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Energy metabolism and glycolysis of normal human term placental trophoblast in two-sided culture was investigated during differentiation from cytotrophoblast to syncytiotrophoblast, because glycogen metabolism is abnormal in several trophoblast related pregnancy diseases, including pre-eclampsia. After initial recovery of energy and cytoplasmic NADH/NAD+ redox by 24 h of culture, measures of cellular energy state, [ATP], [ADP], [ATP]/[ADP] ratio, ([ATP] + [ADP] + [AMP]), [ATP]/([ATP] + [ADP] + [AMP]) and energy charge remained essentially constant until 72 h, despite periods of increased energy turnover. At 24 h there was a burst of glycogenolysis, and glycolysis indicated by increased lactate production, which coincided with formation of syncytium. Subsequently, there was no resynthesis nor further breakdown of glycogen. At 48 h, oxygen consumption temporarily increased substantially, without increased glycolysis, during functional differentiation of the syncytiotrophoblast. Glucose uptake was constant and largely from the basal (in vivo fetal facing) side. Lactate output into the basal fetal medium was twice as fast as that into the microvillous (maternal) medium, and oxygen uptake was also asymmetrical. The results show that before and after differentiation substantial relatively constant aerobic glycolysis occurs, but that during increased energy demand cytotrophoblast depends on both glycolytic and aerobic energy production whereas syncytiotrophoblast relies on aerobic metabolism.

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To help determine physiologically important routes by which zinc (Zn) is acquired by human fetal vascular endothelium, the authors incubated cultured umbilical vein endothelial cells with 65Zn(II)-tracer labeled human fetal whole serum, ultrafiltrate (containing low molecular mass serum zinc complexes), and dialyzed serum (containing protein-bound zinc). Zinc from whole serum and from both serum fractions entered a rapidly labeled cellular compartment, removable by edetic acid (EDTA), representing Zn bound to the outside cell surface, and accumulatively, an EDTA-resistant compartment-probably largely internalized Zn. Entry of Zn into the EDTA-resistant pool from both serum fractions was strongly temperature-dependent, and was not via the EDTA-sensitive pool. Entry from the ultrafiltrate was resolvable into high affinity saturable, and non- (or hardly-) saturable components. Transfer from the dialyzed serum fraction was not significantly saturable, but only partially accounted for by nonspecific pinocytosis. Thus, Zn is obtained by fetal vascular endothelium partly from low molecular mass serum species, probably through at least one carrier-mediated membrane transport system; but also from Zn complexed with serum protein, via at least one metabolism-related route.

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Criteria for a successful model for the study of trans-syncytiotrophoblast transfer include isolating substantially pure trophoblast cells from placental villous tissue, and obtaining from them phenotypical villous syncytial syncytiotrophoblast during culture. For studies involving the basal membrane, including overall transfer, basal uptake and output, and controls acting at the basal membrane, a two-sided model is required with a separate compartment of culture medium in contact with the basal cell surface. All current methods of isolating cytotrophoblast, the precursor of syncytiotrophoblast, derive from the original tissue trypsinization method of Thiede (1960), which produces cultures of villous cytotrophoblast cells contaminated with other placental cell types. Lessons learned from successful and unsuccessful development of the model over 35 years are outlined, and recently established methods for purifying the isolated mixed cells discussed. These include sedimentation and centrifugation methods, immunological and receptor binding methods, and more selective release of trophoblast cells from tissue. Immuno flow cytometric cell sorting methods are potentially capable of isolating subpopulations of various phenotypical trophoblast types. We conclude that satisfactory methods are now available for isolating and purifying cytotrophoblast from early or late gestation human placenta.

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The conditions necessary for producing syncytical syncytiotrophoblast are examined. Tissue disaggregation conditions, culture media composition, different extracellular matrices and the influence of placental gestational age are all assessed. The importance of evaluating the biochemical and functional differentiational state of the cells is also stressed. Evidence is summarized that syncytiotrophoblast in culture is morphologically and ultrastructurally very similar to syncytiotrophoblast in vivo, and what is so far known biochemically is largely consistent with what is known in vivo. Studies published to date on microvillous membrane uptake and release and relationships with intracellular metabolism using syncytiotrophoblast in conventional culture are outlined from the point of view of the advantages and potential of this model. The present state of development of the two-sided model is assessed, mentioning factors to be considered such as the supporting membrane to be used, accounting for passive diffusion and paracellular leak components of transport and dealing with quantitative effects in kinetic studies of the presence of the supporting membrane. It is concluded that satisfactory methods are now in place for preparing pure villous syncytial syncytiotrophoblast in culture from cytotrophoblast derived from term (but not early) placentae, suitable for studying microvillous membrane transport and relationships with intracellular metabolism. Cytotrophoblast from early gestational age placenta may require different conditions to form true syncytiotrophoblast. A two-sided model for studies of overall transfer, basal transport and basal control mechanisms is now available and possibly with some development should be a good model for such investigations.

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Uptake of zinc into placental villous syncytiotrophoblast is the first step in its transfer from mother to fetus. To help characterise physiologically significant pathways of zinc accumulation by these cells, we incubated cultured layers of syncytiotrophoblast cells derived from human near-term placental tissue with serum ultrafiltrate (containing the zinc complexed with low molecular mass serum constituents), dialysed serum (containing the zinc bound to the serum proteins) and whole serum, each of whose endogenous zinc was tracer-labelled with 65Zn(II). Zinc label from both fractions of serum readily entered a rapidly labelled EDTA-sensitive cellular compartment, probably representing zinc bound to the outside cell surface and in accumulative fashion, an EDTA-resistant compartment, probably consisting largely of internalised cellular zinc. Movement of zinc into the EDTA-resistant pool was strongly temperature-dependent and did not occur via the EDTA-sensitive pool from either serum source. Transfer of zinc from the low molecular mass serum fraction into the EDTA-resistant pool was saturable, the concentration giving half-maximal rate being 1.2 mumol/l nonprotein-bound zinc. No nonsaturable component was detected. Zinc from the serum protein-bound fraction entered by a saturable component, already saturated at physiological total protein-bound zinc concentration, and by an apparently nonsaturable component, not appreciably accounted for by nonspecific fluid-phase endocytosis. The results show that zinc is acquired by placental syncytiotrophoblast from the low molecular mass serum zinc pool probably by a carrier-mediated process, and at least as importantly, from the zinc bound to serum protein, possibly by an endocytic mechanism.

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The hypothesis was examined that altered maternal zinc metabolism is involved in the low birthweight associated with raised maternal serum alpha-fetoprotein (MSAFP). Mothers with clinically normal pregnancy and normally formed infant but with raised MSAFP (> 90th percentile) had lower than normal plasma zinc concentration, raised leucocyte zinc and disturbed zinc-albumin relationship. They delivered offspring with lower birthweights than did women with normal MSAFP concentration, due both to shorter pregnancy and slower intrauterine growth. These results and others identify a subgroup of mothers associated with low infant birthweight, those with raised MSAFP, who have altered zinc distribution.

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We examined the effects of epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on the uptake of AIB by and its transfer across near-term human placental syncytiotrophoblast in two-sided culture. Pre-incubation of the trophoblast cell layer with either EGF (50ng/ml) or IGF-I (100ng/ml) on the apical (microvillous, in vivo maternal-facing) side reduced rates of unidirectional microvillous-to-basal transtrophoblast AIB transfer, increasing AIB retention within the cells. EGF on the basal (fetal-facing) side of the cell layer enhanced AIB uptake from the microvillous side but also increased overall mediated permeability in both directions. IGF-I at the basal surface, however, increased AIB uptake across the microvillous membrane, and induced a backflux from the cells into the basal medium dependent upon basal AIB concentration, suggesting that in vivo IGF-I on the fetal side enhances maternal-to-fetal placental transfer. The ideas are consistent with current concepts of maternal-placental and fetal-placental interactions regulating pregnancy and fetal development.

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The single copy endogenous retrovirus locus ERV-3 is known to be primarily expressed in the placenta. The absence of expression of this gene in choriocarcinoma cell lines has led to speculation that this may be a defect associated with this abnormality. We show here that ERV-3 is not normally expressed in the cytotrophoblast from which these tumour cells are derived but is expressed in normal syncytiotrophoblast. The conservation of the ERV-3 open reading frame for env in ape and old world monkey species and its tight regulation and site of expression suggest a functional role for this gene in this tissue.

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We describe the culture of human term placental trophoblast cells on cell-free amniotic membrane, with medium on both sides. Over the course of 2 days, the isolated cells, initially simple, mononucleated and probably cytotrophoblast, form a confluent layer of multinucleated syncytial cells with morphological and immunocytochemical properties of syncytiotrophoblast. This layer becomes polarized with respect to morphology, alkaline phosphatase distribution and hCG secretion. Contamination with amnion cells, and with other cell types that are present in placental tissue, was less than 1 per cent. A preliminary investigation of the permeability properties of the preparation showed that the trophoblast cell layer, rather than the amniotic membrane, was rate-limiting to transtrophoblast transfer, but that possible effects of the supporting membrane should be considered. The transtrophoblast transfer of D-glucose and the non-metabolisable analogue, 3-O-methyl-D-glucose (3OMG), had saturable and non-saturable/leak components in both directions, indicating that carrier-mediated processes were involved. The non-metabolisable amino acid 2-aminoisobutyrate (AIB) was both accumulated within the trophoblast cells, and transferred by saturable and non-saturable processes from the microvillous side, but no saturable accumulation or transfer was observed from the basal side, at the concentrations tried. The results suggest that this model may prove suitable for studies of transtrophoblast transfer.

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Trophoblastic cells, of at least 95 per cent purity by immunofluorescence and morphological criteria, were obtained from human term placenta by a simple trypsinisation method without the additional purification steps or complex culture conditions used by others. The differentiation of these cells was followed over four days in culture by fluorescence immunocytochemistry, by scanning and transmission electron microscopy and by light microscopy. The results support the idea that the isolated cells are cytotrophoblast and that these differentiate during this time into cells with characteristics of villous syncytiotrophoblast. This process involved first the formation of a multicellular layer of mononucleated cells, then the development of a syncytium of multinucleated cells and, not necessarily concurrently, functional differentiation. This may be a useful model for the study of syncytiotrophoblast function.

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1. To investigate the possible effect of iron ingestion on maternal zinc status, one group of women was given 94 mg of iron per day as ferrous sulphate with multivitamins during the second and third trimesters of their pregnancies and another, control, group was given a placebo of multivitamins without iron. 2. The subjects given iron developed significantly lower plasma zinc concentrations than those in the control group. This effect on zinc was maximal by 6 weeks, whilst that on maternal iron status was slower. 3. There was no parallel decrease of zinc concentration in maternal mixed leucocytes, or of plasma heatlabile alkaline phosphatase activity, suggesting that there was a redistribution of zinc between plasma and tissues. 4. The results indicate that iron supplementation during pregnancy alters the disposition of zinc in the mother.

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We examined the possibility that in preeclampsia complicated by fetal growth retardation, placental energy state is low either because of impaired glycolysis or because of ischemia resulting from reduced maternal placental blood flow. Concentrations of pyruvate and lactate, but not of glycogen and glucose, were significantly low in placentas of mothers with severe preeclampsia, supporting previous indirect evidence of inhibited glycolysis. Nevertheless, direct measurements of adenine nucleotide concentrations did not indicate reduced placental energy level in the preeclamptic placentas. This along with a lack of change of the ratio of lactate/pyruvate concentration (an indication of the redox state of cytoplasmic reduced nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide) is also evidence against the hypothesis of general placental ischemia leading to energy deficiency. However, as glycolysis is an important source of precursors, particularly pyruvate, for synthesis of amino acids and lipids, these results suggest that there is a significant metabolic abnormality in placentas of mothers with severe preeclampsia.

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The perfused human placental cotyledon was examined with respect to its viability, metabolic state, and performance. During the ischemic period before the start of perfusion, tissue adenosine triphosphate concentration and other measures of energy state fell rapidly to about half of the estimated in vivo value. During the subsequent perfusion, energy levels remained relatively stable but did not recover appreciably toward in vivo values. A very low transplacental leakage of inulin and a small cellular potassium loss indicate relative intactness of membrane function, but there were differences from the in vivo state in levels and balance of metabolic regulators adenosine triphosphate, adenosine monophosphate, and adenosine triphosphate/adenosine monophosphate ratio, and a more reduced cytoplasmic reduced nicotinamide adenine dinucleotide/ionized nicotinamide adenine dinucleotide couple. However, rates of oxygen and glucose consumption and lactate production and the maintenance of physiologic upward maternal-to-fetal concentration gradients of amino acids lead us to conclude that despite differences in energy and metabolic states the perfused cotyledon remains substantially intact and functions in certain respects comparably with the in vivo state.

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Using an ultrafiltration technique, apparent non-protein bound (NPB) zinc concentrations in plasma were found to be 2.2 +/- 0.2 (SEM) microgram Zn/l (10 observations) in normal males, 1.6 +/- 0.3 (10) micrograms/l in normal females and 1.2 +/- 0.2 (10) micrograms/l in pregnant mothers during their 16th week of gestation. These values are about 0.2% of the total plasma zinc concentrations, at least five-fold less than previous estimates. In amniotic fluid, the NPB-zinc concentration was 12.6 +/- 0.4 (10) micrograms/l, 5-10 times that of normal plasma, though the total zinc concentration (100 +/- 30 micrograms/l) was only one tenth that of plasma. When plasma or amniotic fluid samples were ultrafiltered without precaution against CO2 loss, their NPB-zinc concentrations increased, suggesting that pH changes alter zinc binding. The low concentration of NPB-zinc in plasma explains the low urinary excretion of zinc observed by others and would be expected to restrict the entry of zinc into cells.

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Angiotensins I, II, and III, renin substrate, and des-Asp1-angiotensin I were injected as a bolus into either the maternal or fetal circulation of human placental cotyledons perfused in vitro. All drugs tested produced dose-related increments in fetal perfusion pressure when injected into the fetal circulation, with the order of potency being angiotensin I approximately equal to angiotensin II approximately equal to angiotensin III greater than or equal to des-Asp1-angiotensin I greater than or equal to renin substrate. The responses to all the drugs could be blocked by the competitive inhibitor of angiotensin II, (Sar1, Ala8)-angiotensin II, but only the actions of angiotensin I, renin substrate, and des-Asp1-angiotensin I could be blocked by angiotensin converting enzyme inhibitor. When the agents were injected into the maternal circulation, only angiotensins II and III caused dose-related increments in fetal perfusion pressure. Possibly, the placenta may be the main site of conversion of angiotensin I to angiotensin II in the fetoplacental unit, and angiotensin II produced by the placenta could act locally to control fetoplacental blood flow.

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A simple, specific, and reliable method has been developed for the determination of L-lysine in blood plasma and tissue. The L-lysine in the sample is decarboxylated enzymatically, and fluorescamine is added to a pentan-1-ol extract of the cadaverine formed. This produces a stable product which is measured fluorometrically.

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