The Stomatin-like Protein 2(SLP-2) Is Involved In Endometrial Stromal Cell Proliferation And Differentiation During Decidualization In Mouse And Human

Background: Endometrial stromal cell proliferation and decidualization play a critical role in establishing and maintaining normal pregnancy. Decidualization itself is governed by an array of modulators of embryonic and maternal origin. However, the molecular mechanisms underlying decidualization still remain poorly understood. Both our previous serial analysis of gene expression analysis and recent RNA sequencing showed that Slp-2 was differentially expressed through the peri-implantation stage. Overexpression of slp-2 has been recently found in several cancer cells, regulating cell growth, cell adhesion and tumorigenesis. Hence, we hypothesize that Slp-2 plays a novel role in peri-implantation in mice and humans—participating the endometrial stromal cell proliferation and decidulization.Methods: 1. Establishment of animal model and sample collection: Mice(Kunming White outbred strain; 8-week-old) were obtained from the Laboratory Animal Centre, Chongqing Medical University(Chongqing, China) and housed in the specific pathogen-free facility in a temperatureand light-controlled environment(14 h light/10 h dark) with free access to regular food and water. Pregnancy mice: Adult female mice were mated with fertile males.The day of the postcoital vaginal plug designated as pregnant day 1(D1). The mice were sacrificed on D1-D8 for the uterus and embryo tissues collection. Pseudopregnancy mice : Adult female mice were mated with vasectomized males The day of the postcoital vaginal plug designated as day of pseudopregnancy day 1(PD1). The mice were sacrificed on PD1-PD8 fot the uterus tissues collection. Artificially induced decidualization: on PD4, one side of the uterine horn was infused with corn oil to artificially induce decidualization, and the other side of the uterine horn was served as the control. The mice were sacrificed on PD8,and the uterus tissues were collected. 2. The role of Slp-2 in endometrial stromal cell proliferation and decidualization: 1) Detection of the expression of Slp-2 in normal pregnancy: examine Slp-2 on D1, D4, D5, D6, D8 by RT-q PCR, Western Blot and immunohistochemistry. 2) Detection of the expression of Slp-2 in pseudopregnancy pregnancy: examine Slp-2 on PD1, PD4, PD5, PD6, PD8 by RT-q PCR, Western Blot and immunohistochemistry. 3) Detection of the expression of Slp-2 in artificial decidualization: examine Slp-2 and decidualization related marker gene dtprp between artificial groups and common groups by RT-q PCR, Western Blot and immunohistochemistry. 3. Examination of the expression of Slp-2 in proliferation phase, secretory phase and decidualization human endometrium by RT-q PCR, Western Blot and immunohistochemistry. 4. Mouse and human uterine endometrial stromal cells isolation: Isolate and culture stomal cells from mouse endometrium on PD4 and human endometrium in proliferation phase and secretory phase and identify purity by immunofluorescence. 5. In vitro induced decidualization: add E2, P4 to mouse and human endometrial stomal cells to induce decidualization in vitro. Detect the expression of dtprp, IGFBP1, PRL, Desmine and Slp-2. 6. Evaluate cell proliferation using EDU cell proliferation kit.Results: 1. Slp-2 expression in mouse endometrium in early pregnancy: immunohistochemistry results showed slp-2 was expressed in glandular and luminal epithelium on D1 and D4, the primary decidualization zone(PDZ)in implantation site(IS) on D5 and the secondary decidualization zone(SDZ)in implantation site on D6 and D8. The staining intensity of Slp-2 antibody in glandular and luminal epithelium in the inter-implantation on D5 and D6 is similar to D1. RT-q PCR and Western Blot results show slp-2 up-regulated SLP-2 that in implantation site on D5, D6 and D8. 2. The Slp-2 expression pattern in glandular and luminal epithelium on PD1 and PD4 is similar to that on D1 and D4. No signal can be detected in stromal cells in the endometrium on PD5, PD6 and PD8. 3. Slp-2 was up-regulated in artificially induced decidualization by using the immunohistochemistry, Western Blot and RT-q PCR. 4. RT-q PCR and Western blot results showed that mouse and human related decidualization marker genes dtprp, IGFBP1, PRL and Desmin were up-regulated in vitro decidualizationn. 5. RT-q PCR and Western Blot results showed compromised dtprp,IGFBP1 and PRL down-regulation after knocking down slp-2 gene.6. Cell proliferation was significantly repressed under SLP-2 RNAi treatment, compared with control.Conclusions: 1. Slp-2 is a key factor during normal decidualization progression. Lack of Slp-2 inhibits decidualization. 2. Slp-2 is related to endometrium stomal cells decidualization through influencing stomal cells proliferation and differentiation.