Endothelin receptor expression in human decidua

The endothelins are signalling peptides that act via two receptors, ET(A) and ET(B). In the human endometrium, endothelin receptors have been demonstrated in glands and stroma and have been shown to vary during the course of the menstrual cycle. The present study was undertaken to determine whether or not expression of endothelin receptors changes during pregnancy or after administration of exogenous progestagens. The expression of the receptors was correlated with the appearance of basement membrane components during decidualization of the endometrial stroma. Decidual specimens (n = 15) were obtained during the first trimester of pregnancy and 10 at term. Sixteen pairs of endometrial biopsies were obtained from women with menorrhagia before and after exposure to exogenous progestagens. A total of 15 hysterectomy specimens were used as controls for the expression of stromal basement membrane proteins in the absence of decidualization. Autoradiography was carried out with selective ligands for ET(A) ([125I]-PD 151242) and ET(B) ([125I]-BQ3020). The distribution of ligand binding was then compared with the distribution of laminin alpha2 light chain and collagen IV. ET(A), ET(B), laminin alpha2 light chain, and collagen IV were expressed in stromal decidual cells in the first trimester of pregnancy. ET(B) was also found on endometrial glandular epithelium. Quantitative macro-autoradiography and multiple regression analysis demonstrated a highly significant positive correlation (P < 0.001) between expression of ET(B) and laminin alpha2 light chain. In the third trimester qualitative examination suggested a reduction of ET(A) in the stroma. Progestagen-induced decidua exhibited a similar pattern to that found in first trimester decidua. This study has demonstrated up-regulation of ET(B) during the progesterone- dependent process of decidualization and suggests a paracrine or autocrine role for endothelins in the decidua.      

Expression of interleukin-11 during the human menstrual cycle: coincidence with stromal cell decidualization and relationship to leukaemia inhibitory factor and prolactin

Interleukin-11 (IL-11) is crucial in the decidualization response of the uterine stroma to the implanting blastocyst in the mouse. This study examined the localization and expression of IL-11 in human endometrium throughout the menstrual cycle and of prolactin and leukaemia inhibitory factor (LIF) in secretory phase endometrium. The mRNA expression of IL-11 receptor alpha and the signalling component, gp130, in endometrial tissue were also determined. Immunoreactive IL-11 was highest in the secretory phase and present in decidualized stromal cells, glandular epithelial cells, endothelial and smooth muscle cells, and the mRNA expression was verified by in-situ hybridization. Decidual cells showed the most intense staining. IL-11 receptor alpha and gp130 mRNA were detected throughout the cycle with minimal variation. Expression of IL-11 mRNA and protein preceded that of prolactin. While immunoreactive prolactin was found in stromal, decidual and glandular epithelial cells, prolactin mRNA was confined to decidual cells. In contrast, endometrial LIF expression preceded IL-11 but was largely confined to the glandular epithelium. The sequence of appearance of LIF, IL-11 and prolactin suggests a synchronized role for each in the differentiation of the endometrium. The cyclical changes and cell type specific expression of IL-11 suggests a potential role in the decidualization of stromal cells.     

Differential effects of interleukin-1beta and transforming growth factor-beta1 on the expression of the inflammation-associated protein, ADAMTS-1, in human decidual stromal cells in vitro

BACKGROUND: The pro-inflammatory cytokine, interleukin-1 beta (IL-1beta) promotes the proteolytic degradation of the extracellular matrix (ECM) of maternal decidua, a critical step in pregnancy that is counterbalanced by the expression of the anti-inflammatory cytokine, transforming growth factor-beta 1 (TGF-beta1). Recently, the inflammation-associated protein, ADAMTS-1, a member of the ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin repeats) gene family of metalloproteinases has been assigned a central role in the formation and organization of tissues. In view of these observations, we have hypothesized that ADAMTS-1 contributes to the cytokine-mediated remodelling of decidual ECM. METHODS: The spatiotemporal expression of ADAMTS-1 in human endometrium was examined by immunohistochemistry. A quantitative-competitive (QC)-PCR strategy and western blot analysis was then employed to determine whether IL-1beta and TGF-beta1 regulate ADAMTS-1 mRNA and protein expression levels in primary cultures of stromal cells isolated from first trimester decidua. RESULTS: ADAMTS-1 expression is associated with decidualization of the endometrial stroma in vivo. IL-1beta increased whereas TGF-beta1 decreased ADAMTS-1 mRNA and protein levels in decidual stromal cell cultures in a concentration- and time-dependent manner. These regulatory effects were attenuated by function-perturbing antibodies specific for either cytokine. CONCLUSION: IL-1beta and TGF-beta1 differentially regulate ADAMTS-1 expression in human decidual stromal cells.     

Expression of intermediate filament in endometrial glands changes with the onset of pregnancy and in endometriosis.

Appropriate endometrial differentiation is believed to be a prerequisite for pregnancy success. This study investigates the expression of two intermediate filament proteins, cytokeratin and vimentin, in human endometrium and first trimester decidua and in ectopic endometrium from women with endometriosis. Stromal elements, including vascular endothelial cells, were consistently vimentin-positive and cytokeratin-negative. Surface and glandular epithelial cells of human endometrium co-expressed vimentin and cytokeratin during all stages of the menstrual cycle, but failed to express vimentin after the onset of pregnancy. This suggests that intermediate filaments, and especially vimentin, may have a role to play in the proliferation and/or differentiation of the endometrial glands during decidualization. Ectopic endometrium showed a staining pattern similar to normal endometrium.     

Progesterone-dependent release of transforming growth factor-beta1 from epithelial cells enhances the endometrial decidualization by turning on the Smad signalling in stromal cells

Endometrial decidualization results from the differentiationof stromal cells in an ovarian steroid-sensitive manner. Humanendometrial tissues obtained from fertile women at various stagesof the menstrual cycle were subjected to immunohistochemistryto localize the components of the transforming growth factor-beta(TGF-ß) system. TGF-ß receptor-I and -IIexpression was higher in stromal cells than in epithelial cellsduring the secretory phase while no such variation was observedduring the proliferative phase. The expression of phosphorylatedSmad3 (pSmad2/3), an activated form of a component of the TGF-ßsignalling pathway, and translocation of pSmad2/3 from the cytoplasmto the nucleus were more pronounced in secretory endometrium.In coculture of human endometrial epithelial with stromal cells,each isolated from the proliferative endometrium, administrationof progesterone stimulated decidualization as well as TGF-ßsignalling activation in stromal cells. Progesterone also significantlyelevated the concentration of TGF-ß1 in the coculturemedium. Careful manipulation of the coculture, i.e. selectiveaddition and omission of the cellular components, showed thatthis progesterone-induced increase in secretion of TGF-ß1come mainly from epithelial cells. Moreover, administrationof TGF-ß1 (10 ng/ml) directly to cultured stromalcells enhanced the expression of prolactin as well as pSamd2/3even without progesterone. Taken together, our present datasupport the notion that progesterone induces stromal decidualizationindirectly, i.e. by enhancing the expression and secretion ofTGF-ß1 from epithelial cells. The secreted, epithelial-derivedTGF-ß1 then acts on adjacent stromal cells, at leastin part, to turn on Smad signalling that could lead to stromaldecidualization.     

Galectin fingerprinting in human endometrium and decidua during the menstrual cycle and in early gestation

The emerging functionality of the sugar code via cell surface glycans and endogenous lectins ascribes pertinent roles in cell physiology to the carbohydrate signals of cellular glycoconjugates. To initiate monitoring of endogenous lectins in human endometrium, we focused on a family of growth/adhesion-regulatory lectins, i.e. galectins. Comprehensive fingerprinting was performed on samples throughout the menstrual cycle and in decidua. The endometrium (n = 30) and decidua (n = 7) were collected from patients undergoing hysterectomy for benign reasons and from induced abortions. Measurements by RT-PCR and then by multiprobe RNase protection assay with total endometrial and decidual tissue and with epithelial cells, stromal cells and CD45-positive cell fractions (n = 16), isolated by the use of antibody-coated magnetic beads, revealed a predominant expression of galectins-1 and -3. Protein analysis was performed by immunocytochemistry with monoclonal and polyclonal antibodies (n = 40). Galectin-1 was localized mainly in stromal cells, whereas galectin-3 was predominantly found in epithelial cells. Expression of galectin-1 increased significantly in the late secretory phase endometrium and in the decidual tissue. Expression of galectin-3 increased significantly during the secretory phase of the menstrual cycle. Cycle-dependent expression of galectin-1 in stromal cells and galectin-3 in epithelial cells suggest these lectins to be involved in the regulation of different endometrial cellular functions.     

Decidual expression and localization of human surfactant protein SP-A and SP-D,and complement protein C1q

Surfactant proteins SP-A and SP-D, and complement protein C1q are soluble innate immune pattern recognizing molecules. SP-A, SP-D and C1q have an overall similar structure composed of an N-terminal triple-helical collagen region that is followed by a trimeric globular domain. While SP-A and SP-D belong to the collectin family (collagen containing lectin), C1q is the first recognition subcomponent of the classical pathway of the complement system. Recently, SP-A, SP-D and C1q have been considered to play important roles in early and late pregnancy. However, their expression in early human decidua has not been examined. Here, we investigated whether SP-A, SP-D and C1q are expressed within first trimester decidua in humans and their expression is associated with trophoblasts and decidual stromal cells. Decidual samples from women undergoing elective vaginal termination of pregnancy during first trimester were obtained from 25 subjects. Immunohistochemical studies using anti-human SP-A, anti-human SP-D and anti-human C1q antibodies were performed on decidual tissue sections along with anti-vimentin and cytokeratin-7 antibodies to identify stromal cells and trophoblasts. The expression was also examined by immunostaining and PCR using decidual and stromal cells. C1q expression was significantly higher when compared to SP-A and SP-D in the first trimester human decidua. Double immunostaining revealed that all stromal cells and trophoblasts expressed SP-A, SP-D and C1q, while only few invasive trophoblasts expressed C1q. Thus, expression of SP-A, SP-D and C1q in human decidua during first trimester suggests potential role of SP-A, SP-D and C1q during the early stages of pregnancy including implantation, trophoblast invasion and placental development.     

Expression and subcellular distribution of the active form of c-Src tyrosine kinase in differentiating human endometrial stromal cells

Decidual growth factors and locally produced cytokines are thought to activate specific phosphorylation signalling pathway(s), thereby eliciting a variety of decidual functions. We have previously reported the activation of c-Src tyrosine kinase during ovarian steroid-induced decidualization of cultured human endometrial stromal cells. As chicken c-Src is known to be activated upon dephosphorylation of tyrosine 527 (Y527, corresponding to Y530 in human), we here employed a monoclonal antibody, clone 28, directed against the active form of human c-Src whose Y530 is dephosphorylated, and investigated whether c-Src became dephosphorylated at Y530 and thereby activated during decidualization. We found that the active form of c-Src was up-regulated and demonstrated increased kinase activity during in-vitro decidualization. Immunohistochemistry revealed that decidual cells in early pregnancy decidua were intensely stained with clone 28 when compared with the stromal cells in the non-pregnant endometrium. Moreover, the active form of c-Src translocated from a perinuclear region to the cytoplasm upon decidualization. Thus, the Y530 dephosphorylation, kinase activation, and subcellular translocation of c-Src may be intracellular signalling events associated with decidualization in vivo as well as in vitro.     

Fas antigen (CD95) mediates cell survival signals to regulate functional cellular subpopulations in normal human endometrial stromal cells

Fas antigen (CD95) is expressed on almost all types of human cells and is believed to mediate receptor-specific apoptotic signals. In human endometrial tissues, high Fas and Fas ligand expressions and Fas-mediated apoptosis in endometrial epithelial cells have been discussed in many reports but no study has examined Fas-mediated signals in endometrial stromal cells. In this study we investigated Fas expression and Fas-mediated signals of normal human endometrial stromal cells. Flow cytometric analysis revealed Fas antigen expression on the stromal cells and their Fas expression was enhanced by 8-Br-cAMP, a strong inducer of decidualization. Neither short-term nor long-term cultures with anti-Fas IgM affected proliferation or viability of the stromal cells. Anti-Fas IgM alone affected neither viable cell numbers nor PRL release of unstimulated stromal cells. However, anti-Fas IgM dose-dependently stimulated viable cell numbers of stromal cells co-stimulated with 8-Br-cAMP and anti-Fas IgM, whereas PRL secretion of the co-stimulated cells was not affected. Anti-Fas IgM dose-dependently stimulated viable cell numbers of 8-Br-cAMP-stimulated cells but did not affect PRL secretion of 8-Br-cAMP-induced decidualized cells. These results indicate that Fas antigen on human endometrial stromal cells cannot mediate receptor-specific apoptotic signals, and that Fas-mediated signals stimulate survival of 8-Br-cAMP-stimulated non-decidualized stromal cells. Thus, stimulation of Fas-antigen on the endometrial stromal cells enhances anti-apoptotic/survival signals in certain stromal cells, autoregulating functional cellular subpopulations of human endometrial stromal cells in a paracrine manner.     

Insulin-like growth factor binding protein-1 induces decidualization of human endometrial stromal cells via alpha5beta1 integrin

Progesterone is known to induce decidualization of human endometrial stromal cells in vitro. Decidualized stromal cells produce insulin-like growth factor binding protein-1 (IGFBP-1) as well as prolactin (PRL). In this study, we tested the possibility that IGFBP-1 directly stimulates endometrial stromal cell decidualization. Endometrial stromal cells were obtained from normal menstruating patients with uterine myoma at hysterectomy. Stromal cells were cultured for up to 4 weeks with estradiol (E(2)) and/or medroxy progesterone acetate (MPA) in the presence or the absence of IGFBP-1 and, LR(3)-IGF-I (an IGF-I analogue) that binds to the IGF-I receptor but has reduced affinity for IGFBPs. Decidualization of endometrial stromal cells was evaluated by morphological changes and PRL release into culture media. The binding of IGFBP-1 to endometrial cells was analysed using a biosensor. MPA and E(2) induced decidualization of stromal cells, while LR(3)-IGF-I inhibited decidualization by MPA and E(2) as well as PRL and IGFBP-1 secretion into medium. IGFBP-1 induced decidualization of stromal cells in the absence of MPA and E(2) in the medium. IGFBP-1-induced decidualization was not inhibited by the addition of LR(3)IGF-1 but was inhibited by the addition of an RGD peptide, however, the RGD peptide had no effect on decidualization when added alone. The binding analysis showed that IGFBP-1 bound to the surface of endometrial stromal cells and an anti-alpha5beta1 integrin antibody inhibited its binding. These results suggest that IGFBP-1 produced by endometrium can mediate progesterone-induced decidualization possibly by interacting with alpha5beta1 integrin on the surface of endometrial stromal cells.     

Dipeptidyl peptidase IV as a differentiation marker of the human endometrial glandular cells.

To investigate the involvement of membrane-bound peptidases in the human endometrial function, we examined the expression of dipeptidyl peptidase (DPP) IV and its enzyme activity. Immunohistological studies revealed that DPP IV was detected on human endometrial glandular cells and endometrial surface epithelium, but not on endometrial stromal cells or decidual cells in the first trimester of pregnancy. DPP IV expression on glandular cells and surface epithelium was weak in the proliferative phase, began to increase gradually in the early secretory phase, and was strong in mid-to late secretory phase and in the first trimester of pregnancy. DPP IV enzyme activity was detected histochemically in glandular cells and surface epithelium in the mid-secretory phase, and became stronger in the late secretory phase, but was rarely detected in the proliferative phase and early secretory phase. During the first trimester of pregnancy DPP IV enzyme activity in glandular cells and surface epithelium was slightly weaker than in the late secretory phase. Endometrial stromal cells and decidual cells, however, had no detectable DPP IV enzyme activity at any time throughout the menstrual cycle or during the first trimester of pregnancy. These findings indicate that DPP IV is a differentiation marker for glandular cells and surface epithelium and that active DPP IV is present in both areas during the peri-implantation period and thereafter.     

Plasma membrane and vesicular monoamine transporters in normal endometrium and early pregnancy decidua

The uterus is innervated by adrenergic sympathetic fibres, and the endometrium has a capability for endogenous monoamine synthesis. Extracellular monoamine levels are regulated primarily through re-uptake by specific membrane-bound transporter proteins dopamine transporter (DAT), norepinephrine transporter (NET) and serotonin transporter (SERT). Intracellular storage of monoamines involves vesicular transporter proteins (VMAT1 and VMAT2). This study explored gene expression of the monoamine transporters in normal endometrium throughout the menstrual cycle and early decidua. In-situ hybridization histochemistry revealed three general classes of expression patterns: (i). epithelial expression of NET mRNA; (ii). increasing stromal expression of VMAT2 mRNA in the proliferative phase; and (iii). increasing epithelial expression of VMAT2 mRNA during the secretory phase. Real time PCR showed low expression levels of NET in all phases of the endometrial cycle and a higher expression of VMAT2 mRNA in the mid-secretory phase. Our results suggest that several monoamine transporters may have menstrual cycle phase-specific functions in endometrial biology by maintaining adequate levels of monoamines. Re-uptake and regulated release of monoamines may also modulate several steps of the reproductive processes such as embryo implantation and decidua formation.     

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.     

Maternal Hoxa10 is required for pinopod formation in the development of mouse uterine receptivity to embryo implantation.

Hoxa10 is a homeobox gene that is expressed both during the embryogenesis of the genitourinary tract and in the adult reproductive tract. Maternal Hoxa10 expression is necessary for endometrial receptivity to blastocyst implantation. The mechanism by which Hoxa10 induces endometrial development to a state of receptivity is unknown as HOXA10-deficient endometrium appears histologically normal. We altered the expression of Hoxa10 in the uterus of cycling adult female mice and examined the uterus at the time of implantation by transmission electron microscopy for alterations in epithelial morphology. Pinopods are projections on the surface of the uterine endometrial epithelial cells that develop transiently at the time of endometrial receptivity. Blocking Hoxa10 expression by transfection of Hoxa10 antisense into the cycling mouse uterus before implantation dramatically decreased pinopod number. Constitutively expressing Hoxa10 in the uterus just before the normal time of pinopod formation resulted in increased pinopod number. Therefore, Hoxa10 is necessary for pinopod development. Hox genes have been implicated in both the regulation of cellular proliferation and the determination of developmental fate. Hoxa10 exemplifies this dual role in the uterus by regulating both endometrial stromal cell proliferation and epithelial cell morphogenesis. Taken together, these results demonstrate that maternal Hoxa10 has an essential role in pinopod development and this function of Hoxa10 likely contributes to endometrial receptivity for the purpose of blastocyst implantation.     

Expression of integrin alpha5 and integrin beta4 and their extracellular ligands fibronectin and laminin in human decidua during early pregnancy and its sex steroid-mediated regulation

The reorganization of the human endometrium is termed decidualization, which includes endometrial cell proliferation, differentiation, integrin switching and extracellular matrix (ECM) remodeling during early pregnancy. The present study aimed to investigate distribution patterns, staining intensity and sex steroid-mediated regulation of integrin alpha5 (CD49e), integrin beta4 (CD49f) expression and their ligands fibronectin and laminin during decidualization. Human tissue samples were evaluated in two groups, those collected in early days and those collected in advanced days of the first trimester. Correlating immunostaining was found between laminin and integrin beta4, and between fibronectin and integrin alpha5. The expression of fibronectin was higher than that of laminin in the early days (p < 0.05). Temporal and spatial immunostaining of integrin beta4 and alpha5 in the apical pole of luminal and glandular cells was observed as pregnancy progressed (p < 0.05). In vitro results showed that human chorionic gonadotropin (hCG) stimulated laminin expression, downregulated integrin beta4 expression, whereas estradiol decreased fibronectin expression by Ishikawa cells. hCG suppressed fibronectin expression in endometrial stromal cells in culture. Our results suggest that fibronectin is responsible for induction of decidual cell differentiation, and different temporal and spatial expression of the integrins may play a role in implantation. Our in vitro results suggest that regulation of extracellular matrix remodeling and integrin switching are at least partially regulated by reproductive hormones.