Research On Mouse Endometria Proteome Of Different Stages In Blastocyst Implantation

ObjectiveProcess of blastocyst invadeing endometrium is called implantation. It is complex and subtile, and related to blastocyst adhering,differentiation and synchronizing development of endometrium. The blastocyst couldn't successfully implant until endometrium achieve reception status by a series of complicated changes. But the mechanism is unclear. Some researches at present cannot explain the whole process due to considering only single protein. We analyzed the difference of mice endometria proteome to different stages of blastocyst implantation by technologies correlated to proteome, it would be helpful to approach the mechanism of blastocyst implantation from level of proteomeMethods1. Construct the NIH mouse pregnant animal model, endometrium were isolated from the mouse at pregnant d3,d5,d7, then frozen at -80℃respectively.2. proteins of the mouse endometrial tissue from pregnant d3,d5,d7 were separated by Two-dimensional gel electrophoresis (2-DE). These proteins were stained with Coomassie Brilliant Blue and analysised by PDQuest Software after3. Twelve differential expressed proteins were excised by dot-gel digestion with trypsin. Peptide mass fingerprinting (PMF) can be obtained through analysis of fragment length with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Then PMF was submitted to an appropriate software (Mascot: Peptide Mass Fingerprint) for identifying the protein. According to the probability of each score (from http://www.matrixscience.com/cgi/search_form), we can evaluate if there is signifcant to each results. The threshold is usually acceptable if there is less than 5% probability of a random incident ,and the incident is significant (p<0.05).4. Collectting mouse endometrium at pregnant d3,d5,d7, to measure mRNA and level of Annexin A1 expression by RT-PCR,Western blotting technologies. By immunohistochemical methods and in situ hybridization, detection of the distribution of Annexin A1 and Annexin A1 mRNA in mouse endometria were detected and semi-quantitative analysis were carried out.Results1. 2D-PAGE profile showed that protein molecular weight (Mw) of mouse endometrial is concentrated mainly in the area 14.4~116KDa and isoelectric points (PI) located in between PH4.0 and PH 8.0. By comparing proteins atlas at pregnant d3, d5 and d7(analyzing with PDQuest7.3 software), the results showed that protein atlas at pregnant d3, d5, d7 is broadly consistent . The protein spots number is 713,736,673 respectively. Comparation between pregnant d3, d7 and pregnant d5 using atals of pregnant d5 as a reference, matching rate is 68%,61% respectively. The total number of differential expression protein above 3 fold is 105. Compared to pregnant d3, there are 24 proteins up-regulating and 7 proteins down-regulation above 3 fold at pregnant d5 ; compared to pregnant d7, there are 32 proteins up-regulating and 19 proteins down-regulating above 3 fold at pregnant d5. Compared to pregnant d7, there are 15 proteins up-regulating and 8 proteins down-regulation above 3 fold at pregnant d3.2. Twelve differential expressed protein spots were identified by MALDI-TOF-MS. By database searching, five proteins of them were got significant result(p<0.05). They are apolipoprotein A1(Apo-AI),Annexin A1,Glutathione transferase omega-1(GSTO1),Serine protease inhibitor A3M(SPA3M),Alpha-enolase(ENOA).3. By analysis of RT-PCR, western blotting, immunohistochemistry and in-situ hybridization, these results showed that expression of Annexin A1 mRNA and Annexin A1 is the strongest at pregnant d5, stronger at pregnant d3, weaker at pregnant d7. Annexin A1 mainly locates in glandular epithelium and luminal epithelium in endometria.Conclusion1. Apo-AI can be detected in early pregnant period and continued until late period of implantation. The result suggested that Apo-AI might play an important role for promoting implantation. Expression of Annexin A1 at pregnant d3 and d5 was significantly higher than that of pregnant d7. It suggested that this protein might make endometrium reach a state of embryo implantation by regulating process of apoptosis, inflammatory response and many other ways. GSTO1-1 promotes possibly embryo implantation through regulation of local immunity. Serine protease inhibitor A3M can be expressed in whole process of embryo implantation, and the strongest at pregnant d5. Suggestting that it be possible to inhibit the activity of protease to prevent excessive matrix degradation. It may be involved in the embryo implantation. Alpha-enolase's expression is the highest at pregnant d3, it indicates that the protein may be involved in extracellular matrix degradation and prepare for the invasion of trophoblast cells.2. Annexin A1 expressed in glandular epithelium and luminal epithelium of mouse endometrium in defferent stages of implantation, and the expression has strongly time limited characteristics. Express of pregnant d5(implantation widow) is the maximum. So we infer that Annexin A1 may participate in embryo adhesion to endometrium and decidualization.3. The results of this study showed that most of differential expressed proteins of the endometria in peri-implantation stage are involved in immune response and uterine stromal degradation. These proteins may play an important role in regulating endometrium to achieving the best receptive state for implantation. Because expression peak and time of Annexin A1 is highly coincident to embryo implantation. It implies that Annexin A1 is closely related to the embryo implantation.