Excretion of fetal biliverdin by the rat placenta-maternal liver tandem

Fetal liver immaturity is accompanied by active heme catabolism. Thus fetal biliary pigments must be excreted toward the mother by the placenta. To investigate biliverdin handling by the placenta-maternal liver tandem, biliverdin-IXalpha was administered to 21-day pregnant rats through the jugular vein or the umbilical artery of an in situ perfused placenta. Jugular administration resulted in the secretion into maternal bile of both bilirubin and biliverdin (3:1). However, when biliverdin was administered to the placenta, most of it was transformed into bilirubin before being transferred to the maternal blood. Injecting Xenopus laevis oocytes with mRNA from rat liver or placenta enhanced their ability to take up biliverdin, which was inhibited by estradiol 17beta-d-glucuronide. The expression of three OATP isoforms in this system revealed that they have a varying degrees of ability to transport biliverdin (Oatp1/1a1 > Oatp2/1a4 > Oatp4/1b2). The abundance of their mRNA in rat trophoblast was Oatp1/1a1 > Oatp4/1b2 > Oatp2/1a4. The expression of biliverdin-IXalpha reductase in rat placenta was detected by RT-PCR/sequencing and Western blot analysis. The relative abundance of biliverdin-IXalpha reductase mRNA (determined by real-time quantitative RT-PCR) was fetal liver > placenta > maternal liver. Common bile duct ligation in the last week of pregnancy induced an upregulation of biliverdin-IXalpha reductase in maternal liver but had no effect on fetal liver and placenta. In conclusion, several members of the OATP family may contribute to the uptake of fetal biliverdin by the rat placenta. Before being transferred to the mother, biliverdin is extensively converted into bilirubin by biliverdin-IXalpha reductase, whose expression is maintained even though bilirubin excretion into maternal bile is impaired.     

Endocrine regulation of phosphorus and calcium metabolism during pregnancy in domestic ruminants

In pregnant domestic ruminants (cows, ewes, goats) foetal plasma calcium and inorganic phosphorus concentrations are higher than those measured in the dam. The foetus regulates its own calcaemia and phosphataemia. Changes in maternal plasma calcium levels have no significant effect on foetal calcaemia. Calcium and phosphorus are transported from the dam to the foetus according to a one-way process, the transport from the foetus to the dam being negligible. An important part of the calcium transferred to the foetus comes from the maternal skeleton. The true molecular mechanisms involved in placental transport of calcium are still unknown. This is an active transport, stimulated by vitamin D metabolites (of maternal, foetal or placental origin) and maternal prolactin. Maternal calcitonin protects the skeleton of the pregnant (and lactating) female ruminant against excessive demineralization, partly by modulating placental transport of calcium during periods of intense mineralization of foetal skeleton.     

Hormonal regulation of the Menkes and Wilson copper-transporting ATPases in human placental Jeg-3 cells

Copper deficiency during pregnancy results in early embryonic death and foetal structural abnormalities including skeletal, pulmonary and cardiovascular defects. During pregnancy, copper is transported from the maternal circulation to the foetus by mechanisms which have not been clearly elucidated. Two copper-transporting ATPases, Menkes (ATP7A; MNK) and Wilson (ATP7B; WND), are expressed in the placenta and both are involved in placental copper transport, as copper accumulates in the placenta in both Menkes and Wilson disease. The regulatory mechanisms of MNK and WND and their exact role in the placenta are unknown. Using a differentiated polarized Jeg-3 cell culture model of placental trophoblasts, MNK and WND were shown to be expressed within these cells. Distinct roles for MNK and WND are suggested on the basis of their opposing responses to insulin. Insulin and oestrogen increased both MNK mRNA and protein levels, altered the localization of MNK towards the basolateral membrane in a copper-independent manner, and increased the transport of copper across this membrane. In contrast, levels of WND were decreased in response to insulin, and the protein was located in a tight perinuclear region, with a corresponding decrease in copper efflux across the apical membrane. These results are consistent with a model of copper transport in the placenta in which MNK delivers copper to the foetus and WND returns excess copper to the maternal circulation. Insulin and oestrogen stimulate copper transport to the foetus by increasing the expression of MNK and reducing the expression of WND. These data show for the first time that MNK and WND are differentially regulated by the hormones insulin and oestrogen in human placental cells.     

Maternally-inherited Grb10 reduces placental size and efficiency

The control of foetal growth is poorly understood and yet it is critically important that at birth the body has attained appropriate size and proportions. Growth and survival of the mammalian foetus is dependent upon a functional placenta throughout most of gestation. A few genes are known that influence both foetal and placental growth and might therefore coordinate growth of the conceptus, including the imprinted Igf2 and Grb10 genes. Grb10 encodes a signalling adapter protein, is expressed predominantly from the maternally-inherited allele and acts to restrict foetal and placental growth. Here, we show that following disruption of the maternal allele in mice, the labyrinthine volume was increased in a manner consistent with a cell-autonomous function of Grb10 and the enlarged placenta was more efficient in supporting foetal growth. Thus, Grb10 is the first example of a gene that acts to limit placental size and efficiency. In addition, we found that females inheriting a mutant Grb10 allele from their mother had larger litters and smaller offspring than those inheriting a mutant allele from their father. This grandparental effect suggests Grb10 can influence reproductive strategy through the allocation of maternal resources such that offspring number is offset against size.     

Foetal and Maternal RNA labelled with <Superscript>3</Superscript>H-Cytidine

BIOCHEMICAL analysis of in vivo RNA metabolism in the rat foetus has been hindered by inability to adequately label foetal RNA^1–5. In contrast, in vitro studies of RNA metabolism in mammalian blastocysts have not been hampered by this restriction as uptake of isotopic uridine is adequate^6–8. The inability of the foetus to incorporate labelled uridine administered to the mother may be due to dependence on the maternal circulation and placental transport. Our experiments show that rat foetal and maternal RNA can be labelled to yield a high specific activity RNA by administering ³H-cytidine to the pregnant animal. Moreover, they indicate that placental transport is not the limiting factor in the use of labelled uridine as a precursor for foetal RNA. Although labelled cytidine has been reported to cross the rat placenta in later stages of pregnancy³, its value in labelling foetal RNA has not been demonstrated previously.     

Mammalian viviparity: a complex niche in the evolution of genomic imprinting

Evolution of mammalian reproductive success has witnessed a strong dependence on maternal resources through placental in utero development. Genomic imprinting, which has an active role in mammalian viviparity, also reveals a biased role for matrilineal DNA in its regulation. The co-existence of three matrilineal generations as one (mother, foetus and post-meiotic oocytes) has provided a maternal niche for transgenerational co-adaptive selection pressures to operate. In utero foetal growth has required increased maternal feeding in advance of foetal energetic demands; the mammary glands are primed for milk production in advance of birth, while the maternal hypothalamus is hormonally primed by the foetal placenta for nest building and post-natal care. Such biological forward planning resulted from maternal–foetal co-adaptation facilitated by co-expression of the same imprinted allele in the developing hypothalamus and placenta. This co-expression is concurrent with the placenta interacting with the adult maternal hypothalamus thereby providing a transgenerational template on which selection pressures may operate ensuring optimal maternalism in this and the next generation. Invasive placentation has further required the maternal immune system to adapt and positively respond to the foetal allotype. Pivotal to these mammalian evolutionary developments, genomic imprinting emerged as a monoallelic gene dosage regulatory mechanism of tightly interconnected gene networks providing developmental genetic stability for in utero development.     

Placental structure in gestational diabetes mellitus

The placenta is a transitory organ, located between the mother and the foetus, which supports intrauterine life. This organ has nutritional, endocrine and immunologic functions to support foetal development. Several factors are related to the correct functioning of the placenta including foetal and maternal blood flow, appropriate nutrients, expression and function of receptors and transporters, and the morphology of the placenta itself. Placental morphology is crucial for understanding the pathophysiology of the organ as represents the physical structure where nutrient exchange occurs. In pathologies of pregnancy such as diabetes mellitus in humans and animal models, several changes in the placental morphology occur, related mainly with placental size, hypervascularization, higher branching capillaries of the villi and increased glycogen deposits among others. Gestational diabetes mellitus is associated with modifications in the structure of the human placenta including changes in the surface area and volume, as well as histological changes including an increased volume of intervillous space and terminal villi, syncytiotrophoblast number, fibrinoid areas, and glycogen deposits. These modifications may result in functional changes in this organ thus limiting the wellbeing of the developing foetus. This review gives an overview of recurrent morphological changes at macroscopic and histological levels seen in the placenta from gestational diabetes in humans and animal models. This article is part of a Special Issue entitled: Membrane Transporters and Receptors in Pregnancy Metabolic Complications edited by Luis Sobrevia.     

The existence during gestation of an immunological buffer zone at the interface between maternal and foetal tissues.

In mammalian pregnancy the trophoblast normally constitutes an uninterrupted boundary of foetal tissue in immediate contact with maternal tissue, including blood in some species, and is the decisive immunological barrier to rejection of the foetus as an allograft. The ability of the trophoblast to function as a barrier evidently results from its capacity to resist immunological attack by either alloantibody or alloimmune cells and to prevent immunocompetent cells from reaching and damaging the foetus but, as yet, there is no general agreement regarding the means by which it exercises these functions. In view of the dramatic hormonal changes that occur during pregnancy and the undisputed involvement of trophoblast in these endocrine events, the possibility exists of an interaction between the hormones of pregnancy and the immunological phenomena. The present account furnishes evidence that endocrine activity at the maternal surface of the trophoblast, the presumptive site of the immunological frontier between foetus and mother, may be a factor in its local survival at implantation. The placental hormones so far known that are capable of blocking the antigen receptor sites of the mother's lymphocytes and thus preventing the latter from reacting with the foetal antigens are the glycoprotein, human chorionic gonadotrophin (HCG) and the polypeptide hormone, human chorionic somatomammotrophin (HCS) or human placental lactogen (HPL), both of which are specific to the human placenta. The origin of these hormones, their spatial distribution and their probable interaction with placental steroid hormones are discussed. It is argued that the place of highest concentration of these hormones is on the surface of the syncytial microvilli and the adjacent caviolae of the apical plasma membrane, as well as on the surfaces of the persisting cytotrophoblastic cells of the basal plate (cytotrophoblastic shell), the cell islands and the septa-precisely where the immunological challenge of the foetal allograft to the maternal host occurs. An explanation is offered for the continuing production of the voluminous quantities of these hormones during human pregnancy.     

Ultrastructural changes in the placenta of the ewe following foetal infusion of cortisol.

The placental changes which followed continuous infusion of cortisol into the sheep foetus in the later stages of gestation were, like the hormonal changes, broadly similar to those of spontaneous parturition. There was, however, a premature separation of foetal and maternal tissues in certain areas of the placental cotyledons, and this separation appeared to protect the foetal epithelium from the degenerative changes which normally take place in the short space of time between the birth of the lamb and the delivery of the foetal membranes. The results suggest that an experimental model in which premature labour is induced by the administration of cortisol to the foetus is probably incomplete, and that additional factors almost certainly contribute to the cascade phenomenon of spontaneous parturition.     

The specificity of biliverdin reductase. The reduction of biliverdin XIII isomers

The substrate specificity of the different molecular forms of biliverdin reductase (bilirubin:NAD(P)+ oxidoreductase, EC 1.3.1.24) using biliverdin XIII alpha, XIII beta and XIII gamma was examined. It was found that molecular form 1 (the major form in normal rat liver) reduced biliverdin XIII alpha at a much higher rate than the other two isomers. Molecular form 2 (the minor form) reduced isomers XIII alpha and XIII beta at similar rates, while molecular form 3 (the major form induced by CoCl2 treatment) reduced the XIII beta isomer at a slightly higher rate than the XIII alpha isomer. Molecular forms 2 and 3, both reduced isomer XIII gamma more slowly than they reduced the XIII alpha and XIII beta isomers. These results are similar to those obtained previously using biliverdins IX alpha, IX beta and IX gamma, suggesting that biliverdin reductase specificity is related to the type of the isomer rather than to the series (IX or XIII) of the isomer.     

Preparation and properties of crystalline biliverdin IX alpha. Simple methods for preparing isomerically homogeneous biliverdin and [14C[biliverdin by using 2,3-dichloro-5,6-dicyanobenzoquinone.

Amorphous isomerically pure biliverdin IX alpha is readily prepared in more than 70% yield by dehydrogenation of bilirubin with 2,3-dichloro-5,6-dicyanobenzoquinone in dimethyl sulphoxide under carefully controlled conditions. Crystalline biliverdin IX alpha and amorphous [14C]biliverdin can be obtained similarly in more than 40+ yield. The pure crystalline pigment was characterized by elemental analysis, methylation, chemical and enzymic reduction to bilirubin, i.r.- and u.v.-visible-absorption spectroscopy, n.m.r. spectroscopy and field-desorption mass spectrometry, and its solubility was determined. Under certain conditions, dehydrogenation, gave biliverdin contaminated with III alpha and XIII alpha isomers as a result of disproporationation of bilirubin. Formation of non-IX alpha isomers depends on the concentrations of the reagents and the order in which they are mixed, and occurs under neutral anaerobic conditions. Free-radical reactions probably are responsible, suggesting that the first step in the deydrogenation of bilirubin with 2,3-dichloro-5,6-dicyanobenzoquinone in dimethyl sulphoxide is formation of a bilirubin cation radical, rather than hydride ion abstraction.     

Up regulation of the maternal immune response in the placenta of cattle naturally infected with Neospora caninum

Neospora caninum is an intracellular protozoan parasite which is a major cause of abortion in cattle worldwide. It forms persistent infections which recrudesce during pregnancy leading to foetal infection and in a proportion of cases, abortion. The mechanisms underlying abortion are not understood. In this study, recrudescence of a persistent infection in eight naturally infected cows occurred between 20 and 33 weeks of gestation. Animals were killed at the time of recrudescence and parasites were detected in the placentae and foetuses. An active maternal immune response consisting of an infiltration of CD4+ and CD8+ T cells and a 46-49 fold increase in interferon-γ and interleukin-4 mRNA was detected. Other cytokines, notably interleukin-12 p40, interleukin-10 and tumour necrosis factor-α were also significantly increased and Major Histocompatibility Class II antigen was expressed on maternal and foetal epithelial and stromal fibroblastoid cells. Significantly, despite the presence of an active maternal immune response in the placenta, all the foetuses were alive at the time of maternal euthanasia. There was evidence of parasites within foetal tissues; their distribution was restricted to the central nervous system and skeletal muscle and their presence was associated with tissue necrosis and a non-suppurative inflammatory response involving lymphocytes and macrophages, irrespective of the gestational age of the foetus. Whilst an active maternal immune response to a pathogen in the placenta is generally considered to be damaging to the foetal trophoblast, our findings suggest that the presence of a parasite-induced maternal immune response in the placenta is not detrimental to foetal survival but may contribute to the control of placental parasitosis.     

The excretion pattern of biliverdin and bilirubin in bile of the small skate (Raja erinacea)

Summary Bile pigment composition (biliverdin, bilirubin and their conjugates) was analyzed in stored gallbladder bile and newly synthesized hepatic bile from the small skate (Raja erinacea). During a five day period of captivity, gallbladder volume remained relatively constant while bilirubin and biliverdin content increased two to three fold. Biliverdin which accounted for 50% of the pigments did not increase as a percentage of tetrapyrroles during this period. The relative proportion of bilirubin and its conjugates also remained constant, averaging 65% for bilirubin monoglucuronide, 30% for bilirubin diglucuronide and 5% for unconjugated bilirubin as measured by HPLC methods. Intravenous administration of biliverdin resulted in significant increases in the biliary excretion of both biliverdin and all bilirubin tetrapyrroles. Insignificant quantities of³H-biliverdin were detected in hepatic bile following the intravenous administration of³H-bilirubin. These studies indicate that the small skate excreted both biliverdin and bilirubin conjugates in bile and that the biliverdin was not produced by in vitro oxidation of bilirubin or its metabolites.     

Impact of foetus and mother on IFN-gamma-induced indoleamine 2,3-dioxygenase and inducible nitric oxide synthase expression in murine placenta following Toxoplasma gondii infection

IFN-gamma production is a hallmark of acute infection with the protozoan parasite Toxoplasma gondii. The tryptophan-catabolising enzyme indoleamine 2,3-dioxygenase (IDO), as well as inducible nitric oxide synthase (NOS2) are induced by IFN-gamma and can play extremely diverse roles in immune regulation, defence against pathogens and physiological homeostasis. We investigated the regulation of these two central enzymes in the placenta during acute infection of pregnant female mice. Using IFN-gamma receptor knockout (IFNgammaR-/-) mice, we showed that IDO is not constitutively expressed in term placentas. In contrast, NOS2 expression was observed, largely dependent on IFN-gamma signalling. Upon infection with the avirulent PRU strain of T. gondii, IDO mRNA expression was induced in an IFNgammaR-dependent manner. Surprisingly, NOS2 mRNA was severely suppressed. Importantly, we showed in crossing experiments of heterozygote (IFNgammaR+/-) mothers with IFNgammaR-/- males and vice versa that IDO expression largely depends on the presence of IFN-gamma receptors on foetal cells, and to a lesser extent on maternal cells. Immunohistochemical analysis localised foetal IDO production to invasive trophoblasts within the maternal part of the placenta. The placental vascular endothelium only stained positive when the mothers possessed functional IFN-gamma receptors. In contrast, placental NOS2 expression, but also its suppression following infection, seems to be largely dependent on IFN-gamma signalling in maternal cells. Neither factor appears to regulate placental T. gondii growth, as we observed no difference in parasite numbers between (+/-) and (-/-) foetuses. Taken together, our results demonstrate the crucial role of the foetus in placental IDO, but not NOS2, production following T. gondii infection.     

Enzymic basis of deranged foetal flavin-nucleotide metabolism consequent on immunoneutralization of maternal riboflavin carrier protein in the pregnant rat.

A comparison of the kinetic and other parameters of enzymes of flavin-nucleotide metabolism in the whole foetus vis-à-vis the maternal liver in the pregnant rat revealed relatively lower activities of foetal flavokinase and FAD pyrophosphorylase. Passive immunoneutralization of the maternal riboflavin carrier protein suppresses foetal FAD pyrophosphorylase rather selectively. Additionally, although the activities of foetal nucleotide pyrophosphatase and FMN phosphatase were unchanged owing to immunoneutralization, higher activities of these enzymes in the whole foetus as compared with the maternal liver may be responsible for the drastic depletion of FAD levels that precipitates foetal degeneration.     

DNA methylation changes in genes coding for leptin and insulin receptors during metabolic-altered pregnancies

The overwhelming rates of obesity worldwide are a major concern due to the elevated medical costs associated and the poor quality of life of obese patients. In the recent years, it has become evident that the intrauterine milieu can have a long-term impact on the foetus health. The placenta is a highly dynamic organ; whose primary function is to carry nutrients from the mother to the foetus and to remove waste products from the foetus. Any alteration in maternal circulating metabolites elicits a response in order to ensure the developing foetus an adequate growth environment. This response can be translated into epigenetic modifications in coding genes for metabolic-related receptors located in the placenta and foetal tissues. The most studied receptors involved in the metabolic sensing are the leptin and the insulin receptors. A maternal metabolic disease-like state can alter the expression of these receptors in different organs, including placenta. There is evidence that these alterations not only affect the expression level of these receptors, but there are also differences in epigenetic marks in regulatory elements of these genes that may become permanent despite the mother's treatment. This review provides evidence about possible mechanisms involved in the foetal programming of metabolic diseases originated from the pre-natal environment that could contributive to increasing levels of obesity in the world.     

Materno-foetal exchanges and utilisation of nutrients by the foetus: comparison between species

Several general features of nutrient uptake and utilisation by foetuses are similar among mammalian species. Nevertheless, there are also differences linked mainly to differences in placental permeability. Glucose and lactate are the main energetic substrates of the foetus. In normal conditions, the oxidation of carbohydrates accounts for about 75, 60 and 50% of oxygen uptake in the foetal pig, foal and lamb, respectively, and acetate accounts for about 10% in ruminants. Acidic amino acids are synthesised by the foetus, whereas neutral and basic amino acids are transported from the placenta. As shown by the high urea level in foetal blood, amino acids are partly involved in the oxidative metabolism of foetuses; their contribution is higher in ruminants than in humans, horses and pigs. Fatty acids cross the haemochorial placenta of rodents, rabbits and primates, and are incorporated into the foetal lipids, whereas their uptake by ruminant, pig and horse foetuses is very low.     

Synthesis of biliverdin and bilirubin 1-O-acyl-beta-D-glucopyranuronic acids.

Biliverdin and bilirubin mono- and di-beta-glucuronides were prepared by nucleophilic substitution of the 1-O-mesyl derivative of alpha-ethoxyethyl-protected glucuronic acid (compound II) with the tetrabutylammonium salts of biliverdin and bilirubin. Removal of the acetal-protecting groups by mild acid treatment yielded biliverdin glucuronides, which were reduced to bilirubin glucuronides. Depending on reaction conditions the pure beta-anomers or mixtures highly enriched in the beta-anomers were obtained. The biliverdin and bilirubin glucuronides were identical with pigments derived from bile. They were characterized as the IX alpha isomers and the beta-anomers by alkaline hydrolysis, n.m.r. spectroscopy, hydrolysis with beta-glucuronidase and conversion into dipyrrolic azopigments. Model reactions of the 1-O-mesylate (II) with other nucleophiles also were performed, i.e. the acetate anion and various alcohols.     

Tissue levels of bilirubin and biliverdin in the sea lamprey, Petromyzon marinus L., before and after biliary atresia

1. Liver, intestine, kidney, muscle and epidermis from larvae, juvenile adults and upstream migrants of the sea lamprey, Petromyzon marinus L., were assayed for the presence of biliverdin and bilirubin. Urine was also examined for these bile pigments in juveniles and upstream migrants. 2. Bilirubin concentration increased dramatically in the liver and caudal intestine following loss of larval bile ducts while biliverdin levels were highest in the liver of upstream migrants and rose sharply in the caudal intestine immediately following the atresia. 3. Small amounts of bile pigment were present in larval kidneys but high concentrations were found in this organ in upstream migrants. The urine of the latter possessed biliverdin. 4. Mucus of the epidermis may be a vehicle for transport and release of bilirubin in upstream migrants. 5. These data indicate that lampreys utilize different avenues for bile pigment storage and elimination over the course of their life cycle.     

Synthesis, chromatographic purification, and analysis of isomers of biliverdin IX and bilirubin IX.

Neutral solvent systems were developed to isolate the alpha, beta, gamma, and delta isomers of biliverdin IX dimethyl ester by TLC. The individual free acids of biliverdin IX were obtained by saponification of the corresponding dimethyl esters. The bilirubin IX isomers were prepared by reducing the corresponding biliverdin IX isomers with NaBH3CN. Starting from a pure biliverdin IX dimethyl ester, the corresponding free acid of biliverdin IX or bilirubin IX was available within 3-4 h. Preparation of spectrally pure bile pigment required final TLC on acid-cleaned neutral TLC plates. The absorption spectra of the free acids and dimethyl esters of biliverdin IX in methanol showed a broad band at about 650 nm and a sharp band at about 375 nm. The long-wave-length band was extremely sensitive to the presence of strong acid. A 10-fold molar excess of HCl caused a 35- to 50-nm shift of the absorption maximum to longer wavelengths and near doubling of the maximum absorption. The molar absorption coefficients of biliverdins were identical for each free acid and dimethyl ester pair. In each case, Beer's law was followed in both methanol and acidified methanol. Methanol also proved to be a suitable solvent for spectroscopic determination of the non-alpha isomers of bilirubin IX. The wavelength of maximum absorption and molar absorption coefficient of each dipyrrolic ethyl anthranilate azo pigment derived from the various bilirubin IX isomers are also reported.