| ObjectiveImplantation is an important stage during mammal reproductive processes. Up to now, it is not clear about the molecular mechanism of the embryo implantation. Endometrium experiences morphological and biochemical changes which result from changes of protein expression during this stage. A number of proteins were involved in the implantation. In the present study, we analyzed the proteome from the implantation sites and the peri-implantation sites on Day 5 of gestation in NIH mice by using comparative proteomics. The aim of this work is to acquire knowledge about the mechanism involving in the embryo implantation.Methods1. Animals and tissue preparation On Days 5 of pregnancy, the implantation sites were separated from the periimplantation sites and immediately stored in liquid nitrogen until processed. The tissues were also fixed by immersion in 10ï¼… neutral buffered formalin for 12-24 h and processed for paraffin embedding by the standard method. The matched virgin mouse endometrium was analyzed as the control. 2. Two-dimensional gel electrophoresis and analysis of gel-separated proteinsThe endometrium proteins 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 using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Coomassie Blue Staining of two-dimensional gel electrophoresis gels were subjected to analysis using GS-710 Calibrated Densitometer and PDQuest 6.0 Software. 3. MALDI-TOF mass spectrometry analysis of the target protein Target protein spots were excised from the gel and the 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. The peptide mass fingerprint was then submitted to an appropriate software (Mascot: Peptide Mass Fingerprint) in order to identify the protein (http://www.matrixscience.com/cgi/search_form.). Score is -10*Log(P), where P is the probability that the observed match is a random event. Protein scores greater than Top Score are significant (p<0.05).4. Further verification of target proteins at protein and mRNA level RT-PCR,Western Blot and immunohistochemical analysis were used to further verify some target proteins which were identified unambiguously by sequence database searching .Results1. 2-D PAGE analyses 2-D PAGE profile showed that the proteins in the endometrium were located mostly in the range of mass 14.4~75.4 kDa and isoelectric points (pI)4~8. 723,891 and 924 protein spots with a molecular mass 10.0~97 kDa range and isoelectric points (pI)3~10 were catalogued from Coomassie Brilliant Blue-stained gels of the virgin endometrium,the peri-implantation sites and the implantation sites respectively. The number of matched protein spots among three gels was about 78%. At least 60 protein spots were up-regulated or down-regulated by over two folds, when compared to each other among three gels.2. MALDI-TOF and sequence database search13 differentially expressed protein spots were analysed by MALDI -TOF-MS. 5 protein spots of them were identified unambiguously by sequence database searching. They are Peroxiredoxin 1 (Prx 1), Nucleoside diphosphate kinase B(DNPK B/nm23-M2), Apolipoprotein A-I (Apo-AI), Galectin-1 respectively, among which, 2 protein spots were identified as Apo-AI. DNPK B/nm23-M2, Apo-AI and Galectin-1 showed up-regulated expression in the endometrium during the blastocysts adhesiveness compared with the control group, especially in the implantation sites. Prx 1 was significantly higher in the peri-implantation sites compared with the implantation sites and the control group.3. Level of Galectin-1 mRNA in the endometriumLevel of Galectin-1 mRNA was significantly increase in the implantation sites and the peri-implantation sites compared with the control group (p<0.01), the implantation sites was more significantly highe...
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