Cultures Of Endometrial Epithelial Cells Research Articles

Neutral Endopeptidase Expressed by Decidualized Stromal Cells Suppresses Akt Phosphorylation and Deoxyribonucleic Acid Synthesis Induced by Endothelin-1 in Human Endometrium

Endothelin-1 (ET-1) in human endometrium has been proposed to have a potential paracrine role, for its receptors are also present within this tissue. In addition, the expression of ET-1 varies during the menstrual cycle, and therefore, ET-1 may be involved in the cyclic change of the human endometrium, such as proliferation and decidualization. However, neither the inactivation of ET-1 in the endometrium nor the paracrine effect of ET-1 on endometrial cells has been determined. We investigated the production of ET-1 and the presence of neutral endopeptidase (NEP), which cleaves and inactivates ET-1, in primary cultured human endometrial cells. We found primary cultured endometrial epithelial cells, not stromal cells, to be the major source of ET-1. Western blot analysis and RT-PCR demonstrated that NEP was predominantly expressed by endometrial stromal cells. We also demonstrated that ET-1 stimulated the phosphorylation of Akt and DNA synthesis in endometrial stromal cells via the ET(A) receptor and phospahtidylinositol-3 kinase signaling pathways. The effect of ET-1 was regulated by NEP expressed by stromal cells. We also found that conditioned medium containing ET-1 from endometrial epithelial cell culture stimulated phosphorylation of Akt via the ET(A) receptor. In conclusion, ET-1 has a paracrine effect of Akt phosphorylation and cell proliferation on endometrial stromal cells, which occurs via the ET(A) receptor and phospahtidylinositol-3 kinase signaling pathways, and is regulated by cell-surface NEP.

A Bicarbonate Boost to Fertility

Wang et al. linked cystic fibrosis transmembrane conductance regulator (CFTR)-mediated bicarbonate transport in endometrial epithelia to sperm capacitation. Sperm capacitation, which depends on environmental factors in the female reproductive tract, is a process critical to the ability of sperm to fertilize eggs. Cystic fibrosis is a genetic disease associated with mutations of CFTR, a cyclic adenosine monophosphate (cAMP)-activated anion channel found in epithelial tissues. The ensuing defect in chloride secretion, and consequent production of thick sticky mucus, leads to impaired respiratory and digestive function and has been presumed to underlie reduced fertility in women with cystic fibrosis. Although CFTR also mediates bicarbonate secretion, and bicarbonate has been associated--through activation of adenylyl cyclase--with sperm capacitation in vitro (see Sutton et al. ), the physiological significance of these observations has been unclear. Wang et al. used electrophysiological, pharmacological, and antisense analysis, together with fluorimetric measurements of intracellular pH and ion substitution, to implicate CFTR in bicarbonate transport across mouse endometrial epithelial cell cultures. Capacitation of mouse sperm was enhanced by conditioned medium from endometrial epithelial cultures or by bicarbonate. The ability of conditioned medium to enhance sperm capacitation was inhibited by antisense against CFTR. Conditioned medium from a pancreatic duct cell line that expressed CFTR promoted capacitation, whereas medium from a pancreatic line expressing a mutant CFTR that was defective in bicarbonate secretion did not. Capacitation by the latter was rescued by bicarbonate, or by transfecting cells with wild-type CFTR. These data point to a physiological role for bicarbonate transport by CFTR, as well as support a role for bicarbonate in mediating capacitation--and thereby contributing to fertility--in vivo. X. F. Wang, C. X. Zhou, Q. X. Shi, Y. Y. Yuan, M. K. Yu, L. C. Ajonuma, L. S. Ho, P. S. Lo, L. L. Tsang, Y. Liu, S. Y. Lam, L. N. Chan, W. C. Zhao, Y. W. Chung, H. C. Chan, Involvement of CFTR in uterine bicarbonate secretion and the fertilizing capacity of sperm. Nat. Cell Biol . 5 , 902-906 (2003). [Online Journal] K. A. Sutton, M. K. Jungnickel, H. M. Florman, Of fertility, cystic fibrosis and the bicarbonate ion. Nat. Cell Biol . 5 , 857-859 (2003). [Online Journal]

In vitro regulation of beta1 and beta3 integrin subunits in endometrial epithelial cells from normal endometrium.

To evaluate the effect of prostaglandin E2 (PGE2) and interleukin-1beta (IL-1beta) on integrin expression. DESIGN Cultures of endometrial epithelial cells from normal endometrium. All endometrial specimens were obtained from the Obstetrics and gynecology Department of the Catholic University, Rome, Italy. Four patients were normal menstrual cycles undergoing operative laparoscopy for non-endometrial problems. Endometrial samples were collected by uterine courettings. Immunocytochemistry for beta1 and beta3 integrin subunits. PGE2 clearly enhances both beta1 and beta3 integrin subunit expression. IL-1beta seem to slightly increase only beta3 subunit expression. In light of the critical role played by eicosanoids in endometrial differentiation, we suggest that PGE2 is also involved in local paracrine regulation of integrin expression.

Human peripheral blood mononuclear cells enhance cell-cell interaction between human endometrial epithelial cells and BeWo-cell spheroids.

Previously, it was reported that T-lymphocytes derived from non-pregnant mice promote murine embryo implantation. In order to examine the immunological regulation of endometrial receptivity in humans, the effects of peripheral blood mononuclear cells (PBMCs) on endometrial epithelial cell (EEC) function were monitored by a newly developed attachment assay using primary human EEC culture and BeWo cell-derived spheroids. EECs isolated from 25 women in the mid- and late proliferative and early, mid- and late secretory phases were subjected to monolayer culturing. Spheroids were constructed from BeWo cells, a human choriocarcinoma cell line, by incubation with continuous rolling, after which their interaction with cultured EECs was studied. The mean (+/- SEM) number of attached spheroids was significantly higher (P < 0.01) in the EEC culture derived from women in the mid-secretory phase (90 +/- 2.9%) than the other groups (ranging from 0 to 5.8 +/- 3.7%), which is in agreement with the existence of a so-called 'implantation window'. After 72 h co-culture of EECs with PBMCs, the number of attached spheroids significantly increased in the EEC cultures derived from the late proliferative and early secretory phases [65.0 +/- 21.7 versus 5.0 +/- 2.0% (P < 0.05) and 83.1 +/- 4.1 versus 4.4 +/- 1.9% (P < 0.01)]. This attachment assay appears to be a useful method with which to assess endometrial receptivity. Functional change of EECs induced by PBMCs suggests possible regulation of endometrial receptivity by immune cells.

Leptin up-regulates the expression of beta 3 (β3) integrin, and interleukin 1 beta (IL-1β) up-regulates the expression of leptin and leptin receptor by human endometrial epithelial cell cultures (EEC).

Objective: EEC alone or co-cultured with human embryos express leptin and leptin receptor (JCEM, 2000) and IL-1 up-regulates leptin secretion by EEC but not by endometrial stromal cells (ESC) (Fertil Steril, 2000). In the present investigation the effects of leptin on the expression of β3 integrin were investigated and compared with IL-1β effects. IL-1β regulatory effects on leptin and leptin receptor expression at protein level in EEC and ESC cultures were also assessed.

Leptin upregulates beta3-integrin expression and interleukin-1beta, upregulates leptin and leptin receptor expression in human endometrial epithelial cell cultures.

Human endometrium and endometrial epithelial cells (EECs) either cultured alone or cocultured with human embryos express leptin and leptin receptor. This study compares the effect of leptin with that of interleukin-1beta (IL-1beta) on the expression of beta3-EEC integrin, a marker of endometrial receptivity. Both cytokines increased the expression of beta3-EEC at concentrations in the range of 0.06-3 nM; however, leptin exhibited a significantly greater effect than IL-1beta. We also determined the regulatory effects of IL-1beta on leptin secretion and on the expression of leptin and leptin receptor at the protein level in both EEC and endometrial stromal cell (ESC) cultures. In EEC cultures, IL-1beta upregulated secretion of leptin and expression of both leptin and leptin receptors. No effect of IL-1beta was found in the ESC cultures. However, leptin exhibited marginal upregulation of leptin receptor. The upregulation of beta3-integrin and leptin/leptin receptor expression by IL-1beta in EEC cultures indicates that both cytokines may be implicated in embryonic-maternal cross-talk during the early phase of human implantation. Our present data also raise the possibility that leptin is an endometrial molecular effector of IL-1beta action on beta3-integrin upregulation. Thus, a new role for leptin in human reproduction as an autocrine/paracrine regulator of endometrial receptivity is proposed.

Expression of nuclear factor kappa B in human endometrium; role in the control of interleukin 6 and leukaemia inhibitory factor production.

Expression of the rel-A component of nuclear factor kappa B (NFkappaB) by human endometrial cells was investigated by immunocytochemical analysis of cryostat sections cut from endometrial biopsy material and of cultured endometrial epithelial cells. In-vivo expression of rel-A was low in epithelial cells in endometrium obtained during the proliferative phase of the cycle, but increased in these cells during the secretory phase and was maximal at the time of implantation. In-vivo expression of rel-A by stromal cells did not vary greatly throughout the cycle, but showed a slight peak at the time of ovulation. In contrast similar expression of rel-A was seen in short-term cultures of epithelial cells prepared from both proliferative and secretory endometrium. Addition of the NFkappaB inhibitor SN50 (5 microg/ml) to confluent cultures of endometrial epithelial cells inhibited interleukin (IL)-1alpha (10 ng/ml) and tumour necrosis factor alpha (TNFalpha) (10 ng/ml) stimulated IL-6 (P < 0.001 and P < 0.01 respectively) and LIF (P < 0.01 and P < 0.05 respectively) production. The proteasome inhibitor MG132 (0.3 and 3 micromol/l) also caused a dose-dependent decrease in IL-1alpha and TNFalpha-stimulated IL-6 (P < 0.001 and P < 0.001 respectively) and leukaemia inhibitory factor (LIF) (P < 0. 001 and P < 0.001 respectively) production by endometrial epithelial cells. The results support the hypothesis that NFkappaB mediates signalling between IL-1 and TNFalpha receptors and the expression of LIF and IL-6 in endometrial epithelial cells.

Promegestone (R5020) and mifepristone (RU486) both function as progestational agonists of human glycodelin gene expression in isolated human epithelial cells.

One of the most abundant protein products of human secretory endometrium is glycodelin, a glycoprotein previously referred to as PP14. Although the precise function of this protein is unknown, its unique glycosylation pattern is believed to affect immunomodulatory activity during human embryonic implantation and inhibition of sperm-egg binding after ovulation. Having confirmed the expression of glycodelin in secretory endometrial glands, we used purified endometrial epithelial cell cultures to demonstrate the hormonal regulation of glycodelin synthesis and secretion. The findings were corroborated by transiently transfecting glycodelin gene promoter-reporter constructs into human epithelioid HeLa and Ishikawa cells. Our results indicate that glycodelin protein production by endometrial epithelial cells is directly up-regulated 4- to 9-fold by progestins and antiprogestins in vitro. Transcriptional regulation of the glycodelin gene promoter expressed in HeLa cells is progesterone receptor-dependent. As observed in the primary endometrial cells, progestins and antiprogestins both act as agonists on the in vitro expression of this endometrial gene. Our findings provide insight into the regulation of this abundant endometrial protein and raise interesting questions about the physical nature of the interaction of agonist- and antagonist-bound progesterone receptors with the glycodelin gene promoter.

Bovine endometrial epithelial cells as a model system to study oxytocin receptor regulation.

Endometrial epithelial cell cultures were established from bovine uterine tissue collected during the oestrous cycle from commercially slaughtered animals. These cells were shown to express moderately high levels of oxytocin receptors (OTR) (up to 30000 per cell) after about one week in culture. These receptors have been characterized at the molecular, pharmacological and functional level and shown to be identical to those expressed in the bovine endometrium in vivo. Preliminary experiments to investigate the regulation of the OTR and its gene using this system, have shown that expression is to a large degree constitutive, the receptors being spontaneously upregulated during culture. Sex steroids at concentrations close to or above the serum limits observed in vivo appeared to have no effect, although the cells were shown to express mRNA for the specific steroid receptors throughout culture. Only the blastocyst product, interferon-tau, showed a significant effect, downregulating both OTR and their gene transcripts in the cultured endometrial epithelial cells. Although more extensive studies are necessary, these results support the view that the OTR gene is controlled in part at least by a combination of constitutive and inhibitory elements.

Endometrial connexin expression in the mare and pig: evidence for the suppression of cell-cell communication in uterine luminal epithelium.

This investigation examines the relationship between implantation strategy and gap junction protein expression in uterine endometrium. The pattern of gap junction and connexin protein expression was analyzed in porcine and equine endometrium from cycling and pregnant animals using electron microscopy and immunocytochemistry. Functional analysis of cell-cell communication was also monitored by laser cytometry in primary cultures of endometrial epithelial cells. Gap junctions were detected in endometrial stroma of cycling and pregnant animals, which was correlated with immunoreactive Cx43 within stromal fibroblasts and vascular elements. No Cx26, Cx32, or Cx43 immunostaining was detected in luminal endometrial epithelium in either the mare or the pig at any stage of the estrous cycle or pregnancy. In contrast, endometrial glands of the mare exhibited a spatiotemporal pattern of Cx43 expression in the apicolateral plasma membrane which, when present, colocalized with the tight junction-associated protein, ZO-1. Uterine glandular Cx43 expression in mares was present from day 3 postovulation through day 14 of diestrus and until day 23 of pregnancy, whereas Cx43 was absent within uterine glands during seasonal anestrus, estrus, and after day 30 of pregnancy. Primary cultures of equine endometrial epithelial cells expressed both immunoreactive Cx43 and significant gap junction-mediated intercellular communication (GJIC) which was rapidly upregulated by 1.0 mM 8-bromo-cAMP or blocked with 1.0 mM octanol. No GJIC or connexin protein was detected in cultured porcine epithelial cells despite incubation with a variety of agents, including 8-bromo-cAMP, steroid hormones, retinoic acid, and/or prolactin. Junctional communication in endometrial epithelium of domestic farm animals is different than that reported for species exhibiting invasive implantation. The absence of GJIC in uterine luminal epithelium of the gilt and mare may be involved in limiting trophoblast invasiveness.

Vectorial Secretion of Vascular Endothelial Growth Factor by Polarized Human Endometrial Epithelial Cells

Objective: To analyze directional vascular endothelial growth factor (VEGF) secretion in polarized human endometrial epithelial cell cultures. VEGF has distinct distribution patterns in human endometrium. Stromal cells are diffusely positive for VEGF messenger RNA and protein, whereas glandular epithelium shows focal VEGF immunostaining at the apical surface. The epithelial distribution suggests that VEGF is secreted into gland lumina, potentially influencing the nutrition and/or apposition of the developing blastocyst. Design: Controlled in vitro study of protein secretion by polarized endometrial epithelial cells established on polyethylene filters. Setting: University hospital. Patient(s): Endometrial biopsies were obtained from healthy, ovulatory women undergoing elective surgery. Intervention(s): Primary endometrial epithelial cells were cultured. Main Outcome Measure(s): VEGF mRNA and protein production were measured in polarized cells. The vectorial secretion of VEGF was determined. Result(s): VEGF production by endometrial epithelial cells was verified by Northern blotting and immunoassays of conditioned media. The mean (±SD) apical secretion of VEGF was 3.9 ± 1.4 ng per 10 5 cells every 48 hours and the mean (±SD) basal secretion was 0.8 ± 0.2 ng per 10 5 cells every 48 hours. In contrast, the apical and basal secretion of a soluble cellular isoform of fibronectin were 2.76 ± 0.96 ng per 10 5 cells every 48 hours and 2.64 ± 1.79 ng per 10 5 cells every 48 hours, respectively. Conclusion(s): VEGF is secreted preferentially into the lumina of endometrial glands. Apical VEGF may be an endometrial signal for blastocyst development or implantation.

Ion transport by human endometrial epithelia in vitro.

Human glandular endometrial epithelial cells were cultured on porous tissue culture inserts to form tight, confluent layers. These layers generated time-dependent modifications in the ionic composition of both apical and basolateral solutions. Increases in sodium and chloride concentrations in the basolateral fluid were accompanied by reciprocal decreases in the concentrations of these ions in the apical fluid. The potassium concentration was increased in the apical, while decreased in the basolateral, solution. The total calcium concentration was slightly elevated in the apical, as compared with the basolateral fluid, while there were no alterations in pH. The endometrial layers demonstrated a significant transepithelial potential difference, and when this value was substituted in the Nernst equation a prediction of the passive distribution of ions across the cells was possible, indicating that none of the ions were in equilibrium. Addition of the sodium channel blocker amiloride to the medium bathing the cell layers reduced the modifications in ionic composition of apical and basolateral solutions. The data are consistent with other data indicating an amiloride-sensitive sodium-absorptive function for the endometrial epithelium. The ability of these primary cultures of endometrial epithelial cells to reduce the sodium while increasing the potassium concentration of the apical fluid is qualitatively in agreement with the low sodium and high potassium concentrations reported for human uterine fluid. The data suggest a role for the endometrial epithelium in generating and maintaining the distinctive ionic composition of the intra-uterine environment.

Uteroglobin, an apically secreted protein of the uterine epithelium, is secreted non-polarized from MDCK cells and mainly basolaterally from Caco-2 cell

A complete cDNA encoding rabbit Uteroglobin was constructed and expressed in MDCK and Caco-2 cells. The MDCK cells secrete Uteroglobin in approximately equal amounts to the apical and the basolateral side, whereas the Caco-2 cells secrete Uteroglobin mainly to the basolateral side. Both MDCK and Caco-2 cells thus secrete Uteroglobin in a non-sorted manner. It has, however, previously been shown that Uteroglobin is secreted exclusively at the apical membrane in primary cell culture of endometrial epithelial cells [S.K. Mani et al. (1991) Endocrinology 128, 1563-1573]. This suggests that either the endometrial epithelium has an apical default pathway or recognises a sorting signal not recognised by MDCK cells and Caco-2 cells. Our data thus show that a soluble molecule can be secreted at the apical, the basolateral or both membranes depending on the cell type.

Identification and characterization of an early estrogen-regulated RNA in cultured guinea-pig endometrial cells

A cDNA library was prepared from quiescent guinea-pig endometrial glandular epithelial cells stimulated for 2 h with estradiol-17β (E 2) in the presence of cycloheximide. It was screened by differential hybridization for estrogen-regulated sequences. Six recombinants containing E 2-regulated sequences were identified. One of them, called gecl was then characterized by Northern blot hybridization. The gec1 mRNA was 1800 bases in size. A 2-fold increase in the gec1 mRNA level was achieved at 120 min after E 2 treatment. The E 2 action on gec1 gene required the presence of cycloheximide. The cloned gec1 cDNA was 1 kb in size. The sequence so far determined did not show similarity with well characterized genes. This is the first report on a cloned cDNA probe of early estrogen-induced mRNA in a primary culture of endometrial epithelial cells.

Specific effect of oestrone sulphate on protein synthesis and secretion by cultured epithelial cells from guinea-pig endometrium

Patterns of induced protein synthesis and secretion in guinea-pig endometrial epithelial cell cultures in response to oestrone sulphate alone and oestrone sulphate plus progesterone were investigated. Epithelial cells were cultured for 3 days in growth medium, then washed three times in a steroid-free medium. For each experiment, anticytokeratin immunostaining was used to discriminate the epithelial cells from the stromal cells. Only experiments in which the control dishes displayed more than 80% of anticytokeratin-immunostained cells were further processed. After this period oestradiol-17 beta (20 nmol/l; control), oestradiol-17 beta (20 nmol/l) plus progesterone (0.5 mumol/l), oestrone sulphate (1 mumol/l) or oestrone sulphate (1 mumol/l) plus progesterone (0.5 mumol/l) were added to the medium for 48 h. An immunocytochemical progesterone receptor assay showed that oestradiol-17 beta increased the progesterone receptor content of cells, and progesterone added to cultured cells in the presence of oestradiol-17 beta induced a significant increase in oestrogen sulphotransferase activity assessing the hormone responsiveness of the cultured cells. In these culture conditions and after 16 h of incubation, oestradiol-17 beta induced a 1.7-fold increase in [3H]thymidine incorporation into DNA, and [35S]methionine incorporation into cellular proteins was linearly increased up to 8 h. Biochemical changes induced by the different hormone treatments were studied by labelling the proteins with a 6-h pulse of [35S]methionine. The proteins present in the medium and in cells were analysed by two-dimensional polyacrylamide gel electrophoresis, followed by fluorography.(ABSTRACT TRUNCATED AT 250 WORDS)