Effect Of Galectin-3on Cell Apoptosis At The Time Of Formation Of Receptive Endometrium

Embryo implantation involves invasion of trophoblastic cells into the uterine epithelium and the underlying changeable stroma that undergoes a complex process of proliferation and migration. In this process, there is only a restricted time (’window of endometrial receptivity) when the implantation might occur during the menstrual cycle. However, the regulation machine in the formation of receptive endometrium is still unclear. During "embryo implantation", the process of trophoblastic cells invading into receptive endometrium, which includes targeting, adhering to endometrium and implanted into endometrium, is similar with the process of tumor cell invasion. The process of trophoblastic cells into endometrium experiences apoptosis of endometrial epithelial cells and decidualization of endometrial estromal cells.Our group screened a large number of possible molecules related to receptive endometrium. Galectin-3is one of them. Previous studies in our group indicated that Galectin-3increased largely in secretary phase of menstrual cycle and decreased in eutopic endometrium from patients with endometriosis. Decreased expression of Galectin-3in endometrium might be related to the defect in the formation of receptive endometrium. Currently, the investigators focus on the role of Galectin-3in tumor cells. Galectin-3is involved in multiple biological functions of tumor cells such as proliferation, adhesion, invasion, and apoptosis. These cell biological funcetions are cucial in the formation of receptive endometrium, too. We hypothesizes that Galectin-3takes part in the embryo implantation by affecting receptive endometrium. Our previous results showed intra-cellular Galectin-3had the ability to regulate the proliferation and adhesion of endometrial cells. However there is no report about the role of Galectin-3related to other biological function in endometrial cells. Apoptotic signals and anti-apoptotic signals regulate the cell microenviroment, disorder of which may lead to failure of embryo implantion. Thus, we planed a series of experiments on apoptotic effect of Galectin-3on endometrial epithelial cells.Based on preliminary work, our study further verified the effect of Galectin-3in embryo implantation and its effect on apoptosis of endometrial cells by adopting Bewo and RL95-2cell lines to mimic trophoblastic and endometrial cells during ’embryo implantation’. Our study has three parts as the followings.1. The role of Galectin-3in embryo implantionObjectives:To verify the role of Galectin-3in embryo implanationMethods:Galectin-3mRNA and protein expression were detected in mouse endometria of early pregnancy by real-time PCR and western blot. Galectin-3siRNA was tranfected into mouse uterus in vivo and its effects were verified by immunofluorescence and Western blot. Pregnant mice were scarified on the ninth day of pregnancy and implanted embryos were counted.Results:The expression of Galectin-3mRNA and protein in the pregnant group was higher than that in the non-pregnant group, and mRNA and protein expression reached their maximum levels on the fourth day and second day, respectively. Immunohistochemistry results showed that Galectin-3protein presented in luminal epithelium and glandular epithelium, and reached its maximum on the sixth day to eighth and the second to fourth day after pregnancy, respectively. The number of implanted embryos decreased substantially when Galectin-3was knocked-down in mouse endometrium.Conclusions:Increased Galectin-3expression after pregnancy is required for embryo implantation. Decreased Galectin-3in endometrium might lead to spontaneous abortion.2. The anti-apoptotic effect of Galectin-3in endometrial epithelial cells under regulation of estrogen and progesteroneObjectives:To investigate the anti-apoptotic effect of Galectin-3in endometrial epithelial cellsMethods:RL95-2cells were treated with a series of concentrations of staurosporine. MTT were applied to detect cell viability, and acridine orange staining and AnnexinV/PI staining were used to detect apoptosis rate. Galectin-3mRNA and protein levels were detected by real-time PCR and Western blot. RL95-2cells treated with Galectin-3siRNA together with staurosporine, and then the apoptosis rate was evaluated. A series of concentrations of17β-estrodial and progesterone were added into culture media, and Galectin-3mRNA and protein levels were investigated by real-time PCR and Western blot. RL95-2cells were tranfected with Galectin-3siRNA, and17β-estrodial or progesterone was added into culture media together with staurosporine. The apoptosis rate was calculated.Results:Decreased cell viability, increased apoptosis and Galectin-3mRNA and protein expression were detected after the treatment with staurosporine. Compared to negative siRNA treated RL95-2cells, the apoptosis of RL95-2treated with Galectin-3siRNA increased significantly after adding staurosporine. Galectin-3expression was regulated by17β-estrodial or progesterone. Compared with negaitve siRNA, estrogen or progesterone treated RL95-2cells with Galectin-3knock-down had a higher apoptosis rate.Conclusions:Intra-cellular Galectin-3, regulated by estrogen and progesterone, has anti-apoptic effect in endometrial epithelial cells.3. Hormonal regulation of Galectin-3expression in trophoblasts and the effects of extra-cellular GaIeciton-3on apoptosis of endometrial cells.Objectives:To investigate the hormonal regulation of Galection-3and the effect of extra-cellular Galectin-3on apoptosis of endometrial cells.Methods:We used Bewo and RL95-2cells as a model of trophoblastic and endometrial epithelial cells respectively to mimic the in vivo model of embryo implantation. We treated Bewo cells with a series of concentrations of17β-estrodial, progesterone and human chorioic gonadotropin (hCG), and detected Galectin-3mRNA and protein levels by real-time PCR, Western blot and ELISA. Then, we treated RL95-2cells with different concentrations of recombinant Galectin-3protein, and detected cell proliferation by BrdU and apoptosis rate by AnnexinV/PI stainging. We investigated integrin β1/β3expression on RL95-2after the treatment with recombinant Galectin-3protein by Flow cytometry. We further cultured RL95-2cells with Bewo cells to validate the pro-apoptotic effect of Galectin-3secreted by trophoblastic cells on endometrial cells.Results:All concentrations of17β-estrodial, progesterone and human chorioic gonadotropin could increase expression and secretion of Galectin-3in Bewo cells. Recombinant Galectin-3protein induced apoptosis of RL95-2cells and up-regulated integrin β1expression. By co-culturing Bewo and RL95-2cells, we found that secreted Galectin-3by Bewo cells induced apoptosis of RL95-2cells and Galectin-3antibody reversed this phenomenon.Conclusions:The expression and secretion of Galectin-3are regulated by hormones. The secreted Galectin-3in maternal-fetal interface induces apoptosis of endometrial epithelial cells through up-regulating integrin β1.