Human Placental Syncytiotrophoblast Research Articles

Serine Racemase and D-Serine Transport in Human Placenta and Evidence for a Transplacental Gradient for D-Serine in Humans

To investigate the possible role of human placenta in providing D-serine to the developing fetus. Expression of serine racemase in placenta was determined by reverse transcriptase polymerase chain reaction and northern analysis and confirmed by subsequent cloning. The transport of D-serine by human ATB(0) was characterized by expressing the cloned cDNA transiently in mammalian cells using the vaccinia virus expression system. D-serine levels in maternal and fetal blood were measured by fluorescence high-performance liquid chromatography (HPLC) after derivatization of the amino acids with o-phthaldialdehyde and N-tertiary-butyloxycarbonyl-L-cysteine. mRNA for serine racemase was detected in placenta. ATB(0) was capable of d-serine transport, and the transport process is obligatorily dependent on sodium (Na+) with a Na(+):substrate stoichiometry of 1:1 and saturable with a Michaelis-Menten constant of 310 +/- 30 microM. Furthermore, studies have shown that ATB(0) is not expressed in the maternal-facing brush border membrane of human placental syncytiotrophoblast. The circulating concentration of D-serine in maternal serum is 5.8 +/- 0.5 microM, and the corresponding value in the fetal serum is 14.6 +/- 1.2 microM, indicating a two- to three-fold higher concentration of D-serine in the fetus than in the mother. We speculate that D-serine is synthesized in human placenta by the racemization of L-serine and that ATB(0), expressed on the basal membrane of the syncytiotrophoblast, mediates the efflux of D-serine into the fetal circulation in exchange for other amino acids in fetal blood.

Overexpression of the human neonatal Fc-receptor α-chain in trophoblast-derived BeWo cells increases cellular retention of β2-microglobulin

Major histocompatibility complex (MHC) class I and MHC class I-type molecules such as the neonatal Fcγ-receptor, FcRn, are heterodimers consisting of a transmembrane α-chain non-covalently associated with β2-microglobulin (β2m). Human placental villous syncytiotrophoblast (STB) lacks MHC class I molecules, but express hFcRn that mediates materno-fetal transmission of immunoglobulin G (IgG). Trophoblast-derived BeWo cells that are used to study placental IgG transport likewise express β2m and low levels of hFcRn α-chain. The contribution of FcRn α-chain in retention and subcellular distribution of β2m in STB and BeWo cells is unclear. To investigate this issue, we increased expression of hFcRn α-chain in BeWo cells (BeWo/hFcRn) by cDNA transfection. Overexpressed hFcRn protein exhibited the characteristic pH-dependent IgG binding and association with β2m. In comparison to parental BeWo cells, β2m mRNA levels in BeWo/hFcRn cells were not significantly altered, but total cell-associated β2m protein was increased by 120%. Treatment of BeWo and BeWo/hFcRn cells with brefeldin A, an inhibitor of the secretory pathway, abrogated this effect, demonstrating that hFcRn α-chain expression retained otherwise secreted β2m. Flow cytometry revealed that β2m plasma membrane expression was unaffected by α-chain overexpression whereas by fluorescence microscopy a preferential staining of β2m in peripheral endosomes was observed.

Estrogen receptor-mediated down-regulation of corticotropin-releasing hormone gene expression is dependent on a cyclic adenosine 3',5'-monophosphate regulatory element in human placental syncytiotrophoblast cells.

Placental CRH plays a major role in the mechanisms controlling human pregnancy and parturition. Understanding how placental CRH production is regulated is therefore of importance. Previously we have shown that placental expression of CRH peptide and mRNA are inhibited by estrogens, in contrast to the stimulatory effects of estrogen on hypothalamic CRH production. Our current study found that in placental cells cotransfected with a CRH promoter construct and an estrogen receptor-alpha expression vector results in a differential regulation whereby 17beta-estradiol (E2) decreased and the putative pure estrogen antagonist, ICI 182780, increased CRH promoter activity. Sequential deletion of the CRH promoter indicated that the region between -248 and -213 bp was essential for the effect of both E2 and ICI 182780. This region contains a consensus cAMP regulatory element (CRE) that is a requirement for E2- and ICI 182780-mediated activity because the CRE motif can confer E2 inhibition on a heterologous promoter such as rabbit beta-globin. Mutation of the CRE resulted in a complete reversal of E2 and ICI 182780 regulatory effects. In summary, our results demonstrate that a consensus CRE is required for the action of estrogen receptor ligands in human placental syncytiotrophoblast cells.

Non-Gastric H +/K +ATPase is Present in the Microvillous Membrane of the Human Placental Syncytiotrophoblast

In humans, the non-gastric H +/K +ATPase (ATP1AL1) has previously been shown to be expressed in the epithelia of skin, kidney and colon. In this study we tested the hypothesis that the non-gastric H +/K +ATPase is localized to the syncytiotrophoblast, the transporting epithelium of the human placenta. Microvillous (MVM) and basal plasma membranes (BM) of the syncytiotrophoblast were isolated from term placenta and membrane proteins were separated using SDS–PAGE. The ATP1AL1 protein was identified as a 114 kD band in both MVM and BM by Western blot, however, the protein was more abundant in the MVM. Using immunocytochemistry H +/K +ATPase protein was localized in MVM but not BM. We constructed primers specific for ATP1AL1 and performed RT–PCR on RNA isolated from human placenta and human kidney. A product of the expected size could be detected in both tissues after 30 cycles of amplification. The sequence identity of this 517 nucleotide product was confirmed by sequencing and found to be identical to the human non-gastric H +/K +ATPase. The activity of this proton pump appears to be low in normal healthy placental at term, however, it is speculated that MVM non-gastric H +/K +ATPase may be important in pathological states. In conclusion, non-gastric H +/K +ATPase is present in the microvillous plasma membrane of the transporting epithelia of the human placenta.

Isolation and Purification of Human Placental Plasma Membranes from Normal and Pre-eclamptic Pregnancies. A Comparative Study

Human placental syncytiotrophoblast is the main barrier for materno-fetal exchange. Analysis of transplacental transport involves the study of ion channels in both the maternal-facing microvillous membrane (MVM) and the fetal-facing basal membrane (BM). Difficulties in having access to intact placenta with conventional electrophysiological methods favour alternative methodologies, such as isolation and reconstitution of membranes in artificial lipid systems. Pre-eclampsia is a major health problem of human pregnancy. The search for altered physiological processes in pre-eclamptic placentae requires the investigation of events at both the microvillous and basal surfaces. The aim of this study was to obtain reliable syncytiotrophoblast plasma membranes from human normal (N) and pre-eclamptic (PE) pregnancies. We describe a protocol which allows for the simultaneous isolation of MVM and BM. The purity of the membranes isolated was evaluated using enzymatic assays, binding studies, Western blotting and immunohistochemistry. Enrichment of alkaline phosphatase activity for MVM was 17 to 21-fold, with 13–16 per cent protein recovery, for both N and PE. Enrichment of adenylate cyclase activity for BM was 9-fold for N, and enrichment of dihydroalprenolol binding to β-adrenergic receptors was 12-fold for N and 6-fold for PE, with 14 per cent protein recovery for both N and PE. Cross contamination was low and mitochondrial membrane contamination was negligible. We conclude that MVM and BM isolated from placentae of pre-eclamptic women are similar in enrichment and purity to those of healthy women, thus allowing their use in comparative electrophysiological studies.

USF1 and USF2 mediate inhibition of human trophoblast differentiation and CYP19 gene expression by Mash-2 and hypoxia.

In the human placental syncytiotrophoblast, C(19) steroids are converted to estrogens by aromatase P450, product of the CYP19 gene. When human cytotrophoblasts, which lack the capacity to express aromatase, are cultured in 20% O(2), they spontaneously fuse to form a multinuclear syncytiotrophoblast and CYP19 expression is markedly induced. On the other hand, when cytotrophoblasts are cultured in 2% O(2), syncytiotrophoblast differentiation and induction of CYP19 expression are prevented. We previously observed that expression of the transcription factor Mash-2 (mammalian achaete/scute homologue 2), which is elevated in human cytotrophoblasts and maintained at elevated levels by hypoxia, declines with syncytiotrophoblast differentiation. Overexpression of Mash-2 prevents syncytiotrophoblast differentiation and induction of CYP19 expression. In the present study, we observed that unexpectedly immunoreactive Mash-2 protein was localized predominantly to the cytoplasm of human cytotrophoblasts. Elevated cytoplasmic levels of Mash-2 were maintained when trophoblasts were cultured in 2% O(2) and declined to undetectable levels upon culture in 20% O(2). Previously, we found that Mash-2 inhibited CYP19 promoter activity through sequences within a 350-bp region upstream and within placenta-specific exon I.1 containing three E boxes (E1 at -325 bp, 5'-CACTTG-3'; E2 at -58 bp, 5'-CACATG-3'; and E3 at +26 bp, 5'-CACGTG-3'). In this study, we found that trophoblast nuclear protein binding to these E boxes declined with syncytiotrophoblast differentiation in 20% O(2) and was induced by hypoxia; however, Mash-2 did not appear to bind to any of these E boxes. On the other hand, the basic helix-loop-helix leucine zipper transcription factors upstream stimulatory factors 1 and 2 (USF1 and USF2) did bind to E2 and E3 but not E1. Nuclear levels of USF1 and USF2 and DNA-binding activity declined with syncytiotrophoblast differentiation and were maintained at elevated levels by hypoxia and overexpression of Mash-2, whereas USF1 mRNA levels were unaffected. Finally, USF1 overexpression in cultured human trophoblasts markedly inhibited endogenous CYP19 expression, differentiation of cultured human trophoblast cells, and CYP19 promoter activity. These findings suggest that increased protein levels and DNA binding of USF1 and USF2 mediate the inhibitory effects of hypoxia and of Mash-2 on CYP19 gene expression in human placenta.

Effects of low concentrations of organochlorine compounds in women on calcium transfer in human placental syncytiotrophoblast.

For most Canadians, food represents one of the major sources of environmental contaminants. Among them, organochlorine compounds (OCs) are known to affect calcium (Ca2+) homeostasis. They are neurotoxic by perturbation of Ca2+ channels and pumps, and they interfere with protein kinase C (PKC) and Ca2+ binding protein (CaBP). Ca2+ is an essential element to adequate fetal growth and development. The aim of the present study is to determine the relation between low environmental maternal exposure to OCs, such as polychlorinated biphenyls (PCB 153), Aroclor 1260, p,p'-dichlorodiphenyltrichloroethane (DDT) and p,p'-dichlorodiphenyl-dichloroethane (DDE), Ca2+ levels in serum and placenta, placental Ca2+ transfer, and newborn development. Total Ca2+ and OCs were measured in women's serum samples, as well as in umbilical cord's serum and placenta at term. Placentas were taken for trophoblast cells isolation and Ca2+ incorporation kinetic experiments. Our results were obtained from 30 pregnant women from the southwestern area of Quebec. Concentrations of Aroclor 1260, PCB 153, DDE, and DDT were respectively 6.1, 6.0, 3.1, and 2.9 times lower in the umbilical cord serum than in the mother's serum at term. In the placenta, DDE was accumulated at higher levels than other contaminants. A tendency towards an inverse relation was observed for in OCs found in three compartments and Ca2+ levels in maternal serum and in placental tissues. Maternal Ca2+ concentrations do not influence Ca2+ uptake by syncytiotrophoblast. Only DDE (>/=0.70 mug/l) in maternal serum significantly was associated with a small increase in Ca2+ uptake by syncytiotrophoblast. This study will help us determine if low OC contamination significantly modifies Ca2+ transfer in syncytiotrophoblast.

17beta-estradiol and tamoxifen regulate a maxi-chloride channel from human placenta.

Steroid hormones have been implicated in the modulation of several transport processes, including conductive chloride transport in epithelial cells. Micromolar concentrations of these hormones have been determined in blood of pregnant women. The purpose of this work was to explore the effects of 17beta-Estradiol, a steroid hormone, on the biophysical properties of the Maxi chloride channel present in apical membranes from human placental syncytiotrophoblast. Apical membrane chloride channels from human term placentas were reconstituted in giant liposomes suitable for electrophysiologic studies by the patch-clamp method. Low micromolar concentrations of 17beta-Estradiol inhibit the Maxi chloride channels in excised patches in a potential-dependent manner. The addition of 1 mM 17beta-Estradiol to the bath solution decreased the total current in the patch from 100% control to 71% at -40 mV holding potential and the current was not affected by 17beta-Estradiol at + 40 mV. However, the presence of the hormone did not affect the single-channel conductance, therefore its effect must be due to modulation of its open probability (Po). Interestingly, 17alpha-Estradiol did not change the total current in the patch. Tamoxifen, an antiestrogen, also showed inhibition, but in a voltage-independent manner. Our results suggest that the Maxi Cl- channel from human term placenta may be regulated by direct interaction of both compounds with the channel. From a functional point of view, the control of these channels by steroid hormones may be of great importance in placental physiology and their regulation may help to unravel their possible role in transplacental transport.

Estrogen regulates 11 beta-hydroxysteroid dehydrogenase-1 and -2 localization in placental syncytiotrophoblast in the second half of primate pregnancy.

We recently demonstrated that the 11 beta-hydroxysteroid dehydrogenase enzymes catalyzing cortisol-cortisone reduction (11 beta-hydroxysteroid dehydrogenase-1) and oxidation (11 beta-hydroxysteroid dehydrogenase-2) are located in different regions of the baboon and human placental syncytiotrophoblast. Moreover, there was a 2-fold increase in the ratio of 11 beta-hydroxysteroid dehydrogenase-2 to 11 beta-hydroxysteroid dehydrogenase-1 in syncytiotrophoblast membranes contiguous with the basal membrane (BMm) between mid and late baboon gestation. Our laboratories have also shown that estrogen regulates syncytiotrophoblast functional differentiation. Therefore, the current study determined whether the change in the ratio of 11 beta-hydroxysteroid dehydrogenase-2 to 11 beta-hydroxysteroid dehydrogenase-1 in the BMm was regulated by estrogen. Placentas were obtained on d 165 of gestation (term = d 184) from baboons that were untreated or were treated daily beginning on d 100 with the aromatase inhibitor CGS 20267, which reduced uterine and maternal serum E2 by more than 95% or with CGS 20267 plus E2 benzoate. Western blot analyses and immunofluorescence confirmed that in untreated controls the expression of 11 beta-hydroxysteroid dehydrogenase-1 was abundant in the microvillus membranes and considerably less in the BMm. In contrast, expression of 11 beta-hydroxysteroid dehydrogenase-2 was abundant in more internal regions of the syncytiotrophoblast, including the BMm, but was not detected in the microvillus membranes. The 11 beta-hydroxysteroid dehydrogenase-2 protein level was significantly decreased in the BMm of placentas from estrogen-suppressed baboons, resulting in a 2-fold decrease in the ratio of these enzymes in membranes juxta the fetal blood, and these changes were partially restored by CGS 20267 and E2. In contrast, estrogen had no effect on the ratio of 11 beta-hydroxysteroid dehydrogenase-2 to 11 beta-hydroxysteroid dehydrogenase-1 in whole villous homogenate or the micro-villus membranes. Collectively, these results indicate that estrogen regulates the developmental increase in the ratio of 11 beta-hydroxysteroid dehydrogenase-2 to 11 beta-hydroxysteroid dehydrogenase-1 in syncytiotrophoblast membranes juxta fetal blood, providing the subcellular architectural mechanism responsible for the previously demonstrated estrogen-dependent switch in transplacental glucocorticoid metabolism that regulates maturation of the primate fetal pituitary-adrenocortical axis.

Expression of the mRNAs and Proteins for the Na+/H+ Exchangers and Their Regulatory Factors in Baboon and Human Placental Syncytiotrophoblast

In polarized epithelial cells of several organ systems, e.g. the kidney, a family of Na+/H+ exchangers (e.g. Na+/H+ exchanger-1 and -3) and their regulatory proteins, Na+/H+ exchanger regulatory factor and Na+/H+ exchanger-3 kinase A regulatory protein play a major role in regulating Na+/H+ exchange integral to cellular homeostasis. Because the primate placenta regulates exchange of Na+ and H+ between the mother and fetus critical to fetal-placental homeostasis, the current study determined whether Na+/H+ exchanger-1 and -3 were compartmentalized and associated with expression of Na+/H+ exchanger regulatory factor and Na+/H+ exchanger-3 kinase A regulatory protein in baboon and human syncytiotrophoblast. Using RT-PCR, single 413-bp Na+/H+ exchanger-1 and 190-bp Na+/H+ exchanger-3 products were expressed by baboon and human syncytiotrophoblasts. The 104-kDa Na+/H+ exchanger-1 protein was detected by Western blot in microvillus membranes and to a much lesser extent in the basal membranes of the baboon and human syncytiotrophoblasts. In contrast, the 85-kDa Na+/H+ exchanger-3 protein was detected primarily in membranes contiguous with the basal membranes of the syncytiotrophoblast of both species. Differential localization of Na+/H+ exchanger-1 and -3 was confirmed by immunocytochemistry. The Na+/H+ exchanger-3 regulatory protein, Na+/H+ exchanger-3 kinase A regulatory protein, resided almost exclusively in the basal membranes, whereas Na+/H+ exchanger regulatory factor was localized primarily to the microvillus membranes in the baboon and human syncytiotrophoblast. Collectively, these results are the first to show that the baboon and human term placental syncytiotrophoblast expressed the mRNAs and proteins for Na+/H+ exchanger-1 and -3 and their regulatory factors and that Na+/H+ exchanger-1 and Na+/H+ exchanger regulatory factor resided primarily in the microvillus membranes, whereas Na+/H+ exchanger-3 and Na+/H+ exchanger-3 kinase A regulatory protein were localized to membranes contiguous with the basal membranes and to the basal membranes, respectively. We conclude that a complete Na+/H+ exchange system is present in the baboon and human term placental syncytiotrophoblast and suggest that the primate placenta exhibits polarity with respect to the capacity for regulation of Na+/H+ exchange between the placenta and the maternal and fetal circulations.

Automated quantitation of cell-mediated HIV type 1 infection of human syncytiotrophoblast cells by fluorescence in situ hybridization and laser scanning cytometry.

Infection of human placental syncytiotrophoblast cells with HIV requires direct contact with infected leukocytes. In vitro investigations into mechanisms regulating placental HIV transmission and into the development of therapeutic interventions have been hampered by difficulties inherent in quantitating HIV levels in cocultures of infected lymphocytes and adherent multinucleated syncytiotrophoblast cells. Here, we have used fluorescence in situ hybridization (FISH) for the direct detection of HIV-1 RNA within syncytiotrophoblast cells combined with laser scanning cytometry (LSC) to quantitate HIV levels exclusively in the syncytiotrophoblast cells. HIV-1-infected lymphocytic MOLT-4 cells were cocultured with primary human syncytiotrophoblast cells. Lymphocytic cells were identified with an anti-vimentin antibody and Cy5. HIV RNA was localized by in situ hybridization, using a digoxigenin-labeled riboprobe detected by Oregon Green, and nuclei were stained with 7-aminoactinomycin D. The three-color cocultures were analyzed by LSC to remove unwanted cell populations and quantitate HIV expression levels. The total HIV RNA level (green fluorescence integral) in each colony was normalized for cell size by dividing by the total DNA content (red fluorescence integral). The nuclear-normalized fluorescence integral was 2.3 times higher in infected cocultures than in uninfected cultures. When cocultures were incubated with 10 microM AZT, the green/red fluorescence integral value was significantly lower than that of cocultures incubated in the absence of AZT, corresponding to a 78% reduction in fluorescence. Laser scanning cytometry can be used to quantitate cell-mediated HIV infection in syncytiotrophoblast cells and should allow drug assessment studies and studies aimed at understanding the mechanism of virus entry into trophoblast cells to be carried out.

Localization and developmental regulation of 11beta-hydroxysteroid dehydrogenase-1 and -2 in the baboon syncytiotrophoblast.

We previously showed that the messenger RNA and protein levels of the 11ss-hydroxysteroid dehydrogenase (11betaHSD) enzymes catalyzing glucocorticoid reduction (11betaHSD-1) and oxidation (11betaHSD-2) increased with advancing baboon gestation and concluded that the estrogen-regulated change in placental cortisol metabolism from reduction at midgestation to oxidation near term is not simply the result of a change in the relative concentrations of these two enzymes. Therefore, in the current study we determined whether 11betaHSD-1 and -2 are located in different regions of the baboon and human syncytiotrophoblast and whether there is a developmental change in their localization with advancing baboon gestation. Western blot analyses, immunofluorescence, and electron microscopic immunocytochemistry indicated that 11betaHSD-1 expression was abundant in microvillus membranes (MVM) juxta the maternal circulation, and their levels are significantly lower, but detectable, in more internal regions of the syncytiotrophoblast, including membranes contiguous with the basal membrane (BM(m)) facing the fetal vasculature in both the human and baboon. In contrast, in both species 11betaHSD-2 expression was limited in the MVM and extensive throughout the remainder of the syncytiotrophoblast, including the BM(m). In the baboon, the relative mean (+/-SE) concentrations (arbitrary densitometric units per microgram protein) of 11betaHSD-1 in the MVM were similar at mid (i.e. day 100; 38,859 +/- 3,484; n = 3) and late (i.e. day 180; 43,561 +/- 1,784; n = 3) gestation (term = day 184) and exceeded (P < 0.01) respective values for 11betaHSD-2 by approximately 16-fold. In contrast, levels of 11betaHSD-1 in the BM(m) declined (P < 0.05) by approximately 50% between mid (7,099 +/- 758) and late (4,013 +/- 738) gestation, whereas levels of 11betaHSD-2 in this fraction increased. Thus, the ratio of 11betaHSD-2 to 11betaHSD-1 in the BM(m) at midgestation (1.22 +/- 0.10) was increased (P < 0.05) 2-fold in late gestation (2.66 +/- 0.05). Collectively, these findings indicate that the 11betaHSD-1 and -2 enzymes are localized to different membrane fractions of the baboon and human placental syncytiotrophoblast. Moreover, we propose that the developmental increase in the ratio of 11betaHSD-2 to 11betaHSD-1 in membranes facing fetal blood near term is consistent with and perhaps the subcellular mechanism responsible for the previously demonstrated switch in transplacental glucocorticoid metabolism from reduction at midgestation to oxidation late in gestation and appears to be responsible for the activation/maturation of the fetal pituitary-adrenocortical axis.

Polarized release of matrix metalloproteinase-2 and -9 from cultured human placental syncytiotrophoblasts.

The large increase in placental surface area and fetal villous vascular development in the third trimester of pregnancy requires degradation and reformation of the placental basal lamina. Degradation is carried out by matrix metalloproteinases (MMPs) secreted by adjacent cells. Although the gelatinases, MMP-2 and MMP-9, which are released by extravillous cytotrophoblasts (CTs) are believed to play crucial roles in early placental expansion, neither has been reported in third trimester villous trophoblasts nor has appropriate (basolateral) release of any MMP by the highly polarized syncytiotrophoblast (ST) been demonstrated. We demonstrated villous trophoblast expression of both MMP-2 and MMP-9 by in situ immunohistochemistry and by Western blot analysis and zymography of lysates and culture supernatants of highly purified villous CTs. We also found that epidermal growth factor (EGF)-stimulated CT differentiation into ST and stimulation by the phorbol diester, PMA, both increase MMP-9 secretion. The direction of MMP release was determined with confluent cultures of ST on porous membranes. We found that >90% of MMP-2 and MMP-9 were released from the basolateral surface. We conclude that villous STs express and release gelatinases from their basolateral surfaces in a regulated manner and suggest that such polarized release may be important to villous tissue remodeling.

Implication of ATP and sodium in arachidonic acid incorporation by placental syncytiotrophoblast brush border and basal plasma membranes in the human.

The human placental syncytiotrophoblast is the main site of exchange of nutrients and minerals between the mother and her fetus. In order to characterize the placental transport of some fatty acids, we studied the incorporation of arachidonic acid, a fetal primordial fatty acid, in purified bipolar syncytiotrophoblast brush border (BBM) and basal plasma membranes (BPM) from human placenta. The basal arachidonic acid incorporation in BBM and BPM was time dependent and reached maximal values of 0.75+/-0.10 and 0.48+/-0.18 pmol/mg protein, respectively, after 2.5 min. The presence of adenosine triphosphate (ATP) (3 m m) increases significantly the maximal incorporation of arachidonic acid by sixfold (4.75+/-0.35 pmol/mg) and ninefold (4.40+/-0.84 pmol/mg) in BBM and BPM, respectively. Moreover, an increase in the arachidonic acid incorporation was also obtained in the presence of sodium where the values achieved 7.68+/-0.98 (10x) and 6.53 pmol/mg (13.6x) for BBM and BPM, respectively. We also showed that the combination of both Na(+)and ATP increases significantly the maximal incorporation of arachidonic acid in BPM to 7.89+/-0.15 pmol/mg protein, while in BBM it did not modify its incorporation (8.18+/-0.25 pmol/mg protein), as compared to the presence of sodium alone. Our results demonstrate that arachidonic acid is incorporated by both placental syncytiotrophoblast membranes, and is ATP and sodium-linked. However, different mechanisms seem to be involved in this fatty acid incorporation through BBM and BPM, since the presence of Na(+)or ATP increased it, while the association of these two elements increased it only in BPM. We also demonstrated by osmolarity experiments that both membranes bind arachidonic acid, potentially involving one or more fatty acids binding proteins.

The role of the Brambell receptor (FcRB) in liver: protection of endocytosed immunoglobulin G (IgG) from catabolism in hepatocytes rather than transport of IgG to bile.

The Brambell receptor (FcRB) mediates functions of both immunoglobulin G (IgG) transport, transmitting immunity from mother to young, and IgG protection, making IgG the longest surviving of all plasma proteins. Reflecting its role as transport receptor (termed FcRn, for neonatal rat intestine, the tissue from which it was first cloned), FcRB is expressed antenatally in the rabbit, mouse and rat fetal yolk sac and in human placental syncytiotrophoblasts, and neonatally in the intestinal epithelium of mice and rats. Reflecting its role as protection receptor (FcRp), FcRB is expressed in the vascular endothelium throughout life, where it protects IgG from the on-going catabolic activities of this tissue. FcRB detected in hepatocytes was hypothesized to mediate transport of IgG from serum to bile, thus potentially extending the transport expression (FcRn) of this receptor beyond the perinatal period. Our results show serum-to-bile transport of IgG to be unaffected in mice functionally deleted for FcRB. Accordingly, the hypothesis is rejected that FcRB functions as transport receptor (FcRn) in liver. The default conclusion is that FcRB in hepatocytes functions as FcRp, serving to protect IgG from catabolism in hepatocytes that accompanies the endocytic activity of these cells. We conclude that there remains to date no evidence of an FcRn-like transport function of the Brambell receptor beyond the perinatal period, after which the FcRp function of the receptor predominates, paralleling the endocytic activities of the associated tissues.

Development and polarization of cationic amino acid transporters and regulators in the human placenta.

We have investigated L-arginine transport systems in the human placental syncytiotrophoblast across gestation using purified microvillous (MVM) and basal (BM) plasma membrane vesicles. In MVM from first-trimester and term placentas, L-arginine transport was by systems y(+) and y(+)L. In BM (term placentas), however, there was evidence for system y(+)L only. The Michaelis constant of system y(+)L was significantly lower (P < 0.05) in first-trimester compared with term MVM and lower in term MVM compared with BM (P < 0.05). There was no functional evidence for system b(0+) in term MVM or BM. Cationic amino acid transporter (CAT) 1, CAT 4, and 4F2hc were detected using RT-PCR in placentas throughout gestation. rBAT was not detected in term placentas. An approximately 85-kDa and an approximately 135-kDa protein was detected by Western blotting in MVM under reducing and nonreducing conditions, respectively, consistent with the 4F2hc monomer and the 4F2hc-light chain dimer, and their expression was significantly higher (P < 0.05) in term compared with first-trimester MVM. These proteins were not detected in BM despite functional evidence for system y(+)L. These data suggest different roles for 4F2hc in the development and polarization of cationic amino acid transporters in the syncytiotrophoblast.

Cytoadherence of Plasmodium falciparum-infected erythrocytes in the human placenta.

In Plasmodium falciparum-parasitized pregnant women, erythrocytes infected by mature stages of the parasite sequester into placental intervillous spaces. The presence of parasites in the placenta causes maternal anaemia and low birth weight of the infant. In-vitro studies suggest placental sequestration may involve the cytoadherence of infected erythrocytes to chondroitin sulphate A (CSA) and/or intercellular adhesion molecule 1 (ICAM-1) expressed by human placental syncytiotrophoblast. We identified P. falciparum receptors expressed on the surface of human syncytiotrophoblast using immunofluorescence of placental biopsies from Cameroon, a malaria-endemic area. In all placentas, a strongly positive staining was observed on the syncytiotrophoblast for CSA, but not for ICAM-1, vascular endothelium cell adhesion molecule-1, E-selectin, nor CD36. The cytoadherence ability of parasites from pregnant women and nonpregnant subjects was assessed on in-vitro cultured syncytiotrophoblast. Parasites from pregnant women bound to the trophoblast via CSA but not ICAM-1. Parasites from nonpregnant hosts either did not bind to the trophoblast culture or bound using ICAM-1. Our data support the idea that placental sequestration may result from cytoadherence to placental trophoblast and that pregnant women are parasitized by parasites that differ from parasites derived from nonpregnant host by their cytoadherence ability.

Expression of the 11β-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 11β-hydroxysteroid dehydrogenase (11β-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 11β-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 11β-HSD-2, it remains to be determined unequivocally whether 11β-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 11β-HSD-1. The 11β-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 11β-HSD-1 to the syncytiotrophoblast was confirmed by immunocytochemistry following antigen retrieval. These results show that both human and baboon placental syncytiotrophoblast expressed the 11β-HSD-1, as well as the 11β-HSD-2, proteins. Because 11β-HSD-1 can function as a reductase, the expression of 11β-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.

Expression of Endogenous Retrovirus ERV-3 Induces Differentiation in BeWo, a Choriocarcinoma Model of Human Placental Trophoblast

Differentiation-related expression of endogenous retrovirus ERV-3envin the normal human placental syncytiotrophoblast suggests a role in placental development. The choriocarcinoma cell line BeWo, a model of trophoblast differentiation, is maintained in an undifferentiated state and undergoes differentiation upon the addition of forskolin. The expression of ERV-3envmRNA increased after 48h forskolin treatment, concurrently with increased intercellular fusion and production of human chorionic gonadotropin (β-hCG) mRNA, a hormonal differentiation marker for trophoblast. Over expression of ERV-3envinduced differentiation of BeWo characterized by decreased cell growth, differentiation-related morphologic changes, and induction of β-hCG mRNA. These results support the first known role for the expression of an endogenous retrovirus in trophoblast differentiation.

Gene expression of nitric oxide synthase in cultured human term placental trophoblast during in vitro differentiation

The human placental syncytiotrophoblast is derived from differentiating cytotrophoblasts and is in contact with maternal blood. This endothelial function positions the trophoblast to regulate maternal-fetal exchange and to influence circulatory dynamics through paracrine interactions in the placenta. Two isoforms of nitric oxide synthase (NOS) are expressed in placenta, and northern analysis, reverse transcription-polymerase chain reaction (RT-PCR), and immunocytochemistry were used to correlate expression of the type II, inducible NOS (iNOS) and the type III, endothelial NOS (eNOS) with state of differentiation in cultured trophoblast from term placentae. It was also tested whether cytokines known to induce NOS in other cell systems would induce iNOS in human trophoblast. The mRNA for eNOS was detected by RT-PCR, but not by Northern analysis, in cultures grown for 24 h when cytotrophoblasts were dominant. In contrast, eNOS mRNA was abundant in cultures grown for 72 h when syncytiotrophoblast was present. Immunocytochemical staining for eNOS protein showed specific fluorescence in a few cells in cultures at 24 h, but the vast majority of cells expressed eNOS at 72 h. The iNOS isoform was expressed neither basally in any trophoblast culture nor was this isoform induced in cultures exposed to interleukin-1, tumour necrosis factor-α, interferon-γ and lipopolysaccharide. The in vitro pattern of trophoblast eNOS expression models the in vivo pattern of eNOS expression described for villous trophoblast. The results suggest that eNOS plays a role in human trophoblast differentiation and function.