P. M. Coan

The Placenta and Intrauterine Programming

Intrauterine programming is the process by which the structure and function of tissues are altered permanently by insults acting during early development. In mammals, the placenta controls intrauterine development by supplying oxygen and nutrients, and by regulating the bioavailability of specific hormones involved in foetal growth and development. Consequently, the placenta is likely to have a key role in mediating the programming effects of suboptimal conditions during development. This review examines placental phenotype in different environmental conditions and places particular emphasis on regulation of placental nutrient transfer capacity and endocrine function by insults known to cause intrauterine programming. More specifically, it examines the effects of a range of environmental challenges on the size, morphology, blood flow and transporter abundance of the placenta and on its rate of consumption and production of nutrients. In addition, it considers the role of hormone synthesis and metabolism by the placenta in matching intrauterine development to the prevailing environmental conditions. The adaptive responses that the placenta can make to compensate for suboptimal conditions in utero are also assessed in relation to the strategies adopted to maximise foetal growth and viability at birth. Environmentally‐induced changes in placental phenotype may provide a mechanism for transmitting the memory of early events to the foetus later in gestation, which leads to intrauterine programming of tissue development long after the original insult.

Developmental Dynamics of the Definitive Mouse Placenta Assessed by Stereology

Abstract The mouse is an excellent model for studying the genetic basis of placental development, but analyses are restricted by the lack of quantitative data describing normal murine placental structure. This study establishes a technique for generating such data, applies stereological techniques on systematic uniform random sections of placentas between E12.5—E18.5 of gestation (E1.0 = day of the vaginal plug), and considers the results in the context of development of the labyrinth zone. Half of each placenta was wax embedded and exhaustively sectioned to determine absolute volumes of the labyrinth zone (Lz), junctional zone (Jz), and decidua using the Cavalieri principle. The other half was resin embedded and 1-μm sections were used to generate all volume, surface, and length densities within the Lz. Maximum placental volume is reached by E16.5, whereas the Lz volume fraction increases until E18.5 at the expense of the Jz and decidua. Within the Lz, the absolute volume and surface area of maternal blood spaces (MBS) expand rapidly between E14.5 and E16.5, with no increase thereafter. In contrast, fetal capillary development is linear and continues for longer than that of the MBS. The interhemal membrane separating maternal and fetal circulations undergoes thinning prior to expansion of maternal and fetal surface areas, achieving a harmonic mean thickness of 4.39 μm by E18.5. The specific diffusion capacity for oxygen of the interhemal membrane is maximal by E16.5, which may be necessary to support rapid fetal growth until the end of gestation.

Placental efficiency and adaptation: endocrine regulation

Size at birth is critical in determining life expectancy and is dependent primarily on the placental supply of nutrients. However, the fetus is not just a passive recipient of nutrients from the placenta. It exerts a significant acquisitive drive for nutrients, which acts through morphological and functional adaptations in the placenta, particularly when the genetically determined drive for fetal growth is compromised by adverse intrauterine conditions. These adaptations alter the efficiency with which the placenta supports fetal growth, which results in optimal growth for prevailing conditions in utero. This review examines placental efficiency as a means of altering fetal growth, the morphological and functional adaptations that influence placental efficiency and the endocrine regulation of these processes.

Origin and characteristics of glycogen cells in the developing murine placenta.

The junctional zone (Jz) of the mouse placenta consists of two main trophoblast populations, spongiotrophoblasts and glycogen cells (GCs), but the development and function of both cell types are unknown. We conducted a quantitative analysis of GC size, number, and invasion of cells into the decidua across gestation. Furthermore, we identified markers of GC function to investigate their possible roles in the placenta. While the spongiotrophoblast cell volume doubles, and cell number increases steadily from E12.5 to E16.5, there is a remarkable 80‐fold increase in GC numbers. This finding is followed by a notable decrease by E18.5. Surprisingly, the accumulation of GCs in the decidua did not fully account for the decrease in GC number in the Jz, suggesting loss of GCs from the placenta. Glucagons were detected on GCs, suggesting a steady glucose release throughout gestation. Connexin31 staining was shown to be specific for GCs. GC migration and invasion may be facilitated by temporally regulated expression of matrix metalloproteinase 9 and the imprinted gene product, Decorin. Expression of the clearance receptor for type II insulin‐like growth factor (IGF‐II), IGF2R, in a short developmental window before E16.5 may be associated with regulating the growth effects of IGF‐II from glycogen cells and/or labyrinthine trophoblast on the expansion of the Jz. Thus stereology and immunohistochemistry have provided useful insights into Jz development and function of the glycogen cells. Developmental Dynamics 235:3280–3294, 2006.

Adaptations in placental phenotype support fetal growth during undernutrition of pregnant mice

Undernutrition during pregnancy reduces birth weight and programmes adult phenotype with consequences for life expectancy, but its effects on the phenotype of the placenta, responsible for supplying nutrients for fetal growth, remain largely unknown. Using molecular, morphological and functional analyses, placental phenotype was examined in mice during restriction of dietary intake to 80% of control from day 3 of pregnancy. At day 16, undernutrition reduced placental, but not fetal, weight in association with decreased junctional zone volume and placental expression of glucose transporter Slc2a1. At day 19, both placental and fetal weights were reduced in undernourished mice (91% and 87% of control, respectively, P < 0.01), as were the volume and surface area of the labyrinthine zone responsible for placental nutrient transfer (85% and 86%, respectively, P < 0.03). However, unidirectional materno‐fetal clearance of tracer glucose was maintained and methyl‐aminoisobutyric acid increased 166% (P < 0.005) per gram of undernourished placenta, relative to controls. This was associated with an 18% and 27% increased placental expression of glucose and system A amino acid transporters Slc2a1 and Slc38a2, respectively, at day 19 (P < 0.04). At both ages, undernutrition decreased expression of the placental specific transcript of the Igf2 gene by 35% (P < 0.01), although methylation of its promoter was unaffected. The placenta, therefore, adapts to help maintain fetal growth when its own growth is compromised by maternal undernutrition. Consequently, placental phenotype is responsive to environmental conditions and may help predict the risk of adult disease programmed in utero.

Adaptations in placental nutrient transfer capacity to meet fetal growth demands depend on placental size in mice

Experimental reduction in placental growth often leads to increased placental efficiency measured as grams of fetus produced per gram of placenta, although little is known about the mechanisms involved. This study tested the hypothesis that the smallest placenta within a litter is the most efficient at supporting fetal growth by examining the natural intra‐litter variation in placental nutrient transfer capacity in normal pregnant mice. The morphology, nutrient transfer and expression of key growth and nutrient supply genes (Igf2P0, Grb10, Slc2a1, Slc2a3, Slc38a1, Slc38a2 and Slc38a4) were compared in the lightest and heaviest placentas of a litter at days 16 and 19 of pregnancy, when mouse fetuses are growing most rapidly in absolute terms. The data show that there are morphological and functional adaptations in the lightest placenta within a litter, which increase active transport of amino acids per gram of placenta and maintain normal fetal growth close to term, despite the reduced placental mass. The specific placental adaptations differ with age. At E16, they are primarily morphological with an increase in the volume fraction of the labyrinthine zone responsible for nutrient exchange, whereas at E19 they are more functional with up‐regulated placental expression of the glucose transporter gene, Slc2a1/GLUT1 and one isoform the System A family of amino acid transporters, Slc38a2/SNAT2. Thus, this adaptability in placental phenotype provides a functional reserve capacity for maximizing fetal growth during late gestation when placental growth is compromised.

Differential regulation of imprinting in the murine embryo and placenta by the Dlk1-Dio3 imprinting control region.

Genomic imprinting is an epigenetic mechanism controlling parental-origin-specific gene expression. Perturbing the parental origin of the distal portion of mouse chromosome 12 causes alterations in the dosage of imprinted genes resulting in embryonic lethality and developmental abnormalities of both embryo and placenta. A 1 Mb imprinted domain identified on distal chromosome 12 contains three paternally expressed protein-coding genes and multiple non-coding RNA genes, including snoRNAs and microRNAs, expressed from the maternally inherited chromosome. An intergenic, parental-origin-specific differentially methylated region, the IG-DMR, which is unmethylated on the maternally inherited chromosome, is necessary for the repression of the paternally expressed protein-coding genes and for activation of the maternally expressed non-coding RNAs: its absence causes the maternal chromosome to behave like the paternally inherited one. Here, we characterise the developmental consequences of this epigenotype switch and compare these with phenotypes associated with paternal uniparental disomy of mouse chromosome 12. The results show that the embryonic defects described for uniparental disomy embryos can be attributed to this one cluster of imprinted genes on distal chromosome 12 and that these defects alone, and not the mutant placenta, can cause prenatal lethality. In the placenta, the absence of the IG-DMR has no phenotypic consequence. Loss of repression of the protein-coding genes occurs but the non-coding RNAs are not repressed on the maternally inherited chromosome. This indicates that the mechanism of action of the IG-DMR is different in the embryo and the placenta and suggests that the epigenetic control of imprinting differs in these two lineages.

Disproportional effects of Igf2 knockout on placental morphology and diffusional exchange characteristics in the mouse

Both complete knockout of the Igf2 gene (Igf2null+/−) and knockout of its placental specific transcript alone (Igf2P0+/−) lead to fetal growth restriction in mice. However, in the Igf2null+/− this growth restriction occurs concurrently in gestation with placental growth restriction, whereas, placental growth restriction precedes fetal growth restriction in the Igf2P0+/− mouse. Previous studies have shown that the Igf2P0+/− placenta has proportionate reductions in its cellular compartments and its diffusional exchange characteristics. Yet, nothing is known about the structural development or diffusional exchange characteristics of the Igf2null+/− mouse. Hence, this study compares the structural properties (using stereology) and diffusional exchange characteristics (using measurement of permeability–surface area product, P.S, of three inert hydrophilic tracers) of the Igf2null+/− and the Igf2P0+/− placenta to identify the role of Igf2 in the development of the labyrinthine exchange membrane and its functional consequences. Our data show disproportionate effects of complete Igf2 ablation on the compartments of the placenta, not seen when the placental‐specific transcript alone is deleted. Furthermore, although the theoretical diffusing capacity (calculated from the stereological data) of the Igf2null+/− placenta was reduced relative to control, there was no effect of the complete knockout on permeability surface area available for small hydrophilic tracers. This is in contrast to the Igf2P0+/− placenta, where theoretical diffusion capacity and P.S values were reduced similarly. Total ablation of the Igf2 gene from the fetoplacental unit in the mouse therefore results in a disproportionate growth of placental compartments whereas, deleting the placental specific transcript of Igf2 alone results in proportional placental growth restriction. Thus, placental phenotype depends on the degree of Igf2 gene ablation and the interplay between placental and fetal Igf2 in the mouse.

Placental-specific Igf2 deficiency alters developmental adaptations to undernutrition in mice.

The pattern of fetal growth is a major determinant of the subsequent health of the infant. We recently showed in undernourished (UN) mice that fetal growth is maintained until late pregnancy, despite reduced placental weight, through adaptive up-regulation of placental nutrient transfer. Here, we determine the role of the placental-specific transcript of IGF-II (Igf2P0), a major regulator of placental transport capacity in mice, in adapting placental phenotype to UN. We compared the morphological and functional responses of the wild-type (WT) and Igf2P0-deficient placenta in WT mice fed ad libitium or 80% of the ad libitium intake. We observed that deletion of Igf2P0 prevented up-regulation of amino acid transfer normally seen in UN WT placenta. This was associated with a reduction in the proportion of the placenta dedicated to nutrient transport, the labyrinthine zone, and its constituent volume of trophoblast in Igf2P0-deficient placentas exposed to UN on d 16 of pregnancy. Additionally, Igf2P0-deficient placentas failed to up-regulate their expression of the amino acid transporter gene, Slc38a2, and down-regulate phosphoinositide 3-kinase-protein kinase B signaling in response to nutrient restriction on d 19. Furthermore, deleting Igf2P0 altered maternal concentrations of hormones (insulin and corticosterone) and metabolites (glucose) in both nutritional states. Therefore, Igf2P0 plays important roles in adapting placental nutrient transfer capacity during UN, via actions directly on the placenta and/or indirectly through the mother.

Ultrastructural changes in the interhaemal membrane and junctional zone of the murine chorioallantoic placenta across gestation

The mouse is an extremely useful experimental model for the study of human disease owing to the ease of genetic and physiological manipulation. A more detailed knowledge of murine placental development will, we hope, increase our understanding of the pathogenesis of placentally related complications of human pregnancy. The murine placenta consists of two main fetally derived compartments: the labyrinthine zone and the junctional zone. Exchange in the labyrinthine zone takes place across an interhaemal membrane comprising an outer layer of cytotrophoblast cells and two inner layers of syncytial trophoblast. The cytotrophoblast layer thins as gestation advances, and in addition becomes highly perforated after embryonic day (E)12.5. Furthermore, as gestation advances cytotrophoblast nuclear volume and DNA content increase, suggesting the formation of labyrinthine trophoblast giant cells. The syncytial layers become increasingly microvillous, enlarging the surface area for exchange. Separate basement membranes support the syncytium and the fetal capillary endothelium throughout gestation, although these appear to fuse where the capillaries are closely approximated to the trophoblast. The junctional zone consists of two principal trophoblast cell types, spongiotrophoblasts and invasive glycogen cells, yet the functions of each remain elusive. Spongiotrophoblasts vary in their appearance even when not fully differentiated, but a striking feature is the extensive endoplasmic reticulum of the more mature cells. Early glycogen cells are distinguished by the presence of electron‐dense glycogen granules, and large amounts of surrounding extracellular matrix. Later the accumulations of glycogen granules occupy almost all the cytoplasm and there are few organelles. This is the first study to use both scanning and transmission electron microscopy in an ultrastructural description of murine placental development and is complementary to contemporary genetic investigations.