Role Of TRAIL Gene During The Decidualization Process In Pregnant Mice

The invasion of embryonic trophoblast cells into unterine endometrium is a critical step for human and mammal reproduction. Proliferation of trophoblast cells and endometrial decidualization may be precisely regulated by a'crosstalk'among trophoblast cells, endometrial epithelial cells and stromal cells. Decidua is a special tissue derived from endometrial proliferation and differentiation stimulated by decidual inducing factors. In mice, embryonic adhesion induces subepithelial stromal cells to differentiate into mature decidual cells. The decidual cells gradually extend to surround the implanting blastocys, and continue to differentiate and degenerate in the same direction at the same time. These degenerative cells are finally phagocytized by adjacent trophoblast cells or macrophages. The balance between decidual cells proliferation and apoptosis is critical to the appropriate tissue remodeling of the maternal decidua and the accruate invasion of trophoblastic cells. It is indicated that decidual cells degradation is a cell apoptosis process. In view of the similarity between tumor invasion and trophoblast invasion, the underlying mechanism has been one of the hot research field in reproduction. TRAIL, tumor necrosis factor related apoptosis-inducing ligand, is an important cytokine involved in cancer prevention and treatment in recent years. Our previous study in mice showed that TRAIL was expressed in preimplantation uterine epithelium and decidual cells in a specific spatial and temporal pattern. Meanwhile, one of TRAIL receptors, mouse killer (MK) displayed the corresponding expression with TRAIL, suggesting that TRAIL signaling might be involved in decidual cells apoptosis after implantation and participate in the endometrium decidualization process in mice.To address this point, multiple approaches including genetic engineering techniques, primary cell culture, Western blotting, immunocytochemistry, immunofluorescence, MTT assay, fluorescent quantitative,flow cytometry, intra-uterine injection et al. were utilized to investigate the function and molecular mechanism of TRAIL on endometrial decidualization process.PartⅠExpressions of TRAIL gene in mouse uterine decidual cells Pancreatin digestion and mixture enzymes digestion werecomparatively tested to isolate and culture the primary mouse endometrial stromal cells whose purity was examined by immunofluorescent staining of vimentin and cytokeratin. The results indicated that pancreatic digestion could harvest more stromal cells with (95.8±0.3)% of purity at less cost of money and time. The optimal pH value for cell culture cell was 7.0 by counting the cell adhesion rate after 2h of culture. MTT assay was performed to draw the cell growth curve and logarithmic growth phase was found located between 12-48 h of culture. Moreover, both TRAIL mRNA and protein were positively detected by RT-PCR and Western blotting in stromal cells after induced with estriadiol, progesterone plus cAMP treatment.PartⅡRegulation of TRAIL expression by ovary steroid hormonesTo investigate the regulatory effect of ovary steroid hormones of progesterone and estradiol on TRAIL gene expression in mice, ovariectomized mice were randomly divided into four groups treated by s.c. injection of progesterone(0.2mg/mouse), estradiol(100ng/mouse), progesterone combination with estradiol and sesameoil(0.1ml/mouse), respectively. Uteri were collected at 0, 1, 2, 4, 6, 12, and 24 h after treatment, and TRAIL mRNA and protein expression levels were detected by fluorescent quantitative PCR and Western blotting. The results indicated either progesterone or estradiol alone, or combination could up-regulate expressions of both the TRAIL mRNA and protein levels at a time-dependent pattern(p<0.05).PartⅢRole of TRAIL on the endometrial decidualizationon in miceTo explore TRAIL effect on mouse decidualization, both TRAIL over-expression and siRNA eukaryotic vectors and retrovirus were generated. Briefly, TRAIL full-length cDNA sequence was cloned into the vector pSEB-HUS, named pSEB-HUS-TRAIL, which was comfirned by DNA sequencing. Four interfering sequences targeting TRAIL gene were designed with Dharmacon's siDESIGN software and subcloned into the pSEB-HUS-TRAIL vectors, which were named as SipSEB-TRAIL1, SipSEB-TRAIL2, SipSEB-TRAIL3, SipSEB-TRAIL4. SipSEB-TRAIL3 was screened out to be the most efficient siRNA vector for next application.Mouse stromal cells were transfected with pSEB-HUS-TRAIL and SipSEB-TRAIL3, respectively followed by decidualization induction with progesterone, estradiol plus cAMP. 72h after treatment, TRAIL mRNA and protein levels were detected by RT-PCR and Western blotting. MTT assay was performed to investigate the cell growth activity and proliferation capacity, Flow cytometry was used to examine cell cycle distribution and apoptosis rate. The combined results of TRAIL over-expression and interfering study in DSC demonstrated that TRAIL over-expression was capable of markedly inhibiting DSC proliferation by blocking most of cells at G0/G1 phase with the apoptosis rate of DSC was notably increased. Over-expression of TRAIL was capable of increasing the expression levels of prolactin mRNA and protein, while interfering of TRAIL exerted a reversal effect.Moreover, over-expression and interfering retrovirus of TRAIL were injected into mice uterine horns on d3.5 of pregnancy. The weight of uterine decidua was measured and the number of implantation sites were recorded on d7 and d8. In the group injected with over-expression retrovirus of TRAIL, the weight of decidua tissue was decreased while the interfering group increased markedly. Interestingly, the number of implantation sites significantly reduced in both groups, possibly due to disturblence of balance between proliferation and apoptosis of decidual endometrial stromal cells.Finally, TRAIL receptors DcR1, DcR2, DR5 in decidualizing stromal cells were examined by RT-PCR,showing that DcR1 mRNA level was significantly increased at 72h of decidualization(p<0.05)while DcR2 had no changes at all (p>0.05). MK mRNA expression level was gradually induced with the progression of decidualization of stromal cells in culture.Furthermore, protein of pro-caspase-8 and Bcl-2 in decidualizing stromal cells infected with the TRAIL over-expression vector were detected by Western blotting and.both proteins were dramatically decreased(p<0.05).Overall, the role of TRAIL was suggested to induce apoptosis rate of decidual stromal cells possibly through binding its membrane receptor MK leading to downregulation of Bcl-2 expression via mitochondria pathway.In conclusion, the expression of TRAIL gradually increased both the mRNA and protein levels together with the progression of decidualization of stromal cells in culture,TRAIL expression in mice uteri was regulated by P4 and E2. TRAIL was capable of inducing DSC apoptosis possibly through binding its membrane receptor MK leading to downregulation of Bcl-2 expression via mitochondria pathway and blocking the endometrial decidualization process in pregnant mice.