In vitro decidualization of human endometrial stromal cells.

Stromal cells isolated from proliferative human endometrium undergo morphologic and biochemical changes when exposed to a mixture of ovarian hormones, acquiring characteristics of decidual cells. In addition to the previously reported progestin-induced secretion of prolactin (PRL) by explants of human proliferative endometrium, and of PRL and laminin by stromal cells in culture, "in vitro" induction of several other decidual cell products was demonstrated in the present study, using cultures of stromal cells isolated from proliferative endometrium. Incubation of stromal cells with a mixture of estradiol, medroxyprogesterone acetate and relaxin, at a concentration reported to yield maximal stimulation of PRL production, resulted in changes from elongated to rounder cells, approx. 90% of which showed immunostaining for PRL under these conditions. Immunocytochemical procedures were carried out on cytospins of decidual cells isolated from decidual tissue adherent to fetal membranes collected at delivery (positive controls), and on stromal cells cultured in Lab-Tek chamber-slides, in the absence (negative controls) or in the presence of added hormones. Antibodies to 24K (a heat-shock protein also named HRP27), desmin (present in intermediate filaments), p29 (a protein associated with the estrogen receptor), and PP12 (an insulin growth factor-1 binding protein), did not react with stromal cells isolated from proliferative endometrium but showed immunostaining of the rounder cells obtained after hormonal treatment when tested with the peroxidase-labeled second antibody complex. In another series of similar experiments, in which the same decidualization end-points were employed, changes in 24K, desmin and PP12 expression were obtained by adding to the insulin-containing medium PRL instead of the hormonal mixture, a finding suggesting sequential steps during the decidualization process.     

Direct effect of gonadotropins on decidualization of human endometrial stromal cells

Decidualization of stromal cells isolated from proliferative human endometrium was achieved by adding to the culture medium human gonadotropins (FSH, FSH + LH, hCG). In addition to changes in the morphology of the stromal cells to the decidual phenotype, decidualization was evident from the expression of prolactin (PRL), demonstrated immunocytochemically, by Western blotting analysis, and by measuring its output into the medium through solid phase enzyme immunoassay. Gonadotropins also induced cAMP formation in the endometrial stromal cells under the same experimental conditions. This finding suggests that the mechanism by which gonadotropins promote decidualization of human endometrial stromal cells in vitro involves the introduction of cAMP, a compound that we have found to elicit the expression of PRL in this system. PRL is likely to be a key intermediate in the process of decidualization since it is by itself capable of inducing differentiation of the endometrial stromal cells to the decidual phenotype. Awareness of direct actions of gonadotropins on the endometrial cells and, in particular, of the decidualizing effects of FSH (Metrodin), FSH + LH (Pergonal) and hCG may contribute to the understanding of physiologic as well as pathophysiologic conditions relevant to endometrial functions and fertility.     

In vitro decidualization of rat endometrial stromal cells

The induction of the decidualization of endometrial stromal cells is possible in an in vitro cell culture system. However, thus far, methods differ according to species or cell type, and a more stable or universal system has not yet been developed. The purpose of the present study has been to establish an in vitro decidualization system in primary cultured rat endometrial stromal cells (RES). The RES were treated with medroxyprogesterone acetate and dibutyryl-cyclic adenosine monophosphate (MPA treatment), estradiol and progesterone, or arachidonic acid. After 24 h of treatment, cells responded to all of the stimulations by expressing desmin mRNA. However, decidual/trophoblast prolactin-related protein (dPRP) mRNA was only expressed in the MPA-treated cells. Desmin and dPRP mRNA were not expressed after MPA treatment of the RES derived from immature rat uteri. However, mRNA from both desmin and dPRP were expressed in RES derived from gonadotrophin-injected immature rats. The expression of matrix metalloproteinase-2 (MMP-2) and MMP-9 mRNA did not change after the decidual treatment of RES examined by real-time polymerase chain reaction. However, the results of gelatin zymography showed that the active forms of MMP-2 and MMP-9 significantly increased after in vitro decidualization (P < 0.05). We conclude that MPA treatment is the most effective method for stimulating decidualization in RES. Use of this system has revealed that sexual maturation and gonadotrophins are important for RES with regard to decidualization. Furthermore, the activity of MMP-2 and MMP-9 might increase during decidualization without a corresponding increase of the expression of these genes. This research was supported by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (JSPS; no. 18580282, to N. Yamauchi).     

Phospholipase D1 as a key enzyme for decidualization in human endometrial stromal cells

Using primary cell cultures of human endometrial stromal cells (ES cells), we investigated the role of phospholipase D (PLD) in 8-Br-cAMP-induced decidualization, which involves morphological and biological differentiation processes. When treated with 0.5 mM 8-Br-cAMP for 12 days, ES cells were transformed into a decidualized morphology and produced significant amounts of prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP1). Simultaneously, the activity and expression levels of PLD1 increased. In addition, removal of 8-Br-cAMP from decidualized ES cells restored the undifferentiated state, and this was accompanied by decreases in PLD1 promoter activity and PLD1 expression. Overexpression of dominant negative (DN)-PLD1 inhibited the morphological changes induced by 0.5 mM 8-Br-cAMP, whereas PLD1 overexpression induced morphological changes in the absence of 0.5 mM 8-Br-cAMP treatment. Moreover, knockdown of PLD1 by siRNA and blockage of PLD by treatment with 0.3% 1-butanol decreased PRL/IGFBP1 mRNA expression, whereas PLD1 overexpression increased PRL/IGFBP1 mRNA expression. Treatment of ES cells with phosphatidic acid (PA) for 3 days induced PRL mRNA expression and morphological changes, which implies that PA is an end-product of PLD activation-induced decidualization. In addition, pretreatment of ES cells with mepacrine decreased PRL/IGFBP1 expression and inhibited morphological change, whereas pretreatment with propranolol caused no changes, as compared to cAMP-treated cells, which suggests that PA induces decidualization through phospholipase A2 (PLA2G1B). Taken together, these results suggest that PLD1 regulates 8-Br-cAMP-induced decidualization through PLA2G1B, and that PLD1 upregulation is essential for the decidualization of ES cells.     

Epidermal growth factor receptors in human breast and endometrial carcinomas

The incidence and levels of epidermal growth factor receptor (EGFR) were studied in 67 breast tumors and 22 endometrial carcinomas. Estrogen receptors (ER) were also measured in all samples and progesterone receptors (PR) were analyzed in 57 breast samples and 21 endometrial tumors. A high level of EGFR expression is found in both breast and endometrial carcinomas, although the incidence of EGFR content is greater in breast carcinomas. 36% of breast tumors had EGFR at levels 3-49.5 fmol/mg membrane protein, whereas this percentage of positivity was 27% for endometrial tumors. In 51% of breast carcinoma and 73% of endometrial tumors, there was a positive ER content, whereas 53% of breast tumors and 62% of endometrial carcinomas were positive. A clear inverse relationship between EGFR content and ER and PR status has been observed in breast tumors. Our data confirm the previously described inverse correlation between expression of EGFR and estrogen receptors in human breast cancer. We also show here that there is a similar inverse relationship between EGFR content and ER levels in endometrial tumors.     

Epidermal growth factor inhibits cytokine-induced apoptosis of primary human trophoblasts

In the placenta, as in other organs, the development and maintenance of the differentiated phenotype depend on a balance between cell proliferation, maturation, and death. We are interested in the mechanisms that regulate the survival and differentiation of placental trophoblasts and have recently demonstrated that the inflammatory cytokines tumor necrosis factor alpha (TNFα) and gamma interferon (IFNγ) act in concert to induce apoptotic cell death in normal cytotrophoblasts in culture. In this report we show that exposure to epidermal growth factor (EGF), a 6,700 dalton polypeptide that is abundantly expressed in maternal and fetal tissues, blocks the in vitro TNF/IFN-induced cytotoxicity of human cytotrophoblasts and syncytiotrophoblasts from normal term placentas. This antagonistic effect is dose-related (10⁻¹⁰ M EGF, half-maximal) and proceeds via the interruption of an early step in the cytokine-induced apoptotic response. These observations suggest a novel role for EGF in normal placental development and indicate that the interplay between EGF, TNFα, and IFNγ may determine the rate of trophoblast growth and renewal during gestation. © 1996 Wiley-Liss, Inc.     

Dehydroepiandrosterone inhibits glucose flux through the pentose phosphate pathway in human and mouse endometrial stromal cells, preventing decidualization and implantation

Endometrial stromal cells (ESC) must undergo a hormone-driven differentiation to form decidual cells as a requirement of proper embryo implantation. Recent studies from our laboratory have demonstrated that decidualizing cells require glucose transporter 1 expression and an increase in glucose use to complete this step. The present study focuses on the glucose-dependent molecular and metabolic pathways, which are required by ESC for decidualization. Inhibition of glycolysis had no effect on decidualization. However, blockade of the pentose phosphate pathway (PPP) with pharmacologic inhibitors 6-aminonicotinamide or dehydroepiandrosterone (DHEA), and short hairpin RNA-mediated knockdown of glucose-6-phosphate dehydrogenase, the rate-limiting step in the PPP, both led to strong decreases in decidual marker expression in vitro and decreased decidualization in vivo. Additionally, the studies demonstrate that inhibition is due, at least in part, to ribose-5-phosphate depletion, because exogenous nucleoside administration restored decidualization in these cells. The finding that PPP inhibition prevents decidualization of ESC is novel and clinically important, because DHEA is an endogenous hormone produced by the adrenal glands and elevated in a high proportion of women who have polycystic ovary syndrome, the most common endocrinopathy in reproductive age women. Together, this data suggest a mechanistic link between increased DHEA levels, use of glucose via the PPP, and pregnancy loss.     

Leukemia inhibitory factor enhances endometrial stromal cell decidualization in humans and mice

Adequate differentiation or decidualization of endometrial stromal cells (ESC) is critical for successful pregnancy in humans and rodents. Here, we investigated the role of leukemia inhibitory factor (LIF) in human and murine decidualization. Ex vivo human (H) ESC decidualization was induced by estrogen (E, 10⁻⁸ M) plus medroxyprogesterone acetate (MPA, 10⁻⁷ M). Exogenous LIF (≥50 ng/ml) induced STAT3 phosphorylation in non-decidualized and decidualized HESC and enhanced E+MPA-induced decidualization (measured by PRL secretion, P<0.05). LIF mRNA in HESC was down-regulated by decidualization treatment (E+MPA) whereas LIF receptor (R) mRNA was up-regulated, suggesting that the decidualization stimulus ‘primed’ HESC for LIF action, but that factors not present in our in vitro model were required to induce LIF expression. Ex vivo first trimester decidual biopsies secreted >100 pg/mg G-CSF, IL6, IL8, and MCP1. Decidualized HESC secreted IL6, IL8, IL15 and MCP1. LIF (50 ng/ml) up-regulated IL6 and IL15 (P<0.05) secretion in decidualized HESC compared to 0.5 ng/ml LIF. In murine endometrium, LIF and LIFR immunolocalized to decidualized stromal cells on day 5 of gestation (day 0 = day of plug detection). Western blotting confirmed that LIF and the LIFR were up-regulated in intra-implantation sites compared to inter-implantation sites on Day 5 of gestation. To determine the role of LIF during in vivo murine decidualization, intra-peritoneal injections of a long-acting LIF antagonist (PEGLA; 900 or 1200 µg) were given just post-attachment, during the initiation of decidualization on day 4. PEGLA treatment reduced implantation site decidual area (P<0.05) and desmin staining immuno-intensity (P<0.05) compared to control on day 6 of gestation. This study demonstrated that LIF was an important regulator of decidualization in humans and mice and data provides insight into the processes underlying decidualization, which are important for understanding implantation and placentation.     

Inhibition of cAMP-mediated decidualization in human endometrial stromal cells by IL-1beta and laminin.

Both IL-1 and laminin are reported to inhibit progesterone receptor-mediated decidualization signals in endometrial stromal cells, but the effects of these two inhibitors on cAMP-mediated decidualization signals remain unknown. Furthermore, the interaction between IL-1-stimulated and laminin-stimulated signals has not been analyzed yet. In this study, interactions between these two decidualization-inhibitory signals and their effects on 8-Br-cAMP-mediated decidualization in human endometrial stromal cells were investigated. Prolactin release from decidualized stromal cells was measured by enzyme immunoassay in order to quantitate in vitro decidualization. Both IL-1beta and laminin were found to inhibit 8-Br-cAMP-induced decidualization in vitro in human endometrial stromal cells. IL-1beta dose-dependently inhibited decidualization of stromal cells cultured on both laminin-coated and collagen-coated dishes, while laminin inhibited the decidualization of stromal cells cultured both with and without IL-1beta. These results indicate that IL-1beta and laminin additively and mutually inhibit cAMP-mediated decidualization signals, and that they may inhibit these signals through different mechanisms.     

Laminin decreases PRL and IGFBP-1 expression during in vitro decidualization of human endometrial stromal cells

The expression of laminin, a major constituent of endometrial cell basement membranes, is increased during differentiation of human endometrial stromal cells (decidualization). To determine whether laminin plays a role in decidualization, we studied the effects of laminin substrate on the synthesis and release of prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1), two major secretory proteins of decidualized stromal cells. Endometrial stromal cells were plated on laminin as well as several other extracellular matrix (ECM) proteins (types 1 and IV collagen or fibronectin) and on plastic, and cultured in media containing medroxyprogesterone acetate (MPA) and estradiol. Cells cultured on plastic or ECM proteins displayed similar morphological changes indicative of decidualization. However, the release of PRL and IGFBP-1 from cells cultured on plastic and ECM proteins (types 1 and IV collagen and fibronection) was approximately 2.1-fold and 2.8-fold greater respectively, than from cells cultured on laminin. The decrease in PRL and IGFBP-1 expression in cells cultured on laminin was not due to differences in initial cell attachment efficiency or final DNA content. In addition, laminin had no effect on the content of laminin protein or fibronectin mRNA levels, indicating that the effects of laminin on PRL and IGFBP-1 were specific. PGE₂ stimulated the release of PRL and IGFBP-1 from cells cultured on laminin to levels comparable to those from cells cultured on plastic or other ECM proteins. This indicates that the decrease in PRL and IGFBP-1 release by laminin was not due to a generalized unresponsiveness. In contrast to the effects of laminin during decidualization, PRL expression was not altered by laminin in terminally differentiated decidual cells isolated at term. Our results support a role for laminin in selectively regulating PRL and IGFBP-1 gene expression during in vitro decidualization of human endometrial stromal cells. © 1995 Wiley-Liss, Inc.     

Epidermal growth factor inhibits cytoskeleton-related changes in the surface of parietal cells

The effect of epidermal growth factor (EGF) on gastric acid secretion was correlated with the morphological changes of the apical pole of rat parietal cells studied by transmission electron microscopy. Gastric acid secretion was stimulated by histamine, carbachol, pentagastrin, and insulin-induced hypoglycemia, and estimated by continuous recording of pH variations of gastric luminal perfusate. EGF inhibits acid secretion in these conditions. The action of the hormone also results in the arrest or reversal of the changes in shape undergone by parietal cells as they go into secretion. In view of the evidence involving cytoskeletal elements in the generation of these structural alterations, our observations suggest that the action of EGF on gastric acid secretion may be a consequence of a general effect of this hormone on cytoskeletal function.     

Epidermal growth factor and transforming growth factor-alpha stimulate the proliferation of mouse uterine stromal cells

Growth factors produced in the uterine endometrium are considered to be involved in the proliferation of the mouse uterine stromal cells induced by estradiol-17beta (E(2)) and progesterone (P). The effect of epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha), one of EGF-related growth factors, on the proliferation of mouse uterine stromal cells was studied in a serum-free culture. The growth of the uterine stromal cells was measured by MTT assay. EGF was found to increase the number of uterine stromal cells in a dose-dependent manner. The DNA-replicating cells were investigated using the immunocytochemical detection of bromodeoxyuridine (BrdU)-labeled cells. EGF and TGF-alpha increased the percentage of BrdU-labeled cells in a dose-dependent manner. Administration of the combination of E(2) (10(-9) M) and P (10(-7) M) for 2 days increased the percentage of BrdU-labeled cells 2.3-fold. The stimulatory effect of EGF, TGF-alpha and the combination of E(2) and P on DNA replication in the uterine stromal cells was repressed by RG-13022 (10(-5) M, the inhibitor of the EGF receptor tyrosine kinase). RT-PCR analysis of EGF-receptor-, TGF-alpha-, and EGF-mRNA was carried out in the cultured uterine stromal cells, and revealed the expression of those mRNAs. These data supported the hypothesis that uterine endometrial stromal growth induced by sex steroids required the EGF family of ligands such as EGF and TGF-alpha, both produced in the stromal cells, acting for DNA synthesis through EGF receptors.     

Epidermal growth factor stimulate cell proliferation and inhibits prolactin secretion of the human decidual cells in culture

Epidermal growth factor (EGF) has been found to be mitogenic in a variety of tissues. We investigated the biological effect of EGF on early pregnant human decidua and the non-pregnant decidualized human endometrium in the primary cell culture. EGF had a stimulatory action on cell proliferation in the early pregnant decidual cells and an inhibitory effect on prolactin (PRL) secretion from the decidual cells. The addition of progesterone into culture medium suppressed cell proliferation of decidual cells, whereas it enhanced PRL secretion from decidua. The analysis of the specific receptor for EGF in the early pregnant decidua and non-pregnant decidualized endometrium revealed that both tissues had a single component EGF receptor with a dissociation constant of nM order. These results suggest that EGF may play a role in the growth and function of endometrial stromal cells.