The influence of ovarian steroids on ovine endometrial glycosaminoglycans

The ovine endometrium is subjected to cyclic oscillations of estrogen and progesterone in preparation for implantation. One response to fluctuating hormonal levels is the degree of hydration of the tissue, suggesting cyclical alterations in glycosaminoglycan/proteoglycan content. The aim of the present study was to quantitate and characterize glycosaminoglycans in the ovine endometrium during estrogen and progesterone dominant stages. Endogenous endometrial glycosaminoglycan content was determined by chemical analysis and characterized by enzyme specific or chemical degradation. [(35)S]-sulphate and [(3)H]-glucosamine labeled proteoglycans/glycosaminoglycans were extracted by cell lysis or with 4M guanidine-HCl. Extracts were purified by anion exchange and gel chromatography and characterized as above. Estrogen and progesterone dominant endometrium contained 3.2 +/- 0.1 and 2.1 +/- 0.1 mg endogenous glycosaminoglycan/g dehydrated tissue, respectively. Characterization of endogenous glycosaminoglycan showed chondroitin sulphate and hyaluronan contributing over 80%. The major difference between hormonal dominant tissue was a higher estrogenic hyaluronan percentage and a higher progestational keratan sulphate percentage (p < 0.001). Estrogen dominant tissue incorporated 1.6-1.9 fold more radiolabeled proteoglycans/glycosaminoglycans (p < 0.001). Analysis of newly synthesized proteoglycans/glycosaminoglycans revealed a heparan/chondroitin sulphate ratio of 1:2.2-2.5. Keratan sulphate was not detected. Estrogenic hyaluronan was 1.6 fold greater in [(3)H]-labeled tissue. Analysis of labeled proteoglycans/glycosaminoglycans revealed two size classes with apparent molecular weights >2.0 x 10(6) and 0.8-1.1 x 10(5) and a charge class eluting between 0.1-0.5 M NaCl. The greater glycosaminoglycan content (particularly hyaluronan) and synthesis in estrogen dominant tissue supports a role for steroid hormones in endometrial glycosaminoglycan/proteoglycan regulation and consequent tissue hydration. It also suggests a role for these macromolecules in endometrial function and possibly the implantation process.     

Regulated expression of heparin-binding EGF-like growth factor (HB-EGF) in the human endometrium: a potential paracrine role during implantation

Heparin-binding epidermal growth factor (HB-EGF) is a recently identified member of the EGF growth factor family found to be expressed in the uterus of both mouse and human at the time of implantation. In the present study, we investigated the expression patterns of HB-EGF in normal cycling endometrium and compared its expression with the fertility-associated endometrial epithelial biomarkers alpha(v)beta(3) integrin, leukemia inhibitory factor (LIF) and homeobox gene, HOXA-10. RNase protection assay (RPA) using RNA made from endometrium collected from different phases of the menstrual cycle demonstrated increased HB-EGF expression during the mid-secretory phase, a pattern similar to, but slightly preceding the expression of alpha(v)beta(3) integrin and HOXA-10. In vitro studies demonstrated stimulation of HB-EGF expression by estradiol-17beta (E(2)) and progesterone (P(4)) alone or in combination in stromal cells. Combined treatment with E(2) + P(4) was, however, required to stimulate epithelial HB-EGF expression. In vitro experiments demonstrated the ability of HB-EGF to stimulate epithelial expression of the key endometrial proteins including LIF, HOXA-10, and the beta(3) integrin subunit. Each has previously been demonstrated to be an important epithelial biomarker expressed during the implantation window. In addition, conditioned media from endometrial stromal cells treated with E(2) + P(4) + relaxin mimicked the stimulatory effect of HB-EGF on epithelial expression of the beta(3) integrin subunit. The stimulatory effect of the stromal-conditioned medium was blocked by antibodies that neutralize a known receptor for HB-EGF. These data suggest that uterine receptivity may be regulated in part by the stromal-derived HB-EGF.     

The distribution of type-2 chain histo-blood group antigens in normal cycling human endometrium

Summary The blood group ABO(H) determinants are major allogenic antigens in both erythrocytes and tissue of man. These antigens and related carbohydrates are markers of cellular maturation and differentiation in many epithelial tissues and have recently attracted great interest as tumor-associated antigens. Previous studies of endometrial tissues have indicated that glycosylation in this tissue may be related to hormonal stimulation. We have investigated the immunohistochemical distribution of type-2 chain histo-blood group-related carbohydrates in specimens of normal, cycling endometria obtained from hysterectomies on women with known ABO/Lewis erythrocyte type and saliva secretor status. N-acetyllactosamine and Le^x were demonstrated to be uninfluenced by the genetic background. A and Ale^y antigens were exclusively demonstrated in endometria from blood group A individuals, while Le^y was expressed in endometria from blood group 0 individuals mainly. The precursor N-acetyllactosamine as well as the terminal H, A, and ALe^y antigens were shown in only a few cells. In contrast, N-acetyllactosamine substituted by sialic acid and/or fucose residues (Le^x, sialosyl-Le^x, Le^y) were demonstrated in epithelial cells of normal, cycling endometrium, but with both quantitative and qualitative differences in staining relating to the menstrual cycle, indicating that type-2 chain antigens are expressed under both genetic and hormonal influence in human cycling endometrium.     

Progesterone induces nano‐scale molecular modifications on endometrial epithelial cell surfaces

Background information. The endometrial epithelial cell membrane is a key interface in female reproductive biology. Steroid hormones play a predominant role in cyclic changes which occur at this interface during the female menstrual cycle. Specific changes in the morphology of the endometrial epithelial cell surface become apparent with the epithelial transition that drives the switch from a non‐receptive to receptive surface due to the action of progesterone on an oestrogen primed tissue. AFM (atomic force microscopy) allows the high‐resolution characterization of the endometrial epithelial cell surface. Its contact probe mechanism enables a unique imaging method that requires little sample preparation, yielding topographical and morphological characterization. By stiffening the cell membrane, low concentrations of fixatives allow the surface detail of the cell to be resolved while preserving fine ultra‐structural details for analysis. Results. In the present study we use high resolution AFM analysis of endometrial epithelial cells to monitor the effect of progesterone on the nanoscale structure of the endometrial cell surface. High‐resolution imaging reveals similar topographical nanoscale changes in both the Hec‐1‐A and Ishikawa model cell lines. Hec‐1‐B cells, used in the present study as a progesterone receptor negative control, however, exhibit a flattened cell surface morphology following progesterone treatment. Changes in average cell height and surface convolution correlate with increased surface roughness measurements, demonstrating alterations in molecular structure on the cell surface due to hormonal stimulation. Conclusions. Progesterone treatment induces changes to the cell surface as a result of nanoscale molecular modifications in response to external hormonal treatments. AFM provides the basis for the identification, visualization and quantification of these cell surface nanoscale changes. Together these findings demonstrate the utility of AFM for use in reproductive science and cancer biology where it could be applied in both in vitro analysis of protein structure—function relationships and clinical diagnosis.     

The expression pattern of MUC1 glycoforms and other biomarkers of endometrial receptivity in fertile and infertile women

Changes in the surface epithelium of the endometrium, characterized in part by alterations in cell-surface molecules, sex steroid receptors and the appearance of pinopodes, coincide with the window of endometrial receptivity in the menstrual cycle. This study was performed to evaluate the usefulness of hematoxylin and eosin staining, scanning and transmission microscopy, and MUC1 glycoform, sex steroid receptor, and interleukin receptor (type 1) expression as biomarkers of endometrial receptivity using carefully characterized clinical fertile and infertile groups of women. Using a combination of immunohistochemistry and scanning electron microscopy (SEM) called scanning immunoelectron microscopy (SIM), we confirmed that MUC1 mucin was not associated with the endometrial pinopodes, which have been linked with embryo adhesion. We also showed that failure of embryo implantation was associated with an abnormal endometrial expression of MUC1 mucin, and retention of nuclear progesterone receptor (PR) particularly in epithelial cells. Hematoxylin and eosin staining, transmission electron microscopy (TEM), SEM in isolation and immunohistochemistry for interleukin receptor were not shown to be useful markers. Progesterone-dependent regulation of MUC1 appears to be an important factor in determining endometrial receptivity.     

妊娠小鼠子宫内膜LIF基因表达的研究

本文对妊娠第4天(Ⅰ组)、第7天(Ⅱ组)、第10天(Ⅲ组)的小鼠(各20只)子宫内膜LIF基因表达进行了研究。Ⅰ组20只小鼠子宫内膜全部存在LIF基因的表达、Ⅱ组有5只小鼠表达、Ⅲ组仅有1只小鼠表达。文中对不同孕期LIF基因的表达程度与胚胎着床的关系进行了讨论。 Abstract:Leukemia inhibitory factor(LIF)is a glycoprotein with multiple activities and is essential for blastocyst implantation in mouse.We have examined LIF gene expression in mice endometrium on day 4(group Ⅰ),day 7(group Ⅱ),day 10(group Ⅲ)of pregnancy.In group Ⅰ all had LIF gene expression,5 mice had LIF gene expression in group Ⅱ,only one mouse had LIF gene expression in group Ⅲ.We discussed the relation between level of LIF gene expression and embryonic implantation.     

CSDC2, a cold shock domain RNA-binding protein in decidualization

RNA-binding proteins (RBPs) have been described for cancer cell progression and differentiation, although there is still much to learn about their mechanisms. Here, using in vivo decidualization as a model, we describe the role of RBP cold shock domain containing C2 (CSDC2) in the endometrium. Csdc2 messenger RNA expression was differentially regulated depending on time and areas of decidua development, with the most variation in antimesometrium (AM) and, to a lesser degree, in the junctional zone (JZ). Immunohistochemistry of CSDC2 showed a preferentially cytoplasmic localization at AM and JZ, and nuclear localization in underneath myometrium and mesometrium (M). Cytoplasmic localization coincided with differentiated, DESMIN-marked areas, while nuclear localization coincides with proliferative zones. Uterine suppression of CSDC2 through intrauterine-injected-specific small interfering RNA (siRNA) led to abnormal decidualization in early pregnancy, with more extended antimesometrial area and with poor M development if compared with control siRNA-injected animals. These results suggest that CSDC2 could be a regulator during decidua development.     

Normal human endometrium in cell culture

Summary Separation of human endometrium into its epithelial and stromal components has been achieved through collagenase digestion and has permitted a study of these two cell populations under specific experimental culture conditions. The stromal cell populations showed a progesterone response, were easily handled in culture, and displayed a limited in vitro life span typical of human diploid fibroblasts. In contrast, epithelium only survived in shortterm primary culture and showed no clear hormone response. High-density epithelial cultures remained viable for longer periods in culture. Comparisons between resurfacing endometrial epithelial cells in vivo and epithelial cells migrating from explants in vitro suggested that this initial epithelial migration in vitro was the counterpart of the repair response in vivo. We are much in debt to Dr. R. C. Hallowes (Department of Pathology, Imperial Cancer Research Fund) for his guidance and encouragement throughout the course of this work. We also gratefully acknowledge Dr. P. N. Riddle (Time-Lapse Cinematography Unit, Imperial Cancer Research Fund) for carrying out the time-lapse cinematography; Mrs. Lyn Rolph (Stereoscan Unit, Bedford College, University of London) for assisting with the SEM; and Mr. G. D. Leach for his competent help with the photography.     

Extracellular matrix proteins secreted from both the endometrium and the embryo are required for attachment: A study using a co‐culture model of rat blastocysts and Ishikawa cells

Integrins are expressed in a highly regulated manner at the maternal‐fetal interface during implantation. However, the significance of extracellular matrix (ECM) ligands during the integrin‐mediated embryo attachment to the endometrium is not fully understood. Thus, the distribution of fibronectin in the rat uterus and blastocyst was studied at the time of implantation. Fibronectin was absent in the uterine luminal epithelial cells but was intensely expressed in the trophoblast cells and the inner cell mass suggesting that fibronectin secreted from the blastocyst may be a possible bridging ligand for the integrins expressed at the maternal‐fetal interface. An Arg‐Gly‐Asp (RGD) peptide was used to block the RGD recognition sites on integrins, and the effect on rat blastocyst attachment to Ishikawa cells was examined. There was a significant reduction in blastocyst attachment when either the blastocysts or the Ishikawa cells were pre‐incubated with the RGD‐blocking peptide. Thus, successful attachment of the embryo to the endometrium requires the interaction of integrins on both the endometrium and the blastocyst with the RGD sequence of ECM ligands, such as fibronectin. Pre‐treatment of both blastocysts and Ishikawa cells with the RGD peptide also inhibited blastocyst attachment, but not completely, suggesting that ECM bridging ligands that do not contain the RGD sequence are also involved in embryo attachment. J. Morphol. 2013. © 2012 Wiley Periodicals, Inc.     

A role for two‐pore potassium (K2P) channels in endometrial epithelial function

The human endometrial epithelium is pivotal to menstrual cycle progression, implantation and early pregnancy. Endometrial function is directly regulated by local factors that include pH, oxygen tension and ion concentrations to generate an environment conducive to fertilization. A superfamily of potassium channels characterized by two‐pore domains (K2P) and encoded by KCNK genes is implicated in the control of the cell resting membrane potential through the generation of leak currents and modulation by various physicochemical stimuli. The aims of the study were to determine the expression and function of K2P channel subtypes in proliferative and secretory phase endometrium obtained from normo‐ovulatory women and in an endometrial cancer cell line. Using immunochemical methods, real‐time qRT‐PCR proliferation assays and electrophysiology. Our results demonstrate mRNA for several K2P channel subtypes in human endometrium with molecular expression of TREK‐1 shown to be higher in proliferative than secretory phase endometrium (P < 0.001). The K2P channel blockers methanandamide, lidocaine, zinc and curcumin had antiproliferative effects (P < 0.01) in an endometrial epithelial cancer cell line indicating a role for TASK and TREK‐1 channels in proliferation. Tetraethylammonium‐ and 4‐aminopyridine‐insensitive outwards currents were inhibited at all voltages by reducing extracellular pH from 7.4 to 6.6. Higher expression of TREK‐1 expression in proliferative phase endometrium may, in part, underlie linked to increased cell division. The effects of pH and a lack of effect of non‐specific channel blockers of voltage‐gated potassium channels imply a role for K2P channels in the regulation of human endometrial function.