Muscarinic cholinergic receptor subtypes expression by human placenta

The presence of a cholinergic system in the placenta is suggested by several data, but no information is available concerning cholinergic receptor expression by placenta. The present study was designed to investigate muscarinic cholinergic receptors in sections of human placenta using a radioligand binding techniques with [3H]N-methyl scopolamine ([3H]NMS) as a ligand. [3H]NMS was bound to sections of human placenta in a manner consistent with the labelling of muscarinic cholinergic receptors. The dissociation constant (Kd) value was 0.1 +/- 0.03 nM and the maximum density of binding site (Bmax) value was 10.82 +/- 0.09 fmol/mg of tissue. The binding was time-, temperature- and concentration-dependent, belonging to one class of high affinity sites. Analysis of [3H]NMS displacement curves by compounds acting on the different subtypes of muscarinic cholinergic receptor subtypes suggests that human placenta expresses the four subtypes (M1-M4) of muscarinic cholinergic receptor assayable with radioligand binding assay techniques. The demonstration of muscarinic cholinergic recognition sites in human placenta may contribute to define the possible significance of placental cholinergic system. Moreover, human placenta can be used as an easily obtainable human source of M1-M4 muscarinic cholinergic receptor subtypes.     

Glycaemic regulation of glucose transporter expression and activity in the human placenta

To determine whether the expression and activity of glucose transporters in human trophoblast are regulated by glucose, syncytiotrophoblast cells, choriocarcinoma cells, and villous fragments were incubated with a range of glucose concentrations (0-20 mM, 24 h). Expression of GLUT1 and GLUT3 glucose transporters was measured by immunoblotting, while glucose transporter activity was determined by [3H]2-deoxyglucose uptake in the cultured cells. GLUT1 expression in syncytial cells was enhanced following incubation in absence of glucose, reduced by incubation in 20 mM glucose but was not altered by incubation at 1 or 12 mM glucose. Transporter activity was inversely related to extracellular glucose over the entire range of concentrations tested (0-20 mM). Incubation of villous fragments in 20 mM glucose produced a limited suppression of GLUT1 expression, but no effects were noted following incubation at 0 or 1 mM glucose. Neither GLUT1 expression in JAr and JEG-3 choriocarcinoma cells nor transport activity in JEG-3 cells was affected by extracellular glucose concentration. Unlike syncytial cells, JAr, JEG-3 and BeWo all expressed GLUT3 protein in addition to GLUT1. These results show that while syncytiotrophoblast GLUT1 expression is altered at the extremes of extracellular glucose concentration, it is refractory to glucose alone at lower concentrations. By contrast, an inverse relationship exists between glucose transporter activity and extracellular glucose. This suggests that there are post-translational regulatory mechanisms which may respond to changes in extracellular glucose concentration.     

Demonstration of the expression of CD95 ligand transcript and protein in human placenta

Thrombopoietin (TPO) or Mpl ligand is the primary physiological regulator of platelet production. This cytokine is the most potent stimulator of the proliferation and differentiation of MK progenitor and precursor cells in vitro. It also acts additively or synergistically with several cytokines on progenitor cells from various hematopoietic lineages, including the primitive stem cells. The factor is an extremely potent thrombocytopoietic agent when administrated to normal animals, and it accelerates platelet and erythropoietic recovery in several models of myelosuppression. Phase I/II clinical trials are ongoing with no detectable adverse effects. Mpl ligand does not induce platelet aggregation, but it lowers the platelet sensitivity to physiological dose of agonists. In experimental mouse models, high and chronic dose of Mpl ligand results in myelofibrosis. TPO is constantly produced by the liver and the kidney; its plasmatic clearance occurs by binding to its receptor expressed on megakaryocytes and platelets. However, the full spectrum of the biological effects of this new cytokine is not fully understood, in particular its the role in the terminal stage of platelet production. In the near future, it is likely that new insights will be obtained in the physiopathological mechanisms underlying abnormal platelet production in human.     

Endoglin (CD 105) expression in human lymphoid organs and placenta

Endoglin (CD 105) is a cell surface antigen widely expressed on vascular endothelium, syncytiotrophoblast, some tissue macrophages, certain culture cells (including early leukemic B-lineage) and some endothelial cell lines. Though its relation to the transforming growth factor-beta (TGF-beta) receptor system is well documented, its function and detailed pattern of expression still remain to be clarified. We examined the differential tissue distribution of endoglin in human lymphoid organs and placenta with several anti-CD 105 monoclonal antibodies (mAbs) using an indirect immunoperoxidase technique, and performed semi-quantitative measurements using an image-analyzing system for comparison. Arterial, venous and capillary endothelia in these organs were reactive with anti-CD 105 mAbs at varying intensities. Interestingly, a distinctly stronger staining pattern was observed in the high endothelial venules (HEVs) which may indicate a special role for endoglin in lymphocyte trafficking. Syncytiotrophoblast expressed endoglin strongly on their apical cell membrane. Extravillous trophoblasts at certain locations selectively expressed endoglin on their cell membranes, suggesting a special role for this surface antigen during trophoblast differentiation.     

Unexpected expression of glucose transporter 4 in villous stromal cells of human placenta

Glucose transporter 4 (GLUT4) protein expression was characterized in human and rodent term placentas. A 50-kDa protein was detected, by immunoblotting, in term human placenta at levels averaging 25% of those found in white adipose tissue. It was also present, albeit at lower levels, in mouse and rat placentas. The specificity of the 50-kDa signal was established by using skeletal muscle and placental tissues obtained from GLUT4-null mice as controls. Indirect immunohistochemistry, performed in human placentas, showed that intravillous stromal cells were conspicuously labeled by GLUT4 and revealed colocalization of GLUT4 transporters with insulin receptors. This study provides the first evidence that the insulin-responsive GLUT4 glucose transporter is present in human and rodent hemochorial placentas. Placental GLUT4 gene and protein levels were not modified in human pregnancy complicated by insulin-dependent diabetes mellitus. The significance of the high level of GLUT4 protein in human placenta remains to be elucidated, because, so far, this organ was not considered to be insulin-sensitive, with regard to glucose transport.     

Spatiotemporal expression of C-CAM in the rat placenta

We investigated the expression of the immunoglobulin superfamily cell adhesion molecule, C-CAM, in developing and mature rat placenta. By immunohistochemical staining at the light microscopic level, no C-CAM-expression was seen before Day 9 of gestation, when it appeared in the trophoblasts of ectoplacental cones. On Day 10.5, spongiotrophoblasts and invasive trophoblasts around the maternal vessels of the decidua basalis were stained positively. On Day 12.5, C-CAM was detected in the spongiotrophoblasts of the junctional layer, but labyrinth trophoblasts and secondary giant trophoblasts were not stained. On Day 17.5, C-CAM was found only in the labyrinth and lacunae of the junctional layer. At this stage, both the labyrinth cytotrophoblasts of the maternal blood vessels and the endothelial cells of the embryonic capillaries were strongly stained. Placental tissues from gestational Days 12.5 and 17.5 were analyzed by immunoelectron microscopy to determine the location of C-CAM at the subcellular level. On Day 12.5, positive staining of the spongiotrophoblasts was observed, mainly on surface membranes and microvilli between loosely associated cells. On Day 17.5, staining was found primarily on the microvilli of the maternal luminal surfaces of the labyrinth cytotrophoblasts, and both on the luminal surface and in the cytoplasm of endothelial cells of the embryonic vessels. RT-PCR analysis and Southern blotting of the PCR products revealed expression of mRNA species for both of the major isoforms, C-CAM1 and C-CAM2. Immunoblotting analysis of C-CAM isolated from 12.5-day and 14.5-day placentae showed that it appeared as a broad band with an apparent molecular mass of 110-170 kD. In summary, C-CAM was strongly expressed in a specific spatiotemporal pattern in trophoblasts actively involved in formation of the placental tissue, suggesting an important role in placental development. In the mature placenta, C-CAM expression was confined to the trophoblastic and endothelial cells lining the maternal and embryonic vessels, respectively, suggesting important functions in placental physiology.     

Mechanisms of drug transfer across the human placenta

In this review we summarized literature data on the mechanisms of human placental drug transport studied in the isolated perfused placental cotyledon, placental membrane vesicles or trophoblastic cell cultures. Overall human placental drug transport rarely exceeds the transfer of flow-dependent and membrane-limited marker compounds. Interestingly, relatively often placental drug transfer appeared to be much smaller, indicating impaired trans-placental transport, depending on the physico-chemical characteristics of the drug or placental factors such as tissue binding or metabolism. Although in perfusion studies overall human placental drug transport occurs by simple diffusion, at the membrane level several drug transport systems have been found, mainly for drugs structurally related to endogenous compounds.     

Cell type-specific expression of 17 beta-hydroxysteroid dehydrogenase type 2 in human placenta and fetal liver

The enzymatic actions of the 17 beta-hydroxysteroid dehydrogenase (17 beta HSD) isozymes are crucial in steroid hormone metabolism/physiology. The type 1 isozyme catalyzes the conversion of the biologically inactive C18 steroid, estrone, to the active estrogen, 17 beta-estradiol, and the enzyme is predominantly expressed in the syncytiotrophoblast of the placenta and the granulosa cells of the ovary. 17 beta HSD type 2 is highly expressed in placenta, liver, and secretory endometrium and catalyzes the conversion of bioactive estrogens and androgens to biologically inactive 17-ketosteroid counterparts. The expression pattern of 17 beta HSD type 2 protein was determined in human term placenta and fetal liver by immunohistochemical analysis using monoclonal antibodies directed against distinct epitopes of the 17 beta HSD type 2 protein. In placenta, the protein was detected in the endothelial cells of fetal capillaries, but not in cytotrophoblasts or syncytiotrophoblast. There was dichotomous immunostaining seen among pairs of cotyledonary vessels and chorionic vessels. In the liver, on the other hand, staining was detected in the hepatocytes, but not in the cells lining blood vessels. We conclude that the cell type-specific localization of 17 beta HSD type 2 is in accord with the proposed physiological role of the enzyme, namely to protect tissues, in this case the fetus, from bioactive estrogen and androgen.     

In vitro perfusion studies of the human placenta. IV. Some characteristics of the glucose transport system in the human placenta

Cross infection has become a serious risk to hospitalized patients. Potential sources of infection by anaesthetic apparatus and equipment and the danger arising from disregard of proper asepsis are discussed. Prophylactic and hygienic measures to minimize these hazards are reviewed. Since patients receiving intensive therapy are particularly are risk very high hygienic standards are a "must" in these units. The need for thoroughly and regularly checking all equipment for contamination is emphasized.     

Fc gamma receptors in human placenta

This review summarizes the status of our knowledge on the structure, expression and function of Fc gamma R in the placenta. The discovery in syncytiotrophoblast of an MHC class I--related FcR, of the type responsible for intestinal uptake of milk IgG in suckling rats and mice is also described.     

Progesterone induces calcitonin gene expression in human endometrium within the putative window of implantation

The human endometrium acquires the ability to implant the developing embryo within a specific time window that is thought to open between days 19-24 of the secretory phase of the menstrual cycle. During this period the endometrium undergoes pronounced structural and functional changes induced by the ovarian steroids, estrogen and progesterone, that prepare it to be receptive to invasion by the embryo. The identification of reliable biochemical markers to assess this critical receptive phase in the context of the natural cycle remains one of the major challenges in the study of human reproduction. Our previous studies in a rat model system demonstrated that the expression of calcitonin, a peptide hormone involved in calcium homeostasis, is transiently induced by progesterone in the glandular epithelium at the onset of implantation. Attenuation of calcitonin synthesis in the uterus during the preimplantation phase by administration of calcitonin antisense oligodeoxynucleotides severely impairs implantation of rat embryos, suggesting that this peptide hormone plays a critical role in uterine receptivity. To investigate whether calcitonin is also expressed in the human endometrium during implantation, we monitored the spatio-temporal expression of calcitonin on various days of the menstrual cycle. Our studies employing RT-PCR showed that calcitonin messenger ribonucleic acid is expressed in human endometrium during the postovulatory midsecretory phase (days 17-25) of the menstrual cycle, with maximal expression occurring between days 19-21. Very little calcitonin expression was detected in the endometrium in either the preovulatory proliferative (days 5-14) or the late secretory (days 26-28) phase. In situ hybridization and immunocytochemical analyses localized the calcitonin expression predominantly in the glandular epithelial cells of the endometrium. Our studies further showed that calcitonin expression in the human endometrium is under progesterone regulation. Treatment of women with an antiprogestin, mifepristone (RU-486), drastically reduced calcitonin expression in the endometrium. Collectively, these findings reveal that progesterone-induced expression of calcitonin in the secretory endometrium temporally coincides with the putative window of implantation in the human.     

Ultrastructure of perfusion-fixed fetal capillaries in the human placenta

The ultrastructure of human placental capillaries was investigated using perfusion fixation and the freeze-fracturing technique. The capillaries have a continuous endothelium especially rich in microfilaments, whereas micropinocytotic vesicles are exceedingly scarce. The endothelial cells are connected by three types of junctions: (1) zonulae occludentes characterized by 2 to 4 focal regions of membrane contact in thin-sectioned specimens and an equal number of ridges on the membrane E-face in freeze-fractured specimens; (2) small gap junctions associated with the zonula occludens. (3) attachment plaques resembling zonulae adhaerentes in their fine structure. Endothelial cells are provided with long, circularly oriented pseudopodial extensions, which may be responsible for intermittent constrictions of the vessel lumen. These findings indicate that diaplacental transport at the level of the fetal capillary is controlled by the cytoplasm of the endothelial cells and probably occurs only to a very limited extent by way of micropinocytotic vesicles.     

Expression of vascular endothelial growth factor and placenta growth factor in human placenta

Normal development and function of the placenta requires invasion of the maternal decidua by trophoblasts, followed by abundant and organized vascular growth. Little is known of the significance and function of the vascular endothelial growth factor (VEGF) family, which includes VEGF, VEGF-B, and VEGF-C, and of placenta growth factor (PIGF) in these processes. In this study we have analyzed the expression of VEGF and PIGF mRNAs and their protein products in placental tissue obtained from noncomplicated pregnancies. Expression of VEGF and PIGF mRNA was observed by in situ hybridization in the chorionic mesenchyme and villous trophoblasts, respectively. Immunostaining localized the VEGF and PIGF proteins in the vascular endothelium, which was defined by staining for von Willebrand factor and for the Tie receptor tyrosine kinase, an early endothelial cell marker. VEGF-B and VEGF-C mRNAs were strongly expressed in human placenta as evidenced by Northern blot analysis. These data imply that VEGF and PIGF are produced by different cells but that both target the endothelial cells of normal human term placenta.     

Glutamine transport in human and rat placenta

Glutamine plays an important role in fetal nutrition. This study explored the transport of [3H]glutamine into apical and basal predominant membrane vesicles derived from rat and human placenta. Na+-dependent glutamine transport was present in both apical and basal predominant vesicles derived from 20- and, to a lesser degree, 14-day gestation rat placenta. Amino-acid transport systems A, ASC-like, B(o,+) (in apical membrane vesicles) and, perhaps, y+L were involved in Na+-dependent glutamine transport. Na+-dependent glutamine uptake into human placental microvillus and basolateral membrane vesicles also occurred via several distinct transport activities. Glutamine transport via system N was not detected in either rat or human placental preparations. Na+-dependent glutamine transport in the rat was more pronounced in basal as compared to apical membrane vesicles. Conversely, in the human preparations, activity was significantly higher in microvillus as compared to basolateral membrane vesicles. It is concluded that Na+-dependent glutamine transport occurs through a variety of transport agencies in both the rat and human placenta. Transport varies with ontogeny and between species.     

Histochemical localization of nitric oxide synthase in the human placenta

OBJECTIVES: To study localization, distribution and activity of nitric oxide synthase (NOS) in the human placenta and to speculate the action of nitric oxide (NO) during pregnancy. METHODS: NADPH-diaphorasc histochemical method was used to indicate the distribution, localization and activity of NOS in the placentae of normal term pregnancy (n = 21). Severe pregnancy induced hypertension (PIH) (n = 15), intrauterine growth retardation (IUGR) (n = 2) and villi of early pregnancy (n = 16). RESULTS: The NOS reactive product called blue formazan was found in most of syncytiotrophoblast (STr) and located in top of cells in severe PIH and IUGR placentae. In normal term placentae the blue formazan was found in STr and located in base of cells. The number of blue formazan was more than that of PIH and IUGR placentae. Endothelial cells of most capillaries of the terminal villi in severe PIH and IUGR placentae were found to be deposited with blue formazan, but in normal term pregnancy, blue formazan was found only on rare occasion. CONCLUSION: Distribution and localization of NOS in placentae of normal term pregnancy, severe PIH and IUGR cases and early villi are specific. The NOS activity of STr in severe PIH placentae and IUGR placentae are lower than that of normal term placentae. The distribution and localization of NOS within the STr in severe PIH placentae and IUGR placentae are different from those in normal term placentae. The low activity of NOS secreted by placenta may be relative to the pathogenesis of PIH and IUGR.     

A microscopical study of wound repair in the human placenta

In order to fulfill its many functions as the selective interface between maternal and fetal circulations it is imperative that the human placenta remains intact and in good operational order. That damage of some sort occurs during its short but extremely active life seems inevitable given the dynamic environment in which the placenta exists, and evidence has accumulated that disruption is indeed a regular event. The implications of such damage, one could speculate, may impact on functions such as transport and hormone secretion as well as mutual protection against attack by maternal and fetal immune systems. Consequently, it would seem a theoretical necessity for discontinuities in the placenta surface to be repaired as soon as possible. We have used a combination of ex vivo observation, in vitro modelling, immunohistochemistry and correlative microscopy to provide evidence for a wound response in the placenta and to begin dissecting the detail of how this may operate. Evidence for small lesions caused by fusion and subsequent tearing of the syncytiotrophoblast in vivo, as well as plugging of such wounds by underlying cells is shown. We also identify a putative role for migratory cytotrophoblasts in the healing of larger scale injuries and demonstrate that certain molecules, common to wound repair in other tissues, appear to be involved in placenta repair also. Taken together these results clearly show that the human placenta is capable of a degree of self-maintenance by activating what appears to be an endogenous wound healing mechanism.