The ruminant placental trophoblast binucleate cell: an evolutionary breakthrough.

Viviparity and the development of a placenta are two of the major reasons for the success of the mammals in colonizing all habitats, both terrestrial and aquatic. The placenta is an apposition of fetal to maternal tissue which serves two main, but competing functions: to maximize oxygen transfer and the acquisition of nutrients from the mother, but to minimize immunological rejection by the maternal immune system. This has resulted in the evolution of four main types differing in the degree of loss of the maternal uterine epithelial (UE) barrier: epitheliochorial, synepitheliochorial, endotheliochorial, and hemochorial, all providing a successful safe balance between the needs of mother and fetus. Epitheliochorial is the least invasive, a simple apposition and microvillar interdigitation of the apices of uterine epithelium and trophoblast. It is suggested to have evolved as a response to the increase in the size of the animal to provide a sufficiently long gestation to produce a single altricial (run/swim-soon-as-born) neonate as in the Cetartiodactyla. The mother needs to have good control of the fetal demands so the UE barrier is maintained. However, in the synepitheliochorial placenta, characteristic of all ruminants, the fetus has evolved a means of increasing, or at least maintaining, demand without the need for invasion. This has been achieved by the development of the trophoblast binucleate cell which, uniquely, can fuse with a UE cell to form fetomaternal hybrid tissue. This can maintain some maternal barrier function but also deliver fetally synthesized immunomodulatory and metabolic messages to the maternal circulation. This review provides the evidence for this remarkable evolutionary step and also considers an alternative explanation for the formation of the structure of the ruminant placenta.

Asymmetric expression of proteins in the granules of the placentomal Binucleate cells in Giraffa camelopardalis†.

Mature granulated trophoblast binucleate cells (BNC) have been found in all ruminant placentas examined histologically so far. BNC are normally fairly evenly distributed throughout the fetal villus and all their granules contain a similar variety of hormones and pregnancy associated glycoproteins (PAGs). Only the Giraffe is reported to show a different BNC protein expression, this paper is designed to investigate that. Gold labelled Lectin histochemistry and protein immunocytochemistry were used on deplasticised 1 µm sections of a wide variety of ruminant placentomes with a wide range of antibodies and lectins. In the Giraffe placentomes, even though the lectin histochemistry shows an even distribution of BNC throughout the trophoblast of the placental villi, the protein expression in the BNC granules is limited to the BNC either in the apex or the base of the villi. Placental lactogens and Prolactin (PRL) are present only in basally situated BNC: PAGs only in the apical BNC. PRL is only found in the Giraffe BNC which react with many fewer of the wide range of antibodies used here to investigate the uniformity of protein expression in ruminant BNC. The possible relevance of these differences to ruminant function and evolution is considered to provide a further example of the versatility of the BNC system.

Glycosylation and immunocytochemistry of binucleate cells in pronghorn (Antilocapra americana, Antilocapridae) show features of both Giraffidae and Bovidae.

Although the pronghorn (Antilocapra americana) resembles an antelope, its nearest relatives are the giraffe and okapi. In this study we have examined the placentae of 6 pronghorns using lectin- and immunocytochemistry to identify giraffid and bovid features. Binucleate cells (BNC) of the placenta exhibited features intermediate between those of the giraffe and bovine; Dolichos biflorus agglutinin binding - strong in the bovine BNC and absent in the giraffe - was evident in only a subpopulation of BNC while binding to blood vessels, as in the giraffe. Binding of Phytolacca americana agglutinin resembled that of the giraffe and okapi whereas many other glycans were found in all four clades. PAG antigens were similar to bovine and okapi but not giraffe. In summary, although the pronghorn outwardly resembles an antelope, placental BNC show giraffid features. Although each clade has its own individual characteristics, there are far more similarities than differences between them, emphasizing the common ancestry of all four clades.

Immunocytochemistry of the placentas of giraffe (Giraffa cameleopardalis giraffa) and okapi (Okapi johnstoni): comparison with other ruminants.

INTRODUCTION: The trophoblast binucleate cell [BNC] is central to the structure and function of all ruminant placentas so far investigated. The Giraffidae are considered to form a separate family within the ruminant suborder. METHODS: The structure and function of two [mid and late pregnant] giraffe placentas and two term okapi placentas have been investigated immunocytochemically. RESULTS: Their major characteristics: polycotyledonary epitheliochorial structure, sequential glucose transport using two transporter isoforms, expression of water transporters in the interplacentomal [IP] and placentomal [P] trophoblast and restriction of calcium transport to the IP trophoblast are similar to those of the ruminant family Bovidae. . Giraffe and okapi also show characteristic ruminant trophoblast binucleate cells (BNC) which migrate and fuse with individual uterine epithelial cells as in the cow. However, there are many fewer BNC, of limited distribution, when compared with other ruminants so far investigated. The giraffe and okapi BNC also show a different range of proteins, Pregnancy Associated Glycoproteins (PAGs) and glycans which clearly distinguish the Giraffidae from the Bovidae. CONCLUSIONS: The results support a separate giraffid family derived from a common ancestry, possessing subpopulations of BNC with potentially different functions.

The binucleate cell of okapi and giraffe placenta shows distinctive glycosylation compared with other ruminants: a lectin histochemical study.

The placenta of ruminants contains characteristic binucleate cells (BNC) with a highly conserved glycan structure which evolved early in Ruminant phylogenesis. Giraffe and Okapi placentae also contain these cells and it is not known whether they have a similar glycan array. We have used lectin histochemistry to examine the glycosylation of these cells in these species and compare them with bovine BNC which have a typical ruminant glycan composition. Two placentae, mid and near term, from Giraffe (Giraffa camelopardalis) and two term placenta of Okapi (Okapia johnstoni) were embedded in resin and stained with a panel of 23 lectins and compared with near-term bovine (Bos taurus) placenta. Significant differences were found in the glycans of Giraffe and Okapi BNC compared with those from the bovine, with little or no expression of terminal αN-acetylgalactosamine bound by Dolichos biflorus and Vicia villosa agglutinins which instead bound to placental blood vessels. Higher levels of N-acetylglucosamine bound by Lycopersicon esculentum and Phytolacca americana agglutinins were also apparent. Some differences between Okapi and Giraffe were evident. Most N-linked glycans were similarly expressed in all three species as were fucosyl residues. Interplacentomal areas in Giraffe and Bovine showed differences from the placentomal cells though no intercotyledonary BNC were apparent in Okapi. In conclusion, Giraffidae BNC developed different glycan biosynthetic pathways following their split from the Bovidae with further differences evolving as Okapi and Giraffe diverged from each other, affecting both inter and placentomal BNC which may have different functions during development.

Functional immunocytochemistry of Tragulus placenta: implications for ruminant evolution.

INTRODUCTION AND METHODS: Tragulus, the mouse deer, is considered the most primitive ruminant, with a diffuse placenta grossly quite unlike the cotyledonary type of the other ruminants. This immunocytochemical investigation of placental transporters was designed to elucidate possible mechanisms of evolution to the cotyledonary form. RESULTS AND DISCUSSION: Tragulus expresses several of the major transport systems characteristic of the ruminants: the trophoblast binucleate cell (BNC) dynamics, the requirement for two isoforms, GT1 and GT3, for glucose transport, the provision of Aquaporin 3 for water control, and uterine milk and histiotrophic secretion from uterine glands. However whereas the expression of the 9 kD Calcium Binding Protein (9 CBP) for calcium transport in ruminants is restricted to the intercotyledonary trophoblast with its areolae, Tragulus, having no intercotyledonary area, expresses 9 CBP throughout the villus trophoblast. There is some localised development of areolar-like structures in the mid term Tragulus but it is insignificant at term. The strong expression of Glucose Transporter 1 (GT1) in the BNC granules is unique to Tragulus. CONCLUSION: Tragulus relies on essentially similar transport and BNC dynamics as the other ruminants. Thus the evolutionary pressures driving the development of the cotyledonary placenta probably lie in the increase in body size and the consequent need for a larger placental area to ensure sufficient glucose for the fetus. The delivery in Tragulus of GT1 to the maternal facing side may be this species unique solution to maintain the glucose supply.

The trophoblast binucleate cell is the source of maternal circulating C-type natriuretic peptide during ovine pregnancy.

Maternal plasma concentrations of C-type natriuretic peptide (CNP) and a co-secreted bioinactive amino-terminal fragment (NTproCNP) are elevated during ovine pregnancy. Although the uteroplacental unit has been implicated as a likely source of CNP, the relative contributions of specific uterine and placental tissues, and identity of the cellular site/s of production remain unknown. Therefore, we measured CNP and NTproCNP in intercaruncular uterine tissue and maternal (caruncle) and fetal (cotyledon) placental tissues throughout gestation. Concentrations of CNP forms in placental tissues greatly exceeded those in intercaruncular uterine tissue throughout pregnancy (P < 0.05). Mean caruncular concentrations (CNP 32 ± 4, NTproCNP 56 ± 6 pmol g(-1)) peaked at day 60 whereas in the cotyledon there was a progressive increase in CNP forms to peak values (CNP 66 ± 6, NTproCNP 134 ± 9 pmol g(-1)) at day 100-135 followed by a sharp decline just prior to term (day 143). At term CNP gene expression was 6-fold greater in placental tissue compared with intercaruncular uterine tissue. Changes in maternal plasma concentration of CNP forms closely followed those in cotyledonary tissue whereas fetal plasma levels fell progressively throughout gestation. Immunohistochemistry revealed staining in binucleate cells (BNC) and around placental blood vessels. CNP's localization to the BNC suggests a novel endocrine role during pregnancy, in addition to its paracrine actions within the placental vasculature. The function of CNP in maternal circulation remains to be determined, but as proposed for other BNC products, may involve manipulation of maternal physiology and placental function to favour fetal growth.

Functional studies of the placenta of the lizard Mabuya sp. (Scincidae) using immunocytochemistry.

Most lizards lay eggs. However viviparity has evolved in the Squamata on many separate occasions by the process of extended retention of the egg coincident with gradual loss of the eggshell. This process is linked to reduction of the amount of yolk which is coupled with development of placental nutrient transfer. The family Scincidae currently show a range of multiple independent origins of viviparity and placentation along this pathway, and the genus Mabuya shows one of the most structurally complex placentas. This study investigates the transport potential of the different areas of the Mabuya placenta using immunocytochemistry to localize the systems in place for calcium, glucose and water transfer. The localization of these transporters demonstrated restricted distributions in the specialized areas of this morphologically complex placenta.

The glycosylation pattern of secretory granules in binucleate trophoblast cells is highly conserved in ruminants.

The binucleate trophoblast cells (BNCs) in the ruminant placenta are a unique feature of this taxon. These cells produce several secretory proteins and transfer these across the fetomaternal barrier into the dam. We used lectin histochemistry with a panel of 24 lectins to characterise the glycosylation pattern of BNC secretory granules in a variety of ruminants. Seven species out of three ruminant families were thus investigated: greater malayan chevrotain (Tragulidae); fallow deer, red deer, chinese water deer (Cervidae); and domestic goat, springbok, impala (Bovidae). BNC granules in all species studied strongly expressed tri-/tetraantennary complex N-glycans and bisecting N-acetylglucosamine [GlcNAc] as shown by binding of leuco- and erythroagglutins of Phaseolus vulgaris respectively. The presence of terminal N-acetylgalactosamine [GalNAc]) in BNC granules is shown by intense staining with lectins from Dolichos biflorus, Vicia villosa and Wisteria floribunda. Terminal galactose or GalNAc was also present, bound by Glycine max agglutinin. Treatment of slides with neuraminidase strongly intensified staining of Erythrina cristagalli lectin (ECA) to terminal lactosamine in all species studied; this was otherwise absent except in goat. Sambucus nigra-1 lectin bound to BNC granules in all species except in Impala, indicating the presence of abundant alpha2,6 linked sialic acid. These results indicate that these unusual highly branched glycans, with bisecting GlcNAc and terminal GalNAc are a general feature of BNC granules in Ruminants, including the most basal Tragulid branch. It therefore appears that the specific glycosylation pattern of BNC granules evolved early in ruminant phylogenesis, together with the appearance of BNC. The conserved glycan structure in BNC secretory granules indicates that this pattern of glycosylation is likely to be of considerable functional importance for the secretory glycoproteins of ruminant BNC.

Cardiac and vascular disease prior to hatching in chick embryos incubated at high altitude.

The partial contributions of reductions in fetal nutrition and oxygenation to slow fetal growth and a developmental origin of cardiovascular disease remain unclear. By combining high altitude with the chick embryo model, we have previously isolated the direct effects of high-altitude hypoxia on growth. This study isolated the direct effects of high-altitude hypoxia on cardiovascular development. Fertilized eggs from sea-level or high-altitude hens were incubated at sea level or high altitude. Fertilized eggs from sea-level hens were also incubated at high altitude with oxygen supplementation. High altitude promoted embryonic growth restriction, cardiomegaly and aortic wall thickening, effects which could be prevented by incubating eggs from high-altitude hens at sea level or by incubating eggs from sea-level hens at high altitude with oxygen supplementation. Embryos from high-altitude hens showed reduced effects of altitude incubation on growth restriction but not on cardiovascular remodeling. The data show that: (1) high-altitude hypoxia promotes embryonic cardiac and vascular disease already evident prior to hatching and that this is associated with growth restriction; (2) the effects can be prevented by increased oxygenation; and (3) the effects are different in embryos from sea-level or high-altitude hens.

Developmental programming of the ovine placenta.

The pattern of intrauterine growth and size at birth, in particular, programmes the structure and function of tissues later in life in many species, which has important implications for the incidence of adult-onset generative diseases in human populations. In mammals, the main determinant of intrauterine growth is the placental supply of nutrients which, in turn, depends on the size, morphology, transport characteristics and endocrine function of the placenta. However, compared to somatic tissues, little is known about the developmental programming of the placenta. This review examines the epigenetic regulation of placental phenotype with particular emphasis on the nutrient transfer capacity of the ovine placenta and environmental factors shown to cause developmental programming of other tissues. Overall, the placenta is responsive to environmental factors and uses a number of different strategies to adapt its phenotype to help support fetal growth during adverse intrauterine conditions. It is, therefore, not just a passive conduit for nutrient transfer to the fetus but alters its nutrient supply capacity dynamically to optimise fetal nutrient acquisition. Thus, the placental epigenome provides both a memory of environmental conditions experienced during development and an index of the future well being of the offspring.

A light and electron microscopical study of the Tragulid (mouse deer) placenta.

The Tragulidae are the living relics of the basal ruminant stock. They have a diffuse placenta, with no aggregations of the placental villi into localised placentomes characteristic of all other ruminants. Despite this difference, this ultrastructural and immunocytochemical investigation demonstrates that in Tragulus the trophoblast binucleate cell (BNC) plays the same central role in development and structure as in all other ruminants. It shows an identical development and ultrastructure, produces granules reactive with bovine placental lactogen and pregnancy associated glycoprotein antibodies, and migrates when mature through the trophoblast tight junction to fuse into a mosaic of syncytial plaques from which the granules are released to the mother and which have replaced the uterine epithelium. Unlike the persistent plaques in the sheep and goat placenta, in Tragulus they are transient, dying by apoptosis with the fragments phagocytosed by the trophoblast. This brings the trophoblast into direct endotheliochorial apposition to maternal tissue until BNC migration and fusion replace the dead plaque. This intimate fetomaternal confrontation has not been shown in any other ruminant, and could be a relic of the evolutionary development of the synepitheliochorial from the original basic eutherian endo- or hemo-chorial placenta.

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

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

Functional significance and cortisol dependence of the gross morphology of ovine placentomes during late gestation.

The gross morphological appearance of ovine placentomes is known to alter in response to adverse intrauterine conditions that increase fetal cortisol exposure. The direct effects of fetal cortisol on the placentome morphology, however, remain unknown, nor is the functional significance of the different placentome types clear. The present study investigated the gross morphology of ovine placentomes in relation to placental nutrient delivery to sheep fetuses during late gestation and after experimental manipulation of the fetal cortisol concentration. As fetal cortisol levels rose naturally toward term, a significant decrease was observed in the proportion of the D-type placentomes that had the hemophagous zone everted over the bulk of the placentomal tissue. When the prepartum cortisol surge was prevented by fetal adrenalectomy, there were proportionately more everted C- and D-type placentomes and fewer A-type placentomes with the hemophagous zone inverted into the placentome compared with those of intact fetuses at term. Raising cortisol concentrations by infusion before term reduced the incidence of D-type placentomes and lowered the proportion of individually tagged placentomes that became more everted during the 10- to 15-day period between tagging and delivery. Cortisol, therefore, appears to prevent hemophagous zone eversion in ovine placentomes during late gestation. The distribution of placentome types appeared to have no effect on the net rates of placental delivery of glucose and oxygen to the fetus under normal conditions. When fetal cortisol levels were raised by exogenous infusion, however, placental delivery of glucose, but not oxygen, to the fetus, measured as umbilical uptake, was reduced to a greater extent in fetuses with a higher proportion of C- and D-type placentomes. The gross morphology of the ovine placentomes is, therefore, determined, at least in part, by the fetal cortisol concentration and may influence placental nutrient transfer when fetal cortisol concentrations are high during late gestation. These findings have important implications for the placental control of fetal growth and development, particularly during adverse intrauterine conditions.

Light and electron microscope immunocytochemical studies of the distribution of pregnancy associated glycoproteins (PAGs) throughout pregnancy in the cow: possible functional implications.

Pregnancy associated glycoproteins (PAGs) comprise a large group of placental antigens of the aspartic proteinase family. Phylogenetic analysis indicated that the PAGs form two distinct groups, one of ancient origin and one produced by a more recent series of gene duplications. This paper summarises the molecular biological and biochemical studies which have been used to purify and raise antibodies against specific PAGs and groups of related PAGs and their use in light and electron microscope immunocytochemistry to demonstrate that the ancient PAG group has a similar distribution at the placental fetomaternal interface (microvillar junction, MVJ) in cows and pigs. This localization suggests either a possible role in binding the surfaces together and/or in establishment of an immunological barrier. The more recently evolved PAG group, absent in the pig, exhibited no significant localization to the MVJ but was restricted to the trophoblast binucleate cell (BNC) granules in the cow. Furthermore, these PAGs bind to newly formed maternal uterine connective tissue to which they are delivered by BNC migration and fusion with uterine epithelial cells. At this location in the developing maternal villi of the placentomes, they are ideally positioned to manipulate the maternal immune system to facilitate a successful pregnancy.

Localisation of glucose transport in the ruminant placenta: implications for sequential use of transporter isoforms.

The facilitative glucose transporters 1 and 3 are the major routes for glucose transport across placental membranes. Using light and electron microscope immunocytochemistry on acrylic sections this study shows a similar pattern of expression from mid to late pregnancy in all four ruminants examined [cow, deer, ewe and goat]. GT1 and GT3 are localised on different membrane layers of the synepitheliochorial placental barrier and glucose must utilise both isoforms sequentially to pass from the maternal to fetal circulations. It is suggested that this arrangement is designed to support the high glucose utilisation by the multilayered placenta in the ruminant.

Placentation in the African elephant, Loxodonta africanus: III. Ultrastructural and functional features of the placenta.

Successful transfer of nutrients to the elephant fetus during pregnancy relies on a variety of placental modifications. Our light and electron microscopical investigations show that the structure is endotheliochorial from implantation to term, with unicellular, never syncytial trophoblast. Light and electron microscope immunocytochemistry shows the restriction of the glucose transporter 1 isoform to the basolateral surfaces of the trophoblast, with the glucose transporter 3 restricted to the apical plasmalemma of the trophoblast. Glucose transport to the fetus therefore requires a sequential use of both isoforms. Light and electron microscope cytochemistry indicate the presence of iron deposits only in the haemophagous zones confirming their iron transport function. No trophoblast areas with high concentrations of Calcium binding protein, specialised for Calcium transport were found. In situ hybridisation demonstrated the presence of IGF-II mRNA in the trophoblast from the earliest stage, with TGFbeta1 and HGF-SF mRNA expressed subsequently but only IGF-II and HGF mRNA present in the second half of pregnancy. The results are briefly discussed in terms of placental growth and function and indicate that the elephant placenta is another example of a unique solution to the variety of problems posed by a resident fetus.