Isolation of highly enriched apical plasma membranes of the placental syncytiotrophoblast

The human placenta is a complex organ whose proper function is crucial for the development of the fetus. The placenta contains within its structure elements of the maternal and fetal circulatory systems. The interface with maternal blood is the lining of the placenta, that is a unique compartment known as the syncytiotrophoblast. This large syncytial structure is a single cell layer in thickness, and the apical plasma membrane of the syncytiotrophoblast interacts directly with maternal blood. Relatively little is known about the proteins that reside in this unique plasma membrane or how they may change in various placental diseases. Our goal was to develop methods for isolating highly enriched preparations of this apical plasma membrane compatible with high-quality proteomics analysis and herein describe the properties of these isolated membranes.     

Placenta-derived soluble MHC class I chain-related molecules down-regulate NKG2D receptor on peripheral blood mononuclear cells during human pregnancy: a possible novel immune escape mechanism for fetal survival

Mammalian pregnancy is an intriguing immunological phenomenon where the semiallogeneic fetus is not rejected. Tolerance toward the fetus involves a number of mechanisms associated with modifications of the immune status of the mother. In this study, we strongly suggest a novel mechanism for fetal evasion of maternal immune attack, based on the engagement and down-regulation of the activating NK cell receptor NKG2D on PBMC by soluble MHC class I chain-related proteins A and B (collectively termed MIC). A similar immune escape pathway was previously described in tumors. We found that MIC mRNA was constitutively expressed by human placenta and could be up-regulated upon heat shock treatment. Our immunomorphologic studies showed that the MIC expression in placenta was restricted to the syncytiotrophoblast. Immunoelectron microscopy revealed a dual MIC expression in the syncytiotrophoblast: on the apical and basal cell membrane and in cytoplasmic vacuoles as MIC-loaded microvesicles/exosomes. Soluble MIC molecules were present at elevated levels in maternal blood throughout normal pregnancy and were released by placental explants in vitro. Simultaneously, the cell surface NKG2D expression on maternal PBMC was down-regulated compared with nonpregnant controls. The soluble MIC molecules in pregnancy serum were able to interact with NKG2D and down-regulate the receptor on PBMC from healthy donors, with the consequent inhibition of the NKG2D-dependent cytotoxic response. These findings suggest a new physiological mechanism of silencing the maternal immune system that promotes fetal allograft immune escape and supports the view of the placenta as an immunoregulatory organ.     

Endocytic and transcytotic processes in villous syncytiotrophoblast: role in nutrient transport to the human fetus

The supply of nutrients to the developing fetus is a major function of the human hemochorial placenta, a placenta type in which the fetal chorion is in direct contact with the maternal blood. At term, nutrients have to be transported across two cell layers in chorionic villi, the syncytiotrophoblast (STB) and fetal endothelial cells. The STB is a continuous syncytium covering the entire surface of chorionic villi. This polarized epithelium is specialized in exchange processes and membrane trafficking between the apical membrane facing the maternal blood and the basal membrane facing the fetal endothelium. To meet placental and fetal requirements, the STB selectively takes up and transports a variety of nutrients, hormones, growth factors and cytokines and also transfers passive immunity to the fetus by receptor-mediated transcytosis. In this review in vivo and in vitro systems currently used to study STB functions are discussed and the potential mechanisms of transplacental IgG, iron, lipoprotein and glucose transport are presented. As revealed in this article, the placenta is a tissue where intensive cell biological research is required to unravel endocytic trafficking pathways in a highly specialized cell such as the STB.     

Ultrastructural development of chorioallantoic placenta in the Indian Miniopterus bat, Miniopterus schreibersii fuliginosus (Hodgson).

The chorioallantoic placental interhemal membrane of Miniopterus schreibersii fuliginosus has been described electron-microscopically. Morphologically there are three main types of placentae which develop in chronological sequence. They are (1) primary placenta, (2) secondary placenta and (3) tertiary placenta. In neural groove and limb-bud embryos the primary placenta consists of the following elements which separate the maternal and fetal circulations: (1) a continuous ectoplasmic layer, (2) intrasyncytial lamina, (3) syncytiotrophoblast, (4) cytotrophoblast, (5) basal lamina, (6) mesenchyme and (7) fetal endothelium. The primary placenta degenerates until term when it consists of a thin syncytiotrophoblastic layer resting on basal lamina. Mesenchyme does not show the presence of fetal capillaries. The secondary placenta is formed in early limb-bud embryos. The electron microscope has revealed that the placenta is of the endotheliomonochorial type and (1) consists of a well-developed maternal endothelium, (2) the trophoblast surrounding the maternal blood tubule is cellular, not syncytial as previously thought and the apical plasma membrane of these trophoblastic cells is in direct contact with the discontinuous interstitial membrane, (3) basal lamina, (4) mesenchyme and (5) fetal endothelium. Tertiary placenta at full term stage is of the hemodichorial type having the following elements: (1) thin ectoplasmic layer, (2) a thick intrasyncytial lamina, (3) syncytiotrophoblast, (4) cytotrophoblast, (5) basal lamina, (6) mesenchyme and (7) fetal endothelium. The definitive chorioallantoic placental barrier in this bat thus differs from the organization earlier proposed by Chari and Gopalakrishna [Proc. Indian Acad. Sci. 93: 463-483, 1984] on the basis of light-microscopic observations: (1) the absence of maternal endothelium in the primary placenta from the neural groove and early limb-bud embryos, (2) the existence of only cellular trophoblast in the secondary placenta throughout the gestation and (3) the presence of well-developed hemodichorial tertiary placenta is the unique feature of the interhemal membrane in higher Chiroptera.     

Localization of erythrocyte/HepG2-type glucose transporter (GLUT1) in human placental villi

Summary The syncytiotrophoblast covering the surface of the placental villi contains the machinery for the transfer of specific substances between maternal and fetal blood, and also serves as a barrier. Existence of a facilitated-diffusion transporter for glucose in the syncytiotrophoblast has been suggested. Using antibodies to erythrocyte/HepG2-type glucose transporter (GLUT1), one isoform of the facilitated-diffusion glucose transporters, we detected a 50 kD protein in human placenta at term. By use of immunohistochemistry, GLUT1 was found to be abundant in both the syncytiotrophoblast and cytotrophoblast. Endothelial cells of the fetal capillaries also showed positive staining for GLUT1. Electron-microscopic examination revealed that GLUT1 was concentrated at both the microvillous apical plasma membrane and the infolded basal plasma membrane of the syncytiotrophoblast. Plasma membrane of the cytotrophoblast was also positive for GLUT1. GLUT1 at the apical plasma membrane of the syncytiotrophoblast may function for the entry of glucose into its cytoplasm, while GLUT1 at the basal plasma membrane may be essential for the exit of glucose from the cytoplasm into the stroma of the placental villi. Thus, GLUT1 at the plasma membranes of syncytiotrophoblast and endothelial cells may play an important role in the transport of glucose across the placental barrier.     

Early patterning of the chorion leads to the trilaminar trophoblast cell structure in the placental labyrinth

The labyrinth of the rodent placenta contains villi that are the site of nutrient exchange between mother and fetus. They are covered by three trophoblast cell types that separate the maternal blood sinusoids from fetal capillaries--a single mononuclear cell that is a subtype of trophoblast giant cell (sinusoidal or S-TGC) with endocrine function and two multinucleated syncytiotrophoblast layers, each resulting from cell-cell fusion, that function in nutrient transport. The developmental origins of these cell types have not previously been elucidated. We report here the discovery of cell-layer-restricted genes in the mid-gestation labyrinth (E12.5-14.5) including Ctsq in S-TGCs (also Hand1-positive), Syna in syncytiotrophoblast layer I (SynT-I), and Gcm1, Cebpa and Synb in syncytiotrophoblast layer II (SynT-II). These genes were also expressed in distinct layers in the chorion as early as E8.5, prior to villous formation. Specifically, Hand1 was expressed in apical cells lining maternal blood spaces (Ctsq is not expressed until E12.5), Syna in a layer immediately below, and Gcm1, Cebpa and Synb in basal cells in contact with the allantois. Cebpa and Synb were co-expressed with Gcm1 and were reduced in Gcm1 mutants. By contrast, Hand1 and Syna expression was unaltered in Gcm1 mutants, suggesting that Gcm1-positive cells are not required for the induction of the other chorion layers. These data indicate that the three differentiated trophoblast cell types in the labyrinth arise from distinct and autonomous precursors in the chorion that are patterned before morphogenesis begins.     

Role of MDR1 and MRP1 in trophoblast cells,elucidated using retroviral gene transfer

Natural differences in expression and retroviral transduction techniques were used to test the hypothesis that MDR1 P-glycoprotein (P-gp) and MRP1 (multidrug resistance-related protein) contribute to xenobiotic handling by placental trophoblast. RT-PCR and Western blotting in placenta, primary cytotrophoblast cell cultures, and BeWo, JAr, and JEG choriocarcinoma cell lines showed that MRP1 was ubiquitously expressed, whereas MDR1 was absent or minimally expressed in BeWo and JEG cell lines. In syncytiotrophoblast, P-gp was localized predominantly to the microvillous, maternal facing plasma membrane, and MRP1 to the basal, fetal facing plasma membrane. Functional studies showed that cyclosporin A-sensitive accumulation of [³H]vinblastine by cells containing both transport proteins was significantly different from those expressing predominantly MRP1. Retroviral gene transfer of MDR1 to BeWo cells confirmed that this difference was due to the relative expression of MDR1. Therefore, both P-gp and MRP1 contribute to xenobiotic handling by the trophoblast. Localization of P-gp to the microvillous membrane suggests an essential role in preventing xenobiotic accumulation by the syncytiotrophoblast and, therefore, in protecting the fetus. placenta; multidrug resistance; xenobiotic     

Immunolocalization of glucose transporter GLUT1 in the rat placental barrier: possible role of GLUT1 and the gap junction in the transport of glucose across the placental barrier

GLUT1 is an isoform of facilitated-diffusion glucose transporters and has been shown to be abundant in cells of blood-tissue barriers. Using antibodies against GLUT1, we investigated the immunohistochemical localization of GLUT1 in the rat placenta. Rat placenta is of the hemotrichorial type. Three cell layers (from the maternal blood side inward) cytotrophoblast and syncytiotrophoblasts I and II, lie between the maternal and fetal bloodstreams. GLUT1 was abundant along the invaginating plasma membrane facing the cytotrophoblast and the syncytiotrophoblast I. Also, the infolded basal plasma membrane of the syncytiotrophoblast II was rich in GLUT1. Apposing plasma membranes of syncytiotrophoblasts I and II, however, had only a small amount of GLUT1. Numerous gap junctions were seen between syncytiotrophoblasts I and II. Taking into account the localization of GLUT1 and the gap junctions, we suggest a possible major transport route of glucose across the placental barrier, as follows: glucose in the maternal blood passes freely through pores of the cytotrophoblast. Glucose is then transported into the cytoplasm of the syncytiotrophoblast I via GLUT1. Glucose enters the syncytiotrophoblast II throught the gap junctions. Finally glucose leaves the syncytiotrophoblast II via GLUT1 and enters the fetal blood through pores of the endothelial cells.     

Drugs of abuse and human placenta

Drugs of abuse such as cocaine and amphetamines, when used by pregnant women, exert deleterious effects on the fetus. These drugs produce their effects through inhibition of the serotonin transporter, norepinephrine transporter, and dopamine transporter. The inhibition can occur in the pregnant mother as well as in the fetus. These events contribute to the detrimental effects of these drugs on the fetus. However, the role of placenta, which serves as the link between the pregnant mother and the fetus, in the process remains understudied. It has been assumed that the placenta did not play any direct role in the process except that it allowed the passage of these drugs from maternal circulation into fetal circulation. This was before the discovery that the placenta expresses two of the three monoamine transporters. The serotonin transporter and the norepinephrine transporter are expressed on the maternal-facing side of the syncytiotrophoblast, thus exposed to the inhibitory actions of cocaine and amphetamines if present in maternal blood. Inhibition of these transporters in the placenta could lead to elevation of serotonin and norepinephrine in the intervillous space that may cause uterine contraction and vasoconstriction, resulting in premature delivery, decreased placental blood flow, and intrauterine growth retardation. Thus, the placenta is actually a direct target for these abusable drugs. Since the placental serotonin transporter and norepinephrine transporter are also inhibited by many antidepressants, therapeutic use of these drugs in pregnant women may have similar detrimental effects on placental function and fetal growth and development.     

Do glucose transporters have other roles in addition to placental glucose transport during early pregnancy?

Human placenta regulates the transport of maternal molecules to the fetus. It is known that glucose transport occurs via glucose transporters (GLUTs) in the feto–placental unit. Data on the expression of GLUTs during implantation are very scarce. Moreover, the question of how the decidual leukocytes obtain the energy for their activation during implantation mechanism is still under investigation. We studied the distributions of GLUT1, GLUT3, and GLUT4 in tissue sections of first trimester pregnancies the human maternal–fetal interface. GLUT1 was present in apical microvilli of the syncytiotrophoblast, in cytotrophoblast, and in vascular patterns of the villous core, whereas GLUT3 was localized in cytotrophoblasts of placental villi and in some fetal endothelial cells. Moreover, the proliferating cells of the proximal cell columns were also immunopositive for GLUT1 and GLUT3. We did not observe any positive immunoreactivity for GLUT4 in placental and decidual tissues. Essentially, GLUT3 and also to some extent GLUT1 was present in maternal leukocytes and platelets. In conclusion, our results suggest that the glucose taken up via GLUT1 and GLUT3 from the maternal circulation might not only be needed for placental functions but also for successful implantation by trophoblast invasion, proliferation and also by having a role to support energy for maternal leukocytes.     

Pathogens and the placental fortress

Placental infections are major causes of maternal and fetal disease. This review introduces a new paradigm for placental infections based on current knowledge of placental defenses and how this barrier can be breached. Transmission of pathogens from mother to fetus can occur at two sites of direct contact between maternal cells and specialized fetal cells (trophoblasts) in the human placenta: firstly, maternal immune and endothelial cells juxtaposed to extravillous trophoblasts in the uterine implantation site and secondly, maternal blood surrounding the syncytiotrophoblast (SYN). Recent findings suggest that the primary vulnerability is in the implantation site. We explore evidence that the placental SYN evolved as a defense against pathogens, and that inflammation-mediated spontaneous abortion may benefit mother and pathogen.     

A theoretical analysis of the effect of placental metabolism on fetal oxygenation under conditions of limited oxygen availability.

The purpose of this theoretical paper is to examine the effects of placental metabolism on fetal oxygenation under conditions of limited oxygen availability. Features of the mathematical model used here include: (1) ordinary non-linear differential equations defining the oxygen partial pressure profiles in the maternal and fetal streams for a concurrent flow pattern; (2) the presence of maternal and fetal blood flow shunts; (3) consumption of oxygen by a metabolically active placenta; and (4) modification of the fetal input to the placenta by changing the rate of fetal oxygen consumption in response to changes in the rate of oxygen delivered to the fetus via the umbilical vein. Model parameters were chosen to be well within the range of values cited in the literature. Based on these calculations, we conclude that: (1) under normal conditions, approximately one-half of the fetal uterine-umbilical venous oxygen partial pressure difference can be attributed to placental oxygen consumption; (2) utilization of fetal oxygen to help maintain the metabolic activities of the placenta does not significantly impair fetal oxygenation under normal conditions; (3) consumption of oxygen by the placenta will have a significant detrimental effect on the rate of oxygen delivered to the fetus if oxygen availability is compromised; and (4) for the same rate of maternal oxygen delivered to the placenta, maternal hypoxemia has a significantly greater adverse effect on fetal oxygenation than does maternal anemia.     

Metabolism of glucose by fetus and placenta of sheep. The effects of normal fluctuations in uterine blood flow

The metabolism by the fetus and placenta of [2-3H, U-14C]glucose infused into fetal sheep has been studied. Uptake of glucose from the fetus by the placenta and transfer to the ewe, as well as placental metabolism of glucose to fructose and lactate have been quantified. About two-thirds of the glucose removed from the fetal circulation was taken up by placenta. Less than 15% of this passed back into the maternal circulation, the remainder was converted, at roughly equivalent rates, into lactate and fructose, most of which was transferred back to the fetus. It seems likely that little of this glucose is oxidised by the placenta. This data indicates that there are substrate cycles between the placenta and fetus, one possible function of which is to limit fetal glucose loss back to the mother; lactate and fructose have limited placental permeability. At uterine blood flow rates in the middle of the normal range net glucose uptake by the placenta from the maternal circulation was about 7-fold higher than that from the fetus. About 20% of this was transported to the fetus, 50% was oxidised and much of the remainder converted to lactate and transferred back to the ewe. Labelling patterns in fructose and lactate make it unlikely that this placental pool of glucose mixes freely with that derived from uptake from the fetus. Net movement of glucose across the placenta is markedly influenced by fluctuations in uterine blood flow over the normal range of 500-3000 ml/min. At low flow rates there is net output of glucose from the fetus to the placenta, and in some instances from the placenta to the ewe, i.e. there is evidence of net utero-placental production of glucose to the ewe separate from output by the fetus. There is a close linear relationship between uterine glucose supply (maternal arterial concentration x uterine blood flow) and net balance across the placenta. As uterine supply of glucose falls there is increased uptake by the placenta of glucose from the fetal circulation and corresponding enhanced recycling of fructose and lactate to the fetus. This production of fructose and lactate by the placenta may function to reduce glucose loss from the fetus to the ewe. Hence at high rates of placental uptake of glucose from the fetus placental production of lactate and particularly fructose may approach saturation and allow significant backflow of glucose from the fetus to the ewe. Under these conditions glucose uptake may in part sustain placental oxygen consumption.     

Epigenetic regulation of the placental HSD11B2 barrier and its role as a critical regulator of fetal development

“Fetal programming” is a term used to describe how early-life experience influences fetal development and later disease risk. In humans, prenatal stress-induced fetal programming is associated with increased risk of preterm birth, and a heightened risk of metabolic and neurological diseases later in life. A critical determinant of this is the regulation of fetal exposure to glucocorticoids by the placenta. Glucocorticoids are the mediators through which maternal stress influences fetal development. Excessive fetal glucocorticoid exposure during pregnancy results in low birth weight and abnormalities in a number of tissues. The amount of fetal exposure to maternal glucocorticoids depends on the expression of HSD11B2, an enzyme predominantly produced by the syncytiotrophoblast in the placenta. This protects the fetus by converting active glucocorticoids into inactive forms. In this review we examine recent findings regarding placental HSD11B2 that suggest that its epigenetic regulation may mechanistically link maternal stress and long-term health consequences in affected offspring.     

Time-dependent physiological regulation of ovine placental GLUT-3 glucose transporter protein

We immunolocalized the GLUT-3 glucose transporter isoform versus GLUT-1 in the late-gestation epitheliochorial ovine placenta, and we examined the effect of chronic maternal hyperglycemia and hypoglycemia on placental GLUT-3 concentrations. GLUT-3 was limited to the apical surface of the trophoectoderm, whereas GLUT-1 was on the basolateral and apical surfaces of this cell layer and in the epithelial cells lining the placental uterine glands. GLUT-3 concentrations declined at 17-20 days of chronic hyperglycemia (P < 0.05), associated with increased uterine and uteroplacental net glucose uptake rate, but a normal fetal glucose uptake rate was observed. Chronic hypoglycemia did not change GLUT-3 concentrations, although uterine, uteroplacental, and fetal net glucose uptake rates were decreased. Thus maternal hyperglycemia causes a time-dependent decline in the entire placental glucose transporter pool (GLUT-1 and GLUT-3). In contrast, maternal hypoglycemia decreases GLUT-1 but not GLUT-3, resulting in a relatively increased GLUT-3 contribution to the placental glucose transporter pool, which could maintain glucose delivery to the placenta relative to the fetus when maternal glucose is low.     

Annexin 6 modulates the maxi-chloride channel of the apical membrane of syncytiotrophoblast isolated from human placenta

The syncytiotrophoblast separates the maternal and fetal blood and constitutes the primary barrier for maternal-fetal transport. The Maxi-chloride channel from the apical membrane of the syncytiotrophoblast plays a role in the chloride conductance. Annexins can play an important role in the regulation of membrane events. In this study we evaluate the role of annexin 6 in the Maxichloride channel properties. The results showed that annexin 6 is bound in the apical placenta membranes in a calcium-dependent phospholipid-binding manner but also in a calcium-independent fashion. The neutralization of annexin 6 decreased the total current by 39 +/- 1.9% in the range of +/-80 mV, and the currents decrease with the time. The single-channel slope conductance was decreased from 253 +/- 7.4 pS (control) to 105 +/- 13 pS, and the amplitude decreased by 50%. The open probability was also affected when higher voltage steps were used, changes in either the positive or negative direction induced the channel to close, and the open probability (P(o)) did not decrease. In channels with neutralized annexin 6, it was maintained at 1 at +/-40 mV and at +/-80 mV. These results suggest that endogenous annexin 6 could regulate the Maxi-chloride channel. The results obtained with normal placentae, in which annexin 6 was neutralized, are similar to those described for the Maxi-chloride channel isolated from pre-eclamptic placenta. Together these data suggest that annexin 6 could play an important role in ion transport of the placenta.     

Effects of maturation on RNA transcription and protein expression of four MRP genes in human placenta and in BeWo cells

The placenta is a multifunctional organ that protects the fetus from toxic compounds and the MRPs contribute to this function. The expression of MRP1, MRP2, MRP3, and MRP5 was compared in human placental tissue and in BeWo cells by real-time RT-PCR analysis; protein expression was assessed by Western blot. MRP1 and MRP3 were the most abundantly expressed genes in placenta but only MRP1 was highly expressed in the BeWo cells. Expression of MRP1 increased 4-fold in the third as compared with first trimester placental samples, and increased 20-fold with polarization of BeWo cells. MRP2, MRP3, and MRP5 were weakly expressed both in placenta and BeWo cells. Protein expression followed mRNA quantification for MRP1 and MRP5 but not for MRP2 and MRP3. These data indicated that MRP1 and MRP5 increase with trophoblast maturation, suggesting a particular role for these proteins in the organ functional development.     

Evolutionary implications of fetal and maternal microvillous surfaces in epitheliochorial placentae

According to the “parent-offspring conflict hypothesis” the rapid evolution and diversification of the mammalian placenta is driven by divergent optima of resource allocation between fetus and mother. The fetus has an interest to maximize its resource intake, while the mother has an interest to restrict the transfer of resources, and thus retain resources for subsequent pregnancies. In the epitheliochorial placenta, the contacting fetal and maternal surfaces at the feto-maternal interface are covered with microvilli, which leads to an increase of membrane surfaces available for transport processes. Because membranes are the site of active transport, the conflict hypothesis predicts that the fetal surfaces at the feto-maternal interfaces are larger than the maternal ones. We use transmission electron microscopy and a stereological method to estimate the factors by which the apical fetal and maternal membranes are enlarged by the microvilli. Ten species with an epitheliochorial placenta were studied. Focused ion beam—scanning electron microscopy (FIB-SEM) was used to create three-dimensional models of the interdigitating microvilli of the bovine and porcine placenta. In all species, the fetal surface was larger than the maternal. This was due to a higher number of fetal microvilli and to the presence of membrane folds at the base of the fetal, but not of maternal microvilli. Our results suggest that the ultrastructural morphology of the feto-maternal interface in the epitheliochorial placenta is shaped by conflicting interests between fetus and mother and thus represent a so far neglected arena of the parent-offspring conflict.     

Epigenetic regulation of the placental HSD11B2 barrier and its role as a critical regulator of fetal development

“Fetal programming” is a term used to describe how early-life experience influences fetal development and later disease risk. In humans, prenatal stress-induced fetal programming is associated with increased risk of preterm birth, and a heightened risk of metabolic and neurological diseases later in life. A critical determinant of this is the regulation of fetal exposure to glucocorticoids by the placenta. Glucocorticoids are the mediators through which maternal stress influences fetal development. Excessive fetal glucocorticoid exposure during pregnancy results in low birth weight and abnormalities in a number of tissues. The amount of fetal exposure to maternal glucocorticoids depends on the expression of HSD11B2, an enzyme predominantly produced by the syncytiotrophoblast in the placenta. This protects the fetus by converting active glucocorticoids into inactive forms. In this review we examine recent findings regarding placental HSD11B2 that suggest that its epigenetic regulation may mechanistically link maternal stress and long-term health consequences in affected offspring.