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PubMedDate not supplied

Isolation and characterization of trophoblast from murine placenta.

A discontinuous density gradient centrifugation method, devised to isolate enriched populations of trophoblast from murine definitive placentae, is described. It is concluded that the isolated adherent cells are trophoblast on the basis of the following characteristics: they are fetally derived, as determined by their donor glucose phosphate isomerase phenotype in embryo transfer experiments; epithelial cells, as shown by the presence of cytokeratin filaments and the absence of vimentin; negative for the stage-specific embryonic antigen-I (SSEA-I); and capable of progesterone secretion. Initially, they grew as individual polygonal cells, tending to form tight confluent monolayers with poorly defined intercellular boundaries. They were mono- or binucleate and increased their nuclear size with time. After two days, giant cells appeared to be formed from binucleated cells by nuclear fusion, and multinucleated cells appeared forming syncytia. Some of these cells also seemed to form giant cells. A low percentage (1 to 10 per cent) of contaminating cells, mainly macrophage-like cells, was observed. The isolated cells were a mixture of alkaline phosphatase- (AP-)positive and AP-negative cells, with some of the latter having phagocytic capacity. All were Fc receptor-negative. The possible identity of these cells in relation to trophoblast in the intact placenta is discussed. This method of isolating and characterizing trophoblast cells from the definitive mouse placenta will be a useful tool for studying the biology and immunology of trophoblast.

Animals↗
PubMedDate not supplied

The ultrastructure of the trophoblastic layer of the degu (Octodon degus) placenta: a re-evaluation of the 'channel problem'.

Previous studies on immersion-fixed specimens of the haemomonochorial labyrinthine chorioallantoic placenta of the degu have made visible channels or pores which completely crossed the trophoblastic layer; for the first time hints for an open connection between maternal blood spaces and fetal interstitium were demonstrated in a placenta. This important finding was re-evaluated by means of transmission electron microscopy paying particular attention to the influence of the mode of fixation and of ischaemic periods prior to fixation. Thirty placentae from seven near-term degu (estimated gestational age 80-90 days) were fixed at various times (5-45 min) after maternal death. Placentae were perfused in situ via the aorta/uterine arteries or the umbilical vein with 2.2 per cent phosphate-buffered glutaraldehyde (n = 7, ischaemic periods < 5-45 min) or lanthanum hydroxide/osmium tetroxide (n = 7, ischaemic periods < 5 min), or were immersion-fixed in 2.2 per cent phosphate-buffered glutaraldehyde (n = 16, ischaemic periods 10-45 min). In material with brief ischaemic periods (n = 3, 5-10 min) no open connections between the maternal blood lacunae and fetal interstitium could be detected. Instead, occasional foci of trophoblast reduced to a thickness of 0.1 micron were found. After ischaemic periods exceeding 10 min, the thin trophoblastic diaphragms had partly disappeared resulting in complete transtrophoblastic pores. It is likely, therefore, that visible trophoblastic pores or channels in the degu placenta are ischaemic artefacts. Application of lanthanum hydroxide from the fetal circulation showed that a branching system of membrane-lined tubules (15-50 nm wide) intruded the trophoblast from its basal side, as has been reported for the guinea-pig and human placenta. It remains to be investigated whether these invaginations belong to a continuous transtrophoblastic channel system.

Animals↗
PubMed2001

Murine ectoplacental cone-derived trophoblast cells express chemokine receptors.

Chemokine receptors (CCRs) have been shown to regulate T cell migration and differentiation as well as the establishment of Th1/Th2 bias. Furthermore, T cells and T cell products are essential to trophoblast development. Thus, postulating that chemokines as well as their receptors may be expressed by trophoblast to move T cells into an interaction with the feto-placental unit, we examined whether CCRs are expressed during the early stages of ectoplacental cone (EPC) formation. For this, murine EPC-derived trophoblast were examined for their ability to express CCRs constitutively or inducible by interferon-gamma (IFN-gamma). Immunofluorescence experiments on EPC-derived trophoblast cells showed that CCR3, CXCR4 and CCR5 are significantly expressed. IFN-gamma accelerated the mobilization of intracellular pools of CCR molecules during early cell culture periods (2-6 h) and, in most cases, increased their expression on EPC-derived trophoblast cells. CCR activity could be detected in the culture supernatants of these cells, inversely proportional to cell surface expression, suggesting the existence of rapid endocytosis and recycling mechanisms. This finding indicates that the level of intracellular CCRs may partly be determined in the extracellular matrix, an event that could play an important role towards neutralization of specific T cell/trophoblast interactions during early stages of pregnancy and protect the fetus against harmful maternal immune responses.

Animals↗
PubMed2003

Evidence of a novel isoform of placenta growth factor (PlGF-4) expressed in human trophoblast and endothelial cells.

Placenta growth factor (PlGF), a homodimeric glycoprotein that is homologous to vascular endothelial growth factor (VEGF), is mitogenic to endothelial cells and protects trophoblast from apoptosis. Alternative splicing of mature mRNA gives rise to three known isoforms of PlGF. PlGF is expressed by human trophoblast during normal pregnancy, however, it is not known which isoforms are produced. We have utilized RT-PCR to characterize PlGF isoform expression in normal human trophoblast and umbilical vein endothelial cells (HUVEC). Our results show that PlGF-1, PlGF-2, and PlGF-3 isoforms are expressed by trophoblast and HUVECs. In addition, both cell types also express a novel variant of PlGF, tentatively termed PlGF-4, which has not been previously reported. PlGF-4 consists of the same sequence of PlGF-3, plus the heparin binding domain previously thought to be present only in PlGF-2. Presence of the heparin binding domain in PlGF-4 suggests that this variant would remain cell membrane-associated and thus could influence trophoblast and endothelial cells in an autocrine manner.

Alternative Splicing↗
PubMed2000

Measurements of the dielectric properties of peripheral blood mononuclear cells and trophoblast cells using AC electrokinetic techniques.

The separation of trophoblast cells from the maternal circulation could provide a valuable diagnostic tool for prenatal diagnosis of genetic abnormalities. This has been attempted using antibody methods, but due to non-specificity of the antibodies, maternal cell contamination remains a problem. We have investigated the potential of dielectrophoretic separation methods as a means of isolating trophoblast cells from mixed peripheral blood mononuclear cells. To determine the potential of this method the dielectric properties of trophoblast cells and mixed peripheral blood mononuclear cells were measured using dielectrophoretic crossover and single cell electrorotation methods. Both dielectrophoretic crossover data and electrorotation data gave an average specific membrane capacitance of the peripheral blood mononuclear cells of 11.5 mF m(-2). Trophoblast cells prepared using three different methods had a higher average specific membrane capacitance in the range 13-18 mF m(-2). The differences in capacitance between the cell types could be exploited as the basis of an AC electrokinetic-based system for the separation of trophoblast cells from peripheral blood mononuclear cells.

Cell Membrane↗
PubMed2002

The proneural genes NEUROD1 and NEUROD2 are expressed during human trophoblast invasion.

During early human pregnancy, extravillous trophoblast cells invade the maternal tissue of the uterus in a way similar to invasion by cancer cells. However, the process of trophoblast invasion is regulated in a time and place restricted way, in contrast to cancer invasion. We screened first trimester placental tissue enriched by extravillous invasive trophoblasts for the expression of proneural basic helix-loop-helix (bHLH) transcription factors, which are important controllers of cell fate. Surprisingly, the presence of NEUROD1, NEUROD2 and ATH2 transcripts was found by reverse transcriptase polymerase chain reaction (RT-PCR) analysis in first trimester placentabed. Of these genes, the proneural genes NEUROD1 and NEUROD2 are expressed in different subsets of invasive trophoblasts. NEUROD1 expression is found in interstitial and endovascular invasive cells, while NEUROD2 expression is observed mainly in endovascular invasive cells, respectively. These data suggest that in addition to the involvement of proneural genes in neuron, neurendocrine and pancreas differentiation, these genes are involved in trophoblast differentation during progression of invasion.

Annexins↗
PubMed2003

Epidemiology of gestational trophoblastic diseases.

The epidemiology of gestational trophoblastic diseases is unclear. Problems with collection and interpretation of differing data abound. Hydatidiform mole (HM) is associated with abnormal gametogenesis and/or fertilization. This is further influenced by age, ethnicity and a prior history of an HM suggesting a genetic basis for its aetiology. Whilst a prior HM is significant in the development of trophoblastic neoplasia there is no clear explanation for the development of gestational trophoblastic neoplasia in association with a normal gestation. The development and improvements in suction curettage, termination of pregnancy, contraceptive techniques, diagnostic imaging and biochemical testing have been associated not only with a fall in the birth rate, but also with a reduction in the incidence of trophoblastic diseases. Future study should examine the mechanism of malignant change in normal and abnormal trophoblast.

Abortion, Induced↗
PubMed1998

Expression of allogeneic MHC class I antigens by transgenic mouse trophoblast does not interfere with the normal course of pregnancy.

Mammalian embryos express paternal histocompatibility antigens which make them potential targets for maternal immune responses. Yet, the histoincompatible fetus survives and develops normally. Down regulation of classical MHC antigen expression by trophoblast cells which are in direct contact with maternal circulation has been repeatedly shown. The trophoblast cells are unable to function properly in antigen presentation and do not induce allogeneic rejection reactions. In the present study we have created transgenic mice that express an allogeneic class I transgene whose transcription is controlled by the transferrin receptor promoter. The expression patterns of the transgene product mice from a single transgenic line were studied in each of the typical placental subpopulations. The allogeneic class I antigen was expressed in the allantoic plate region of the trophoblast, and this expression was not restricted to the endothelial region but extended also to the spongiotrophoblast, as well as the major blood vessels and in the endodermal sinuses. In contrast to the normal class I expression, prominent levels of allogeneic H-2 antigens were detected in the labyrinthine trophoblast. The fetal resorption rate in females mated with these transgenic males was not higher then the normal rate, and the embryos survived and developed normally. These data imply that the unusual expression of allogeneic class I antigens in certain trophoblast subpopulations does not affect fetal development.

Animals↗
PubMed2000

Eomesodermin is required for mouse trophoblast development and mesoderm formation.

The earliest cell fate decision in the mammalian embryo separates the extra-embryonic trophoblast lineage, which forms the fetal portion of the placenta, from the embryonic cell lineages. The body plan of the embryo proper is established only later at gastrulation, when the pluripotent epiblast gives rise to the germ layers ectoderm, mesoderm and endoderm. Here we show that the T-box gene Eomesodermin performs essential functions in both trophoblast development and gastrulation. Mouse embryos lacking Eomesodermin arrest at the blastocyst stage. Mutant trophoectoderm does not differentiate into trophoblast, indicating that Eomesodermin may be required for the development of trophoblast stem cells. In the embryo proper, Eomesodermin is essential for mesoderm formation. Although the specification of the anterior-posterior axis and the initial response to mesoderm-inducing signals is intact in mutant epiblasts, the prospective mesodermal cells are not recruited into the primitive streak. Our results indicate that Eomesodermin defines a conserved molecular pathway controlling the morphogenetic movements of germ layer formation and has acquired a new function in mammals in the differentiation of trophoblast.

Amino Acid Sequence↗
PubMed2001

Uterine natural killer cells in a three-dimensional tissue culture model to study trophoblast invasion.

The high numbers of CD56(+) cells with natural killer (NK) functions present in the uterine mucosa during the late secretory phase of the menstrual cycle and during early pregnancy have been considered to be implicated in implantation and in the regulation of trophoblast invasion. A three-dimensional organ culture model was used to study the interactions of these uterine NK cells with Jeg-3 and BeWo choriocarcinoma cells as a model of the invasive trophoblast. For this purpose, fragments of endometrial and decidual tissue were put in close contact with multicellular spheroids of choriocarcinoma cells in small silicon funnels. After the formation of stable contacts, the confrontation cultures were transferred to spinner flasks, cultivated for up to 6 days, and prepared for immunohistochemistry. During 2 days of cocultivation, the first cells started to move forward into the stromal component of the confrontation culture as demonstrated by staining of the choriocarcinoma cells using anti-human cytokeratin. Invasion advanced until, after a total of 6 days, some choriocarcinoma cells had already penetrated deeply into the host tissue. After a cultivation period of 1 week, both the endometrial and decidual tissue fragments still contained several CD56(+) uterine NK cells, and some of them expressed the proliferation-associated marker Ki-67 without any exogenous activation. A few CD56(+) cells were found directly at the invasion front, as well as between the choriocarcinoma cells. These cells also contained the cytolytic granule protein perforin indicating a migration of NK cells with cytolytic potential toward the potentially invasive cells. In conclusion, this human system closely resembles the in vivo conditions during trophoblast invasion and provides an appropriate in vitro model for studying dynamic processes involving various cell types during trophoblast invasion at the experimental level. Moreover, it enables us to study the effects of cytokines and growth factors that possibly regulate trophoblast invasion.

Choriocarcinoma↗
PubMed1998

DOC-2/hDab2, a candidate tumor suppressor gene involved in the development of gestational trophoblastic diseases.

Gestational trophoblastic diseases comprise a spectrum of interrelated diseases including partial mole, complete mole and gestational choriocarcinoma. Using reverse transcriptase PCR (RT-PCR) analysis, we identified higher levels of DOC-2/hDab2 expression in the normal trophoblast cells in culture than in choriocarcinoma cell lines. Subsequent study using immunohistochemistry showed high levels of DOC-2/hDab2 protein expression in normal trophoblast tissues but significantly lower levels of expression in gestational trophoblastic disease tissues, particularly in complete mole and choriocarcinoma. When DOC-2/hDab2 was transfected into the choriocarcinoma cell lines, Jar, JEG and BeWo, the stable transfectants showed significantly reduced growth rate in culture. These data suggest that down regulation of DOC-2/hDab2 may play an important role in the development of gestational trophoblastic diseases.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Protein kinase C in cultured human placental trophoblasts: identification of isoforms and role in cAMP signalling.

The protein kinase C (PKC) superfamily is a family of serine/threonine kinases involved in the regulation of many cell functions. The objective of the present work was to identify the PKC isoenzymes present in human placental trophoblasts and to compare their relative responses to acute and chronic phorbol 12-myristate 13-acetate (PMA) treatment. In addition, the effect of PMA treatment on ligand-stimulated cAMP production was determined. In total extracts prepared from cultured or freshly purified trophoblasts, PKC isoforms alpha, epsilon and zeta were detected. Following acute treatment with PMA, PKC alpha and PKC epsilon were translocated from the cytosol to the particulate fraction. Prolonged treatment (up to 36 h) with PMA resulted in the temporal down-regulation of PKC alpha and PKC epsilon. PKC zeta did not respond to either acute or chronic treatment with PMA. An acute 10 min treatment of the cells with PMA (10(-10)-10(-6) M) enhanced isoprenaline- and adrenaline-stimulated cAMP production. An enhanced response was observed at all concentrations of isoprenaline tested (10(-9)-10(-4) M), suggesting an increased capacity to respond. The acute PMA effect was evident within 2 min and near maximal by 5 min. The acute response to PMA was lost in cells where PKC was down-regulated by prior PMA treatment. Epidermal growth factor, a potential ligand for the activation of PKC in trophoblasts, enhanced isoprenaline-stimulated cAMP production. In summary, activation of PMA-responsive PKCs (PKC alpha or PKC epsilon) appears to enhance ligand-stimulated cAMP production in trophoblasts. This may be a physiologically important example of 'cross-talk' between various signalling pathways in human placental trophoblasts.

Animals↗
PubMed2000

Decidual natural-killer-cell interaction with trophoblast: cytolysis or cytokine production?

At the implantation site, the uterine mucosa (decidua) is infiltrated by large numbers of natural killer (NK) cells. These NK cells are in close contact with the invading fetal trophoblast and we have proposed that they might be the effector cells that control the implantation of the allogeneic placenta. Recent characterization of NK cell receptors and their HLA class I ligands has suggested potential mechanisms by which NK cells might interact with trophoblast. However, what happens as a result of this interaction is not clear. The traditional method for investigating NK cell function in vitro is the protection from lysis of target cells by expression of HLA class I antigens. This might not be an accurate reflection of what happens in vivo. Another function of NK cells is the production of cytokines on contact with target cells. This could be an important outcome of the interaction between decidual NK cells and trophoblast. Decidual NK cells are known to produce a variety of cytokines; trophoblast cells express receptors for many of these cytokines, indicating that they can potentially respond. In this way, decidual NK cells have a significant influence on trophoblast behaviour during implantation.

Cytokines↗
PubMed2003

Involvement of peroxynitrite in LPS-induced apoptosis of trophoblasts.

OBJECTIVE: To examine whether or not peroxynitrite was involved in trophoblastic apoptosis induced by a bacterial endotoxin, lipopolysaccharide (LPS). METHODS: Levels of nitrite/nitrate, stable metabolites of nitric oxide (NO), in culture medium of trophoblasts, were determined using Griess reagents. Trophoblastic apoptosis was identified morphologically and confirmed using in situ nick end labeling technique. The amount of nitrotyrosine, a footprint of peroxynitrite, was quantified by dot blotting. Statistical significance was determined by ANOVA. RESULTS: Treatment of trophoblasts with LPS leads to apoptosis accompanied by formation of NO and nitrotyrosine. Aminoguanidine, an inhibitor of NO synthase (NOS), reduced peroxynitrite formation and prevented apoptosis. Scavengers of peroxynitrite also prevented apoptosis in this culture model. CONCLUSION: Peroxynitrite was involved in trophoblastic apoptosis induced by LPS. Peroxynitrite scavengers or inhibitors of NOS may thus be candidate therapeutic agents for infectious diseases, which is associated with overproduction of NO and peroxynitrite.

Apoptosis↗
PubMed1996

Altered expression of gelatinase and surface-associated plasminogen activator activity by trophoblast cells isolated from placentas of preeclamptic patients.

OBJECTIVE: This study compared in vitro degradative properties of trophoblasts isolated from placentas of preeclamptic patients with those of trophoblasts isolated from placentas of patients with uncomplicated pregnancies. Specifically, the expression of gelatinases, plasminogen activators, and plasminogen activator inhibitor type 1 by these cells was examined. STUDY DESIGN: Gelatinase and plasminogen activator secretion were determined by zymography, whereas antigenic levels of urinary-type plasminogen activator and plasminogen activator inhibitor type 1 in culture-conditioned medium were determined by enzyme-linked immunosorbent assay. We also evaluated expression of cell-surface plasminogen activator activity with a sensitive assay that uses the fluorescent plasmin substrate H-D-Val-Leu-Lys-7-amino-4-methylcoumarin. RESULTS: Cells from both normal and preeclamptic placentas secreted variable levels of gelatinase, of which the most predominant was matrix metalloproteinase-9 (gelatinase B). In 60% of the media conditioned by preeclamptic cells the matrix metalloproteinase-9 present was exclusively of a higher molecular weight (> 92 kd) than the predominant enzyme secreted by trophoblasts isolated from normal placentas. Incubation of the preeclamptic cell culture-conditioned media with p-aminophenylmercuric acetate, an activator of metalloproteinases, resulted in a decrease in the apparent molecular weight of the enzyme, indicating that most of the matrix metalloproteinase-9 contained in these samples was in the inactive form. Although trophoblasts from normal and preeclamptic placentas secreted similar amounts of urinary-type plasminogen activator and plasminogen activator inhibitor type 1, the latter cells expressed significantly less (p < 0.001) cell surface plasminogen activator activity. CONCLUSION: These results suggest that abnormal uteroplacental blood flow in preeclampsia (as a result of either shallow invasiveness of the uterine arteries or excessive fibrin deposition within the intervillous spaces) might result from altered expression of active proteinases by trophoblasts.

Adult↗
PubMed2000

Expression of endothelial nitric oxide synthase by extravillous trophoblast cells in the human placenta.

Previous reports have documented the expression of endothelial nitric oxide synthase (eNOS) expression by the syncytiotrophoblast layer of the villus in the human placenta. In contrast, the underlying villous cytotrophoblast cells do not express the enzyme. Because extravillous cytotrophoblasts have not been as extensively investigated, our objective was to test whether these cells express eNOS. Using both a mouse monoclonal and a rabbit polyclonal antibody, we demonstrated immunoreactive eNOS in trophoblast cell columns emanating from anchoring villi in second trimester placentae. Cytokeratin positive trophoblast cells lying beneath remnant anchoring villi, lining decidual blood vessels and scattered throughout the basal plate of normal term and pre-eclamptic placentae also expressed immunoreactive eNOS. By Western analysis, the monoclonal and polyclonal antibodies were shown to be absolutely and relatively specific for eNOS, respectively. The finding of immunoreactive eNOS expression by extravillous trophoblast cells was substantiated by in situ hybridization. Using riboprobes generated from a bovine eNOS cDNA, we demonstrated specific hybridization in the endothelium of blood vessels in the umbilical cord, thus validating the in situ hybridization methodology, as well as specific hybridization in the extravillous trophoblast cells of the basal plate in normal term placenta. In conclusion, several different populations of extravillous trophoblast cells in the basal plate of the human placenta express eNOS.

Animals↗
PubMedDate not supplied

Phagocytosis of chorion laeve trophoblasts in patients with chorioamnionitis-associated preterm delivery: ultrastructural and enzyme-histochemical observations.

The purpose of the present study was to examine morphological evidence for the presence of phagocytosing chorion laeve trophoblasts in fetal membranes from patients who had undergone chorioamnionitis-related preterm delivery. Chorion laeve trophoblasts from six patients, who underwent preterm delivery (28-34 weeks of gestation) complicated with chorioamnionitis, were analysed using transmission electron microscopy and ultrastructural enzyme-histochemistry for acid phosphatase, and compared with those from gestational age-matched chorioamnionitis-negative controls. Cytosomal cell projections, phagosomes, phagocytosis of degenerated cells and cell debris, attachment or fusion of lysosomes to the phagosomal membranes and phagosomal membranes positive for acid phosphatase staining were characteristically observed much more frequently in trophoblasts with chorioamnionitis than those without. The results indicated that chorion laeve trophoblasts in fetal membranes from patients having undergone chorioamnionitis-related preterm delivery underwent phagocytosis. Such phagocytosing trophoblasts may play a role in the pathophysiology or pathogenesis of infection-related preterm delivery.

Acid Phosphatase↗
PubMedDate not supplied

Recognition of trophoblast HLA class I molecules by decidual NK cell receptors--a review.

During placentation the extravillous trophoblast (EVT) cells migrate through the decidua towards the maternal spiral arteries. The walls of the arteries are then destroyed by trophoblast resulting in an increased blood flow to the fetus. These EVT express HLA-G, HLA-E and HLA-C, an unusual combination of two non-classical and one classical MHC class I molecules. The decidua is infiltrated by distinctive uterine natural killer (NK) cells during the time of trophoblast invasion. These cells express a variety of receptors (CD94/NKG2, KIR and ILT) which are known to recognize HLA class I molecules. There is, therefore, a mechanism for molecular recognition of the placental trophoblast cells. The possible functional consequences of this uterine NK cell-trophoblast interactions are uncertain. One possible result is in an altered NK cell cytokine profile which modulates the invasive proclivity of the EVT. In this way placentation could be controlled.

Decidua↗