Tight Junction Between Endometrial Epithelial Cells: Their Structure, Molecular Composition, Roles In Embryo Implantation And Mechanisms Of Regulation

It has been shown that only 50 to 60 percent of conceptions in women could develop and survive past 20 weeks of gestation. Around 40-50% of conception and pregnancy failed at different time of gestation. Because approximately 75 percent of these failures are due to implantation failure, implantation failure is a main factor resulting in early pregnancy loss. So embryo implantation rate determines the success of the in vitro fertilization (IVF) and the birth rate. However, the mechanisms involved in embryo implantation remain largely unknown. The knowledge about the process of embryo implantation we have is well less than the knowledge about the other process of human reproduction. Although the ART protocol and pharmaceuticals for ovarian stimulation are being renovated and reproved constantly, on the contrary, we nearly have no measure about improving the uterine receptivity.The phenotypes of endometrial luminal and glandular epithelium change dramatically from the proliferative to the mid-secretory phase of the menstrual cycle, when the endometrium becomes receptive for embryo implantation under regulation of ovarian hormones, cytokines, cell growth factors, adhesion molecules. Endometrial luminal and glandular epithelium are composed of the polarity epithelial cells. In embryo implantation window, two types of epithelial cells undergo different changes in morphology. A loss of apico-basal polarity could be observed in luminal epithelial cells, while gland epithelial cells showed the most polarity and secretory activity. The different morphology between two cell populations highlights the functional difference, with the luminal epithelial cells initial contact and attachment with the embryo and the glandular epithelial cells expressing secretory proteins for conceptus recognition, survival, and development.However, little is known about the association of TJ functions with the establish of the endometrial receptivity. The purpose of the present study was 1) to investigate the alteration of TJ morphology and TJ protein in human uterine epithelium during the menstrual cycle; 2) to determine whether TJ function associated with the endometrial receptivity; 3) to determine whether expression of TJ proteins in uterine gland epithelial cells had a correlation with serum 17β-estradiol (E2) and/or progesterone; and 4) to determine whether Occludin regulates the uterine epithelial cell proliferation and differentiation.Part1 Alteration of TJ morphology and TJ proteins in human uterine epithelium during the menstrual cycleObjective:To investigate alteration of TJ morphology and TJ proteins in human uterine epithelium during the menstrual cycleMethods:We examined the dynamically changes of morphology of TJs between adjacent uterine epithelial cells including luminal epithelial cells and gland epithelial cells during the menstrual cycle with transmission electroscope. Then we investigated the location and expression of TJ proteins including Occludin,Zo-1,Claudin-1,-2,-3,-4,-5 in the proliferative and secretary endometrium with immunochemistry techniques, respectively.Results:the morphological analysis of uterine epithelial cells with transmission electroscope showed that the depth of TJ between gland epithelial cells in mid-secretary phase increased about 2-fold along the lateral membrane and present more complex and curled when compared with mid-proliferative phase. However, in the luminal epithelial cells, the TJ depth in mid-secretary phase was reduced significantly compared with the mid-proliferative phase.All examined TJ proteins including Occludin. Zo-1,Claudin-1,-2,-3,-4,-5 with confocal immunofluorescence were located at TJ region in mid-secretary phase GE cells. On the other hand, Claudin-2 and -5 were not expressed in mid/late proliferative phase GE cells. During mid/late proliferative phase, Claudin-1, Claudin-3 and Claudin-4 were faintly stained in the basallaterial membrane, and Zo-1, Occludin localized in punctuate manner at the uppermost portion of the lateral membrane and cytoplasm. The endometrial expression levels of TJ proteins in mid-secretary phase were higher than those in other phases with Western blot analysis. Conclusion:Dynamically changes of structural and molecular components of TJs in uterine epithelial cells were detected during proliferative and secretive phases of the menstrual cycle. In LE cells the TJ depth and expression of TJ proteins were downregulated during mid-secretary phase, while GE cells showed an oppositive result.Part 2 The roles of tight junction between uterine endometrial epithelial cells in the embryo implantationObjective:To investigate the roles of tight junction in regulation of adhesion between uterine endometrial epithelial cells and embryo cells.Methods:Endometrial cell lines RL95-2 and HEC-1A were used as models of receptive and nonreceptive cells, respectively. Attachment assay of JAR spheroids to the endometrial cell was used as embryo implantation model to evaluate the hEEC receptivity to embryo under different cultured conditions. We compared the HEC1-A and RL95-2 cell lines in TJ ultrastructure and TJ protein composition with transmission electron microscope, Western blot and immunofluorescent confocal microscope, respectively. We examined the TJ function by measurement of cell transcellular electrical resistance (TER) with Millicell-ERS. The embryo adhesion rate in HEC1-A and RL95-2 cell lines were examined. The expression of Occludin at the adhesion parts was examined with three-color laser confocal inspection. Then, we investigated the embryo adhesion rate in the HEC1-A cells pretreated with siRNA to interfere Occludin gene and TER during TJ assembled under the calcium switch assay with EGTA. Additionally, we treated HEC1-A cells with interleukin-1βfor 48 hours, and examined the expression level and location of Occludin with Western blot and immunofluorescence, and the adhesion rate of HEC-1A cells to JAR spheroids.Results:The transmission electron microscopy showed that HEC1-A cells, not RL95-2 cells, were polarized and had typical tight junctions at the apex of the lateral membrane cells. In HEC1-A the tight junction proteins Zo-1 and Occludin were expressed in the cell membrane, while they were expressed in the cytoplasm of RL95-2 cells. Western blot results showed that the expression of Zo-1 and Occludin in the HEC1-A was significantly higher than that in RL95-2 (P<0.01). Claudin-4, a component of tight junction skeleton, was strongly expressed in HEC1-A cells, but no expression was detected in RL95-2 cells. The TER of monolayer HEC1-A cells was higher than that of RL95-2 cells (163.0±23.0)Ω×cm2 vs (92.3±11.0)Ω×cm2(P＜0.01). In hEEC and JAR spheroids attachment experiments, the adhesion rate of HEC1-A cells to JAR spheroids was significantly higher than that of RL95-2 cells (P＜0.01). Confocal laser microscopic analysis showed that Occludin might move from the cell membrane into cytoplasm at the adhesion position of HEC1-A cells, remaining disrupted and point-like distribution in the cell membrane. After Occludin siRNA interfering, the expression of Occludin in the membrane was decreased in HEC1-A cells, and meanwhile the embryo adhesion rate increased. However, the peak TER was attained more slowly and its value was lower in cultured HEC-1A cells pretreated with Occludin siRNA during TJ reassembled in calcium switch experiments.Conclusion:HEC-1A cell presents TJ and TJ protein in TJ region, while RL95-2 cell did not express TJ and TJ proteind in cell border. Downregulated Occludin protein expression by siRNA interference in HEC1-A cells results in an increase in the embryo adhesion rate, the translocation of Occludin from the cell membrane into cytoplasm at the adhesion position of HEC1-A cells. These results suggest that down-regulation of the uterine endometrial epithelial cell tight junctions may be related to embryonic adhesion.Part 3 The roles of Occludin in regulation of epithelial cell differentiation and proliferation and the alteration of TJ proteins in cultured human endometrial epithelial cells in the presence of ovarian steroid hormonesObjective:To investigate the effects of E2 and progesterone (P4) on the expression of TJ proteins Occludin and Claudin-4 in the cultured human endometrial epithelial cells and the roles of Occludin in the regulation of epithelial differentiation and proliferation.Method:The endometrial gland epithelial cells in proliferative phase was collected from 6 women who underwent hysterectomy for leomyoma. Endometrial GE cells were isolated and cultured in vitro in the presence of 17β-estradiol (E2,10-8mol/L) or E2 (10-8mol/L)+P4(10-6mol/L) for 48h. The control group received no treatment. The levels of Occludin and Claudin-4 proteins in the cultured human endometrial epithelial cells were analyzed with Western blot. Ishikawa (IK) cells were used in these experiments. IK cells have glandular characteristics and are able to secrete some cellular factors similar to normal endometrial cells. IK cells were widely considered as suitable cell lines for studying functions of normal endometrial glandular epithelial cells. We used IK cells as a GE cell model and treated these cells with Occludin siRNA to downregulate Occludin expression. IK cells apoptosis was examined using H2O2 as apoptogen and analyzed with flow cytometry. The cell proliferation was examined with cell cycle flow cytometry analysis. Glycogen level in IK cells was also analyzed.Results:E2 (lOnM) had little effect on Claudin-4 protein expression and P4 significantly increased the 65 kD/50kD ratio of Occludin band and Claudin-4 protein. Pretreatment of IK cells with Occludin siRNA significantly decreased the cell glycogen level, increased cell proliferative rate and reduced apoptosis sensitivity to H2O2.Conclusion:Progesterone upregulates the protein expression of 65kD Occludin and Claudin-4 in primary cultured endometrial gland epithelial cells, suggesting that progesterone may promote the TJ assembled in uterine GE cells. siRNA targeting human Occludin in IK cells led to downregulate apoptosis and increase cell proliferation, reduce cell glycogen production, suggesting that Occludin may play a role in mediating the progesterone effects on GE cell apoptosis and on secretory activity.