The Expression Of T-lymphoma Invasion And Stromal Cell In Implantation Window Of Pregnancy Mice And Its Effect For Blastocyst Implantation

Background:As a guanine nucleotide exchange factor for Rac1, T-lymphoma invasion and metastasis inducing protein 1(Tiam1) encodes two PH domains and one Db1 homology domain. Overpression of Tiam1 can activate Rac1 of Rho family GTPase. And actived Rac1 play an important role in cytoskeleton rebuilding, cell adhesion and movement, regulation of gene expression, apoptosis, formation of axons and dendrites of nerve cells and cell cycle regulation.Tiam1 is involved in cell migration, assembly of adhesion complexes, tumor invasion, formation of protein complex,membrane localization and protein-protein interactions.Embryo implantation is a complex physiological process regulated by a large number of genes and proteins, and depends on a series of key events including blastcyst migration, apposition and adhesion to the luminal epithelium, extensive degradation and remodeling of extracellular matrix (ECM), invasion of the maternal endometrium by trophoblast cells. The synchronization between development of blastocyst and modifications of the maternal endometrium is crucial for successful embryo implantation.A great many of researches suggested that Tiam1 is involved in tumor cells migration, adhesion and invasion, extracellular matrix degradation. Similiarities between embryo implantation and tumor invasion suggest that Tiam1 might be involved in embryo implantation.Previous studies have shown:①Tiam1 expresses in endometrial of estrus mice.②Tiam1 express in early pregnancy mice endometrial stromal cells and the expression in endometrial of pregnant mice was gradually increased from pregnant day 1, reached a maximum level on pregnant day 5 and then declined on pregnant day 6 and 7. So, we speculated that Tiam1 might promote the movement,localization and adhesion of blastocyst by regulating molecule-mediated cell adhesion,and be involved in the invasion and extracellular matrix degradation processes during embryo implantation.Objective: To explore the expression of Tiam1 in stromal cells of endometrium in early pregnant mice and its effect in blastcyst adhesion in endometrial stromal cells and embryo co-culture system in vitro.Methods:1. The endometria separated from early pregnant mice were incubated with trypsin, and then the stromal cells of endometrium were tottered away. After dense culturing, the cultured cells were observed by light microscopy and the purity was identified by immuocytochemistry.(1) RT-PCR was used for detecting the expression of Tiam1 mRNA in endometrial stromal cells of pregnant mice and un-pregnancy mice during blastocyst implantation.(2) Immunocytochemistry was used for detecting the expression of Tiam1 protein in endometrial stromal cells of pregnant mice and un-pregnancy mice during blastocyst implantation.2. We established endometrial stromal cells of pregnant mice and embryo co-culture system in vitro. The anti-Tiam1 antibodies were diluted to different concentration gradient of 0mg/L, 100mg/L, 200mg/L and 400mg/L by DMEM-F12. And then the endometrial stromal cells of pregnant mice and embryo co-culture system were interfered with by different dilution of anti-Tiam1 antibodies. We count the number of blastocyst adhered in stromal cells in the different antibody concentrations. We also detected the expression of Tiam1 protein in different concentrations of antibodies by immunocytochemistry and Western blot.(1)Immunocytochemistry was used for detecting the expression of Tiam1 protein in endometrial stromal cells of implantation window of pregnant mice treated with different concentration of anti-Tiam1 antibodies.(2)Western blot was used for detecting the expression of Tiam1 protein in endometrial stromal cells of implantation window of pregnant mice treated with different concentration of anti-Tiam1 antibodies.Results:1. Immuocytochemical analyses showed that the endometrial stromal cells were obtained and the purity of stromal cell was 93.8±0.5%. Most cells became adherent in 24 hours after transfer of culture and formed tightly and they shaped long-fusiform after culturing 3 days.(1) RT-PCR results showed that Tiam1 mRNA expressed very highly in endometrial stromal cells of early pregnant mice in the implantation window and little in endometrial stromal cells of un-pregnant mice.(2) Immunocytochemistry analyses showed that the expression of Tiam1 protein were significant in endometrial stromal cells during blastocyst implantation of mice,but Tiam1 protein expressed little in endometrial stromal cells of un-pregnant mice .2. The rate of blastocyst adhesion was 34.41%, 13.82%, 20.62% and 24.00% when the antibody dilution were respectively 0 mg/L,100mg/L,200mg/L and 400mg/L. There is an obvious difference between treatment groups and control group (p<0.05). Immunocytochemistry and Western blot results showed that the expression of Tiam1 protein was gradually decreasing with the increasing concentration of anti-Tiam1 antibody, meanwhile the number of blastocyst adhesion was reducing.Conclusion: The expression of Tiam1 mRNA and protein were found in endometrial stromal cells of pregnant mice during blastocyst implantation but it was little expressed in the endometrial stromal cells of un-pregnancy mice. With the increasing concentration of anti-Tiam1 antibody, the number of blastocyst adhesion was reducing. All the results indicated that Tiam1 might be involved in the process of embryo implantation including blastocyst migration, apposition and invasion.