Endometrial Stromal Cells In Culture Research Articles

Liver receptor homolog-1 is essential for pregnancy

Successful pregnancy requires coordination of an array of signals and factors from multiple tissues. One such element, the liver receptor homolog-1 (Lrh-1, NR5A2), is an orphan nuclear receptor that regulates metabolism and hormone synthesis1. It is strongly expressed in granulosa cells of ovarian follicles and in the corpus luteum of rodents2 and humans. Germline ablation of the Lrh-1 gene in mice is embryo-lethal at gastrulation3. Depletion of Lrh-1 in the ovarian follicle demonstrates that it regulates genes required for both steroid synthesis and ovulation4. To study the effects of Lrh-1 on mouse gestation, we disrupted its expression in the corpus luteum, resulting in luteal insufficiency. Hormone replacement permitted embryo implantation but was followed by gestational failure with impaired endometrial decidualization, compromised placental formation, fetal growth retardation, and fetal death. Lrh-1 is expressed in the mouse and human endometrium. In a human model of primary culture of endometrial stromal cells, depletion of Lrh-1 by siRNA abrogated decidualization. These findings demonstrate that Lrh-1 is necessary for maintenance of the corpus luteum, for promotion of decidualization and for placental formation. It therefore plays multiple, indispensible roles in establishing and sustaining pregnancy.

Testosterone Modulates the Expression of Molecules Linked to Insulin Action and Glucose Uptake in Endometrial Cells

Polycystic ovary syndrome (PCOS) is a common hyperandrogenic disorder associated with insulin resistance. Insulin exerts its metabolic function by activating the PI3K/Akt pathway and favoring glucose uptake. Caveolin-1 is a scaffolding protein which increases insulin receptor (IR) stability. Alternatively, activation of IR increases caveolin-1 phosphorylation on tyrosine-14. Furthermore, endometrial tissue from PCOS patients is proposed to be insulin resistant; however, the particular role of testosterone in modulating the metabolic effects of insulin remains unexplored in endometrial stromal cells. To evaluate whether androgens modulate the response to insulin, T-HESCs cells were stimulated with 100 nM testosterone for 24 h and changes in the protein levels of caveolin-1, IR, and Akt were determined by Western blotting (WB). After testosterone treatment, the consequences of acute insulin stimulation were evaluated by WB analysis of phospho-S473Akt and phospho-Y14Caveolin-1, as well as by measuring glucose incorporation analyzing 2-deoxyglucose (2-DOG) uptake. For cells pretreated with testosterone, higher IR, IRS-1, and caveolin-1 protein levels compared with control conditions were detected. However, in testosterone treated cells acute insulin stimulation did not increase phospho-S473Akt and phospho-Y14caveolin-1 levels and reduced 2-DOG uptake was observed compared to control cells. Our results suggest that testosterone may have a detrimental role on the metabolic effects of insulin in endometrial stromal cell cultures. Thus, the high androgen levels in patients with PCOS may favor insulin resistance observed in endometria from these women.

Effect of deforolimus and VEGF on angiogenesis in endometrial stromal cells following three-dimensional culture

The presence of endometrial tissue outside of the uterine cavity is named endometriosis and is the most common gynecologic disorder in women. Determining the inhibitory effect of a Deforolimus on angiogenesis in a three-dimensional (3-D) culture of human endometrial stromal cells (hEnCs) in vitro. The important mechanism in the pathogenesis of endometriosis is angiogenesis, and deforolimus has been shown to have anti-angiogenic activity. This was an in vitro study of human endometrial stromal cells in 3-D culture of fibrin matrix. Endometrial stromal cells isolated and placed in a 3-D fibrin matrix culture system for angiogenesis with VEGF and inhibit angiogenesis by deforolimus. Finally these cells analyzed by CD31 antibodies. After 3 weeks, in cells treated with VEGF, endothelial cell branching was observed and rudimentary capillary-like structures formed. In the presence of 5μM of deforolimus, angiogenesis was reduced. The deforolimus were shown to be effective in inhibiting the mechanisms of angiogenesis.

Epithelial cell protein milk fat globule–epidermal growth factor 8 and human chorionic gonadotropin regulate stromal cell apoptosis in the human endometrium

To study the regulation of apoptosis in human endometrial cells. The specific aims were to determine whether milk fat globule-epidermal growth factor 8 (MFG-E8), a novel endometrial epithelial protein, modulates caspase activation and DNA fragmentation; and to examine whether hCG, an early embryonic product, regulates Bax and Bcl-2 equilibrium, as well as MFG-E8 expression. Primary cultures of human endometrial epithelial cells (EECs) and endometrial stromal cells (ESCs). Academic center. Ovulatory women aged 21-30 years. Treatment with MFG-E8 and hCG. Apoptotic activity was quantified using a luciferase assay. Deoxyribonucleic acid fragmentation was detected by TUNEL assay. Bax, Bcl-2, and MFG-E8 messenger RNA expression levels were determined by quantitative reverse transcription-polymerase chain reaction. Immunocytochemistry was used to establish cell purity and presence of MFG-E8 and hCG-R (receptor) proteins. Endometrial epithelial cells were cytokeratin(+), vimentin(-), MFG-E8(+), and hCG-R(+), whereas ESC were vimentin(+), cytokeratin(-), MFG-E8(-), and hCG-R(+). Treatment of ESC with MFG-E8 resulted in a 13-fold increase in caspase activity and a 30-fold increase in TUNEL. On the other hand, hCG decreased messenger RNA expression of Bax in ESC. Milk fat globule-epidermal growth factor 8 has proapoptotic activity, suggesting participation in endometrial remodeling via an epithelial-stromal cell paracrine effect. Conversely, pregnancy levels of hCG has opposite effects on stromal cells.

Optimization and Scale-up Culture of Human Endometrial Multipotent Mesenchymal Stromal Cells: Potential for Clinical Application

We have previously identified and purified multipotent mesenchymal stromal cell (MSC)-like cells in the highly regenerative endometrial lining of the human uterus (eMSC) as CD140b⁺CD146⁺ cells. Due to ease of accessibility with minimal morbidity via biopsy, we are proposing to use eMSC in cell-based therapies; however, culture conditions compliant with Good Manufacturing Practice have not been established for eMSC. The aim of this study was to optimize serum-free and xeno-free culture conditions for expansion of eMSC for potential clinical use. Real-time cell assessment (Xcelligence) and MTS viability assays were used to measure attachment and proliferation of freshly isolated, flow cytometry-sorted CD140b⁺CD146⁺ eMSC cultured in several commercially available and in-house serum-free and xeno-free media in combination with five attachment matrices (fibronectin, collagen, gelatin, laminin, and Cell Start-XF®). Comparisons were made with a standard serum-containing medium, DMEM/F-12/10% fetal bovine serum. Under all conditions examined, eMSC attachment and proliferation was greatest using a fibronectin matrix, with Lonza TP-SF® and our in-house DMEM/SF/FGF2/EGF serum-free xeno-product-containing medium similar to serum-containing medium. Hypoxia increased eMSC proliferation in the DMEM/SF/FGF2/EGF serum-free medium. Culture of eMSC for 7 days on a fibronectin matrix in DMEM/SF/FGF2/EGF serum-free media in 5% O₂ maintained greater numbers of undifferentiated eMSC expressing CD140b, CD146, and W5C5 compared to culture under similar conditions in Lonza TP-SF medium. However, the percentage of cells expressing typical MSC phenotypic markers, CD29, CD44, CD73, and CD105, were similar for both media. EMSC showed greater expansion in 2D compared to 3D culture on fibronectin-coated microbeads using the optimized DMEM/SF/FGF2/EGF medium in 5% O₂. In the optimized 2D culture conditions, eMSC retained CFU activity, multipotency, and MSC surface phenotype, representing the first steps in their preparation for potential clinical use.

Müllerian Inhibiting Substance Induces Apoptosis of Human Endometrial Stromal Cells in Endometriosis

Müllerian inhibiting substance (MIS) is produced in Sertoli cells of fetal testis and causes regression of müllerian ducts in male embryos. MIS also can induce the cell cycle arrest and apoptosis in müllerian duct-derived tumors in vivo and in vitro. Our objective was to investigate the expression of MIS type II receptor (MISR II) and whether MIS can inhibit the proliferation and induce apoptosis in primary cultures of endometrial stromal cells (ESC) of endometriosis. In vitro experiments were performed in the university research laboratory. Tissue samples from 12 patients who had undergone evisceration for ovarian endometrial cysts were included in this study. The expression of MISR II in ESC was investigated by immunohistochemistry. The cell viability and apoptosis in ESC treated with MIS was measured by methylthiazoletetrazolium assay and annexin V analysis. The expression of regulatory proteins in ESC treated with MIS was shown by Western blotting. ESC showed specific immunostaining for the MISR II. ESC treated with MIS exhibited 32% growth inhibition (P = 0.0001). The changes in cell cycle distribution after MIS exposure at 72 h demonstrated that S and G(2)M phases were decreased; G(0)G(1) and sub-G(0)G(1) phases were increased. ESC treated with MIS showed 13.72% annexin V-fluorescein isothiocyanate positivity. In the ESCs, which contain defective p16, MIS increased the expression of pocket proteins p107 and p130 and decreased E2F transcription factor 1. The results support a central role for MIS in endometriosis. Although the precise mechanism of MIS-mediated inhibition of ESC growth has not been fully defined, these data suggest that MIS has activity against ESC in vitro and may also be an effective targeted therapy for endometriosis.

Arachidonic acid regulation of the cytosolic phospholipase A2α/cyclooxygenase-2 pathway in mouse endometrial stromal cells

ObjectiveTo investigate the role of arachidonic acid (AA) in mouse endometrial stromal cells.DesignExperimental animal study.SettingUniversity research laboratory.Animal(s)Sexually mature female CD1-strain mice.Intervention(s)Primary culture of endometrial stromal cells.Main Outcome Measure(s)Western blot and real-time polymerase chain reaction for gene expression and/or phophorylation analysis. Luciferase assay for Cox-2 promoter analysis.Result(s)AA-derived prostaglandins play important roles during embryo implantation and decidualization. However, the function of AA itself in reproduction is largely unknown. In this study, exogenous AA stimulated cPLA2α phosphorylation and COX-2 expression, mainly through ERK1/2 in mouse endometrial stromal cells, and p38 inhibitor modestly inhibited cPLA2α phosphorylation induced by AA. The induction of COX-2 by AA was diminished by short interfering RNA against C/EBPβ and inhibitory C/EBPβ (LIP). C/EBPβ binding site at −872–−864 of Cox-2 promoter contributes to Cox-2 promoter activation induced by C/EBPβ transfection. The expression of C/EBPβ protein induced by AA was inhibited by p38 inhibitor, and the phosphorylation of C/EBPβ induced by AA was inhibited by p38 inhibitor and ERK1/2 inhibitor. A nonmetabolized analogue of AA (ETYA) also enhanced cPLA2α phosphorylation and COX-2 expression. The activation of cPLA2α/COX-2 by AA was not inhibited by COX inhibitor indomethacin.Conclusion(s)AA can induce cPLA2α/COX-2 pathway activation in mouse endometrial stromal cells. To investigate the role of arachidonic acid (AA) in mouse endometrial stromal cells. Experimental animal study. University research laboratory. Sexually mature female CD1-strain mice. Primary culture of endometrial stromal cells. Western blot and real-time polymerase chain reaction for gene expression and/or phophorylation analysis. Luciferase assay for Cox-2 promoter analysis. AA-derived prostaglandins play important roles during embryo implantation and decidualization. However, the function of AA itself in reproduction is largely unknown. In this study, exogenous AA stimulated cPLA2α phosphorylation and COX-2 expression, mainly through ERK1/2 in mouse endometrial stromal cells, and p38 inhibitor modestly inhibited cPLA2α phosphorylation induced by AA. The induction of COX-2 by AA was diminished by short interfering RNA against C/EBPβ and inhibitory C/EBPβ (LIP). C/EBPβ binding site at −872–−864 of Cox-2 promoter contributes to Cox-2 promoter activation induced by C/EBPβ transfection. The expression of C/EBPβ protein induced by AA was inhibited by p38 inhibitor, and the phosphorylation of C/EBPβ induced by AA was inhibited by p38 inhibitor and ERK1/2 inhibitor. A nonmetabolized analogue of AA (ETYA) also enhanced cPLA2α phosphorylation and COX-2 expression. The activation of cPLA2α/COX-2 by AA was not inhibited by COX inhibitor indomethacin. AA can induce cPLA2α/COX-2 pathway activation in mouse endometrial stromal cells.

Effects of cell storage and passage on basal and oxytocin-regulated prostaglandin secretion by equine endometrial epithelial and stromal cells

Cell cultures are useful for determining the responses of specific cell types to various factors under controlled conditions and for obtaining a better understanding of in vivo physiologic processes. The aims of the present study were (i) to establish methodologies for isolation, culture and cryopreservation of equine endometrial epithelial and stromal cells; and (ii) to determine the effect of passage and cryopreservation on endometrial cell physiology, based on their basal and oxytocin (OT)-stimulated prostaglandin (PG) release. Epithelial and stromal cells were obtained by enzymatic digestion of equine endometrium collected from Days 2–5 of the estrous cycle (n = 16). Primary epithelial and stromal cells, as well as cryopreserved cells were stimulated with OT (10−7m) for 24 h. The concentrations of PGE2 and PGF2α in the culture medium were measured by enzyme-linked immunosorbent assay (EIA). Oxytocin increased PGE2 and PGF2α release by primary cultures of unfrozen epithelial cells until passage I (P < 0.01) and by the primary culture of unfrozen and cryopreserved/thawed stromal cells until passage IV (P < 0.01). Cryopreserved/thawed stromal cells cultured up to passage IV and unfrozen epithelial cells derived from passage I have physiological properties similar to those observed in primary culture and may be successfully used for in vitro studies of PG secretion.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increases the expression of cyclooxygenase-2 and aromatase cytochrome P450 in human endometrium

To investigate the role of 2,3,7,8-Tetrachlorodibenzo-p-dioxin(TCDD) on eutopic endometrium by stromal cell culture. Prospective experimental study. Human endometrial tissues obtained by hysterectomy specimens from 16 women with estrogen dependent diseases (endometriosis (n = 4), adenomyosis (n = 5), uterine fibroids (n = 7)) and 5 women who had undergone hysterectomy for carcinoma in situ of uterine cervix. Endometrial stromal cells were cultured under the following conditions: DMEM/F12 (10% FBS, 1 nM E2 and 100 nM P4). TCDD (10 nmol/l) and TNF-a (0.1ng/ml) were added according to experimental purposes. The protein expression of aromatase cytochrome P450 and COX-2 was examined by Western blot. In control group, the COX-2 protein in stromal cells cultured with TCDD was significantly increased (control; 0.63 ± 0.19 vs. TCDD; 0.94 ± 0.26, P<0.05) And the aromatase P450 protein in stromal cells cultured with TCDD and TCDD plus TNF-a was significantly increased. (control; 0.60 ± 0.26 vs. TCDD; 0.90 ± 0.36, TCDD plus TNF-a; 0.95 ± 0.43, P<0.05). In study group, the COX-2 protein in stromal cells cultured with TCDD and TCDD plus TNF-a was significantly increased. (control; 0.81 ± 0.26, TCDD; 1.04 ± 0.38, TCDD plus TNF-a; 1.30 ± 0.90, P<0.05). And the aromatase P450 protein in stromal cells cultured with TNF-a and TCDD and TCDD plus TNF-a was significantly increased (control; 0.76 ± 0.18 vs. TNF-a; 0.93 ± 0.34, TCDD; 0.97 ± 0.41, TCDD plus TNF-a; 1.16 ± 0.41 P<0.05). In the stromal cell cultured with TCDD plus TNF-a than TNF-a and TCDD alone, the aromatase protein was significantly increased (P<0.05). Treatment with the TCDD increased aromatase cytochrome P450 and COX-2 on the eutopic endometrium of patients with and without estrogen dependent diseases, by endometrial stromal cells in culture.

Perfusion culture enhanced human endometrial stromal cell growth in alginate-multivalent integrin α5β1 ligand scaffolds

A method to functionalize alginate by introducing monomeric or self-assembling (tetrameric) fibronectin (FN) domains is described, leading to a functional scaffold, which is used for three dimensional (3D) culture of human endometrial stromal cells (EnSCs). EnSCs encapsulated in the functional alginate were cultured under perfusion using the TissueFlex® platform, a multiple parallel microbioreactor system for 3D cell culture. The effect of the novel scaffold and the effect of perfusion were examined. Cell viability, proliferation, and extracellular matrix (ECM) deposition were determined and the results compared with those obtained with cells encapsulated in non-functionalized alginate, and also those without perfusion. Staining for focal adhesions and actin showed maximal cell adhesion only for alginate-tetrameric FN scaffolds under perfusion, associated with a significant increase in cell number over 7 days culture; in contrast to poor cell adhesion and a decrease in cell number for non-functionalized alginate scaffolds (irrespective of perfused/static culture) and 3D static culture (irrespective of the scaffold). Conjugation of alginate to FN was an absolute requirement to attenuate the loss of cell metabolic activity over 7 days culture. ECM deposition for blank alginate and alginate-monomeric FN was similar, but increased around 2-fold and 3-fold for alginate-tetrameric FN under static and perfusion culture, respectively. It is concluded that the requirement for EnSC engagement with multivalent integrin α5β1 ligands and perfused culture are both essential as a first step toward endometrial tissue engineering.

Disruption of gap junctions reduces biomarkers of decidualization and angiogenesis and increases inflammatory mediators in human endometrial stromal cell cultures

Context Uterine decidualization is critical to embryonic implantation and sustained pregnancy. Objective To evaluate the role of gap junction intercellular communications and connexin (Cx) proteins in the morphological and biochemical differentiation of decidualized human endometrial stromal cell (ESC) cultures. Design Translational cell biological study. Setting Academic medical center. Patients Endometrial tissue was provided by five healthy reproductive age women on no hormonal medication, undergoing laparoscopy in the early proliferative phase of the menstrual cycle. Interventions Endometrial biopsy under general anesthesia, establishment and decidualization of ESC with 10 nM 17β-estradiol, 100 nM progesterone and 0.5 mM dibutyryl-cAMP (E/P/c), and manipulation of gap junctions in vitro via a combination of pharmacological or transgenic approaches. Main outcome measures Decidualized ESC evaluated morphologically for epithelioid transformation, gap junctions by dye diffusion and Cx43, prolactin, VEGF and IL-6 expression by RT-PCR, Western and ELISA methods. Results Cx43 accumulation and functional gap junctions between decidualized ESC increase concomitantly with morphological differentiation following E/P/c treatment. Disruption of gap junctions using pharmacological inhibitors or Cx43 shRNA prevents morphological differentiation and inhibits prolactin and VEGF secretion. By contrast, IL-6 secretion from decidualized ESC is augmented by both approaches. Conclusions The findings suggest that decidualized ESC function as a coordinated secretory organ to regulate embryonic implantation via intercellular cooperation mediated by gap junctions. When adjacent cells can communicate through these junctions, decidual prolactin and VEGF secretion appears to be optimized for vascular development of the placental bed. Conversely, when intercellular communications are disrupted, angiogenesis is impaired and an inflammatory state is induced.

Demonstrating the Critical Role of Uterine Erbb Signaling in Fertility.

The establishment of the appropriate embryo-uterine interactions required for successful implantation and pregnancy require the coordinate actions of growth factors and their receptors. The spatiotemporal expression of Erbb ligands and receptors in the periimplantation uterus and embryo suggests that they play a role in reproduction. Numerous Erbb ligands have overlapping expression patterns in the uterus during pregnancy, and gene ablations of individual Erbb ligands do not show a major uterine phenotype due to compensation by other family members. We have identified EGFR (ERBB1) as being induced during the implantation period and hypothesize that EGFR signaling regulates the ability of the endometrium to support embryo implantation. This hypothesis is being tested in vivo using mouse models and in vitro using primary human endometrial stromal (HES) cell culture and a novel high-throughput microscopy assay. In order to circumvent the embryonic lethality of Egfr-/- mice, conditional ablation of Egfr was achieved by crossing an Egfr floxed (Egfr f/f) mouse with a progesterone receptor cre (PRcre) mouse, resulting in Egfr deletion in all PR positive cells (PRcre/+ Egfr f/f; Egfr d/d). Egfr d/d mice are severely subfertile and exhibit reduced implantation and abnormal embryos. Mutant mice demonstrate an ablation of artificial decidualization including defects in morphology, proliferation, apoptosis, differentiation and gene expression. EGFR is induced during in vitro HES cell decidualization and that the use of EGFR knockdown impedes decidualization. Furthermore, these results have been confirmed using a novel, automated image-based microscopy assay. These results strongly indicate that Erbb signaling plays a critical role in female reproduction. Continuation of ongoing studies determining the mechanism of EGFR action and the role of Erbb2 will provide valuable insight and allow further elucidation of complex pathways and the orchestrated molecular interactions vital to reproductive health. This research was supported by NIH Grant RO1CA077530 (to J.P.L.), by NIH R01HD057873 (to JW.J), and by the Eunice Kennedy Shriver NICHD/NIH through cooperative agreement U54 HD07495 as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research. (platform)

Natural Selection of Human Embryos: Impaired Decidualization of Endometrium Disables Embryo-Maternal Interactions and Causes Recurrent Pregnancy Loss

BackgroundRecurrent pregnancy loss (RPL), defined as 3 or more consecutive miscarriages, is widely attributed either to repeated chromosomal instability in the conceptus or to uterine factors that are poorly defined. We tested the hypothesis that abnormal cyclic differentiation of endometrial stromal cells (ESCs) into specialized decidual cells predisposes to RPL, based on the observation that this process may not only be indispensable for placenta formation in pregnancy but also for embryo recognition and selection at time of implantation.Methodology/Principal FindingsAnalysis of mid-secretory endometrial biopsies demonstrated that RPL is associated with decreased expression of the decidual marker prolactin (PRL) but increased levels of prokineticin-1 (PROK1), a cytokine that promotes implantation. These in vivo findings were entirely recapitulated when ESCs were purified from patients with and without a history of RPL and decidualized in culture. In addition to attenuated PRL production and prolonged and enhanced PROK1 expression, RPL was further associated with a complete dysregulation of both markers upon treatment of ESC cultures with human chorionic gonadotropin, a glycoprotein hormone abundantly expressed by the implanting embryo. We postulated that impaired embryo recognition and selection would clinically be associated with increased fecundity, defined by short time-to-pregnancy (TTP) intervals. Woman-based analysis of the mean and mode TTP in a cohort of 560 RPL patients showed that 40% can be considered “superfertile”, defined by a mean TTP of 3 months or less.ConclusionsImpaired cyclic decidualization of the endometrium facilitates implantation yet predisposes to subsequent pregnancy failure by disabling natural embryo selection and by disrupting the maternal responses to embryonic signals. These findings suggest a novel pathological pathway that unifies maternal and embryonic causes of RPL.

Prokineticin 1, homeobox A10, and progesterone receptor messenger ribonucleic acid expression in primary cultures of endometrial stromal cells isolated from endometrium of healthy women and from eutopic endometrium of women with endometriosis

To examine prokineticin 1 (PROK1), homeobox (HOX) A10, and P receptor (PR) messenger ribonucleic acid (mRNA) expression in primary cultures of endometrial stromal cells (ESC) obtained from eutopic endometrial samples of patients with endometriosis and to clarify whether in vitro steroid hormone dependence of PROK1 gene expression is altered in endometriosis. Prospective laboratory study. Tertiary university hospital. Twelve normal women (controls) and 12 patients affected by moderate to severe endometriosis in the midsecretory phase of the menstrual cycle. Endometrial specimens were obtained from control women and from women affected by endometriosis; ESC were isolated from endometrial biopsies, and primary cultures were established. Real-time polymerase chain reaction analysis of PROK1, HOXA10, and PR mRNA expression in ESC after 1-4 days of steroid hormone treatment and after decidual differentiation. Contrary to ESC from control women, in ESC obtained from women affected by endometriosis PROK1 and PR mRNA expression was not induced by 1-4 days of treatment with steroid hormones. Nevertheless, when ESC from both groups of women were differentiated to decidual phenotype, PROK1 mRNA was up-regulated and PR and HOXA10 mRNA were down-regulated to the same extent. Our results provide additional evidence for P resistance in endometriosis.

115. DEVELOPMENT OF POTENT AND STABLE PC6 INHIBITORS TO BLOCK EMBRYO IMPLANTATION FOR FEMALE CONTRACEPTION AND PREVENTION OF HIV

Proprotein convertase (PC) 6, a member of the PC family that activate precursor proteins into their active forms, is a critical endometrial factor for embryo implantation. Blocking PC6 production in mice inhibits decidualisation (a critical process of implantation) and inhibits implantation. PCs including PC6 also play a critical role in HIV infection through cleaving HIV envelope precursor protein gp160 into functional gp120 and gp41. PC inhibitors are demonstrated to inhibit HIV transmission via blocking gp160 cleavage. We hypothesised that PC6 is a potential target for the development of female contraception that could also provide protection from HIV infection. One key requirement to prove this concept in an animal model is a potent PC6 inhibitor that is stable in serum. Polyarginine peptide (polyR) is published to be a potent PC6 inhibitor that also inhibits HIV. We have confirmed that polyR inhibits PC6 in vivo and completely blocks decidualisation of human endometrial stromal cells in culture. However, polyR has short serum half-life. The aim of this current study was to generate polyR derivatives that are potent PC6 inhibitors, with increased serum stability. We modified polyR by either PEGylation with different sized PEG (polyethylene glycol) or cyclization, and tested their potency, stability and utility in vivo. Modifications at both terminals of polyR dramatically reduced its PC6 inhibitory potency. PEGylation at the C-terminal, regardless of the PEG size, had no effect. In silico docking experiments showed that N-terminal PEGylation or cyclization affected the binding of polyR to the PC6 active site, but not C-terminal PEGylation. One of the polyR derivatives, C-30kDa-PEG polyR was confirmed to be as potent as the parental peptide but much more stable in serum. Studies are currently in place to inhibit embryo implantation in mice using C-30kDa-PEG polyR and to determine its ability to inhibit HIV.

Establishment of a new method for isolation and culture of equine endometrial epithelial and stromal cells

In general, in vitro culture is an appropriate model to investigate the physiology, interaction and differentiation of different cell types under controlled and defined conditions (Freshney 1990), for the better understanding of the pathogenesis and the etiology of several diseases (Findlay et al. 1990). Because preliminary tests could be accomplished using this technique one can reduce the number of animal experiments, as well.

SHORT COMMUNICATION: Increased Expression of Glutathione by Estradiol, Tumor Necrosis Factor‐Alpha, and Interleukin 1‐Beta in Endometrial Stromal Cells

Problem The intracellular antioxidant system, based on glutathione (GSH), plays a key role in endometrial detoxification reactions and has been proposed to be involved in the pathogenesis endometriosis. This study was designed to evaluate whether estradiol (E2) and proinflammatory cytokines have any effects on expression of glutathione in endometrial stromal cells (ESCs).Method of study Glutathione levels were measured utilizing high‐performance liquid chromatography following in vitro culture and treatment of ESCs with estradiol, tumor necrosis factor‐alpha (TNF‐α) and interleukin 1‐beta (IL‐1β).Results The GSH level in E2 (10−8 m) treatment group was significantly higher than in the control group at 48 h (P &lt; 0.05). In vitro treatment of ESCs with TNF‐α 10 ng/mL as well as E2 (10−8 m) plus TNF‐α 10 ng/mL for 48 hr also led to a significant increase in GSH level (P &lt; 0.05; P &lt; 0.05, respectively). Both IL‐1β 10 ng/mL and E2 (10−8 m) plus IL‐1β 10 ng/mL for 48 hr increased GSH level significantly (P &lt; 0.05; P &lt; 0.05, respectively) as well.Conclusions These findings might suggest that increased production of estradiol and proinflammatory cytokines in the peritoneal cavity possibly leads to the establishment of endometriosis through increased level of GSH.

Resveratrol inhibits development of experimental endometriosis and induces apoptosis of endometrial stroma

OBJECTIVE: Resveratrol (RES), a polyphenol abundant in red grapes, has antioxidant, anti-proliferative and anti-inflammatory properties. Pathophysiology of endometriosis involves ectopic attachment and proliferation of endometrial tissues, oxidative stress and inflammation. This study evaluated effects of RES on a nude mouse model of endometriosis and on cultures of human endometrial stromal cells (HEC). DESIGN: In vitro and in vivo experiments. MATERIALS AND METHODS: Human proliferative phase endometrial biopsies were established as organ cultures or used for HEC isolation. To establish endometriosis in the ovariectomized nude mouse, endometrial tissues were maintained in 1 nM estradiol (E) for 24 hrs and subsequently injected intraperitoneally. Mice (N=12) were randomly assigned to RES Group or Control Group. RES Group received E (8 μg, silastic capsule implants) plus RES (5.9 mg/kg per day) by gavage for 10 days. Control mice received E+vehicle. The animals were then sacrificed and endometrial implants were measured and stained (TUNEL) for apoptosis. Studies of HEC evaluated effects of RES (30-100 μM) on apoptosis by detection of caspases 3/7. RESULTS: Mice treated with RES had significantly lower number and volume of endometrial implants: Control Group had on average 2.5±0.3 lesions/animal while mice treated with RES had 74% fewer lesions (0.6±0.2/animal; P<0.001). Average volume of implants was 11.5±1.5 mm3/animal in the Control Group and was 88% lower in RES Group (1.4±1.1 mm3; P=0.0003). TUNEL staining of implants from RES Group was significantly greater than that from Control mice. In cultures of HEC, RES induced a concentration dependent wave of apoptosis with maximum expression of caspases 3/7 at 48 hours increased 18-fold above control (P<0.001). CONCLUSIONS: RES significantly reduced the number and size of endometrial implants in the nude mouse model of endometriosis as well as induced HEC apoptosis. The present findings may lead to the development of novel treatments of endometriosis involving RES.

Wnt4 Acts Downstream of BMP2 and Functions via Canonical Beta-Catenin-Dependent Signaling Pathway to Regulate Human Endometrial Stromal Cell Differentiation.

Differentiation of endometrial stromal cells into decidual cells is a prerequisite for successful embryo implantation. Our previous studies have shown that bone morphogenetic protein 2 (BMP2), a morphogen belonging to the TGFβ superfamily, is markedly induced in primary cultures of human endometrial stromal cells as they undergo differentiation in response to a hormonal cocktail containing progesterone, estrogen, and a cyclic AMP analog. Using loss-of-function and gain-of-function approaches we further showed that BMP2 plays a key role during human endometrial stromal cell differentiation. The present study was undertaken to identify the downstream targets of BMP2 in human endometrium during decidualization. Gene expression profiling experiments identified Wnt4 as a downstream target of BMP2 regulation during differentiation of human endometrial stromal cells. To determine the function of Wnt4 during this differentiation process, we employed siRNA targeted to its mRNA. Our study showed that siRNA-mediated down-regulation of Wnt4 expression in the stromal cells efficiently blocked their differentiation, as indicated by the loss of expression of well-known biomarkers of decidualization such as prolactin and insulin-like growth factor binding protein 1. Conversely, adenovirus-mediated overexpression of Wnt4 in human endometrial stromal cells markedly advanced the differentiation program. Interestingly, the stimulatory effect of Wnt4 was accompanied by the accumulation of an active form of β-catenin in the nuclei of decidualizing stromal cells, indicating the involvement of the canonical Wnt signaling pathway in the differentiation process. Furthermore, overexpression of Dickkopf-1 (Dkk1), a known inhibitor of the β-catenin-dependent canonical Wnt signaling pathway, led to a marked suppression of BMP2- and Wnt4-mediated decidualization. We also performed gene expression profiling to identify downstream targets of Wnt4 in human endometrial stromal cells. Our studies identified Foxo1, a member of the Forkhead/Winged helix family of transcription factors, as a downstream target of Wnt4 in decidualized human endometrial stromal cells. Collectively, these studies uncovered a unique pathway involving BMP2, Wnt4, and Foxo1 that operates in the human endometrial stromal cells and is critical for their transformation into decidual cells. (Supported by NIH grants to RNT, FJD, MKB, and ICB). (poster)

MicroRNAs in Endometriosis.Shannon M. Hawkins, M.D., Ph.D.

MicroRNAs are 22-nucleotide molecules which are hypothesized to regulate specific mRNA sequences. MicroRNAs play important roles in differentiation, proliferation, and development. However, their roles in normal and reproductive tract disease states are largely unknown. To investigate the role of microRNAs in the pathogenesis of endometriosis, we have undertaken a multi-platform, genome-wide approach. We collected samples of ectopic and eutopic endometrium from women with and without endometriosis. Using next generation microRNA sequencing and transcriptome analysis in combination with publically available targeting databases, we generated a list of microRNA:mRNA pairs potentially involved in endometriosis. We have begun to validate the functional nature of these microRNA:mRNA pairs in vitro using primary human endometrial stromal cell cultures. Using this next generation technology, we also discovered 47 novel microRNAs in eutopic and ectopic endometrial tissues. In addition, to study the global role of microRNAs in reproductive tissues, we have created a conditional knockout mouse model lacking DICER1, a key enzyme in microRNA synthesis. DICER1 conditional knockout animals are sterile and have uterine defects. We are currently in the process of characterizing the phenotype and the microRNA changes associated. Our goal is ultimately to identify which microRNAs are essential to uterine function. This work has been supported by American Society of Reproductive Medicine-National Institute of Child Health and Disease Reproductive Scientist Development Program (5K12HD000849-19), the Women's Reproductive Health Research Program (5K12HD050128) and the Specialized Cooperative Centers in Reproductive and Infertility Research (5U54HD07495).