| Implantation is a reproductive process exclusive to mammals. The implantation process is currently considered the most relevant limiting factor for successful pregnancy. Its mechanism is very complex, and involved in many aspects. The process requires fine co-ordination of embryo and endometrium, including the synchronization between embryonic and uterine development. The process of implantation is an efficient point to regulate the reproduction. Although much attention has been paid to the study of implantation, many questions, such as the roles of proteinases, cytokines, remain to be solved. The technique of PCR-select Subtraction was applied to identify the mouse genes related to implantation. With the RsaI-digested cDNA of mouse implantation sites on day 4.5 of pregnancy as Tester, and excessive RsaI-digested cDNA of mouse inter-implantation sites on day 4.5 of pregnancy and day 5 embryo as Driver, the hybridization was carried out to get a subtraction cDNA library of mouse uterus implantation site. After PCR amplification and agarose electrophoresis, clones with inserted DNA fragments in different length were selected to sequence. Three new ESTs (EST8, EST60, EST81) were obtained and enlisted in GenBank. The accession numbers were BG797173, BG797174, BG797175 respectively. In addition, EST73 was found to have the same sequence with one part of ISP2 ORF sequence, which was reported by O(Sullivan. With the 32P-labelled targeted EST fragments as probes, Northern blot was carried out. The result showed EST73 was expressed only in uterus on day 4.5 of pregnancy, EST8 and EST 81, besides in uterus implantation sites, expressed in liver and ovary respectively, and EST60 was mainly expressed in epididymis, low expression was also found in uterus implantation sites on day 4.5 of pregnancy. The primers were designed according to the homology of ESTs (EST8, EST73, EST81) with known genes in GenBank. PCR was used to amplify the corresponding cDNAs, and three cDNAs were obtained. Nucleotide BLAST Searches were performed to compare the sequence of cDNAs with that of known genes in GenBank, and they were designated as E4BP4, ISP2, RGS2 cDNA respectively. The sequence of the three cDNAshad been enlisted in GenBank, their accession numbers were AY061760, AF442819 and AF432916. It is reported that ISP2 may be a proteinase associated with zona lysis and implantation. In order to investigate its biological functions, it is necessary to prepare ISP2 antisera. GST expression system was used to express fusion protein GST-ISP2.Inclusion bodies were formed and the fusion protein was presented in pellet, and was solublized only in detergents. Only SDS-PAGE could be used to purify the fusion protein and ISP2 protein. Rabbits were immunized with expressed ISP2 and ISP2-specific antisera were obtained. Using the prepared antisera in the dilution of 1:200 as primary antibody, a specific blot was seen in Western blot analysis. And, in the corresponding immunohistochemical analysis, the expression of ISP2 protein could be observed in mouse endometrial glands during early implantation, which was consistent to the previously reported results of in situ hybridization. The function blocking test using ISP2 antibody is being carried out.In above-mentioned Northern blot, high expression level of RGS2 mRNA was only detected in mouse uterus at implantation sites on 4.5-day of pregnancy and ovary, and significantly different expression level was seen between implantation and interimplantation sites. There is no report on RGS2 related to implantation till now. Northern blot and in situ hybridization techniques were used to investigate the expression pattern of RGS2 in mouse uterus and embryo. Northern blot analysis showed that, the positive signal of RGS2 mRNA could be detected both in implantation sites and inter-implantation sites, and its expression level at implantation sites was much higher than inter-implantation sites on day 5 to 9 of pregnancy. In situ hybridization localized this mRNA predominantly in the de...
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