Expression And Regulation Of Galectin-3 In The Patients With Endometriosis

Endometriosis is a common, benign, estrogen-dependent gynecological disorder,frequently associated with pelvic pain and infertility, affecting～10% women ofreproductive age. Although the association between endometriosis and infertility iswell established, the molecular mechanisms remain to be determined. Multiple factorshave been implicated including extensive pelvic adhesions, distortion of the pelvicanatomy, abnormalities of hormone secretion, molecule alteration in peritoneal fluid,disorders of fertilization and immuno-regulatory dysfunction, endometrialdevelopment and so on. However, recent studies showed that patients withendornetriosis have decreased significantly implantation rates than healthy controls.Futhermore, animal model of endometriosis also demonstrates that eutopicendometrium is a lack of molecular makers of endometrium receptivity. Therefore,we can propose that alterations of eutopic endometrium receptivity may be animportant mechanism of endometriosis associated infertility.Galectin-3 (Gal-3) is a member of a growing family ofβ-galactoside-bindinganimal lectins with evolutionary conserved sequence elements of carbohydrate-recognition domain (CRD), to participate in a variety of biological and pathologicalprocesses, including modulate cell-cell or cells-extracellular matrix interactions. Inrecent years, galectin-3 function in cell adhesion, proliferation, migration has beenrecognized more and more in the fertility field. Our research group demonstrated thatGal-3 was a candidate gene related with endometrial receptivity. However, there isstill a lack of domestic and international journal coverage on how Gal-3 effects onendometrial receptivity. In order to identify the relationship between the alterations ofeutopic endometrium receptivity and infertility, we systematically investigated theexpression and regulation mechanism of Gal-3, to explore the mechanism of infertilityin endometriosis patient. Partâ… Expression of galectin-3 in endometrium of patients withendometriosisObjectives: The aim of the study is to investigate the expression and significance ofGal-3 in human eutopic endometrial tissues of patients with endometriosis.Methods: Proliferative and secretory phase endometrium from women with andwithout endometriosis were studied. Immunohistochemistry and Western blottingwere carried out to detect the protein localization and expression of Gal-3. Gal-3mRNA expression was evaluated by real-time RT-PCR.Results: Gal-3 located in luminal, epithelial and stromal cells of the endometirum ineutopic and control endometrium. There was a significant decrease of Gal-3 stainingin eutopic endometrium compared to controls. Gal-3 immunostaining intensity wasstronger in secretory phase than in proliferative phase either in eutopic endometriumor in control. As compared with the control group, the Gal-3 mRNA expression hadsignificantly greater reduction in eutopic endometrium (P＜0.001). Either in eutopicendometrium or in controls, Gal-3 mRNA expression increased significantly in midsecretory phase than in late proliferative phase. The western blot finding wasconsistent with the Gal-3 mRNA expression.Conclusions: The Gal-3 mRNA and protein expression in the patient withendometriosis is significantly lower than that in the control group.Partâ…¡In vitro study on expression of galectin-3 in endometrialcells of patients with endometriosisObjective: To examine the expression and significance of Gal-3 in endometrialglandular epithelial cells (EEC) and endometrial stromal cells (ESC) of the patientswith endometriosis.Methods: EEC and ESC were enzyme-dispersed and isolated from humanendometrium and cultured. Immunohistochemical staining for cytokeratin 7 and vimentin confirmed the origin of endometrial cells. The levels of Gal-3 mRNA andprotein in cultured human EEC and ESC were analyzed by using RT-PCR andWestern Blotting respectively.Results: The cell purity of at least 90% for both cell types. At the transcriptional level,we observed, a increase in Gal-3 expression in EEC versus ESC either in control or inendometriosis group (P＜0.01). Either in endometriosis or in controls, Gal-3 mRNAexpression in EEC is significantly higher than in ESC(P＜0.01). Gal-3 mRNAexpression in EEC and ESC increased significantly in secretory phase than inproliferative phase, either in endometriosis or in controls (P＜0.01). The western blotfinding was consistent with the Gal-3 mRNA expression that the Gal-3 proteinexpression in the endometriosis group reduced significantly than that in the controlgroup. At the protein level, a decrease was observed in Gal-3 expression in ESCversus EEC either in control or in endometriosis group (P＜0.05).Conclusion: Both EEC and ESC in culture can retain the ability to synthesize andsecrete Gal-3. Cultured EEC expresses significantly more Gal-3 mRNA and proteinthan ESC. The expression of Gal-3 in endometriosis is significantly lower than that inthe control group.Partâ…¢Effect of ovarian steroid hormones on galectin-3 expressionin cultured endometrial epithelial cells from the patients withendometriosisObjective: To investigate the effects of 17β-E₂ and MPA on the expression of Gal-3expression in the cultured human EEC.Methods: EEC isolated and cultured from EMT and control group were treated with17β-E₂ (10-^-10mol/L to 10^-6mol/L) and MPA (10^-9mol/L to 10^-5mol/L) for 24h toinvestigate the effect 17β-E₂ and MPA on Gal-3 transcriptional level. Then theglandular epithelial cells were treated for 24h, 48h, 72h, respectively, with E₂(10^-8mol/L), MPA (10^-7mol/L), E₂(10^-8mol/L)+MPA (10^-7mol/L). The control groupreceived no treatment. The transcriptional level of Gal-3 in the cultured human EECwas analyzed by using RT-PCR. Results: In normal group, 10^-9M(2.748±0.378), 10^-8M(6.767±0.737), 10^-7M (3.732±0.826),10^-6M(2.730±1.023) E₂-treated EEC express Gal-3 significantly morethan untreated group (P＜0.05). 10^-9M(4.560±0.566),10^-8M(6.597±0.735),10^-7M(10.609±0.335), 10^-6M(3.946±0.860), 10^-5M(2.263±0.966) MPA-treatedEEC induced the up-regulation of Gal-3 mRNA level than untreated group (P＜0.01).In the control group, up-regulation of Gal-3 mRNA level in a time-dependent manner,with the strongest effect observed at treated 72h and in the order of MPA treatedalone＞E₂plus MPA treated＞E₂-treated alone (P＜0.01). In EMT group, Gal-3expression was significantly increased in the order of 24h＞48＞72＞unprocessed group(P＜0.01). The estrogen-treated alone, MPA-treated and estrogen plus MPA treateddecreased not with time as shown on the Gal-3 table. Estrogen plus MPA group ishigher than the estrogen processed group and MPA processed group, with thestatistical significance of P＜0.01. However, estrogen processed group and MPAprocessed group had no statistical significance (P＞0.05).Conclusion: Estrogen and MPA up-regulate the Gal-3 expression with dose-dependency. MPA-treated Gal-3 expression has the most significant catalytic effects.Estrogen degreases the boosting effects on MPA in the Gal-3 display. Processed MPAshows significantly increase in Gal-3 from the comparison group, it decreases withprocess time in the endometrial glandular epithelial cells from EMT patients. Gal-3expression in the patients with endometriosis exhibits progesterone insensitivity,which could involve in the mechanisms of endometriosis-related infertility.Partâ…£The regulation of NF-_κB signaling pathway on galeetin-3expression in endometrial epithelial cells from patient withendometriosisObjective: NF-_κB is a general transcript factor, mediating one of the classics signalpathways through membrane receptors and a key in embryo implantation. Thepurpose of the part is to explore whether Gal-3 expression and its upstream regulationin endometrial glandular epithelial cells through the NF-_κB signaling pathway.Methods: EEC isolated and cultured from EMT and control group were treated with MG132 for 1h. Then EEC were treated with E₂ (10^-8mol/L), MPA (10^-7mol/L), E₂(10^-8mol/L)+MPA (10^-7mol/L) for 24h. The control group received no MG132. Theprotein level of Gal-3 in the cultured human EEC was analyzed by using Westernblotting.Results: In EMT group, compare with the blank, MG132 alone treated group expressno Gal-3. The Gal-3 protein expression in EEC is significantly up-regulated in theorder of treated by MG132 plus MPA, MG132 plus E₂ and MG132, E₂plus MPA (P＜0.01). In normal endomtrium, compare with the blank, MG132 alone treated groupexpress no Gal-3. The Gal-3 protein expression in EEC is significantly up-regulatedin the order of treated by MG132 plus E₂, MG132, E₂ plus MPA and MG132 plusMPA (P＜0.01). MPA treated group can up-regulate the Gal-3 expression in EEC ofnormal endometrium, but the up-regulation in EMT group is the weakest. Gal-3expression in estrogen group is higher than in the estrogen plus MG132 processedgroup and MPA processed group is higher than the MPA plus MG132 processedgroup, with the statistical significance of P＜0.01 in EMT group. Gal-3 proteinexpression in the estrogen plus MG132 and MPA plus MG132 is significantly higherthan treated only by MG132 (P＜0.01). Gal-3 protein expression in the estrogen andMPA treated group is significantly higher than untreated group (P＜0.01).Conclusion: Gal-3 expression in EEC is mediated by NF-κB signaling pathway.Estrogen and MPA, the upstream factor on Gal-3, could involve in regulating theGal-3 expression in EEC via NF-κB signaling pathway.