ABSTRACT
Objective To investigate the expression and distribution of leptin in pre-implantation embryo,and its effect on mouse embryo development in vitro. Methods Adult NIH female mice were superovulated and then mated with fertile males of the same strain. On the morning of 1st to 4th d of pregnancy,the oviduct and horn of uterus were dissected out and flushed with DMEM/F12 medium adequately. The embryos collected were subjected to the following procedures: ①Total RNA was isolated from embryos of each stage respectively and mRNA expression of leptin was detected by RT-PCR. ②Immunofluorescence staining was performed on blastocysts to analyze the expression and the distribution of leptin protein under laser confocal microscope. ③Eight-cell embryos were cultured in medium containing leptin antibody at various concentrations to observe the formation and hatching of blastocysts. Results ①Leptin mRNA expression was only detected in blastocyst,and no expression was detected in embryos in other stages. Leptin protein was detected in cytoplasma,membrane of trophoblastic cell and inner cell mass,however no expression was detected in cell nucleus. ②Leptin polyclonal antibody significantly inhibited formation of blastocysts (1∶400,P
ABSTRACT
Objective To investigate the effect of recombinant adenovirus containing CD82/KAI1 on the expressions of integrin ?5,integrin ?3,MMP-9,E-cadherin and ?-catenin in uterine stromal cells.Methods Uterine stromal cells were isolated from pregnant mice during implantation window and cultured primarily.The recombinant adenovirus containing mouse CD82/KAI1 gene was constructed.Protein expression of integrin ?5,integrin ?3,MMP-9,E-cadherin and ?-catenin were detected by immunocytochemical methods in the cultured cells with or without CD82/KAI1 adenovirus transfection.Results When the cells which were cultured for 48 h were transfected with the recombinant adenovirus vector at a titer of 6.5?1012 pfu/ml,CD82/KAI1 were obviously upregulated in the primary uterine stromal cells.In the uterine stromal cells from pregnant mice on the 4 d after gestation,integrin ?5,E-cadherin,?-catenin and MMP-9 were detected,while,integrin ?3 were not.But in the cells of nonpregant rats,no expression of these 5 proteins was found.Compared with the cells transfected with blank adenovirus,the recombinant adenovirus encoding CD82/KAI1 upregulated the expressions of ?-catenin and MMP-9(P0.05)and no integrin ?3 was detected in the stromal cells of pregnant mice on day 4 of gestation.Conclusion The recombinant adenovirus containing mouse CD82/KAI1 gene is successfully constructed.CD82/KAI1 might have certain effect on the expressions of integrin ?5,MMP-9,E-cadherin and ?-catenin in mouse uterine stromal cells before embryo implantation.