| Summary: | 碩士 === 國立陽明大學 === 解剖暨細胞生物學研究所 === 92 === During embryo implantation in mammals, trophoblast cells of the attached blastocyst penetrate the endometrial epithelium of the uterus before invasion into the endometrial stroma. Signaling of apoptosis was demonstrated in endometrial epithelial cells (EEC) surrounding the trophoblast cells; however, the signaling mechanisms leading to apoptosis in EEC remain unclear. Since mitogen-activated protein kinases (MAPKs) were shown to mediate apoptosis in several model systems and found to be activated in the uterus during decidualization, and then evidenced the important role of induction of p38 MAPK-mediated apoptosis involved in outgrowth of trophoblast cells on EEC in a model of human trophoblast-endometrial interactions, rather not ERK. The Fas-Fas Ligand (FasL) system is one of the major for the induction of apoptosis in cells and tissues. However, the Fas/FasL death system was found to be active at the embryo-endometrial interface, and in various cell models, activation of members of the MAPK superfamily was demonstrated to either mediate Fas-induced apoptosis or enhance FasL expression. The objective of this study were to investigate the roles of JNK activation and Fas/FasL system during embryo implantation using a co-culture model by co-culturing BeWo human trophoblast spheroids with RL95-2 human EEC monolayers to mimic the blastocyst-endometrial interaction. First, immunoblotting analysis showed that JNK were activated in EEC after co-culture. However, SP600125 (a JNK inhibitor) inhibited trophoblast outgrowth on EEC monolayers through the suppression of JNK activation in EEC and trophoblast cells. Second, MTT assay showed that Fas and FasL expressed on RL95-2 cells were functional to Fas-mediated apoptosis by using anti-Fas activating Ab, but BeWo cells was not observed. Then, trophoblast spheroid outgrowth on EEC monolayers was not significantly inhibited by anti-Fas neutralizing Ab, but spheroid outgrowth on EEC was notably increased by anti-Fas activating Ab. Furthermore, trophoblast expansion on EEC in the presence of anti-Fas activating Ab caused prominent EEC apoptosis at the spheroid-EEC interface compare with in the absence of anti-Fas activating Ab, as detected by annexin V labeling and valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (VAD-FMK, which binds activated caspases) staining. Then, Z-VAD (pan-caspase inhibitor) appeared to have minimal effect on spheroid outgrowth, whereas Z-VAD significantly inhibited spheroid expansion on EEC monolayers that anti-Fas activating Ab was existed. In addition, SB203580 and SP600125 inhibited spheroid outgrowth enhanced by anti-Fas activating Ab on EEC monolayers, respectively, and p38 MAPK and JNK were both activated in EEC exposed to anti-Fas activating Ab. Then, using VAD-FMK staining technique for detecting caspase activation, marked activated caspase-positive cells along the spheroid-EEC interface in the presence of anti-Fas activating Ab that could be significantly suppressed by SB203580 and SP600125, respectively. Our results based on a model of human trophoblast-EEC interactions establish that trophoblast cells cause activation of JNK in EEC and trophoblast cells. In addition, our results also suggests that Fas/FasL system was not one of major for the mechanism of attachment between trophoblast cells and EEC, but may play an important role in trophoblast invasion, such as using anti-Fas activating Ab in our study. The signal was transferred by activating p38 MAPK and JNK in EEC, respectively, and then inducing apoptosis in EEC at the inter-implantation site and implantation site through the caspase-dependent pathway and caspase-independent pathway, therefore EEC were easy to be displaced and trophoblast spheroids outgrowth were easy successful.
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