Transferrin receptors in the basal plasma membrane of the human placental syncytiotrophoblast

Transferrin was identified in a preparation of human syncytiotrophoblast basal plasma membrane. Such transferrin was shown to be bound to an amphiphilic membrane protein of subunit mol.wt 94 000 by crossed hydrophobic interaction immunoelectrophoresis and immunoprecipitation with antibodies to transferrin. Basal plasma membrane bound 125I-labelled transferrin in a saturable, reversible manner with high affinity (Kd = 2.5 +/- 0.6 X 10(-9) M). The maximum binding capacity (0.9 +/- 0.2 pmol transferrin/mg membrane protein) was approximately a half of that of microvillous membrane. The basal membrane transferrin receptors were similar to microvillous receptors in that their affinity for diferric transferrin was higher than that for apo-transferrin and transferrin dissociation was negligible at pH 5.0 but rapid at pH 7.4. We conclude that syncytiotrophoblast basal plasma membrane possesses a receptor similar, if not identical, to that on the microvillous membrane. These receptors are thus in a position to participate in iron transfer to the fetus or potentially to have alternative functions in the syncytiotrophoblast.     

Transferrin receptor affinity and iron transport in the human placenta

Transferrin receptor activity has been investigated on isolated human placental syncytiotrophoblast microvillous plasma membrane preparations following 3 M KCl washing of membranes to remove endogenous receptor-bound maternal transferrin. The calculated binding parameters for essentially apo- and diferric human transferrin, and also for rabbit transferrin, were closely comparable (ranges: Ka, 3.5 to 5.0 X 10(7) M-1; n, 2.6 to 4.0 X 10(14)/mg membrane protein). Iron transport in pregnancy is thus unlikely to involve a simple process of transferrin displacement following iron release at the placental trophoblast plasma membrane receptor site.     

Localization of alkaline phosphatase and Ca2+-ATPase in the cat placenta

We localized alkaline phosphatase and plasma membrane calcium-ATPase (PMCA) in the cat placental syncytiotrophoblast to address their polarized distribution and their potential as markers for specific plasma membrane purification. We used enzyme- (alkaline phosphatase) and immuno- (PMCA) histochemistry and, for alkaline phosphatase, compared data to observations on the human placenta. Alkaline phosphatase activity in the cat was localized to the decidual cell membranes, to within the associated interstitial space and on the subjacent apical (maternal facing) plasma membrane of the syncytiotrophoblast. Occasional maternal capillaries were positive on their basal surface and there was focal staining within the syncytiotrophoblast. This widespread distribution is less specific than in the human placenta where alkaline phosphatase was restricted to the apical and basal plasma syncytiotrophoblast membranes, with much greater density on the apical membrane. Expression of PMCA in the cat was restricted to the basal membrane of the syncytiotrophoblast only. This specific localization of PMCA is identical to the human placenta and all other species in which its placental localization has been studied. We conclude that the plasma membranes of the cat syncytiotrophoblast show a broadly similar functional polarization to the human and that PMCA would prove a useful marker in isolation of the cat syncytiotrophoblast basal plasma membrane.     

Iron metabolism and placental transfer of iron in the term rhesus monkey (Macaca mulatta): a compartmental analysis.

The kinetics of iron and the transplacental iron transport in the near-term rhesus monkey were investigated by means of injection of 59Fe(III) bound to rhesus monkey transferrin into the maternal or into the fetal circulation. The experimentally obtained 59Fe(III) bound to rhesus monkey transferrin into the maternal or into the fetal circulation. The experimentally obtained 59Fe dissappearance and appearance curves were analyzed. The analyses were based on a 5-compartmental system. Alternative models were considered but found to be inadequate. Very high maternal as well as fetal iron turnover were calculated. The daily reflux of iron in the mother and in the fetus amounted to 75% of the amount of iron cleared from the plasma compartment daily. The calculated iron transport from mother to fetus was about 1.0 mumol/day/100 g fetus. The reverse transplacental iron transport was negligible. A small placental iron pool of exchangeable iron had to be assumed. There were no clear difference in the distribution of 59Fe over the fetal organs when the tracer dose was injected into the maternal or into the fetal circulation. Only one of the experiments showed a preferential labeling of the fetal liver when the label was given to the mother, a preference which would be expected on the basis of the hypothesis of Fletcher and Suter (1969).     

Effects of chronic hypoxia in vivo on the expression of human placental glucose transporters

Birth weight is reduced and the risk of preeclampsia is increased in human high altitude pregnancies. There has been little work to determine whether hypoxia acts directly to reduce fetal growth (e.g. reduced blood flow and oxygen delivery), or via changes in functional capacities such as nutrient transport. We therefore investigated the expression of a primary nutrient transporter, the GLUT1 glucose transporter and two in vitro markers of hypoxia (erythropoietin receptor, EPO-R, and transferrin receptor, TfR) in the syncytial microvillous (MVM) and basal membrane fractions (BMF) of 13 high (3100 m) and 12 low (1600 m) altitude placentas from normal term pregnancies. Birth weight was lower at 3100 m than at 1600 m despite similar gestational age, but none of the infants were clinically designated as fetal growth restriction. EPO-R, TfR and GLUT1 were examined by immunoblotting and maternal circulating erythropoietin and transferrin by ELISA. EPO-R was greater on the MVM (+75%) and BMF (+25%) at 3100 m. TfR was 32% lower on the MVM at 3100 m. GLUT1 was 40% lower in the BMF at 3100 m. Circulating EPO was greater at high altitude, while transferrin was similar, and neither correlated with their membrane receptors. BMF GLUT1 was positively correlated with birth weight at high, but not low altitude. In this in vivo model of chronic placental hypoxia, syncytial EPO-R increased as expected, while nutrient transporters decreased, opposite to what has been observed in vitro. Therefore, hypoxia acts to reduce fetal growth not simply by reducing oxygen delivery, but also by decreasing the density of nutrient transporters.     

Expression of transferrin receptors during differentiation of human placental trophoblast cells in vitro.

In most cell types, transferrin receptor expression is correlated with the proliferation rate, being increased by growth stimulation, or decreased by induction of terminal differentiation. In the human placenta the multinucleated syncytiotrophoblast, in direct contact with maternal blood, is derived by differentiation from mononucleated cytotrophoblast. In this study we examined changes in transferrin receptor expression during in vitro differentiation of trophoblast. Cells cultured in Ham's/Waymouth's medium (HWM) remained primarily mononuclear throughout the study, whereas incubation in keratinocyte growth medium (KGM) led to formation of multinucleate masses within 2-3 days of culture. Cell surface binding of 125I-labelled transferrin increased fivefold between days 1-5 of culture in both media and surface receptors were saturated at 7-14 micrograms/ml (90-200 fM). At saturation, the amount of transferrin bound to syncytiotrophoblast was 37 per cent lower than in cytotrophoblast. Scatchard analysis revealed a reduction in the number of surface transferrin receptors in syncytiotrophoblast compared to cytotrophoblast. A corresponding 29 per cent reduction in the binding of transferrin to intracellular sites was observed in syncytiotrophoblast. Distribution of receptors between surface and intracellular sites was therefore similar in both cytotrophoblast and syncytiotrophoblast. The affinity of transferrin for transferrin receptors was 3.7-fold higher in syncytiotrophoblast when compared to cytotrophoblast. Observed differences between the two cell types were not due to the presence of growth factors or higher iron levels in KGM. Expression of a high number of surface transferrin receptors in syncytiotrophoblast (1.5 x 10(12)/mg protein), along with a high affinity of these receptors for iron-saturated transferrin, could help explain the efficient transport of large amounts of iron from mother to fetus.     

Characteristic differences in immunohistochemical localization of new placental proteins (PP1, PP19, PP21) in the human placenta

The immunohistochemical localization in the human placenta of new placental proteins PP1, PP19, and PP21 was clarified using modified indirect enzyme-labeled antibody method and compared with that of pregnancy-specific beta 1-glycoprotein (SP1). The major results are as follows: positive staining for PP1 was seen at the nucleus and cytoplasm of villous cytotrophoblasts, the X cells at the basal plate, and of chorionic trophoblasts, while the decidua cells and amnion were not stained. PP19 was characteristically seen in the nucleus and cytoplasm of syncytiotrophoblasts. X cells in basal plate, chorionic trophoblasts, and maternal leukocytes. The villous cytotrophoblasts, decidua cells, and amnion were not stained. PP21 localization was found at the microvilli and basal membrane of syncytiotrophoblasts and at the cytotrophoblast plasma membrane of the chorionic villus in early gestation. In late gestation, increased staining was seen at the syncytiotrophoblast microvilli and the villous basement membrane, and moderate staining at plasma membrane of the amniotic epithelium and chorionic trophoblasts. SP1 was found only at the syncytiotrophoblast cytoplasm of chorionic villi. Studies using these four placental proteins simultaneously may therefore provide a new key learning about unknown metabolic functions of trophoblasts.     

Distribution of glutamate transporters in the human placenta

Glutamate metabolism is known to be important for growth and development of the human fetus. The glutamate transporters EAAT1, EAAT2 and EAAT3 are key components of the glutamate-glutamine cycle and responsible for active transport of glutamate over the cell membrane. The placenta is thought to regulate glutamate transport during fetal development. Glutamate transporters have been found in placentae of rats, but their distribution in the human placenta is unknown. Therefore, the distribution of glutamate transporters EAAT1, EAAT2 and EAAT3 were analysed in the human placenta during normal pregnancies ending between 8 and 40 weeks of gestation and in placentae of intrauterine growth restricted infants with gestational ages between 28 and 35 weeks of pregnancy. Using immunohistochemistry, EAAT1 expression was found in the syncytiotrophoblast layer, while EAAT2 was detected in the syncytiotrophoblast layer and in endothelial cells of about 5 per cent of all fetal blood vessels. EAAT3 was observed in the endothelium of the fetal blood vessels in all placentae examined. However, expression was also found in the syncytio- and the cytotrophoblast layer of the fetal villi at 8 weeks of gestational age. The expression patterns of EAAT1, EAAT2 and EAAT3 suggest involvement in active transport of glutamate between the fetal and maternal blood circulation. No differences were found in the distribution of the glutamate transporters between control and IUGR placentae. Our data show specific localization of EAAT1, EAAT2 and EAAT3 in the human placenta during development.     

Differential intracellular localisation of the Menkes and Wilson copper transporting ATPases in the third trimester human placenta

Copper is an essential trace element necessary for normal growth and development. During pregnancy, copper is transported from the maternal circulation to the fetus by mechanisms which have not been clearly elucidated. Two copper transporting ATPases, Menkes (ATP7A; MNK) and Wilson (ATP7B; WND) are known to be expressed in the placenta and are thought to have a role in copper transport to the fetus. In this study, the intracellular localisation of the MNK and WND proteins in the third trimester human placental tissue was investigated in detail using double-label immunohistochemistry and immuno-electron microscopy. MNK and WND were differentially localised within the placenta. MNK was present at the basal side of the syncytiotrophoblast layer and also within the fetal vascular endothelial cells, whereas WND was localised at the microvillous membrane of the syncytiotrophoblast. These data offer some insights into possible differential roles for MNK and WND within the placenta.     

A placental sub-proteome: the apical plasma membrane of the syncytiotrophoblast

As a highly vascularized tissue, the placenta mediates gas and solute exchange between maternal and fetal circulations. In the human placenta, the interface with maternal blood is a unique epithelial structure known as the syncytiotrophoblast. Previously we developed a colloidal-silica based method to generate highly enriched preparations of the apical plasma membrane of the syncytiotrophoblast. Using similar preparations, a proteomics assessment of this important sub-proteome has identified 340 proteins as part of this apical membrane fraction. The expression of 38 of these proteins was previously unknown in the human placental syncytiotrophoblast. Together with previous studies, the current proteomic database expands our knowledge of the proteome of the syncytiotrophoblast apical plasma membrane from normal placentas to include more than 500 proteins. This database is a valuable resource for future comparisons to diseased placentas. Additionally, this data set provides a basis for further experimental studies of placenta and trophoblast function.     

Partitioning of glutamine synthesised by the isolated perfused human placenta between the maternal and fetal circulations

Introduction

Placental glutamine synthesis has been demonstrated in animals and is thought to increase the availability of this metabolically important amino acid to the fetus. Glutamine is of fundamental importance for cellular replication, cellular function and inter-organ nitrogen transfer. The objective of this study was to investigate the role of glutamate/glutamine metabolism by the isolated perfused human placenta in the provision of glutamine to the fetus.

Methods

Glutamate metabolism was investigated in the isolated dually perfused human placental cotyledon. U–¹³C-glutamate was used to investigate the movement of carbon and ¹⁵N-leucine to study movement of amino-nitrogen. Labelled amino acids were perfused via maternal or fetal arteries at defined flow rates. The enrichment and concentration of amino acids in the maternal and fetal veins were measured following 5 h of perfusion.

Results

Glutamate taken up from the maternal and fetal circulations was primarily converted into glutamine the majority of which was released into the maternal circulation. The glutamine transporter SNAT5 was localised to the maternal-facing membrane of the syncytiotrophoblast. Enrichment of ¹³C or ¹⁵N glutamine in placental tissue was lower than in either the maternal or fetal circulation, suggesting metabolic compartmentalisation within the syncytiotrophoblast.

Discussion

Placental glutamine synthesis may help ensure the placenta's ability to supply this amino acid to the fetus does not become limiting to fetal growth. Glutamine synthesis may also influence placental transport of other amino acids, metabolism, nitrogen flux and cellular regulation.

Conclusions

Placental glutamine synthesis may therefore be a central mechanism in ensuring that the human fetus receives adequate nutrition and is able to maintain growth.     

Glucose transporter 1 localisation throughout pregnancy in the carnivore placenta: light and electron microscope studies

Glucose is one of the major fetal nutrients. Maternofetal transfer requires transport across the several placental membranes. This transfer is mediated by one or more of the fourteen known isoforms of glucose transporter. So far only Glucose Transporters 1 and 3 (GT1, GT3) have been shown to be located in placental membranes. GT1 may be the only one on the syncytiotrophoblast (human) or both may be present on the same membrane (rodents) or be required in sequence (ruminants, horses and elephant). This paper shows GT1 to be the only transporter demonstrable by immunocytochemistry in carnivore (cat, dog and mink) endotheliochorial placental membranes. GT1 is invariably present on both apical and basal surfaces of the cyto- and syncytiotrophoblast in all carnivore species examined and the pattern of development is described from implantation to term.     

Maternal serum levels of human chorionic somatotropin correlates with transferrin and erythropoietin in pregnancy.

During pregnancy, plasma levels of erythropoietin (EPO) and transferrin (TSF) show a gradual increase till term, when a slight decrease occurs. Human chorionic somatotropin (hCS) is a peptide hormone with a direct effect on the function of the syncytiotrophoblast and on the mass of functional placental tissue. In order to understand the relationships between the placental functions, maternal erythropoiesis and iron transfer to the fetus, the relationships between EPO and hCS and between TSF and hCS were investigated. A group of healthy pregnant women (n = 190) attending the Obstetric Clinic of the University of Siena were studied. They were between 18 and 43 weeks pregnant. A single blood sample was taken from each patient by radial vein puncture on admission into the clinic before the beginning of any therapy, for the determination of haemoglobin, EPO, ferritin, serum iron, TSF and hCS. The highest correlation was that between hCS and TSF (r = 0.336, p < 0.0001). This correlation was significant in anaemic patients and lost its significance in non-anaemic patients. In our study, we also found a significant relationship between hCS and logEPO (r = 0.195; p < 0.01) which reached an elevated significance over the entire survey, but did not reach statistical significance in anaemic women. We hypothesise that in pregnancy chorionic villi develop a great capacity to respond to pathological maternal events with the result of regulating the transport of iron, the oxygen tension and the maternal-fetal transfer of oxygen through haemoglobin. Evidently, the response of the villi serves to balance the lack of iron in favour of the fetus, probably through a mechanism that links the variations of the iron transport on the maternal side and the increased use on the fetal side through a dynamic and consensual response by the chorionic villi.     

Change in iron transporter expression in human term placenta with different maternal iron status

OBJECTIVES: To better understand the molecular mechanism of human placental iron transfer, the protein and mRNA expression of iron transporters, transferrin receptor 1 (TfR1), ferritin and ferroportin 1 (FP1) were examined simultaneously. STUDY DESIGN: Forty pregnant women, 18 normal, 22 with anemia but all free from other medical conditions, were chosen for the research and their iron status was determined before delivery. Forty human term placentas were collected from these pregnant women with different iron status in the third trimester. The protein and mRNA expression of iron transporters in the placental tissues were assessed by means of Western blot and real-time quantitative PCR. RESULTS: The study showed that the expression of TfR1 mRNA increased significantly (almost three-fold) in the mild anemia group and decreased near to the normal level in the moderate anemia group. The same trend was observed for TfR1 protein expression, but it was not statistically significant. Ferritin protein expression decreased slightly in the mild anemia group but significantly in the moderate anemia group (almost two-fold). L-ferritin mRNA expression increased slightly in the mild anemia group and H-ferritin mRNA reduced gradually with the aggravation of the maternal anemia, but neither showed statistical significance. No significant change in either protein or mRNA expression of FP1 was observed in the maternal anemia groups. CONCLUSION: The expression of TfR1, ferritin and FP1 in the human term placenta tissues showed different trend of change with different maternal iron status. It revealed that a human placenta has the function to protect a fetus through regulating the expression of iron transporters. The expression of FP1 in a human placenta may not be regulated by the iron responsive element/iron regulatory protein (IRE/IRP) system. In addition to FP1, other proteins may be involved in the placental iron efflux either directly or indirectly.     

Diverse calcium channel types are present in the human placental syncytiotrophoblast basal membrane

The functional expression of calcium channels has been scarcely studied in human placental syncytiotrophoblast. We have presently sought to characterize Ca(2+) currents of the healthy syncytiotrophoblast basal membrane using purified basal membranes reconstituted in giant liposomes subjected to patch-clamp recordings. We detected presence of channels with high permeability to Ca(2+) (relative PCa/PK up to 99.5) using K(+) solutions in symmetric conditions. Recordings performed in Ba(2+) gradients showed Ba(2+)-conducting channels in 100% of experiments. Ba(2+) total patch currents were consistently blocked by addition of NiCl(2), Nifedipine (L-type voltage-gated calcium channel blocker) or Ruthenium Red (TRPV5-TRPV6 channel blocker); Nifedipine and Ruthenium Red exerted a synergic blocking effect on Ba(2+) total patch currents. Immunohistochemistry of placental villi sections evidenced presence of alpha(1) subunit of voltage-gated calcium channels and TRPV5-TRPV6 channels in basal and apical syncytiotrophoblast plasma membranes; these three calcium channels were also detected in purified basal and apical fractions using Western blot. These results show the presence of three types of calcium channels in the syncytiotrophoblast basal membrane by both functional and molecular means. These basal membrane calcium channels would not be directly involved in mother-to-fetus Ca(2+) transport, but could participate in other relevant trophoblast processes, such as exocytosis and Ca(2+) transport regulation.     

Neutral Amino Acid Uptake by Microvillous and Basal Plasma Membrane Vesicles from Appropriate- and Small-for-Gestational Age Human Pregnancies

Fetal growth depends upon nutrient availabilty in the maternal circulation as well as appropriate transfer across the placenta via known transport mechanisms in the maternal- and fetal-facing plasma membranes of the syncytiotrophoblast. In this study, microvillous (maternal-facing) and basal (fetal-facing) plasma membrane vesicles were isolated from the placentas of appropriate-for-gestational age (AGA) and small-for-gestational age (SGA) human pregnancies and the uptakes of tritiated leucine, alanine, and glycine were determined. There was no significant difference in the uptakes of either alanine or leucine by the microvillous and basal vesicles from AGA and SGA pregnancies. In contrast, glycine uptake by both microvillous and basal membrane vesicles from SGA pregnancies was significantly less than vesicles from AGA pregnancies. Glycine is delivered to the umbilical circulation in amounts only minimally in excess of its accretion into protein resulting in a narrow margin of safety for fetal growth. The relative decrease in glycine uptake by SGA vesicles may reflect compromised in vivo availability with resultant depletion of marginal reserves and impaired fetal growth.     

Downregulation of Placental Syncytin Expression and Abnormal Protein Localization in Pre-eclampsia

Development of placentation and successful pregnancy depend on co-ordinated interactions between the maternal decidua and myometrium, and the invasive properties of the fetal trophoblast. Syncytin, a protein encoded by the envelope gene of a recently identified human endogenous defective retrovirus, HERV-W, is highly expressed in placental tissue. Previously, we have shown that the major site of syncytin expression is the placental syncytiotrophoblast, a fused multinuclear syncytium originating from cytotrophoblast cells. Here we present the first evidence that in pre-eclampsia, syncytin gene expression levels are dramatically reduced. Additionally, immunohistochemical examination of normal placentae and placentae from women with pre-eclampsia reveals that the syncytin protein in placental tissue from women with pre-eclampsia is localized improperly to the apical syncytiotrophoblast microvillous membrane as opposed to its normal location on the basal syncytiotrophoblast cytoplasmic membrane. Our previous results suggest that syncytin may mediate placental cytotrophoblast fusion in vivo and may play an important role in human placental morphogenesis. The present study suggests that altered expression of the syncytin gene, and altered cellular location of its protein product, may contribute to the aetiology of pre-eclampsia.     

Expression of the 11beta-hydroxysteroid dehydrogenase types 1 and 2 proteins in human and baboon placental syncytiotrophoblast.

We have shown that the placenta, via metabolism of maternal cortisol and cortisone by the 11beta-hydroxysteroid dehydrogenase (11beta-HSD) enzymes types 1 and 2 in the syncytiotrophoblast, regulates the maturation of the fetal pituitary adrenocortical axis in the baboon. Because the timing and regulation of fetal adrenal development by fetal ACTH in the human seem to parallel that in the baboon, we propose that the placental 11beta-HSD-1 and -2 system also has a role in regulating the development of the fetal pituitary adrenocortical axis during human pregnancy. However, although the human placenta has been shown to express the 11beta-HSD-2, it remains to be determined unequivocally whether 11beta-HSD-1 protein is present in the human placental syncytiotrophoblast. To answer this question, enriched fractions of syncytiotrophoblast were prepared from human and baboon term placentae and proteins probed with polyclonal antibodies directed to amino acids 22-36 or 66-77 of human 11beta-HSD-1. The 11beta-HSD-1 was detected by Western blot analysis as a 32-kDa protein in human and baboon syncytiotrophoblast and as a 34-kDa protein in adult baboon liver. Localization of the 11beta-HSD-1 to the syncytiotrophoblast was confirmed by immunocytochemistry following antigen retrieval. These results show that both human and baboon placental syncytiotrophoblast expressed the 11beta-HSD-1, as well as the 11beta-HSD-2, proteins. Because 11beta-HSD-1 can function as a reductase, the expression of 11beta-HSD-1 in human syncytiotrophoblast would be consistent with the ability of this tissue to convert cortisone to cortisol and provide a means by which transplacental transport of cortisol could regulate the fetal pituitary adrenocortical axis in the human, as recently shown experimentally in the non-human primate baboon model.     

Presence of CLA-1 and HDL binding sites on syncytiotrophoblast brush border and basal plasma membranes of human placenta.

It is now known that rapid placental and fetal development is associated with elevated levels of circulating high density lipoprotein (HDL) in pregnant women. The main structure implicated in the maternal-fetal exchange is the syncytiotrophoblast, composed of a brush border membrane (BBM), facing the mother, and a basal plasma membrane (BPM), facing the fetus. In order to understand the mechanisms controlling the placental and fetal supplies of cholesterol, we purified both BBM and BPM and verified the presence of HDL binding sites in these membranes. Binding studies using(125)I-HDL(3)show a single affinity binding site on BPM which has a dissociation constant (K(d)) of 3.45+/-0.43 microg protein/ml and a maximal binding capacity (B(max)) of 5.46+/-1.69 microg protein/mg membrane proteins. In BBM, we observed two affinity binding sites, one with a K(d)of 0.62+/-0.03 microg protein/ml and another one with a K(d)of 6.57+/-0.87 microg protein/ml. Their B(max)values were 0.54+/-0.11 and 2.34+/-0.39 microg of HDL(3)/mg membrane proteins, respectively. CLA-1, a putative HDL-receptor of 85 kDa, was detected on both BPM and BBM, together with two apo A-l binding sites of 110 and 96 kDa on BPM and BBM, respectively. These results provide further evidence that human placenta possesses specific sites for HDL binding, underlining the important role of maternal HDL in the transfer of cholesterol from the maternal circulation to the placenta and the fetus.