometrial cells.2. Immunofluorescence and laser confocal microscopy assay were performed to localize S100A11in sub-cellular levels of endometrial cells.3. Immune electron microscopy assay were to perform to observe the sublocalization of S100A11in endometrial cells4. Single-cell microfluorimetry was performed to investigate the contribution of S100A11to the increase of intracellular Ca2+in human endometrial cells.5. Ryanodine and thapsigargin were administrated to induce Ca2+influx in endometrial cells.Results:1.mRNA and protein expression levels of Sl00All were not regulated by E2, P4and hCG, but upregulated by EGF.2. S100A11was mainly located in the endoplasmic reticulum of endometrial cells.3. EGF-pormoted cytosolic Ca2+transients were inhibited in the human endometrial cells treated with specific siRNA targeting S100A11.4. The EGF-enhanced Ca2+uptake, release and store were significantly inhibited in the human endometrial cells treated with siRNA targeted S100A11.Conclusion:Endometrial S100A11is up-regulated by EGF; S100A11in endoplasmic reticulum of endometrial cells mediates EGF-induced intracellular Ca2+release, uptake and store, which is the potential molecular mechanism of EGF-induced interaction of embryo and uterus during the process of embryo implantation.