Identification And Functional Study Of Embryonic And Endometrial Factors Involved In Embryo Implantation Process

Embryo implantation is a critical step in reproductive process specifically owned by mammalian and human being, and is the most relevant limiting factor for successful pregnancy. In order to establish an active interaction between the implanting embryo and the maternal endometrium during a very limited timespan defined as the "implantation window", the embryo should synchronously develop to the blastocyst stage and escapte from the zona pellucida, when the endometrium becomes receptive. Such an exquisite coordination involves the regulation of hormones and various non-hormonal molecules produced by embryonic and maternal tissues, including growth factors, cytokines, proteinases.With a view to further understand the precise molecular mechanism underlying embryo implantation and to find out new targets for the development of novel fertility regulation techniques, our lab used the mouse as a model to identify the novel implantation-related genes and explored the roles these genes played in embryo implantation. The mouse uterine tissues of implantation sites and inter-implantation sites were respectively collected at the time of implantation (Day 5 of pregnancy), and the pre- and post-implantation embryos were also collected on Day 4 and Day 5 of pregnancy. Subsequently, the techniques of cDNA subtracted hybridization and DD-RT-PCR were respectively applied to screen the novel factors involved in mouse embryo implantation process. A large number of EST fragments of genes differentially expressed between the implantation and inter-implantation sites, or between the pre- and post-implantation implantation embryos, were screened out. However, having been short in labor power and research funds, just a small part of the EST fragment pools were sequenced for the further analysis, and finally 5 novel implantation-related genes were identified. The full-length cDNA of these genes were successively cloned and registered in the GenBank. They are E4BP4(AY061760), RGS2(AF432916), ISP2(AF442819), EMO-1 (AY238936) and EMO-2(AY372183).The results of GenBank Blast searches and references review showed that, E4BP4 is an IL3-regulated nuclear transcript factor, and no report on E4BP4 related to implantation was found; RGS2 participates in the regulation of G-protein signaling pathway and is involved in the regulation of intercellular Ca²⁺ mobilization and T cell proliferation, but there was also no report on RGS2 related to implantation; ISP2 was a newly-identified mouse uterine specific gene, and its expression was regulated by progesterone and sharply up-regulated during the period of implantation, but the roles of ISP2 played in implantation was still unclear; EMO-1 was highly homologous to mammalian Sec63 gene which was reported to be involved in the post-translation modification of various proteins; EMO-2 was a new gene and partly homologous to the human hypothetical protein MGC50372, and no report on EMO-2 and its homologous gene was found. MNSFP was a previously identified novel implantation-related factor that needed to be further investigated in our lab, and is a lymphokine produced by suppressor T cell. As it was reported that MNSFβ could inhibit the immune response in an antigen-nonspecific suppressive manner by inhibiting the secretion of IL-4 and production of Th2 cells, MNSFβ was presumed to participate in the formation of immune tolerance between embryo and maternal endometrial tissues.As the anti-ISP2 antibody and anti-MNSFβ antibody were not commercial available, we therefore prepared the recombinant ISP2 protein, as well as fusion proteins MNSFβ-hCGβ and GST-MNSFβ, respectively, and immunized animals by using these expressed products as the antigen to respectively prepare the anti-ISP2 and anti-MNSFβ antibodies. Meanwhile, the antibodies respectively against the EM0-1NTP, EMO-1CTP, EMO-2NTP and EMO-2CTP were also prepared in our lab. All of these antibodies were used in the subsequent IHC study and in vivo loss of function experiments.The uterine expression patterns of E4BP4, RGS2, ISP2, EMO-1 and EMO-2 during the period of embryo implantation were respectively investigated by in-situ hybridization and/or IHC. The results showed that, the uterine expression of these 5 genes were up-regulated during the implantation process, but the expression localization of these gene were not the same. Particularly, the high expression level of EMO-2 was embryo-independent. Furthermore, the expression pattern of ISP2 protein was well consistent with the ISP2 mRNA as previously reported.In order to further investigate the roles of ISP2, MNSFβ, EMO-1 and EMO-2 played in embryo implantation process, and to explore whether or not the potential target(s) for the development of novel contraceptive could be found among these genes, the antibody-blocking experiments were performed in mice. The results showed that, the antibodies against ISP2, MNSFβ, EMO-1 and EMO-2 caused a dose-dependent inhibitory effects on mouse embryo implantation in vivo. Consistently, the previous antisense RNA-blocking experiments of EMO-1 and EMO-2 also showed the inhibitory effects on embryo implantation. D5 pregnant mouse uterine tissues treated and non-treated by ISP2-antibody or MNSFβ were respectively collected. IHC analysis was carried out to observe whether or not the expression of uPA and MMP-9 proteins would be effected by the treatment of anti-ISP2 antibody. cDNA array technique was used to identify the up- and down-regulated genes in MNSFβ-antibody treated uterine tissues.In order to develop the techniques related to the development of knockout mouse model, which could be used to deeply investigate the inhibitory effects and the underlying mechanism caused by the function-deficiency of these implantation-related genes, we established a germ-line transmission using C57BL/6J embryonic stem cell lines that can be applied to generate transgenic or gene knock-out mice.To sum up, it could be concluded that, ISP2, MNSFβ, EMO-1 and EMO-2 play the critical roles in mouse embryo implantation, and consequently are potential targets for the development of novel contraceptive product. However, the mechanisms and the potential interaction among these implantation-related factors are still needed to be further explored.