The Expression And Regulation Of EBP50in Mouse Uterus During Blastocyst Implantation

Objectives:Embryo implantation is a crucial process for successful pregnancy.Till now the mechanism is still unclear. EBP50(ezrin-radixin-moesin-binding protein-50-kDa)a is scaffold protein which has been proved to play an important role in cancer development.Implantation and cancer has the similar progression,so in this article,we design this experiment.The aim of this research is to investigate whether EBP50locate in the mouse uterus during the peri-implantation period, and its expression pattern.We also utlize mouse models to detect if the expression of EBP50is influnced by the implanting embryo or steriod hormon,we also want to explore the mechnisms of EBP50during early pregnancy.Methods:1. The female mice were caged overnight with fertile males of the same strain to induce normal pregnancy. The presence of a vaginal plug or sperm was considered to be day1of pregnancy (D1). In normal pregnant mice, uteri were collected from D1-D4, and the uteri together with embryo were collected from D5-D8.2.Pseudopregnancy, artificial decidualization, steroid hormone treatment models, delayed and activated implantation models were made.3.Immunohistochemistry staining and western blot was used to examine the spatiotemporal expression and regulation of EBP50protein in the mouse uterus.Results:1. On D2-D4of pregnancy, the immunostaining of EBP50was mainly detected in the apical membrane of the glandular and luminal epithelium. On D5of pregnancy, at the implantation site (IS), further away from the implantation site, we observed a stronger immunostaining signal mainly localized in the nuclei. Immunostaining was also observed in the glandular and luminal epithelium At the inter-implantation site (INT), the immunostaining was similar to that on D2-D4. On D6-D8, a positive signal was seen in decidual cells as well as in glandular and luminal epithelia, the EBP50staining strength in the epithelium was markedly higher than that in the decidual cells. Western blot analyses showed that at the IS, the EBP50expression level was lower than that at the INT.2. EBP50signal was mainly found in the uterine glandular and luminal epithelia during D1-D5of pseudopregnancy.3. Immunohistochemical analyses showed that during delayed implantation, the main signal appeared in the uterine glandular and luminal epithelia. After implantation was activated by estrogen treatment and the embryos had implanted, EBP50expression was found in the nuclei of stroma cells as well as the glandular and luminal epithelia which resembled the expression on IS of D5. From Western blot analyses, we observed that after activated implantation, the activated implantation sites had lower levels of EBP50expression compared with that at the implantation sites (P<0.05).4. In the model of experimentally induced decidualization, In the oil-infused uterus, strong signals were detected in the luminal and glandular epithelia, and a relatively weaker staining was observed in the induced decidual cells. The expression level of EBP50in the decidualized uterus was clearly lower than that in the nonstimulated uterus on D8of pseudopregnancy (P<0.05).5. EBP50expression was only seen in the luminal and glandular epithelia of the ovariectomized mouse uterus, without any steroid hormone treatment.In the estrogen-treated group, in addition to the apical staining of epithelial cells, we also detected staining in the stroma cell nuclei. Furthermore, in the progesterone-treated group and in the estrogen plus progesterone-treated group, EBP50expression was primarily present in the luminal and glandular epithelia, the staining was darker in the apical part compared with that in the cytoplasm. Western blot analyses showed that the estrogen-treated group had higher levels of EBP50protein expression than the other groups (P<0.05).Conclusions:Collectively, we suppose that in the peri-implantation stage, EBP50mainly locating in the apical cell membrane maintains the normal cells’shape and function; after embryo attaching the uteri epithelia, EBP50shifts in nuclei and participate in the transformation of stroma cells to decidual cells; when the decidual cells have formed, EBP50moves in cytoplasm to promote proliferation and differentiation.