| Objective:To investigate the potential role ofβ-TrCP,β-catenin, IκBαduring early pregnancy, RT-PCR, immunohistochemistry, Loser scanning confocal microtechnic, Western blotting have been applied to detectβ-TrCP,β-catenin, IκBαmRNA and protein expression, distribution and dynamic change in the endometria of mice from pseudopregnancy and day 1 to day 7 of pregnancy, respectively.Methods:1. The NIH mouse models of early pregnancy and pseudopregnancy were established respectively. The endometria of mice from day 1 to day 7 of pregnancy were used to following study. Pregnant mice (D1, D2, D3, D4, D5, D6, D7) were randomly divided into 7 groups and pseudopregnant mice were control group. Both pseudopregnant and pregnant mice endometria were collected, and then immediately stored at -80℃for further test.2. After injecting Trypan blue into pregnant mouse body through tail vein on the day 5 of pregnancy, we collected the uterus and endometria of implantation site and peri-implantation site, respectively.3. The expression ofβ-TrCP mRNA,β-catenin mRNA, IκBαmRNA in the endometria of early pregnant and pseudopregnant mice was deteced by RT-PCR.4. The expression ofβ-TrCP,β-catenin, IκBαprotein in the uterus and endometria of early pregnant and pseudopregnant mice was detected by Western blotting, immunohistochemistry.5. The expression ofβ-TrCP and IκBαprotein in the uterus of pseudopregnant mice, implantation site and peri-implantation site of pregnant mice was detected by Loser Scanning Confocal microtechnic.Result:1. After injecting Trypan blue into pregnant mouse body through tail vein, the blue-dyed zone of implantation site can be observed obviously, which can be easily distinguished with peri-implantation site.2. The spatio-temporal differences of expression ofβ-TrCP,β-catenin, IκBαmRNA in mice endometria during early pregnancy were shown. The expression level ofβ-TrCP reached a maximum on day 4 of pregnancy (the window of implantation). On the contrary, the expression level ofβ-catenin, IκBαreached a minimum on day 4 of pregnancy respectively.3. By immunohistochemistry, the positive expression ofβ-TrCP,β-catenin, IκBαprotein has been observed in endometria of pregnant and pseudopregnant mice. The expression ofβ-TrCP protein gradually increased from day 1 to day 4 of pregnancy and reached its summit on day 4 of pregnancy. The lower expression ofβ-TrCP was observed in luminal epithelium, glandular epithelium of uterus of pseudopregnant mice, but gradually increased in luminal epithelium, glandular epithelium on day 1, day 2, day 3 of pregnancy, and in stromal cells on day 4 of pregnancy. From day 5 of pregnancy, the expression in stromal cells gradually decreased. The expression ofβ-catenin, IκBαprotein was lower in uterus of pseudopregnant mice, and gradually increased from day 1 to day 3 of pregnancy. However, the expression reached a minimum on day 4 of pregnancy, and gradually increased from day 5 of pregnancy. The expression ofβ-catenin and IκBαlocated at stroma cells.4. The result from indirect immunofluorence histochemistry shown thatβ-TrCP, IκBαprotein expressed in uterus of pseudopregnant mice, implantation site and peri-implantation site of pregnant mice. The expression ofβ-TrCP protein in implantation site was stronger than that in peri-implantation site(p<0.05). On the contrary, the expression of IκBαprotein in peri-implantation site was stronger than that in implantation site(p<0.05). The green fluorescence signal ofβ-TrCP located at cytoplasm of glandular epithelium, luminal epithelium, also in stomal cell. The red fluorescence signal of IκBαlocated at cytoplasm and nucleus of glandular epithelium, luminal epithelium and stomal cell. 5. The result of Western blotting was consistent with the result of the immunohistochemistry and indirect immunofluorence histochemistry.Conclusion:1.β-TrCP,β-catenin, IκBαmRNA and protein have been observed in the endometria of pseudopregnant and pregnant mice, and the spatio-temporal differences of expression ofβ-TrCP,β-catenin and IκBαin mice endometria during early pregnancy were observed.2. As the member of SCF (Skp1-Cull-F-box) complex,β-TrCP participated in ubiquitination degradation of phosphorylation ofβ-catenin and IκBαby the proteasomes. The higher level expression ofβ-TrCP leads to the lower level expression ofβ-catenin and IκBαin cytoplasm.β-TrCP is the negative regulation factor of Wnt/β-catenin signal pathway as well as the positive regulation factor of NFκB signal pathway. The fact of the higher level expression ofβ-TrCP on day 4 and day 5 of preganancy was consistent with the inhibition of Wnt/β-catenin signal pathway and the temporal activation of NFκB signal pathway during the implantation window phase.3. The fact that the expression level ofβ-TrCP protein in implantation sites was obviously higher than in peri-implantation sites indicated thatβ-TrCP may participate in the invasion process of embryo implantation.
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