Distribution of type-I interferon-receptors in human first trimester and term placental tissues and on isolated trophoblast cells

PROBLEM: Type-I interferon (IFN) is the protein recognizing pregnancy in ruminants. Although IFN is secreted in early pregnancy, its role is not still clear in other species. Like other cytokines, IFN exerts its biological functions through specific membrane receptors. We have investigated the potential action of IFN in human pregnancy by studying the distribution of the receptors in the human placenta. METHOD: Reactivity to monoclonal antibodies (mAbs) to the type-I IFN-receptor (R) was analyzed by immunohistochemistry in human placental tissues and in cytospins of first trimester trophoblast cells. RESULTS: Type-I IFN-R immunoreactivity was observed mostly in first trimester villous cytotrophoblasts and in the cytotrophoblast cell columns. Trophoblast in the decidua, the epithelium of the uterine glands, and most of the isolated trophoblast cells were also immunoreactive. CONCLUSION: The expression of type-I IFN-R in the highly proliferating and migrating trophoblast suggests that this cytokine has a role in trophoblast growth and invasion.     

Identification of ''tissue'' transglutaminase binding proteins in neural cells committed to apoptosis

Mice in which the gene that encodes the receptor (R) for leukemia inhibitory factor (LIF) has been deleted show abnormal growth and development of the placenta. This indicates that LIF plays an important role in placental development. The expression of LIF-R and LIF was examined in human trophoblast and decidua using in situ hybridization and immunocytochemistry. LIF-R mRNA and immunoreactivity was localized in villous and extravillous trophoblast throughout pregnancy, and in endothelial cells of the fetal villi. Strong expression of mRNA encoding LIF was detected in decidual leukocytes, which are abundant at the implantation site. Extravillous trophoblast, which invades the maternal decidua, therefore expresses LIF-R as it moves past decidual leukocytes, which express LIF mRNA. The effect of LIF on cultured human trophoblast was examined in vitro. Recombinant human LIF had no effect on [3H]thymidine incorporation by purified extravillous trophoblast, nor on expression of integrins alpha1, alpha5, or beta1 by isolated trophoblast. These results identify fetal endothelial cells and all cells of the trophoblast lineage as targets for the action of LIF in human placenta. Although its effects on trophoblast are not yet clear, LIF appears to mediate interactions between maternal decidual leukocytes and invading trophoblast. LIF may also play a critical role in controlling angiogenesis in the placental villi, since human fetal endothelial cells express LIF-R, and mice lacking a functional LIF receptor gene show altered vascular development in the placenta.     

Prevention of spinal anesthesia-induced hypotension in the elderly: comparison between preanesthetic administration of crystalloids, colloids, and no prehydration

The expression of tissue transglutaminase (tTG) was studied during the formation of the normal human placenta and in molar pregnancies and choriocarcinoma, in order to correlate its expression with the functional characteristics of the recognized trophoblast cell types. tTG expression was found to be developmentally regulated. Before 6-7 weeks' gestation, only the chorionic villous cytotrophoblast expresses tTG. Thereafter the overlying syncytiotrophoblast becomes positive. tTG expression is gradually downregulated in the intermediate trophoblast cells emerging from the tips of the chorionic villi invading the uterine tissue. In the decidual wall, the intermediate trophoblast does not express tTG, whereas scattered syncytial cells, the placental bed giant cells, express tTG. Villi from complete hydatidiform mole (CHM) show tTG expression in both the cyto- and the syncytiotrophoblast. The intermediate trophoblast cells from CHM show heterogeneous tTG expression, with a majority of negative cells, whereas extravillous syncytia always express tTG. In choriocarcinoma, the tumour cells show heterogeneous tTG expression, with a majority of positive cells. Analysis of tTG protein and mRNA in placental extracts by Western and Northern blotting did not provide evidence for expression of the truncated form of tTG found in some cell types. The regulated expression of tTG in the normal placenta suggests that the enzyme is involved in important trophoblastic functions and may participate in the control of invasion.     

The expression of stem cell factor and its receptor, c-kit in human endometrium and placental tissues during pregnancy

Stem cell factor (SCF) and its receptor Kit regulate the proliferation and survival of early hematopoietic cell types as well as germ cells and melanocytes. As SCF augments the effects of several hematopoietic growth factors that are produced in reproductive tissues during pregnancy and also plays an important role in cell migration, proliferation, and survival, we studied the expression and localization of this receptor/ligand in human endometrial and placental tissues. Kit was detected by Western blot analysis in early decidual and placental tissues (8-19 weeks gestation) and in term placenta. Immunohistochemistry localized Kit mainly in trophoblast and to a lesser extent in scattered cells in the placental villous core and decidual stroma. Ribonuclease protection assay showed that SCF messenger ribonucleic acid (mRNA) expression increased 3-fold in decidua from early pregnancy compared to proliferative and secretory endometrium (P < 0.05). Placental tissues expressed 4- to 8-fold higher levels of SCF mRNA compared to decidus (P < 0.05). Isolated placental villous core expressed 7-fold higher levels of SCF mRNA than did trophoblast (P < 0.05). Thus, SCF and its receptor Kit are expressed in human endometrium and placental tissues during pregnancy, and the pattern of receptor/ligand expression suggests that endometrial and placental villous core SCF may have a paracrine effect on trophoblast through the receptor Kit.     

Endothelin-1 and endothelin receptors are present in the sheep uterus and conceptus at implantation

Previous studies have demonstrated that endothelin is present in the ovine endometrium and increases at around the expected time of implantation. To characterize further uterine endothelin at the time of establishment of pregnancy in sheep, endothelin was measured by radioimmunoassay in uterine flushings obtained during the oestrous cycle and in pregnant ewes up to the time of implantation (day 16). During the oestrous cycle, the highest amounts of endothelin were present in uterine flushings on day 14 (1.1 +/- 0.2 ng endothelin/uterus). During early pregnancy, basal levels of endothelin (0.5-0.6 ng endothelin/uterus) were present in uterine flushings for the first 10 days and then increased on day 14 to levels similar to those found at the equivalent stage of the oestrous cycle. On days 15 and 16 of pregnancy, endothelin content in the uterine lumen increased to significantly (P < 0.05) higher concentrations (2.9 +/- 0.4 ng endothelin/uterus) when compared with the non-fertile cycle. The principal isoform present in flushings at the time of implantation was endothelin-1, as determined by reverse-phase HPLC. Endothelin was released principally by purified endometrial epithelial cells in culture, with barely detectable amounts released by endometrial stromal cells or conceptus tissue, which is consistent with the epithelium being the principal source of endothelin in the uterine lumen. Endothelin binding sites were present in endometrium and myometrium, as demonstrated by specific binding of 125I-labelled endothelin-1, which was saturable and displaced by endothelin-1. Both endothelinA and B sub-types of receptors were present as demonstrated by the biphasic displacement of 125I-labelled endothelin-1 binding by the specific endothelinB agonist BQ3020. These were localised principally on luminal and glandular epithelium and in the vasculature of the endometrium and myometrium as shown by autoradiography. Endothelin receptors were also present on the conceptus obtained at the time of implantation. In the day 20 conceptus, endothelin immunostaining was localised principally in the heart, in trophoblast in uninucleate but not in binucleate cells, and in fetal membranes. This immunostaining of the conceptus may represent binding to receptor sites. It is concluded that endothelin-1 is present in the uterine lumen and may play an important role in the paracrine regulation of the conceptus and endometrium at the time of rapid embryo development, implantation and early placentation.     

Expression of laminin and nidogen genes during the postimplantation development of the mouse placenta

The expression patterns of laminin A, B1, B2, and nidogen genes were identified by in situ hybridization in postimplantation mouse extraembryonic tissues and maternal decidua during the period when the chorioallantoic placenta is established. Laminin and nidogen genes were not coordinately expressed either in the decidua or in trophoblast cells, indicating that these genes are regulated independently in these cell types during the establishment of the placenta. Laminin A mRNA was absent from the decidua except in the outer layer of cells adjacent to the myometrium and in the central decidual zone adjacent to the remnant of the uterine epithelium on Day 9. At this stage laminin B1, B2, and nidogen genes were strongly expressed in these cells and also in other regions of the decidua. Laminin B1 mRNA was present at higher levels in the decidua capsularis than in the decidua basalis, while nidogen mRNA showed highest expression in the decidua basalis. Laminin B2 mRNA was produced uniformly throughout the decidua at very high levels, suggesting that laminin B2 chains may be an important component of the decidual matrix. By Day 11, the nidogen gene was expressed only in endothelial cells lining the maternal blood spaces within the decidua. Laminin B1 and nidogen mRNAs were found at high levels within trophoblast giant cells at all stages, while laminin A mRNA was detected in trophoblast giant cells at later stages and laminin B2 mRNA was not produced in high levels by these cells. The patterns of gene expression show a very high degree of regional specialization, suggesting that the extracellular matrices in different regions of the decidua and extraembryonic membranes are likely to be composed of quite different ratios of laminin and nidogen polypeptides.(ABSTRACT TRUNCATED AT 250 WORDS)     

TNF-alpha messenger RNA and protein expression in the uteroplacental unit of mice with pregnancy loss

An elevated expression of TNF-alpha in embryonic microenvironment was found to be associated with postimplantation loss. In this work, we examined the pattern of TNF-alpha expression at both the mRNA and the protein level as well as the distribution of TNF-alpha receptor mRNA in the uteroplacental unit of mice with induced (cyclophosphamide-treated) or spontaneous (CBA/J x DBA/2J mouse combination) pregnancy loss. RNase protection analysis demonstrated an increase in TNF-alpha mRNA expression in the placentae of mice with pregnancy loss compared with that in control mice. TNF-alpha messages were localized to the uterine epithelium and stroma as well as the giant and spongiotrophoblast cells of the placenta. The intensity of the hybridization signal in placentae of mice with pregnancy loss was substantially higher than that in control mice. The up-regulation of TNF-alpha mRNA was accompanied by an increase in the expression of TNF-alpha receptor I mRNA in the same cell populations. The elevation of TNF-alpha production was also demonstrated at the protein level. Western blot analysis showed an increased level of the 18- and 26-kDa TNF-alpha protein species in the uteroplacental unit of mice with pregnancy loss. Immunostaining revealed TNF-alpha-positive leukocytes located in the uterus and placenta. Finally, we found that immunization of mice with cyclophosphamide-induced pregnancy loss while decreasing the resorption rate in these females resulted in a decline in TNF-alpha expression at the fetomaternal interface. These data clearly suggest an involvement of TNF-alpha in pathways leading to both spontaneous and induced placental death.     

Immunohistochemistry of matrix metalloproteinases (MMP), their substrates, and their inhibitors (TIMP) during trophoblast invasion in the human placenta

The invasion of extravillous trophoblast cells into the maternal endometrium is one of the key events in human placentation. The ability of these cells to infiltrate the uterine wall and to anchor the placenta to it as well as their ability to infiltrate and to adjust utero-placental vessels to pregnancy depends, among other things, on their ability to secrete enzymes that degrade the extracellular matrix. Most of the latter enzymes belong to the family of matrix metalloproteinases. Their activity is regulated by the tissue inhibitors of matrix metalloproteinases. We have studied the distribution patterns of matrix metalloproteinases-1, -2, -3, and -9 and their inhibitors TIMP-1 and TIMP-2 as compared to the distribution of their substrates along the invasive pathway of extravillous trophoblast of 1st, 2nd, and 3rd trimester placentas by means of light microscopy on paraffin and cryostat sections as well as at the ultrastructural level (only 3rd trimester placenta). The comparison of different methods proved to be necessary, since the immunohistochemical distribution patterns of these soluble enzymes are considerably influenced by the pretreatment of tissues. All three methods revealed immunoreactivities of both, proteinases and their inhibitors, not only intracellularly in the extravillous trophoblast but also extracellularly in its surrounding matrix, the distribution patterns depending on the stage of pregnancy and on the degree of differentiation of trophoblast cells along their invasive pathway. Within the extracellular matrix, immunolocalization of matrix metalloproteinases as well as their inhibitors showed a specific relation to certain extracellular matrix molecules.     

Decreased expression of PAI-2 mRNA and protein in pregnancies complicated with intrauterine fetal growth retardation

An increase in plasma plasminogen activator inhibitors (PAIs), fundamentally PAI type 2 (PAI-2), has been described in normal pregnancy probably because the placenta is the main source of the high plasma levels of this protein. Although we have previously described plasmatic alterations of these inhibitors in pregnancies complicated with intrauterine fetal growth retardation (IUGR), no reports have been published about placental PAI-2 mRNA expression. In the present study, the placental PAI-2 expression determined in pregnancies complicated with IUGR and in severe preeclamptic patients was compared with that of normal pregnancies in order to identify the placental cell types expressing PAI-2 and to determine whether the production of PAI-2 is altered in placentas from IUGR. In situ hybridization analyses show that the syncytiotrophoblasts are the cells with the greatest PAI-2 expression in placenta. We report that the significant decrease in plasma and placental PAI-2 levels in IUGR groups is fundamentally due to a diminished expression of PAI-2 mRNA in placenta.     

Epithelial cell polarity and embryo implantation in mammals

At embryo implantation we are confronted with the fact that uterine and trophoblast epithelium make contact via their apical cell membranes. This epithelium-epithelium adhesion leading to definitive attachment of the embryo to the uterine wall, however, is far from being trivial and has been called a cell biological paradox. It has been proposed that some of the molecular events involved in epithelium-to-mesenchyme transformation might play a role in the interaction between uterine cells and trophoblast. As a mechanism to achieve uterine epithelium adhesiveness for trophoblast it is postulated that uterine cells partially modulate their epithelial phenotype. Data from recent in vitro experiments give support to this hypothesis and suggest that loss of apical-basal cell polarity might prepare the apical cell pole of uterine epithelium for cell-to-cell contact with trophoblast in vivo.     

The dependence of amino acid pair correlations on structural environment

The two main groups of placental proteins of ruminants are discussed in this paper: chorionic somatomammotropins (placental lactogens) and pregnancy-specific (-associated) proteins. Placental lactogens belong to the prolactin and growth hormone family. They stimulate mammogenesis, fetal growth and maternal metabolism. Pregnancy-specific proteins and pregnancy-associated glycoproteins belong to the aspartic proteinase family like pepsin, cathepsin D and E. These two groups of proteins are secreted in the maternal circulation by the binucleate cells after their migration to and fusion with the uterine cells. Their profiles were determined by radioimmunoassay (RIA). Further investigations are in progress to relate secretory profiles with alterations of the trophoblastic function such as those occurring in embryonic mortality, abortion, and fetal distress. The endocrine function of the primate and equine placenta is also discussed.     

Aspects of calcium transport by the ovine placenta: studies based on the interplacentomal region of the chorion

An in vitro technique for the measurement of calcium uptake into the maternal-facing fetal chorionic membrane (apical trophoblast) was used to study the relationship between calcium uptake and stage of pregnancy in the sheep. The effects on calcium uptake of varying calcium concentration and temperature of the incubation medium, of adding calcium channel blockers or heavy metals (lanthanum and nickel) or calcium ionophore/agonist were also studied. The data indicate a saturable calcium uptake process, plateauing after 15 min incubation. This uptake remained constant throughout the last third of gestation until a significant fall in uptake was noted during the final week prior to parturition. This uptake was not due to extracellular cellular diffusion since there was no significant uptake of tritiated inulin over the same period in each case. Calcium uptake in this system was also shown to be a temperature dependent process which was abolished at temperatures of 0-4 degrees C. A decrease in calcium concentration to 0.12 mM in the incubation medium also caused a corresponding decrease in calcium uptake to 21 per cent of control (1.2 mM). The addition of the heavy metals lanthanum and nickel also significantly reduced calcium uptake as did the calcium channel blockers verapamil, metoprolol and diltiazem. The calcium channel ionophore A23187 increased calcium uptake into the material facing chorion. Although the interplacentomal chorion may not be representative of the whole of the placental unit, it clearly contains a specific calcium uptake process under local physiological control. The blocking of calcium uptake by the specific I-type calcium channel blocker verapamil may indicate the presence of I-type channels of unusually low sensitivity since the concentration needed to block them was much higher than would be required for excitable I-type channels in isolated cells.     

Vascular endothelial growth factor, placenta growth factor and their receptors in isolated human trophoblast

The expression of the angiogenic growth factors, vascular endothelial cell growth factor (VEGF) and placenta growth factor (PIGF) was demonstrated in isolated human term cytotrophoblast and in vitro differentiated syncytiotrophoblast. RNase protection assays demonstrated VEGF expression in both cytotrophoblast and syncytiotrophoblast while prominent PIGF expression was detected in both types of trophoblast by Northern blot analyses. VEGF expression increased approximately eightfold in trophoblast cultured under hypoxic conditions (1 per cent O2) yet PIGF expression decreased 73 +/- 5.5 per cent in the same trophoblast. These results suggest distinct regulatory mechanisms govern expression of VEGF and PIGF in trophoblast. Characterization of the VEGF/PIGF receptors, KDR and flt-1, revealed the presence of flt-1 mRNA in isolated cytotrophoblast and in vitro differentiated syncytiotrophoblast. KDR was not detected in the isolated trophoblast. Exogenous rhVEGF induced c-Jun N-terminal kinase (JNK) activity in the normal trophoblast indicating that the flt-1 receptors on trophoblast are functional. Trophoblast-derived VEGF/PIGF could act in a paracrine fashion to promote uterine angiogenesis and vascular permeability within the placental bed. In addition, presence of function flt-1 on normal trophoblast suggests that VEGF/PIGF functions in an autocrine manner to perform an as yet undefined role in trophoblast invasion, differentiation, and/or metabolic activity during placentation.     

The regulatory aspects of the blood circulation in the placenta in physiological and complicated pregnancies

New data about the development of placental circulation and the features of its regulation in normal and complicate pregnancy are considered in present review. Biochemical mechanisms leading to the dilatation of umbilical-placental circulation as well as the relaxation and contraction of placental vessels smooth muscles are discussed. It is shown that the disturbance of the balance between dilation and constriction factors being the results of injury of endothelial cells causes the spasm of uterine vessels, "delimiting" of maternal circulation from fetal one and proceeds a clinical manifestations of placental insufficiency. The data about the trophoblast autoimmune affection that underly to induction of autoimmune state of pre-eclampsia are described.     

Ultrastructural evidence for loss of the trophoblastic layer in the chorioallantoic placenta of Australian bandicoots (Marsupialia: Peramelidae)

In most marsupials, placentation involves only the yolk sac; however, in the bandicoot family, Peramelidae, a functional chorioallantoic placentation develops in addition (Hill, 1895, 1897, 1900; Flynn, '22, '23). This duality is viewed as having evolutionary significance because most eutheria have both placentae. Furthermore, the bandicoot trophoblast was reported to vanish from the chorioallantoic site in late gestation (Hill, 1897; Flynn, '23); whereas, the eutherian trophoblast is identifiable throughout later pregnancy and may act as an immunological barrier between maternal and fetal genotypes (Kirby '68). Thus we have re-examined this singular chorioallantoic placenta of the bandicoot in plastic sections with light and electron microscopy. A distinctive feature of the bandicoot placentation is the transformation of the uterine simple columnar luminal epithelium into a highly vascular lining composed almost entirely of discrete syncytial masses (homokaryons). Endometrial blood vessels penetrate among the homokaryons to create a rich network of large diameter capillaries at extremely superficial locations near the maternal surface. In the chorioallantoic placenta (7 mm to 10-11 mm crown-rump embryos) the microvillous surface of the maternal homokaryons interdigitates with the microvillous border of the fetal trophoblast with desmosomal interaction. This trophoblast consists of a single layer of tall columnar undifferentiated cells rich in ribosomes-polysomes, poor in cytoplasmic membranes, and with large nuclei that have distinct clumps of heterochromatin and conspicuous nucleoli. It is thus remarkable that these undifferentiated cells disappear as a recognizable layer later in gestation (12 mm crown-rump embryos). Flynn's hypothesis that the trophoblastic cells disappear by fusing with maternal syncytia gains support from the existence of two populations of nuclei in the syncytial masses only at the chorioallantoic site. One population is comparable to that occurring in the homokaryons pf the yolk sac placenta, i.e., pale staining nuclei with little heterochromatin and small peripheral nucleoli. However, the other nuclei resemble those of the trophoblast cells. Since the trophoblastic cells before their disappearance as a layer possess properties associated with potential for further differentiation, the possibility of fusion between the maternal homokaryons and fetal trophoblastic cells to form heterokaryons composed of two genotypes merits fur     

Slowed reaction time during a continuous performance test in children with Tourette''s syndrome

The HRP-1 cell line is derived from normal rat placenta and appears morphologically similar to and retains characteristic expression of cellular markers of labyrinthine trophoblast cells. In this study, monolayers of HRP-1 cells grown on permeable supports were evaluated as a potential in vitro system to study trophoblast transport and metabolism. The cell line was shown to express and retain functional activity of the predominant placental cytochrome P450 isozyme, CYP1A1. Additionally, the HRP-1 cells retain functional activity of angiotensin I converting enzyme and carboxypeptidase N-like enzyme, peptidases characteristic of the trophoblast. The permeation of several hydrophilic, inert markers across the HRP-1 monolayers was observed to be dependent on effective molecular size and to be passive in nature. Functional asymmetry of the HRP-1 cells was illustrated by the predominant permeation of linoleic acid in the apical-to-basolateral direction across the monolayers. Transferrin passage across HRP-1 monolayers was concentration-dependent, was bidirectional, and could be inhibited by unlabeled transferrin, features typical of the trophoblast transport system for transferrin. Collectively, these properties suggest that the HRP-1 cell line may provide a useful tool for evaluating some of the permeability and metabolic properties of the trophoblast.     

Fertility impairment in granulocyte-macrophage colony-stimulating factor-deficient mice

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been identified as a potentially important mediator of intercellular communication in the female reproductive tract, with principal target cells being the large populations of myeloid leukocytes in the cycling and pregnant uterus, the preimplantation embryo, and trophoblast cells of the developing placenta. To determine the physiological significance of this cytokine in reproduction, the fertility of genetically GM-CSF-deficient (GM-/-) mice was examined. Implantation rates were normal in GM-/- mice, and viable pups were produced. However, the mean litter sizes of GM-/- x GM-/- breeding pairs were 25% smaller at weaning than those of GM+/- x GM+/- pairs, due to fetal death late in gestation and early in postnatal life, with a disproportionate loss of male pups. On Day 17 of pregnancy, the mean number of resorbing and malformed fetuses was twice as high in pregnant GM-/- females (21%, vs. 11% in GM+/- females); the mean fetal weight and the mean fetal:placental ratio in surviving conceptuses were diminished by 7% and 6%, respectively; and the number of very small fetuses (< 500 mg) was 9-times as high (23% vs. 2.5%). Mortality during the first 3 wk of life was 4.5-times as high in pups born to GM-/- mothers (9%, vs. 2% in GM+/- females), and diminished size persisted in GM-/- pups, particularly males, into adulthood. The detrimental effect of maternal GM-CSF deficiency was less apparent when GM-/- females were mated with GM+/+ males; litter sizes at birth and at weaning were not significantly smaller than in GM+/- matings, and fetal weights and fetal:placental ratios were also comparable. When polymerase chain reaction was used to genotype embryonic tissue in heterozygote matings, GM-/- fetuses from GM-/- females were found to be smaller than their GM+/- littermates and smaller than GM-/- fetuses gestated in GM+/- females. The size and distribution of uterine granulocyte and macrophage populations were normal during the estrous cycle, during early pregnancy, and in midgestation. Analysis of placental structure revealed that the ratio of labyrinthine to spongiotrophoblast areas was reduced by approximately 28% in GM-/- placentae, and the proportion of vacuolated trophoblast "glycogen cells" in the spongiotrophoblast layer was diminished. Compromised placental function as a result of subtle developmental aberrations may therefore partially account for embryonic growth retardation in GM-CSF-deficient mice. Collectively, these studies show that fetal growth and viability are jeopardized in the absence of maternal GM-CSF. The detrimental effects are most clearly evident when the conceptus is also GM-CSF deficient, suggesting that GM-CSF of either maternal or fetal origin is required for optimal growth and survival of the fetus in mice.