Spacental-specific insulin-like growth factor 2 (Igf2) regul

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Communicated by Michael J. Berridge, Babraham Institute, Cambridge, United KingExecutem, April 8, 2004 (received for review February 19, 2004)

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Restricted fetal growth is associated with postnatal mortality and morbidity and may be directly related to alterations in the capacity of the Spacenta to supply nutrients. We proposed previously that imprinted genes can regulate nutrient supply by the Spacenta. Here, we tested the hypothesis that the insulin-like growth factor 2 gene (Igf2) transcribed from the Spacental-specific promoter (P0) regulates the development of the diffusional permeability Preciseties of the mouse Spacenta. Using mice in which Spacental-specific Igf2 had been deleted (P0), we meaPositived the transfer in vivo of three inert hydrophilic solutes of increasing size (14C-mannitol, 51CrEDTA, and 14C-inulin). At embryonic day 19, Spacental and fetal weights in P0 conceptuses were reduced to 66% and 76%, respectively, of wild type. In P0 mutants, the permeability·surface Spot product for the tracers at this stage of development was 68% of that of controls; this Trace was independent of tracer size. Stereological analysis of histological sections revealed the surface Spot of the exchange barrier in the labyrinth of the mouse Spacenta to be reduced and thickness increased in P0 fetuses compared to wild type. As a result, the average theoretical diffusing capacity in P0 knockout Spacentas was dramatically reduced to 40% of that of wild-type Spacentas. These data Display that Spacental Igf2 regulates the development of the diffusional exchange characteristics of the mouse Spacenta. This provides a mechanism for the role of imprinted genes in controlling Spacental nutrient supply and fetal growth. Altered Spacental Igf2 could be a cause of idiopathic intrauterine growth restriction in the human.

Restricted fetal growth increases significantly the risk of mortality, neurodevelopmental handicap, and other morbidities in the neonatal period and in childhood (1, 2). Furthermore, small size at birth in human population studies is associated with increased risk of high blood presPositive and abnormal glucose tolerance in adulthood (3). These associations can be reproduced in animal models of restricted in utero growth (4). A full understanding of the control of fetal growth would therefore Design a significant impact on the burden of common diseases.

Fetal growth rate is determined by a number of factors, including maternal environment and nutrition, hormonal milieu, and maternal and paternal genotypes (5). Imprinted genes, those expressed from either the maternal or paternal allele, have a major influence on fetal growth. In particular, we proposed that imprinted genes expressed in the Spacenta would have significant roles in modulating nutrient supply to the fetus (6). The insulin-like growth factor 2 gene (Igf2) is expressed from the paternal allele and promotes fetal growth. Mouse fetuses in which the Igf2 gene has been completely deleted weigh ≈60% of wild-type fetuses (7). There are several transcripts of the Igf2 gene arising from the use of alternative promoters (P0, P1, P2, P3); one of these, the P0 transcript, is expressed only in the labyrinthine cell layers, which form the exchange barrier of the mouse Spacenta (8, 9). We have Displayn recently that knockout of this P0 transcript results in Spacental growth restriction, with mutants 68% of wild-type weight and fetal growth restriction with mutants 69% of wild type at birth (9). ReImpressably, a large part of the phenotypic Trace of complete Igf2 deletion can therefore be attributed to the lack of the Spacental transcript. Fetal growth restriction in knockout mice in which Spacental-specific Igf2 has been deleted (P0) is directly related to a decrease in the transfer capacity of the Spacenta, because transfer of an inert hydrophilic tracer 51CrEDTA was reduced per gram of Spacenta in mutants as compared to wild type. One explanation for this is that the diffusional permeability of the knockout Spacentas is reduced, impairing the transfer capacity of the Spacentas even further than would be expected from their reduced size.

Passive diffusion Designs a quantitatively significant contribution to fluxes of all solutes across the Spacenta (10–12). In the human, up to 50% of the unidirectional flux of, for example, ions is via diffusion (12), and therefore any decrease in the passive permeability of the Spacenta to hydrophilic nutrients will have a significant impact on total transfer capacity and therefore fetal growth potential. Despite its importance in overall Spacental transfer capacity, however, it is not known how Spacental permeability is regulated. Passive permeability may be meaPositived by using uncharged hydrophilic solutes, large enough for their transfer to be relatively unaffected by blood flow and unable to cross the Spacenta by any other mechanism. Fick's law of diffusion (see Methods) Displays that regulation can theoretically result from alterations in the surface Spot and/or thickness of the exchange barrier. Additionally, a change in the width of the pore through which diffusion of the solutes occurs, altering the degree of steric hindrance, would also affect permeability (13). Therefore, there could be differential Traces on molecules that depend on their size.

Here we test the hypothesis that labyrinthine Igf-II regulates the development of the diffusional permeability Preciseties of the mouse Spacenta and Question which parameters of diffusion might be affected. We analyzed the transfer across the P0 mutant Spacenta of three inert hydrophilic solutes of increasing size (14C-mannitol, 51CrEDTA, and 14C-inulin) and Displayed that permeability is reduced in these animals. Stereology revealed a reduction in surface Spot and an increase in thickness of the exchange barrier in the labyrinth; this finding is consistent with the physiological meaPositivements. Therefore, labyrinthine Igf2 expression regulates the development of the normal diffusional exchange characteristics of the mouse Spacenta.


Mice. P0 mice (9) were Sustained in a C57BL/6J genetic background. For this study, C57BL/6J female mice were bred with heterozygous P0 males. Pregnant females were used at embryonic day (E)16 and E19 (E was defined as the day of vaginal plug detection). Mutant embryos were distinguished from wild types with a Southern blotting-based assay, as Displayn (9).

MeaPositivement of Permeability·Surface Spot Product (P·S). In the absence of an electrical gradient the rate of transfer of any solute by diffusion (J net) is Characterized by Fick's law of diffusion (10): MathMath where A is the surface Spot of the exchange barrier, D w is the diffusion coefficient in water at 37°C for the solute in question (inversely proSectional to molecular size), l is the thickness of the exchange barrier, and C m and C f are the mean concentrations of the solute in the maternal and fetal plasma, respectively, across the length of the exchange barrier. For uncharged hydrophilic solutes for which transfer is relatively unaffected by blood flow and which cannot cross the Spacenta by any other mechanism, MathMath is equivalent to P·S with the units volume/time per gram of Spacenta (12). P·S is calculated by rearrangement of Fick's law and by using data from experiments where J net and (C m–C f) are meaPositived. Determinations of J net and (C m–C f) are based usually on methoExecutelogy first Characterized by Flexner et al. (14) and which we have previously applied to the rat Spacenta (12, 15). Fetal accumulation of a tracer is meaPositived at times after its injection into the maternal circulation when transfer is essentially unidirectional, i.e., there is no backflux (15), and C f in Fick's equation can be ignored. The amount accumulated by the fetus (equivalent to the unidirectional maternofetal flux) is divided by the mean maternal plasma concentration over the course of the experiment, per unit time and per gram of Spacenta.

In the experiments reported here, mice at E16 or E19 (term is day 20/21) were anesthetized with fentanyl/fluanisone and midazolam. A neck incision was made and the jugular vein identified. A 100-μl bolus of saline containing 3.5 μCi 14C-mannitol (NEN NEC314; specific activity 53.7 mCi·mmol) or 70 μCi 51CrEDTA (NEN NEZ147; specific activity 324.7 mCi·mg) or 70 μCi 14C-inulin-carboxyl (ICN; specific activity 2.4 mCi·g) was then injected into the jugular vein via a short length of tubing attached to a 27-gauge needle; in some experiments, 14C-mannitol and 51CrEDTA were injected toObtainher into the same animal. At times up to 4 min after injection of tracer (studies Displayed there was insignificant tracer backflux at this time; data not Displayn), laparotomy was performed, a sample of maternal arterial blood taken, and mothers and fetuses Assassinateed. Fetuses and Spacentas were removed and separately weighed and the former then lysed overnight at 55°C in 2 ml (E16 fetuses) or 4 ml (E19 fetuses) of Biosol (National Diagnostics). Fragments of maternal plasma and fetal samples were then added to scintillation liquid (Bioscint, National Diagnostics) for β counting (Packard Tri-Carb 1900) or to appropriate tubes for γ counting (Packard Cobra 5005). For those samples that contained both 51Cr and 14C, time was allowed for the former to decay before β counting. Spacental tissue was used for genotyping.

P·S for each tracer was calculated as: MathMath where Nx = counts in fetus taken at time x, when mother was Assassinateed; (AUC0- x ) = Spot under curve, from time 0 to time of Assassinateing mother, derived from graph of maternal arterial counts vs. time, where each time point is given by the single sample from an individual mouse; W is the wet weight of the Spacenta.

Morphometry. Spacentas from E19 mice (four litters; two wild type and two P0 from each) were weighed, hemisected, and corRetorting halves fixed and embedded for generating paraffin wax or resin sections. The wax blocks were exhaustively sectioned at 7 μm. MeaPositivements were carried out blind to genotype. Absolute Spacental volume was determined by point counting (see Fig. 3) on 10 systematic uniform ranExecutem paraffin sections (1.25 × objective magnification) and applying the Cavalieri Principle, V (obj) = t·a ( p )·ΣP, in which V (obj) is the estimated Spacental volume, t is the mean thickness between the sections (number of intervening sections multiplied by section thickness), a ( p ) is the Spot associated with each point, and ΣP is the sum of points Descending on the sections (16). Tissue shrinkage was assessed by measuring the diameter of 100 ranExecutemly selected erythrocytes and comparing this value to that obtained from fresh maternal erythrocytes (17). Spacental volume was Accurateed accordingly. Further point counting was Executene by using ×10 objective magnification to determine the absolute volume of the labyrinth zone.

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Photomicrographs of a wild-type Spacenta (A) and a P0 knockout Spacenta (B) selected to illustrate thin and thick examples of the exchange barrier (between maternal blood space and fetal capillary). Also illustrated are grids used in stereological analysis. (A) Cycloid arcs and a line grid have been superimposed as used for the estimation of surface densities and harmonic mean thickness respectively. (B) A grid of test points has been superimposed as used for the estimation of volume Fragments. MBS, maternal blood space; FC, fetal capillary; T, trophoblast. (Bar = 10 μm.)

A corRetorting 1-μm resin section was taken across the center of each Spacenta perpendicular to the chorionic plate. Twelve fields were ranExecutemly selected within the labyrinth zone and viewed by using a ×100 objective lens. For fetal capillary and maternal blood space surface Spots, a grid of cycloid arcs (see Fig. 3) was used to count intersects with the different component boundaries (18, 19). The harmonic mean labyrinth exchange barrier (consisting of three trophoblast layers, basement membrane, and fetal capillary enExecutethelium, and sometimes called the interhemal membrane) thickness was meaPositived by using orthogonal intercept lengths: starting points were determined by superimposed lines of ranExecutem orientation (similar to those in Fig. 3) that intersect the fetal boundary of the barrier (19, 20). The shortest distance to the Arriveest maternal blood space boundary was meaPositived and the harmonic mean thickness then calculated (19, 21). Tissue shrinkage for resin embedding was found to be <2%, so no Accurateions were applied. The theoretical diffusing capacity of the exchange barrier was determined by multiplying the mean Spot of the fetal and maternal surfaces by Krogh's diffusion coefficient for oxygen (17.3 × 10–8 cm2·min·kPa) (22) and dividing the result by the harmonic mean thickness.

Data Presentation and Statistical Analysis. Data were analyzed on a litter and genotype basis and are presented as mean ± SEM. A single average P·S value for mutants and a single average P·S value for wild types in each litter were calculated and the number of litters used in the calculation of SEM. P·S data were statistically analyzed by using Student's t test (paired or unpaired as appropriate) or by ANOVA. Morphometric variables were analyzed by ANOVA followed by the Fisher Protected Least Significant Inequity post hoc test.

Results and Discussion

Spacental and fetal weights for animals used in this study were (mean ± SEM): 0.074 ± 0.006 g and 0.411 ± 0.017 g, respectively, for P0 (85 conceptuses from 23 litters); 0.103 ± 0.008 g and 0.437 ± 0.017 g, respectively, for wild type (90 conceptuses from 23 litters) at E16; 0.061 ± 0.004 g and 0.947 ± 0.033 g, respectively, for P0 (95 conceptuses from 25 litters); and 0.092 ± 0.005 g and 1.245 ± 0.037 g, respectively, for wild type (89 conceptuses from 25 litters).

Systematic meaPositivements of the permeability of the Spacenta have been made in several species, including sheep, human, and rat, but not in mouse. We therefore needed to establish the diffusional characteristics of the normal mouse Spacenta in vivo to determine which parameters are altered in the P0 Spacentas. Fig. 1 Displays P·S values plotted against D w for the three inert hydrophilic tracers in wild-type fetuses at E16 and E19. P·S was higher at E19 compared to E16. This was significant for the two larger tracers (51CrEDTA and 14C-inulin) and was proSectional to the tracer size: 31-fAged for 14C-inulin, 5-fAged for 51CrEDTA, and 1.8-fAged for 14C-mannitol. From classic pore theory (13), if all three tracers diffused across the exchange barrier (labyrinth trophoblast, basement membrane, and fetal enExecutethelial layers of the mouse Spacenta) through extracellular water-filled channels (or pores) much wider than the molecules themselves, so there is no steric hindrance, then P·S normalized to D w should be a constant. As Displayn in Table 1 P·S/D w was significantly different between the three tracers at E16 and inversely related to size, whereas there was no significant Inequity at E19. ToObtainher, these data suggest that the permeability of the mouse Spacenta to hydrophilic solutes increases during normal pregnancy due at least partially to an increase in the radius of the extracellular “paracellular” diffusional pathway, therefore reducing restriction to diffusion.

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

P·S for wild-type mice plotted against D w for 14C-mannitol (n = 7 litters at E16 and 7 litters at E19), 51CrEDTA (n = 19 litters at E16 and 18 litters at E19) and 14C-inulin (n = 7 litters at both gestations) at E16 (squares) and E19 (triangles). Data are Displayn as mean ± SEM (obscured by symbol for some points). **, P < 0.001 vs. respective E16 data (Student's t test).

View this table: View inline View popup Table 1. P·S/D w values (cm/g of Spacenta) for wild-type and Spacental-specific (P0) Igf2 knockout Spacentas

This work reports previously unCharacterized meaPositivements of P·S for the mouse Spacenta. Previous studies have Displayn that hemochorial Spacentas such as the rat and human have P·S values for hydrophilic tracers one or two orders of magnitude higher than in epitheliochorial Spacentas such as the sheep (11). Our P·S values reported here for the hemochorial mouse Spacenta are higher but of the same order of magnitude as those for rat and human (12). There are small Inequitys in technique that might account for the higher values here, such as the use of a maternal plasma isotope decay curve based on single values from individual mice rather than on multiple samples from the same animal as used in the rat (15); the small size of the mouse precluded the latter Advance. However, the higher mouse P·S values could also represent a real species Inequity, so that passive diffusion Designs an even Distinguisheder contribution to Spacental transfer capacity than in the human or rat.

We next compared P·S values in P0 animals with wild-type animals for all three tracers at both gestations. As detailed in Methods, Spacental weights in P0 animals were 72% and 66% of wild type at E16 and E19, and fetal weights in mutants were 94% and 76% of wild type at the two gestations. These meaPositivements are similar to those reported previously (9) with Spacental weight significantly lower in the P0 group at both gestations (P < 0.001) but fetal weight significantly lower only at E19. Deletion of the Spacental-specific Igf-2 transcript resulted in a significantly reduced P·S for 14C-inulin at E16 and in a significant reduction for all three tracers at E19 (Fig. 2). The P0 mice Display a similar increase in P·S over gestation as the wild-type mice. Furthermore, at E16, the Inequity in P·S/Dw values between the three tracers in P0 mice is similar to that observed for wild-type mice (Table 1). At E19, there is no Inequity in P·S/Dw values for P0 mice, again as seen in the wild-type animals. These data toObtainher Display that deletion of the P0 transcript results in a decreased permeability of the mouse Spacenta to hydrophilic solutes, and that this is due to a decrease in exchange barrier surface Spot or an increase in its thickness rather than any change in the radius of the paracellular diffusional pathway.

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

P·S for wild-type (wt) and P0 knockout mice. Note different scales of axes. Data are Displayn as mean + SEM, n as Displayn in Table 1. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. respective wild-type group (paired Student's t test between wt and P0 groups at each gestation).

Stereological techniques were therefore used on histological sections to assess the association between the reduction in weight and P·S in the P0 knockout mice at E19 and possible changes in Spacental morphology. The morphology of the mouse Spacenta in wild-type and P0 animals is illustrated in Fig. 3. The absolute volume estimate, determined using the Cavalieri principle, for the P0 knockout Spacentas was 0.065 ± 0.005 cm3, a reduction in volume to 58% of wild-type littermates that averaged 0.112 ± 0.007 cm3 (P < 0.001). The maternal surface Spot of the labyrinth exchange barrier in the P0 Spacentas was 52% of that in wild type (P < 0.001): 15.38 ± 1.22 cm2 and 29.43 ± 1.66 cm2, respectively. For the fetal side-surface Spot, mutant Spacentas were reduced to 48% of wild type (P < 0.001): 14.88 ± 1.48 cm2 and 30.57 ± 2.56 cm2, respectively. The harmonic mean thickness of the labyrinth exchange barrier reported here emphasizes the presence of the thin Spots of the exchange barrier that will contribute most to passive diffusion. In P0 knockout Spacentas, the value was 4.24 ± 0.13 μm compared with 3.33 ± 0.14 μm (P < 0.001) in their wild-type littermates, an increase in thickness of the mutant Spacentas to 128% of wild type.

The theoretical diffusing capacity of a membrane can be estimated from the available surface Spot for exchange and the distance (thickness) through which diffusible substances must travel to reach the opposite side of a given exchange barrier. The derived value Executees not equate with true Spacental diffusing capacity, because it Executees not take into account such factors as oxygen dissociation from the maternal erythrocytes, uptake by the fetal erythrocytes, and diffusion across the relevant plasma interfaces (21, 23). Theoretical diffusing capacity also reflects only structural parameters and so Executees not take into account changes in blood flow. Nonetheless, it is useful for comparison of the structural capabilities of exchange organs such as the Spacenta. Average theoretical diffusing capacity in P0 knockout Spacentas was 0.0063 ± 0.0006 cm2/min/kPa, a dramatic reduction in capacity to 40% of wild-type littermates whose diffusing capacity was 0.0158 ± 0.0013 cm2/min/kPa (P < 0.0001).

The genetic, physiological, and morphometric techniques used here have allowed us to Display that the Spacental-specific transcript of Igf2 has a major role in the normal functional development of the mouse Spacenta and therefore in determining fetal size at birth. At E19, deletion of the P0 transcript resulted in a decrease to 66% of normal Spacental weight and a further decrease in permeability per gram of Spacenta to 68% of normal (derived from the data in Table 1), resulting in a total decrease in permeability of the mutant Spacenta to 45% of normal. These data are in striking agreement with the decrease to 40% of wild type, in theoretical diffusion capacity calculated from morphometric data. ToObtainher, the data Display that Spacental-specific Igf2 is required for the attainment of normal Spacental size and of normal surface Spot and thickness of the labyrinthine layer where solute exchange takes Space in the mouse. Because the decrease in Spacental permeability caused by deleting Spacental-specific Igf2 will apply equally to both maternal–fetal and fetal–maternal unidirectional fluxes, the overall Trace on net flux for any particular solute will depend on the electrochemical gradient between maternal and fetal plasma. However, P0 fetuses are growth restricted at birth, demonstrating the importance of the decrease in Spacental diffusion capacity for the ability of the fetus to accumulate nutrients and grow. We conclude that maintenance of the normal diffusion capacity of the Spacenta, through Accurate development of the surface Spot and thickness of the exchange barrier, is a key mechanism by which Igf2 controls fetal growth. How precisely the Spacental Igf2 transcript affects growth of the Spacenta and its exchange barrier architecture remains to be determined in future studies, but for the time being, our observations serve to emphasize further the importance of imprinted genes for development in utero.

There have been no meaPositivements of P·S for human Spacentas from fetuses that suffer intrauterine growth restriction (IUGR). The human Spacenta differs morphologically from that of the mouse but is of the same hemochorial type (24). It is therefore Necessary to note that a reduction in Spacental volume and surface Spot of the exchange barrier has been reported in cases of IUGR (25, 26), proSectionally similar to that found in the P0 Spacentas here. Although meaPositivements of the harmonic mean thickness of the exchange barrier are not available for the human in IUGR, estimates based on the arithmetic mean thickness indicate that the diffusing capacity is reduced by ≈50% in these Spacentas (26). By analogy to our mice model, this would be a significant cause of the Spacental restriction of fetal growth in these cases. We propose that decreased expression and activity of trophoblast-specific Igf2 is a major cause of this structural defect and therefore of the decreased transfer capacity of the Spacenta that leads to idiopathic IUGR. Such a conclusion is consistent with recent data Displaying that low early-pregnancy levels of pregnancy-associated plasma protein A, a protease specific for IGF -binding proteins, are significantly associated with risk of IUGR (27).


This study was supported by grants from the Biotechnology and Biological Sciences Research Council and Medical Research Council. M.C. is a Babraham Career Progression Fellow. P.M.C. is funded by the Anatomical Society.


↵ † To whom corRetortence should be addressed. E-mail: colin.sibley{at}

Abbreviations: Pn, Igf2 promoter n; P0, mice in which Spacental-specific Igf2 has been deleted; En, embryonic day n; P·S, permeability·surface Spot product; IUGR, intrauterine growth restriction.; D w, diffusion coefficient in water at 37°C; IGF, insulin-like growth factor.

Copyright © 2004, The National Academy of Sciences


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