The significance of insulin, insulin-like growth factor I (IGF-I) and glucocorticoids to the early mammalian embryo is clear in that they are key regulators of both mitogenic and metabolic effects during development. In the present study, the temporal sequence of expression of the respective receptor proteins was investigated for the first time in the developing rat utero-embryonic unit between conception and day 12 of gestation using immunocytochemistry. Insulin, IGF-I and glucocorticoid receptor were expressed in embryonic tissues after the start of implantation, and were co-localized in the primary ectoderm, extraembryonic ectoderm as well as in the ectoplacental cone. The parietal endoderm was devoid of glucocorticoid receptor staining, whereas IGF-I receptor was absent in visceral endoderm. After completion of basic organogenesis, the neural tube, notochord, otic placode, Wolffian duct, mesonephros and intestinal tube expressed insulin, IGF-I and glucocorticoid receptor. The glucocorticoid receptor was not expressed in heart tube and dorsal aortae. Considerable amounts of insulin receptor were detected in trophoblast-derived giant cells. In the uterus, luminal epithelium, endometrial stromal and myometrial smooth muscle cells immunoreacted with antisera against insulin, IGF-I and glucocorticoid receptor. Endometrial glands remained negative for the glucocorticoid receptor throughout the gestational period investigated. Uterine hormone receptor expression reached a peak at days 4 and 5 of gestation in endometrial stromal cells and decidua, respectively. In conclusion, the demonstrated ontogenetic pattern of insulin, IGF-I and glucocorticoid receptor expression indicates the potential sites of biological action of the respective ligands, providing supportive evidence for their critical importance during the course of embryogenesis in rats.

The present study compares some phenotypic and physiologic characteristics of microvascular and macrovascular endothelial cells from within one human organ. To this end microvascular endothelial cells from human full-term placenta (PLEC) were isolated using a new method and compared with macrovascular human umbilical vein endothelial cells (HUVEC) and an SV40-transformed placental venous endothelial cell line (HPEC-A2). PLEC were isolated by enzymatic perfusion of small placental vessels, purified on a density gradient and cultured subsequently. Histological sections of the enzyme-treated vessels showed a selective removal of the endothelial lining in the perfused placental cotyledons. The endothelial identity of the cells was confirmed by staining with the endothelial markers anti-von Willebrand factor, Ulex europaeus lectin and anti-QBEND10. The cells internalized acetylated low-density lipoprotein and did not show immunoreactivity with markers for macrophages, smooth muscle cells and fibroblasts. The spindle-shaped PLEC grew in swirling patterns similar to that described for venous placental endothelial cells. However, scanning electron microscopic examination clearly showed that PLEC remained elongated at the confluent state, in contrast to the more polygonal phenotype of HPEC-A2 and HUVEC that were studied in parallel. The amount of vasoactive substances (endothelin-1,2, thromboxane, angiotensin II, prostacyclin) released into the culture medium and the proliferative response to cytokines was more similar to human dermal microvessels (MIEC) derived from non-fetal tissue than to HUVEC. Potent mitogens such as vascular endothelial growth factors (VEGF121, VEGF165) and basic fibroblast growth factor (FGF-2) induced proliferation of all endothelial cell types. Placental growth factors PIGF-1 and PIGF-2 effectively stimulated cell proliferation on PLEC (142 +/- 7% and 173 +/- 10%) and MIEC (160 +/- 20% and 143 +/- 28%) in contrast to HUVEC (9 +/- 8% and 15 +/- 20%) and HPEC-A2 (15 +/- 7% and 24 +/- 6%) after 48 h incubation time under serum-free conditions. These data support evidence for (1) the microvascular identity of the isolated PLEC described in this study, and (2) the phenotypic and physiologic heterogeneity of micro- and macrovascular endothelial cells within one human organ.

Fetal macrosomia may occur even in adequately controlled diabetic mothers. This may reflect the problem of using maternal glycemia as an indicator of fetal glycemia, because the placenta interposed between both compartments has its own glucose metabolism. Here, we propose a model by which the placenta protects the fetus at moderate levels of maternal hyperglycemia. One characteristic feature of the human placenta in diabetes is the increased deposition of glycogen. Neither hyperglycemia nor hyperinsulinemia increase the glycogen content in the trophoblast. Since the glycogen increments in diabetes are predominantly located around fetoplacental vessels, it is tempting to assume a fetal origin of glucose making up the glycogen deposits. In fact, glucose can be transported back from the fetus into the placenta and this reflux is increased in diabetes. Therefore, in conditions of fetal glucose levels exceeding the demand for sustaining fetal growth and metabolism, glucose can be stored in the liver and other fetal tissues. Once these stores are saturated, glucose is extracted from the fetal circulation by the glucose transporters GLUT1 and GLUT3 on cells surrounding the fetoplacental vasculature and stored therein, again in the form of glycogen. These processes might be under the control of fetal insulin, because insulin injected into the fetal circulation increases placental glycogen stores. Fetal macrosomia would then occur only when fetal hyperglycemia exceeds the placental capacity to store excess fetal glucose. Thus, the placental failure to protect the fetus would cause the 'unexplained' phenotypic changes occasionally found in fetuses born to well-controlled diabetic women.

The scarce data available on leukocyte glucose transporter expression are contradictory and nothing is known about its regulation by glycemic state. Therefore, cytospin preparations of blood leukocytes were searched immunocytochemically for the high-affinity glucose transporters GLUT1, 3, and 4. Hypoglycemia-associated quantitative changes in transporter expression were assessed by flow cytometry. Granulocytes and monocytes stained for GLUT1, 3, and 4. Granulocyte GLUT4 levels were increased by 73% (P < 0.05) under hypoglycemic conditions, which was paralleled by a reduction in GLUT1 and a rise in GLUT3. In monocytes, GLUT3 was elevated by 134% (P < 0.05), whereas GLUT1 and GLUT4 remained unaffected upon hypoglycemia. Apart from a minor subpopulation, lymphocytes were negative for these carriers. In conclusion, GLUT1, 3, and 4 are abundantly expressed in granulocytes and monocytes. The differential response of individual isoforms to hypoglycemia may represent a mechanism to protect the cells from the stress of glucose deprivation.

A variety of growth factors promote the complex multistep process of angiogenesis. The mitogenic activity of vascular endothelial growth factors (VEGFs) and placental growth factors (PlGFs), known as cytokines acting predominantly on endothelial cells, was tested on human umbilical vein endothelial cells (HUVEC) and microvascular endothelial cells (MIEC) and compared with the potency of the universally acting basic fibroblast growth factor (FGF-2). The cells were seeded at different cell numbers and incubated with various doses of growth factors for a period of 24-72 h in culture medium +/- serum. Proliferation was determined by measuring the optical density after staining the cells with the tetrazolium salt WST-1.VEGF(121) and VEGF(165) increased the number of HUVEC and MIEC at low and high seeding densities various doses and incubation times. The efficiency of FGF-2 was less pronounced at high seeding densities of the cells under serum-free conditions. PlGF-1 and PlGF-2 stimulated mitogenesis on HUVEC only at low cell numbers and after a short incubation time by 125 +/- 3% and 102 +/- 5% (P < 0.001), respectively. Longer incubation times with the lower seeding density in the absence of FCS did not induce a significant stimulatory effect of the PlGFs. MIEC responded stronger to all growth factors. In particular under serum free conditions, PlGF-1 and PlGF-2 effectively stimulated cell proliferation by 247 +/- 54% (P < 0.01) and 288 +/- 40% (P < 0.05) at low cell numbers, and by 81 +/- 13% (P < 0.05) and 49 +/- 13% (P < 0.01), respectively, at high cell numbers. The addition of fetal calf serum caused a reduced proliferative response of all growth factors on both cell types related to the controls. In conclusion, MIEC and HUVEC differ in their proliferative response to VEGFs, PlGFs and FGF-2.

Efficient transfer of glucose from the mother to the embryonic compartment is crucial to sustain the survival and normal development of the embryo in utero, because the embryo's production of this primary substrate for oxidative metabolism is minimal. In the present study, the temporal sequence of expression of the sodium-independent facilitative glucose transporter isoforms GLUTs 1, 3, 4, and 5 was investigated in the developing rat uteroembryonic unit between conception and Gestational Day 8 using immunohistochemistry. The GLUTs 1, 3, and 4 were expressed in the embryonic tissues after the start of implantation, being colocalized in the parietal endoderm, visceral endoderm, primary ectoderm, extraembryonic ectoderm, and the ectoplacental cone. In the uterus, a faint GLUT1 labeling emerged, but not until Gestational Day 3, in the luminal epithelium, endometrial stroma, and decidual cells. The intensity of GLUT1 staining increased in the latter population with progressing decidualization. Endometrial glands and myometrial smooth muscle cells stained neither for GLUT1 nor for GLUT3 until postimplantation. During all developmental stages examined, GLUT4 was visualized throughout the pregnant rat uterus, as was GLUT3 (with the above-mentioned exceptions). The density of GLUT5 was generally less than the sensitivity of the immunohistochemical detection method in all tissues investigated. In conclusion, the data point to a significant expression of the high-affinity glucose transporters GLUTs 1, 3, and 4 in the rat uteroembryonic unit, providing supportive evidence for an important role of facilitative glucose diffusion during peri-implantation development.

OBJECTIVE The aim of this study was to evaluate the rate of transtubal dissemination of endometrial carcinoma cells by hysteroscopy and the functional viability of disseminated tumor cells by assessing cell adhesion in an in vitro model. METHODS We studied 24 uteri obtained at TAH+BSO in patients with endometrial carcinoma. Further inclusion criteria were negative peritoneal cytology, no involvement of the uterine serosa or extrauterine disease, and endometrial surface involvement >1 cm in diameter. In vitro fluid hysteroscopy was performed with a 5-mm single-flow rigid hysteroscope. A maximum of 150 ml saline was infused at a maximum pressure of 100 mm Hg for a maximum of 3 min. Fluid running off through the tubes was collected. The cell suspension was enriched by a density gradient centrifugation. The isolated cells had a mean viability of 90% as judged by trypan blue exclusion. Viable cells (5 x 10(4) per 2-cm(2) polyvinyl chloride well plate) were cultured with equal parts of Dulbecco's modified Eagle's minimal essential medium and Ham's F-12 for 24 h. The endpoint of the analysis was the adherence of tumor cells to the polyvinyl chloride well plate, which was taken as a proxy for functional cell viability. Cytological evaluation was performed separately by two cytologists blinded to the source and date of the smears. RESULTS Transtubal fluid dissemination was seen in 20 of 24 (83%) uteri. Tumor cells were found in 17 specimens (71%). In 10 (42%) specimens the disseminated tumor cells were functionally viable. CONCLUSIONS Our model suggests that hysteroscopy can cause dissemination of malignant cells into the abdominal cavity from uteri containing endometrial carcinoma and that these cells can be functionally viable and adhere to a matrix.

The human trophoblast secretes endothelin-1 (ET-1) and expresses ET receptors. The present study tested whether the transformed BeWo, JAR and JEG-3 choriocarcinoma cells: (1) secrete endothelin-1 (ET-1); (2) express both ET-A and ET-B receptor subtypes; and (3) have the potential to allow for autologous regulation of ET-receptor proteins. The cells were cultured for 24/48 h with or without 10% FCS and, in experiments on receptor regulation, with ET-1 (5-20 nM and 10 microM). ET-1 secretion was measured by RIA and receptor levels by immunoblotting. All cell types secreted ET-1 albeit at different levels and sensitivity to FCS. All cell lines expressed both ET-A (JEG-3>BeWo=JAR) and ET-B (JEG-3=JAR>BeWo) receptor subtypes, which could be up- and downregulated depending on ET-1 concentration, culture time and FCS presence. It is concluded that BeWo, JAR and JEG-3 choriocarcinoma cells secrete ET-1 and express both ET-A and ET-B receptor subtypes. The receptor levels can be regulated by ET-1. This provides the molecular basis for an autocrine system with the potential of autologous regulation of yet unidentified ET-1-induced functions.

International Journal of Gynecology & ObstetricsVolume 70, Issue S1 p. A72-A72 Uterine cancer Viability and adhesion of hysteroscopically disseminated endometrial cancer cells in vitro M.G. Arikan, M.G. ArikanSearch for more papers by this authorU. Weiss, U. WeissSearch for more papers by this authorO. Reich, O. ReichSearch for more papers by this authorT. Hahn, T. HahnSearch for more papers by this authorH. Pickel, H. PickelSearch for more papers by this authorK. Tamussino, K. TamussinoSearch for more papers by this authorM. Cervar, M. CervarSearch for more papers by this authorG. Desoye, G. DesoyeSearch for more papers by this author M.G. Arikan, M.G. ArikanSearch for more papers by this authorU. Weiss, U. WeissSearch for more papers by this authorO. Reich, O. ReichSearch for more papers by this authorT. Hahn, T. HahnSearch for more papers by this authorH. Pickel, H. PickelSearch for more papers by this authorK. Tamussino, K. TamussinoSearch for more papers by this authorM. Cervar, M. CervarSearch for more papers by this authorG. Desoye, G. DesoyeSearch for more papers by this author First published: 09 December 2003 https://doi.org/10.1016/S0020-7292(00)82718-6AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume70, IssueS12000Pages A72-A72 RelatedInformation

Endothelin receptors have recently been found in non-vascular tissues including the human placenta. The present study investigated developmental changes in location and expression levels of endothelin A and B receptors (ETA-R, ETB-R) in human placentae and isolated trophoblast by comparing first and third trimester tissues. In the first trimester all cells and tissues were immunolabelled for ETA-R and ETB-R, whereas in third trimester placentae the syncytiotrophoblast (ETA-R, ETB-R) and macrophages (ETA-R) were unstained. Immunoblotting for both receptors revealed up to three bands at 33–35, 50 and 75 kDa, respectively, which were differentially present in the first and third trimester. Pre-adsorption of antibodies with oligopeptides used for antigen-generation weakened the immunoreactions. ETA-R protein levels decreased (P< 0.05) in total villous tissue and isolated trophoblast, whereas ETB-R was unchanged. ETB-R transcripts (RT-PCR) prevailed in both stages and tissues, but in contrast to the protein levels its preponderance decreased from first trimester to term in villous tissue (P< 0.01), because of a four to five-fold increase in ETA-R and only a two-fold (P< 0.05) increase in ETB-R mRNA levels (P< 0.01). We conclude that ET receptor location, intracellular processing and expression levels in human villous tissue change between the first and third trimester. This may reflect changing functions of ET-1 during placental development.

Aims/hypothesis. We have recently shown that hyperglycaemia down-regulates the GLUT1 glucose transport system of term placental trophoblast. The reduction in GLUT1 protein alone was, however, not sufficient to explain the decrease in net glucose uptake, suggesting additional mechanisms. Therefore, we hypothesised that hyperglycaemia in vitro leads to a GLUT1 translocation from the trophoblast surface to intracellular sites.Methods. This was tested in our study by determining the subcellular distribution of GLUT1 in human term placental trophoblast (n = 5 placentas) cultured for 48 h with 5 compared with 25 mmol/l d-glucose in vitro using immunogold labelling.Results. Electron microscopic examination of cell profiles showed that 73 % of total GLUT1 molecules reside in the trophoblast plasma membrane under basal conditions. The reduced GLUT1 expression (–20 %; p < 0.05) after culture of the cells with 25 mmol/l glucose was accompanied by an internalisation of plasma membrane GLUT1, resulting in a loss of 40 % (p < 0.05) in cell surface transporter labelling. Western blotting identified a characteristically broad band between 55–65 kDa, confirming the specificity of the GLUT1 antiserum.Conclusion/interpretation. We postulate that in addition to down-regulating human GLUT1 protein concentrations, glucose exerts its autoregulatory effect on hexose transport in term placental trophoblast by altering GLUT1 partitioning between the plasma membrane and intracellular sites in favour of the latter. [Diabetologia (2000) 43: 173–180]

Although glucocorticoids play important roles in development and fetal programming, they are widely used for treatment of a variety of diseases during pregnancy. In various tissues, glucocorticoids down-regulate glucose transport systems; however, their effects on glucose transporters in the placenta are unknown. In the present study, the glucose carrier proteins GLUT1 and GLUT3 were localized in the trophoblast and endothelium of the human, rat, and mouse placenta. Subsequently, it was investigated whether glucocorticoids affect messenger ribonucleic acid and protein expression of these molecules by Northern and Western blotting using 1) human term placental trophoblast cells cultured in the presence or absence of 0.5, 5, and 50 micromol/L triamcinolone; 2) placentas of rats that received a single i.p. dose of 0.38 mg/kg triamcinolone; and 3) placentas of transgenic mice bearing an antisense glucocorticoid receptor gene construct. In all of these systems, both glucose transporters were significantly down-regulated (P < 0.05), with the exception of increased GLUT3 messenger ribonucleic acid and protein levels in transgenic mice. The results demonstrate that triamcinolone is a potent regulator of placental GLUT1 and GLUT3 expression involving the glucocorticoid receptor. We speculate that impaired expression of placental glucose transporters after glucocorticoid administration might contribute to the adverse side-effects, the foremost of which is a growth-retarded fetus, of this treatment during pregnancy.

ABSTRACT Human trophoblast cells were permissively infected by human cytomegalovirus. The kinetics of viral immediate-early, early, and late gene expression was clearly delayed compared to that in fibroblasts. Productive infection was unequivocally proven by the detection of virion particles, infectious virus in trophoblast culture supernatant, and cell-to-cell spread of cytomegalovirus from infected trophoblasts to uninfected fibroblasts. These observations indicate that infected trophoblasts may be involved in maternofetal transmission of human cytomegalovirus.

The gonads of female and male Triops cancriformis specimens from populations of the northern part of the species distribution area were examined by conventional transmission electron microscopy in order to gain insight into the complicated reproductive mode of the species. Ovarian follicles consisted of an oocyte and three nurse cells and were surrounded by a thin layer of follicular cells. Oocytes are initially smaller than nurse cells and contained mitochondria of the cristae type as well as many free ribosomes. The prominent nucleus contained a nucleolus. The cytoplasm of oocytes was filled with yolk globules that were surrounded by membranes of the endoplasmic reticulum. Nurse cells also contained yolk globules. The follicle duct walls consisted of closely packed elongated cells covered by a lamellar basal lamina. No distinct Golgi apparatus was found in the follicle duct cells. The passage of oocytes through the duct was accompanied by a marked flattening of the follicle duct cells. Simultaneously, the oocytes were wrapped in eggshell material before entering the longitudinal oviduct. Testicular lobes were not found in any of the investigated female specimens. In male animals, the epithelial wall of the testicular tubules consisted of both germinal and vegetative cells. Maturing spermatids formed multicellular nests located in clearly delimited cysts in the intercellular space of the testicular epithelium. The lumen of the testicular tubules contained, exclusively, free mature spermatozoa. These characteristics point to the cystic type of spermiogenesis. Mature spermatozoa were non-flagellate, had a regular ovoid shape of 4–5 μm diameter, and an acrosome was not found. Only a negligible amount of spermatozoa (< 5%) showed signs of degeneration. In conclusion, the results provide evidence for a parthenogenetic and/or bisexual reproduction mode rather than a hermaphroditic one in the populations examined.

Three regions of the human placental lactogen (hPL) promoter that contain several half-site motifs that closely resemble the responsive elements for thyroid hormone (TR), all trans retinoic acid (RAR) and 1,25 dihydroxyvitamin D3 (VDR) have been identified and characterized. Transfection studies in BeWo choriocarcinoma cells indicate that site A (nt −979 to −954) is responsive to RARα but not TRβ. Site B (nt −1140 to −1170) is responsive to both RARα and TRβ, and site C (nt −550 to −580) is not responsive to either RARα or TR. These findings, together with the observation that placental cells express retinoid receptors and TRs, strongly suggest a role for these receptors in the regulation of the hPL gene. Site B on the hPL promoter is able to integrate the responses to RA and T3 through a single element.