Induction of superoxide dismutase by decidualization in human endometrial stromal cells

The present study was undertaken to investigate the effect of decidualization on superoxide dismutase (SOD) expression in human endometrial stromal cells (ESC). To induce decidualization, isolated ESC were incubated with medroxyprogesterone acetate (MPA, 10(-6) mol/l) and oestradiol (10(-8) mol/l) for 23 days. Insulin-like growth factor-binding protein-1 (IGFBP-1) was used as a marker of decidualization. SOD mRNA in ESC was significantly increased on day 12 of the hormone treatment (P < 0.01), which was concomitant with the onset of IGFBP-1 mRNA expression, and further increased until day 23 of the treatment in a manner similar to the change in IGFBP-1 expression. To examine the synergistic effect of human chorionic gonadotrophin (HCG) with MPA and oestradiol on SOD and IGFBP-1 expression, ESC were incubated with HCG in the presence or absence of MPA and oestradiol. HCG had no synergistic effect on SOD and IGFBP-1 expression. SOD activities in the decidualized endometrial tissue obtained from patients given oestradiol and progesterone for 7-10 days were significantly higher than those in the non-decidualized endometrial tissue from patients without the hormone treatment (P < 0.01). In conclusion, SOD expression in ESC was induced by MPA and oestradiol accompanied by decidualization, suggesting that SOD may play important roles in decidualization of ESC.     

Differentiation of endometrial stromal cells in vitro: down-regulation of suppression of the cell cycle inhibitor p57 by HOXA10?

Decidualization is a critical step during embryo implantation that is characterized by the differentiation of endometrial stromal cells (ESC) into decidua cells. However, the mechanism of differentiation remains largely unknown. Previously, it has been shown that the null function of homeo box A10 (HOXA10) causes defects in both implantation and decidualization, suggesting that the HOXA10 signalling pathway is likely to be involved in uterine decidualization. In the present study, we determined the expression and subcellular distribution of HOXA10 and its downstream molecule, p57, in ESC during in vitro decidualization induced by a combination of 8-bromo-cAMP and medroxyprogesterone acetate. We demonstrated that the HOXA10 was down-regulated while in contrast, p57 was up-regulated in the process of decidualization. Immunocytochemistry and transient expression of the HOXA10 tagged with green fluorescence protein revealed that there were no differences in the HOXA10 subcellular localization between the induced and non-induced ESC. Our results suggest that the down-regulation of HOXA10 may contribute to increased p57 and that up-regulation of p57 likely plays an important role in ESC differentiation in the process of decidualization. The progesterone receptor pathway may participate in promoting ESC to exit the cell cycle and enter differentiation.     

Endometrial stromal cell decidualization inhibits human chorionic gonadotrophin and human placental lactogen secretion by co-cultured trophoblasts

We have developed an in-vitro co-culture system to examine theinteraction between purified first trimester cytotrophoblastsand purified non-pregnant human endometrial stromal cells (ESC).ESC decidualization is an important step in endometrial maturationand may modulate embryo implantation. In order to investigatethe effects of ESC decidualization on trophoblast function,we examined human chorionic gonadotrophin (HCG), human placentallactogen (HPL), progesterone and oestrogen secretion by trophoblastsco-cultured in contact with ESC, either with or without decidualizationinduced by progesterone. Decidualized ESC inhibited basal HCGand HPL secretion for 3 days during the culture for HCG, andfor 5 days during the culture for HPL (P < 0.01 and P <0.03 respectively). After 5 days of co-culture, decidual transformationof ESC as indicated by prolactin production occurred in thecontrol cultures due to progesterone and oestradiol secretionby the co-cultured trophoblasts, but no significant differencesin HCG or HPL secretion were observed between the two groups.Although the type of trophoblast used in the present study isfar from implantation, our results clearly demonstrated thatHCG and HPL secretion by trophoblasts was inhibited by the presenceof co-cultured decidualized ESC, and suggested that ESC decidualizationmay regulate trophoblast function at the human fetal-maternalinterface.     

Akt as a possible intracellular mediator for decidualization in human endometrial stromal cells

To gain an insight into intracellular mechanisms involved in differentiation of human endometrial cells into decidual cells, we examined the presence of Akt, an emerging intracellular mediator in human endometrial stromal cells (ESC). We explored the mechanisms regulating Akt phosphorylation during the process of progesterone-induced decidualization using a primary cell culture system of ESC. Both Akt and phosphorylated Akt (phospho-Akt) were present in ESC. The total Akt level in ESC cultured for 12 days in the absence of ovarian hormones was similar to ESC treated with estradiol (E(2)) at 10 ng/ml, progesterone at 100 ng/ml or E(2) plus progesterone (E(2)progesterone), whereas the levels of phospho-Akt were markedly decreased with progesterone or E(2) progesterone, compared to control cells. Notably, phospho-Akt levels increased during 12 days of culture in parallel with an increase in total Akt in untreated cells. An increase of phospho-Akt in the E(2) progesterone-treated cells was marginal. The level of phospho-Akt in E(2) progesterone-treated cells was markedly reduced compared to control cells at all time points examined. Treatment of the cells with 8-bromo-cAMP decreased the amount of phospho-Akt in ESC in as short a period as 15 min, while no discernible change was observed in the untreated cells. Conversely, H89, an inhibitor of protein kinase A (PKA), significantly increased the amount of phospho-Akt. The addition of H-89 reversed the decrease in the level of phospho-Akt seen in the cells treated with E(2) progesterone. Thus, we demonstrated the presence of Akt and its phosphorylated form in human ESC. We further suggest that Akt phosphorylation through the cAMP/PKA signal transduction pathway may regulate cellular functions coupled with decidualization.     

Differential regulation of copper-zinc superoxide dismutase and manganese superoxide dismutase by progesterone withdrawal in human endometrial stromal cells.

The present study was undertaken to investigate the role of estrogen and progesterone in the expression of copper-zinc superoxide dismutase (Cu,Zn-SOD) and manganese SOD (Mn-SOD) in human endometrial stromal cells (ESC). ESC were incubated with estradiol (10(-8) mol/l), medroxyprogesterone acetate (MPA, 10(-6) mol/l), or estradiol + MPA for 18 days. MPA significantly increased Cu,Zn-SOD and Mn-SOD mRNA levels and enzyme activities as well as the mRNA level of insulin-like growth factor-binding protein-1 (IGFBP-1), a marker for decidualization. Estradiol only augmented the effects of MPA on Cu,Zn-SOD activity and IGFBP-1 mRNA level, and estradiol alone had no effect. To study the withdrawal of estrogen and progesterone (EP withdrawal), ESC that had been treated with estradiol + MPA for 12 days were washed and then incubated with or without estradiol + MPA for a further 11 days. Cu,Zn-SOD mRNA levels and activities declined after EP withdrawal, while they were gradually increased by the continuous treatment with estradiol + MPA. In contrast, Mn-SOD mRNA levels and activities were not affected by EP withdrawal. IGFBP-1 mRNA levels were significantly increased 4 days after EP withdrawal and decreased thereafter, whereas they were gradually increased by the continuous treatment with estradiol + MPA. In conclusion, Cu,Zn-SOD, Mn-SOD and IGFBP-1 are differently regulated by estrogen and progesterone in human ESC. The decrease in Cu,Zn-SOD after the ovarian steroid withdrawal may be involved in endometrial breakdown.     

Different mechanisms for the induction of copper-zinc superoxide dismutase and manganese superoxide dismutase by progesterone in human endometrial stromal cells

BACKGROUND: The present study was undertaken to investigate the cAMP-dependent regulation of copper-zinc superoxide dismutase (Cu,Zn-SOD) and manganese SOD (Mn-SOD) by ovarian steroids in human endometrial stromal cells (ESC). METHODS and RESULTS: To examine the effect of cAMP on SOD expression, ESC were incubated with dibutyryl-cAMP (db-cAMP, 0.5 mmol/l), forskolin (25 micromol/l), or estradiol (E(2), 10(-8) mol/l) + medroxyprogesterone acetate (MPA, 10(-6) mol/l), for 18 days. E(2) + MPA significantly increased Cu,Zn-SOD activity and mRNA concentrations, whereas db-cAMP and forskolin had no effect. On the other hand, Mn-SOD activity and mRNA concentration were significantly increased by all of these treatments. Insulin-like growth factor-binding protein-1, a marker of decidualization, was clearly induced by db-cAMP, forskolin or E(2) + MPA, accompanied by morphological changes characteristic of decidualization. To study whether the increase in Mn-SOD by db-cAMP or E(2) + MPA was mediated by cAMP-dependent protein kinase A (PKA), ESC were incubated with protein kinase inhibitor (PKI) (10 microg/ml), an inhibitor of PKA, in the presence of db-cAMP or E(2) + MPA. The increase in Mn-SOD activity following db-cAMP or E(2) + MPA was completely inhibited by PKI. CONCLUSIONS: In the process of decidualization, E(2) + MPA increases Mn-SOD expression via a cAMP-dependent pathway. Cu,Zn-SOD is also up-regulated by E(2) + MPA, but via a different pathway from that involving cAMP.     

Mechanisms regulating the expression of indoleamine 2,3-dioxygenase during decidualization of human endometrium

BACKGROUND: Expression of the tryptophan catabolizing enzyme, indoleamine 2,3-dioxygenase, in the mouse placenta has been shown to be critical in preventing immunological rejection of the fetal allograft. To clarify the physiological importance of indoleamine 2,3-dioxygenase in human pregnancy, we have studied how the expression of this enzyme changes during decidualization of human endometrium at both the cell and tissue level. METHODS AND RESULTS: The level of indoleamine 2,3-dioxygenase mRNA expression (determined by RT-PCR) was higher in decidual than in endometrial tissue. Uterine decidual tissue in ectopic pregnancy similarly showed increased mRNA expression. Immunohistochemistry demonstrated that indoleamine 2,3-dioxygenase protein immunoreactivity was found in glandular epithelium and in stromal cells. The intensity of this immunoreactivity was increased in decidualized tissue. In a cell culture model, the level of indoleamine 2,3-dioxygenase mRNA was suppressed specifically by progesterone-induced decidualization of isolated endometrial stromal cells. Indoleamine 2,3-dioxygenase protein abundance (determined by Western blot) was also decreased by progesterone-induced decidualization. However interferon-gamma, a potent stimulator of indoleamine 2,3-dioxygenase gene expression, increased the level of indoleamine 2,3-dioxygenase mRNA and protein in both non-decidualized and in decidualized cells. Indoleamine 2,3-dioxygenase activity (determined by measuring the concentration of tryptophan and its indoleamine 2,3-dioxygenase catabolite, kynurenine) was also decreased by progesterone-induced decidualization but enhanced following interferon-gamma treatment. Expression of other interferon-gamma inducible genes (STAT1 and tryptophanyl-tRNA synthetase) showed the same pattern as that of indoleamine 2,3-dioxygenase in tissue samples, but was not changed by decidualization in the cell culture model. CONCLUSIONS: These data suggest that despite suppression by progesterone, indoleamine 2,3-dioxygenase expression in endometrial stromal cells may increase during decidualization due to stimulation by interferon-gamma secreted by infiltrating leukocytes.     

Progesterone induces the fibulin-1 expression in human endometrial stromal cells

BACKGROUND: By using microarray analysis with human endometrial stromal cells (ESCs), we previously reported that the mRNA for fibulin-1, an extracellular matrix as well as a plasma glycoprotein, is up-regulated by progesterone. In the present study, we tried to clarify the spatial and temporal regulation mechanism of fibulin-1 in the human endometrium. METHODS AND RESULTS: Quantitative analysis with real-time PCR experiments on human endometrial tissues showed significantly higher fibulin-1 mRNA expressions in secretory phase endometria than in proliferative phase. Immunohistochemical studies revealed that the fibulin-1 protein is expressed in the glandular epithelium in proliferative phase endometria, and that expression switched to the stroma in secretory phase endometria. In culture experiments with ESCs, a significant increase of fibulin-1 mRNA expression was observed in cells treated with 6 alpha-methyl-17 alpha-hydroxy-progesterone acetate (MPA) or 8 bromoadenosine 3':5'-cyclic monophosphate (8-Br-cAMP). MPA stimulated the fibulin-1 mRNA expression in a dose-dependent manner, and a progesterone antagonist, RU-486, inhibited the stimulatory effect almost completely. By contrast, beta-estradiol alone did not increase the fibulin-1 mRNA expression. CONCLUSIONS: These results suggest that fiblin-1 is an important molecule that mediates progesterone action in human ESC differentiation towards implantation.     

Endocrinology and paracrinology: The presence of platelet-derived endothelial cell growth factor in human endometrium and its characteristic expression during the menstrual cycle and early gestational period

Decidualized tissues are characterized by the intensive outgrowthof the microvasculature. Several angiogenic factors are assumedto be involved during the drastic change in the vasculatureoccurring in the process of decidualization. We examined thepossible role of platelet-derived endothelial cell growth factor(PD-ECGF), a known angiogenic factor, during the process ofearly decidualization in humans. The expression of PD-ECGF inhuman endometrium was demonstrated by Western blot analysis,a marked increase being found in decidualized endometrium. Immunohistochemicalstaining showed that PD-ECGF immunoreactivity was present mainlyin decidualized endometrial stromal cells. We established aprimary cell culture of human endometrial stromal cells whichwere differentiated into decidualized cells in vitro by theaddition of progesterone. In this cell culture system, progesteroneaugmented the expression of PD-ECGF in a dose-dependent fashion.The addition of progesterone also resulted in an increased releaseof prolactin, a well-known marker for decidualization. Thesefindings suggest that PD-ECGF may play a physiological roleas a possible angiogenic factor in the process of decidualizationof human endometrium.     

Interleukin-11 expression: its significance in eutopic and ectopic human implantation

Embryo implantation and subsequent decidualization, trophoblast invasion and formation of a functional placenta are crucial for establishment and maintenance of pregnancy. Interleukin-11 signalling has been shown to be obligatory for adequate decidualization and trophoblast invasion in mice. Defects in IL-11 signalling in mice result in trophoblast over-invasion and fetal loss. The pathological situation of human tubal pregnancy resembles that of IL-11Ralpha(-/-) mice concerning these symptoms. As our interest is focused on the human early pregnancy, we compared IL-11 expression at the implantation site of ectopic tubal pregnancy (EP) to 1st and 2nd trimester of normal intrauterine pregnancies (IP), and to the normal cycling endometrium. The mRNA expression of IL-11 and IL-11Ralpha was analysed by semiquantitative RT-PCR. Protein expression was detected by western blotting and immunohistochemistry. IL-11Ralpha is expressed constitutively in all tissue specimens analysed. IL-11 is expressed predominantly during follicular and early luteal phase of the menstrual cycle. In IP, IL-11 expression peaks during the 1st trimester and declines from the beginning of the 2nd trimester onwards. In tubal abortions, IL-11 expression is reduced in comparison to vital EP and IP. Cultured primary endometrial and decidual epithelial cells were analysed for hormonal regulation of IL-11 by enzyme-linked immunosorbent assay and RT-PCR. IL-11 is up-regulated by estrogen and down-regulated by progesterone. Overall, our results indicate that in humans, IL-11 signalling is significantly involved in regulation of trophoblast invasion. In the case of tubal abortion, inadequate IL-11 signalling may therefore result in dysregulation of trophoblast invasion.     

Secretory endometrium highly expresses urocortin messenger RNA and peptide: possible role in the decidualization process

BACKGROUND: Urocortin (UCN) gene expression and synthesis have been reported in epithelial and stromal cells of the human endometrium. In this study we evaluated (i) UCN messenger RNA (mRNA) expression and peptide production in uterine specimens collected throughout the endometrial cycle, (ii) UCN secretion after decidualization of cultured human endometrial stromal cells (HESCs) and (iii) the effect of UCN on endometrial decidualization. METHODS: HESCs were isolated from samples of human endometrium collected from healthy patients with normal menstrual cycle and cultured in presence of cAMP, 17-beta-estradiol (E(2)) + medroxyprogesterone acetate (MPA) and UCN. UCN levels were measured in endometrial extracts by an enzyme immunoassay, and changes of endometrial UCN mRNA expression were measured by RT-PCR analysis. RESULTS: UCN peptide concentrations and mRNA expression were highest in the secretory phase of the menstrual cycle (P < 0.001, late secretory versus early and late proliferative phase) and higher in the late than the early secretory phase (P < 0.01). After decidualization of HESC with cAMP or E(2) + MPA, UCN levels rose in parallel with prolactin concentrations by days 6 (P < 0.01, for all). Finally, the addition of UCN to HESCs, with or without E(2) + MPA, induced the release of prolactin. CONCLUSIONS: The evidence that (i) UCN is highly expressed in the secretory phase of the endometrial cycle; (ii) cAMP and E(2) + MPA modulate secretion of UCN and (iii) UCN induces HESCs decidualization together suggest a possible role for UCN in endometrial physiology.     

The presence of platelet-derived endothelial cell growth factor in human endometrium and its characteristic expression during the menstrual cycle and early gestational period

Decidualized tissues are characterized by the intensive outgrowthof the microvasculature. Several angiogenic factors are assumedto be involved during the drastic change in the vasculatureoccurring in the process of decidualization. We examined thepossible role of platelet-derived endothelial cell growth factor(PD-ECGF), a known angiogenic factor, during the process ofearly decidualization in humans. The expression of PD-ECGF inhuman endometrium was demonstrated by Western blot analysis,a marked increase being found in decidualized endometrium. Immunohistochemicalstaining showed that PD-ECGF immunoreactivity was present mainlyin decidualized endometrial stromal cells. We established aprimary cell culture of human endometrial stromal cells whichwere differentiated into decidualized cells in vitro by theaddition of progesterone. In this cell culture system, progesteroneaugmented the expression of PD-ECGF in a dose-dependent fashion.The addition of progesterone also resulted in an increased releaseof prolactin, a well-known marker for decidualization. Thesefindings suggest that PD-ECGF may play a physiological roleas a possible angiogenic factor in the process of decidualizationof human endometrium. angiogenesis/decidualization/human endometrium/PD-ECGF/prolactin     

The inhibitory effect of dienogest, a synthetic steroid, on the growth of human endometrial stromal cells in vitro

Dienogest is a synthetic steroid that has been used as a progestogen in contraceptive pills and is currently being studied for its possible clinical use in the treatment of endometriosis. In this study, we investigated the direct effects of dienogest in differentiation and proliferation of human endometrial stromal cells (ESC) in vitro. After 12 days in the presence of oestradiol (10(-8) mol/l) plus dienogest (10(-6) mol/l), cultured ESC underwent morphological differentiation and produced prolactin, a typical marker for decidualization. By using Northern blot analysis and radioimmunoassay, it was shown that treatment of ESC with oestradiol (10(-8) mol/l) plus dienogest (10(-9) to10(-6) mol/l) led to an increase in the levels of prolactin mRNA and prolactin production in a dose-dependent manner. Additionally, RU-486, a progesterone receptor antagonist, almost completely inhibited dienogest-induced prolactin production. As shown by the thymidine uptake method, there was a dose-dependent inhibition of ESC proliferation with dienogest (P < 0.01, control versus concentrations >10(-7) mol/l). The significant inhibition of ESC proliferation by dienogest (10(-7) mol/l) was partially reversed by RU-486 (10(-6) mol/l). In summary, dienogest directly acts on endometrial tissue in progestogenic response, such as decidualization, increased prolactin production and growth retardation. These data imply that dienogest exerts direct effect in suppressing growth of endometriotic implants.     

Corticotrophin-releasing hormone (CRH) interacts with inflammatory prostaglandins and interleukins and affects the decidualization of human endometrial stroma

The hypothalamic neuropeptide, corticotrophin-releasing hormone (CRH), which is also produced by human endometrium, has been shown to induce its decidualization in vitro. This process, induced mainly by progesterone, has characteristics of an aseptic inflammatory reaction, and is modulated by locally produced pro-inflammatory factors. In humans, prostaglandin E(2) (PGE(2)) enhances while interleukin (IL)-1 inhibits the decidualizing effect of progesterone. The aim of the present work was to test the hypothesis that CRH might affect the decidualization of human endometrium interacting with these factors. Therefore, we studied its effects on the production of pro-inflammatory interleukins IL-1, IL-6 and of PGE(2) from human endometrial stromal cells in primary culture. The results strongly suggest that CRH decidualizes stromal cells, as judged by the appearance of cytokeratins and the production of prolactin, two established markers of decidualization. In parallel to its effect on decidualization, CRH also decreased the production of PGE(2), while it increased the production of IL-1 and IL-6. Exposure of endometrial stromal cells to IL-6 also caused decidualization. The data presented here suggest that endometrial CRH regulates the production of local modulators of decidualization, i.e. PGE(2), IL-1 and IL-6. We postulate that, through the regulation of these factors, CRH acts as a local fine-tuner of decidualization initiated by progesterone.     

Interleukin 11 advances progesterone-induced decidualization of human endometrial stromal cells

Differentiation of endometrial stromal cells into decidual cells is crucial for embryo implantation and placentation. Interleukin (IL)-11 signalling is essential for adequate decidualization in the mouse uterus. We examined the role of IL-11 during progesterone-induced decidualization of human endometrial stromal cells over a 10-12 day period, using prolactin (PRL) production as a decidual marker. These cells produced biologically active IL-11 and expressed IL-11, IL-11Ralpha and PRL mRNA during decidualization. Neutralization of endogenous IL-11 with an anti-human (hu)IL-11 antibody (AB) reduced production of PRL from day 8 and insulin-like growth factor binding protein (IGFBP)-1, another marker of decidualization, from day 10 of culture. Following AB washout, PRL and IGFBP-1 secretion increased. Addition of recombinant (r)huIL-11 (10 or 100 ng/ml) to endometrial stromal cells increased secretion of PRL from day 4 and IGFBP-1 from day 6 compared with progesterone alone. Morphological signs of differentiation accompanied biochemical differentiation in the progesterone-treated cells and were further induced by exogenous rhuIL-11. Our observations demonstrate that human endometrial stromal cells produce biologically active IL-11, which promotes progesterone-induced decidualization. These results suggest that IL-11 has both paracrine and autocrine actions on human endometrial stromal cells and plays an important role in preparing the human endometrium for implantation.     

Isolation of arterial-specific genes by subtractive hybridization reveals molecular heterogeneity among arterial endothelial cells.

Arteries are distinguished from veins by differences in gene expression, as well as in their anatomy and physiology. The characterization of arterial- and venous-specific genes may improve our understanding of cardiovascular development and disease. Here we report the results of a subtractive hybridization screen for arterial-specific genes, and describe in detail the expression of a novel arterial-specific gene, Depp (decidual protein induced by progesterone), using a GFP-Cre knock-in that permits a comparison of both instantaneous and cumulative expression patterns in situ. Several features of Depp expression are noteworthy. First, Depp is expressed in endothelial cells of peripheral tissues, but not in atrial or ventricular endocardial cells of the heart. Very few genes have been reported to discriminate between these two cell types, and therefore this specificity may be useful in generating conditional mutations in other genes implicated in cardiovascular development. Second, Depp reveals an unexpected degree of molecular heterogeneity among arterial endothelial cells. Third, Depp is up-regulated in subsets of endothelial cells, in settings of adult neo-vascularization, including tumor angiogenesis. Taken together, these data reveal unanticipated temporal and spatial heterogeneity among arterial endothelial cells of various tissues and organs, raising new questions regarding the functional significance of this diversity.