Comparison of dexamethasone and aldosterone on ERK-mTOR pathway in genes expression associated with mouse uterine endometrial receptivity during implantation

Abstract

Introduction: Implantation of embryos needs endometrial receptivity. Mineralocorticoids and glucocorticoids play an important role in endometrial receptivity, and influence the implantation window. This study aimed to evaluate fludrocortisone and dexamethasone effects on endometrial receptivity by the morphological study of the endometrium as well as the expression of LIF, Muc1, HAND2, MSX.1, HB-EGF and miRNA Let-7a. Then the mechanisms of fludrocortisone and dexamethasone effects were studied through mTOR and ERK1/2 signaling pathways by using the mTOR pathway inhibitor (PP242). Materials and methods: A total of 60 Bulb/c mice were randomly divided into six groups: one was the control group, and the other five groups received dexamethasone (DEX), PP242, fludrocortisone (FCA), FCA+PP242 and DEX+PP242. Mating was done by vaginal plug and the presence of sperm in the vaginal smear was confirmed on the following morning which was considered as day 1 of post-coitum (dpc). Fludrocortisone, dexamethasone and PP242 were injected on the 4th and 5th day of pregnancy and 2 hours after the last injection, all the mice were euthanized. First, histochemical and histological analysis by H&E and PAS were performed on uterine tissue samples. Second, the endometrial epithelial and the stromal cells were separated manually from myometrium. Finally, Western blot was used to measure the level of proteins, and real-time PCR to evaluate the gene expression. Results: Fludrocortisone injection led to the transformation of epithelial cells to cuboidal form and increasing the proliferation of stromal cells during implantation. However, PP242 and dexamethasone injection resulted in epithelial transformation to columnar form. Fludrocortisone injection increased the expression of LIF, HAND2, HB-EGF, Msx.1, and miRNA Let-7a in a normal ovarian cycle but decreased Muc1, SGK1 and ENAC expression. It also increased 4EBP1, mTOR and ERK1/2 phosphorylation in normal ovarian cycles. In addition, injection of PP242 and dexamethasone inhibited mTOR, ERK1 and 4EBP1 and reduced expression of LIF, HAND2, HB-EGF, Msx.1, and miRNA Let-7a in mice but increased Muc1, SGK1 and ENAC expression. Methods: In this study, we used quantitative real-time PCR (qRT-PCR) to measure CD133 expression in metastasis-derived prostate cancer cell line, LNCaP. The cells were transfected with CD133 siRNA using transfection reagent. The cytotoxic effects of CD133 siRNA, paclitaxel alone and the combination of CD133 siRNA/paclitaxel on LNCaP cells were determined using an MTT assay. The migration and invasive capacity of LNCaP cells after treatment by CD133 siRNA, paclitaxel alone and the combination of CD133 siRNA/paclitaxel were examined by scratch wound healing assay. Subsequently, in order to investigate the effect of CD133 siRNA on CSCs properties in the LNCaP cells after treatment by CD133 siRNA, paclitaxel alone and the combination of CD133 siRNA/paclitaxel, spheroid assay, colony formation assay, and cancer cell proliferation were examined. In addition, Flow cytometry and 4′, 6‐diamidino‐2‐phenylindole staining analysis were used to determine that CD133 siRNA increased the paclitaxel‐induced apoptosis. We also used qRT-PCR to study the effect of CD133 silencing on the AKT/mTOR/C-myc axis and the expression levels of pro-metastatic genes and EMT markers in CD133+ prostate CSCs. Implantation of embryos needs endometrial receptivity. Mineralocorticoids and glucocorticoids play an important role in endometrial receptivity, and influence the implantation window. This study aimed to evaluate fludrocortisone and dexamethasone effects on endometrial receptivity by the morphological study of the endometrium as well as the expression of LIF, Muc1, HAND2, MSX.1, HB-EGF and miRNA Let-7a. Then the mechanisms of fludrocortisone and dexamethasone effects were studied through mTOR and ERK1/2 signaling pathways by using the mTOR pathway inhibitor (PP242). Materials and methods: A total of 60 Bulb/c mice were randomly divided into six groups: one was the control group, and the other five groups received dexamethasone (DEX), PP242, fludrocortisone (FCA), FCA+PP242 and DEX+PP242. Mating was done by vaginal plug and the presence of sperm in the vaginal smear was confirmed on the following morning which was considered as day 1 of post-coitum (dpc). Fludrocortisone, dexamethasone and PP242 were injected on the 4th and 5th day of pregnancy and 2 hours after the last injection, all the mice were euthanized. First, histochemical and histological analysis by H&E and PAS were performed on uterine tissue samples. Second, the endometrial epithelial and the stromal cells were separated manually from myometrium. Finally, Western blot was used to measure the level of proteins, and real-time PCR to evaluate the gene expression. Results: Fludrocortisone injection led to the transformation of epithelial cells to cuboidal form and increasing the proliferation of stromal cells during implantation. However, PP242 and dexamethasone injection resulted in epithelial transformation to columnar form. Fludrocortisone injection increased the expression of LIF, HAND2, HB-EGF, Msx.1, and miRNA Let-7a in a normal ovarian cycle but decreased Muc1, SGK1 and ENAC expression. It also increased 4EBP1, mTOR and ERK1/2 phosphorylation in normal ovarian cycles. In addition, injection of PP242 and dexamethasone inhibited mTOR, ERK1 and 4EBP1 and reduced expression of LIF, HAND2, HB-EGF, Msx.1, and miRNA Let-7a in mice but increased Muc1, SGK1 and ENAC expression.