Proteomic Analysis Between Villus And Decidua Tissues In The First Trimester Pregnancy

ObjectiveThe successful implantation depends on the synchronization development between endometrium and blastocyst. The proteins are the true material base in this process. We analyzed the proteome from the deciduas and the villus in the first trimester pregnancy by using comparative proteomics. The aim of this work is to acquire knowledge about the mechanism involving in the embroy implantation.Methods1. The villous and the deciduas in the first trimester pregnancy were collected and stored in -80℃.2. The villous and the deciduas were separated in the first dimension by isoelectric focusing in linear pH 3~10 immobilized pH gradient (IPG). The separation in the second dimension was performed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Coomassie Blue Staining of two-dimensional gel electrophoresis gels were analysised by PDQuest Software. 3. The target protein spots were excised from the gel and digested with trypsin. The MALDI-TOF mass spectrometer was operated in the reflector mode. A mass list of peptides was obtained for the target protein digest. Then peptide mass fingerprint was submitted to an appropriate software (Mascot: Peptide Mass Fingerprint) for identifying the protein (http://www.matrixscience.com/cgi/search_form).4. The expression of Annexin A1 in villous and deciduas tissues were verified by RT-PCR,Western Blot and Immunohistochemical analysis .Results1. 2-D PAGE profile showed that molecular mass of these proteins in the deciduas and the villus tissues were located mostly in the range of 18.4~116.0 kDa and isoelectric points(pI) distribute in the range of 4~7. The matched rate of protein spots was about 45% between the villus and the deciduals gels by PDQuest Software. About 680 and 621 protein spots were isolated from the villus and the deciduals gels respectively. 60 proteins were significantly up-regulated between the two groups, 33 proteins were significantly up-regulated in the villus compared with the deciduas, 27 proteins were significantly up-regulated in the deciduas compared with the villus.2. Twelve differential proteins were randomly chosen from the 60 proteins, and analysized by MALDI -TOF-MS. Seven protein spots of them were identified clearly by sequence database searching. They are ANXA1, HSP90β, APOA1, VIME, ANXA5, TPM4, PDIA3.3.Among twelve differentially expressed proteins, the five proteins of them expressed highly in villus and seven proteins expressed highly in decidua.4.Ananlysis by RT-PCR, level of ANXA1 mRNA in the villus was significantly up-regulated compared with the deciduas. Western Blot analysis also demonstrated that level of ANXA1 in the villus was up-regulated. ANXA1 was detected in villus by Immunohistochemistry, the result showed that ANXA1 in villus cells were stained cinnamomeous and the cinnamomeous distribution were widespread, especially endochylema of trophoblastic cell were stained strongly. This also can been found in endochylema of endothelial cells and decidual cells, but light staining than trophoblastic cell. Obvious Immunoreactivity was not observed in the control group.ConclusionA great many of proteins involve in the embryo implantation process. We have got initial results from the proteomics analysis between deciduas and the villus tissue in the first trimester pregnancy. Seven protein spots were identified clearly by sequence database searching, they are ANXA1, HSP90β, APOA1, VIME, ANXA5, TPM4, PDIA3.These proteins may play a critical role for embroy implantation.