| MNSFβ, a novel implantation-related factor newly identified in our lab, is a nonspecific immune inhibitory lymphokine produced by suppressor T cell.It was presumed that MNSFP could be related with the participation in the formation of immune tolerance between the embryo and maternal endometrial tissues. The subject mainly studied the function of MNSFβgene and explored the roles the gene played in the embryo implantation so as to further reveal the molecular mechanism of embryo implantation, provide the theoretical basis with the research of infertility mechanism, improvement of the artificial assisted reproductive technology and development of anti-implantation contraceptive drugs and probe for the new potential link or new target molecules to achieve the purposes of fertility regulation. The main results are as follows: 1. As the anti-MNSFβpolyclonal antibody was not commercial, it was prepared at first in the lab. The antibody titers were confirmed as 1:125000 by ELISA. The antibody was used as one of the experimental tools in this study.2. The immunohistochemistry(IHC), RT-PCR and Western blot techniques were applied to confirm that the MNSFβwas expressed in most tissues and organs of the mouse. In the periods of the mouse embryo preimplantation, implantation and pregnancy, the MNSFβexpression had its dynamic changes in its uterine tissues. The results showed that the MNSFβwas lowly expressed on day 4.5 of the pregnancy, the MNSFβexpression gradually increased with the prolongation of the pregnancy time, and the expression of the MNSFβwas much higher in the interimplantation sites than in the implantation sites on day 8.5.3. The primary mouse uterine endometrial cells were isolated by random cycling on day 4.5 pregnant mouse uterine from which blastocysts were flushed. The isolated cells were stromal cells identified by immunofluorescence assays. And the MNSFP was expressed in mouse blastocysts and endometrial stromal cells by the IHC method assay. The mouse endometrial stromal cells were co-cultured with the blastocysts of the day 4.5 pregnancy with the results of the spread, invasion, attachment and fusion of the mouse blastocysts to the endometrial stromal cells similar to implantation process in vivo, the process of which was observed daily under an inverted microscope. MNSFβantiserum was used to inhibit the spreading and adhesive growth of the mouse blastocysts to endometrial stromal cells and induce the death of the embryo, which presented a dose-dependent relationship.4. The transfection of MNSFβexpression plasmid or siRNA into HCC-94 cells and re-suspense of mouse lymphocytes in the culture medium from the different transfected HCC-94 cell supernatants showed that MNSFβcould promote the secretion of IL-4 and inhibit the secretion of TNFa from mouse lymphocytes.In contrast, the secretion of TNFa from lymphocytes sharply increased with the siRNA and MNSFP antibody blocking. The results suggested that MNSFβmight be involved in maternal-fetal immune tolerance.5. The heterozygous MNSFβknockout mice were obtained by mating of the MNSFβconditional gene knockout mice with Cre-mice. The mating experiments confirmed that part of the MNSFβgene defects led to the significant fertility decrease in mice, but only the fertility of the female mice was affected. The male and female ratio of their offspring mice was imbalanced and the homozygous mice could not be obtained.6. The methods of the IHC, Real-time PCR and Western blot also confirmed that the expression level of MNSFβmRNA in the heterozygous mice uterine tissue had no more significant change than that of wild-type mice, but the expression of MNSFP protein level was significantly reduced. In addition, the structure of the heterozygous mice uterine gland-epithelial tissues became very loose and the uterine cavities were significantly widened.7. The observation of the numbers of embryos in the uterus and their develop ment by selecting different times of the embryo implantation and pregnancy (i. e. day 4,5,8,11 and 14 of pregnancy) demonstrated the normality of the num bers of embryos in the uterus of embryo implantation and early embryonic develop ment, but some obvious abnormalities of the embryos appeared:loss of the shap e of fetal mice and formation of clumps shape in the period of the medium-ter m embryo development (day 11 and 14 of pregnancy). The results suggested th e MNSF P was involved in the embryonic development.To sum up, it could be concluded that the MNSFβwas required in th e process of embryo implantation in mice and related with immunosuppressio n. Part of the defects or reduction of the gene expression could cause its dis orders in the implantation of the embryo and development in vitro and in vi vo; therefore, it is possible that the MNSFβbecome the potential target m olecule for the development of a novel contraceptive technology.
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