Non-technical summary
Studies on mice using severe diets show alterations in placental function, and fetal and adult health. However, little is known about the effects of mild dietary variations on the placenta. We investigated placental growth and function in mice fed diets with similar energy, but small differences in protein and sugar content. We show that placental adaptations occur to help support fetal growth: reduced protein leads to increased glucose transport and transporter gene expression in late pregnancy; just prior to term, amino acid transport expression correlated with protein intake; the placental endocrine compartment was smaller with the least dietary protein and somewhat larger with slight reduction in protein. Placentas in mice fed the least protein were better adapted than those exposed to slight protein reduction. These results may provide a good index of conditions in the womb and have important implications for the pre-birth programming of life expectancy.
Abstract
Abstract
Dietary composition during pregnancy influences fetal and adult phenotype but its effects on placental phenotype remain largely unknown. Using molecular, morphological and functional analyses, placental nutrient transfer capacity was examined in mice fed isocaloric diets containing 23%, 18% or 9% casein (C) during pregnancy. At day 16, placental transfer of glucose, but not methyl-aminoisobutyric acid (MeAIB), was greater in C18 and C9 than C23 mice, in association with increased placental expression of the glucose transporter Slc2a1/GLUT1, and the growth factor Igf2. At day 19, placental glucose transport remained high in C9 mice while MeAIB transfer was less in C18 than C23 mice, despite greater placental weights in C18 and C9 than C23 mice. Placental System A amino acid transporter expression correlated with protein intake at day 19. Relative growth of transport verses endocrine zones of the placenta was influenced by diet at both ages without changing the absolute volume of the transport surface. Fetal weight was unaffected by diet at day 16 but was reduced in C9 animals by day 19. Morphological and functional adaptations in placental phenotype, therefore, occur to optimise nutrient transfer when dietary composition is varied, even subtly. This has important implications for the intrauterine programming of life expectancy.
Introduction
In developed countries, the diet is rich in fat and sugar, whereas in developing countries it is more often low in protein and high in complex carbohydrates. These dietary differences during pregnancy not only affect birth weight and neonatal viability but may also have longer term consequences for adult morbidity and mortality globally (Barker, 1994). In experimental animals, manipulations of the protein, fat and carbohydrate content in the maternal diet have all been shown to programme intrauterine development with important sequelae for cardiovascular, metabolic and endocrine function of the adult offspring in several species (Fowden et al. 2006; Warner & Ozanne, 2010). In particular, it is the postnatal consequences of feeding isocalorific low protein-high carbohydrate diets during pregnancy that have been studied most extensively (Langley-Evans, 2000; Reusens & Remacle, 2009). In rats, these low protein diets alter development of a wide range of tissues and lead to hypertension, glucose intolerance, obesity and premature ageing in the adult offspring (Gardner et al. 1997; Erhuma et al. 2007; Chen et al. 2010). In some but not all of these studies, altering the composition of the maternal diet caused intrauterine growth retardation (IUGR), a known risk factor for adult cardiovascular and metabolic dysfunction in experimental animals, and for increased susceptibility to hypertension and type 2 diabetes in human populations (Barker et al. 2002; Fowden et al. 2006).
While prenatal nutritional programming of postnatal tissue and organ function has been well established experimentally (McMillen & Robinson, 2005), the mechanisms involved in utero remain poorly understood. Since the placenta is the principal source of nutrients for fetal growth and development (Fowden et al. 2008), it may be an important contributory factor to intrauterine programming induced by changes in dietary composition during pregnancy. However, compared to fetal tissues, little is known about placental programming per se. Adaptations in placental phenotype are known to occur in response to variations in maternal dietary intake and composition and when placental growth is restricted either naturally or experimentally in several species (Heasman et al. 1998; Roberts et al. 2001; Allen et al. 2002; Doherty et al. 2003; Rutland et al. 2007; Quigley et al. 2008). In pregnant rats, feeding isocalorific diets low in protein has also been shown to both increase and decrease placental weight depending on the degree of protein deprivation and the source of the extra carbohydrate used to maintain the calorific content of the diet (Langley-Evans et al. 1996; Gardner et al. 1997; Langley-Evans, 2000; Fernandez-Twinn et al. 2003; Franko et al. 2009). When dietary protein content is severely restricted to 6% or less in pregnant rats, the nutrient transfer capacity of the placenta is reduced not only by restricting placental growth and vascularity but also by decreasing the placental abundance of specific glucose and System A amino acid transporters (Rosso, 1977a,b; Jansson et al. 2006; Rutland et al. 2007). However, little is known about the consequences for placental phenotype of the less extreme variations in dietary composition more commonly used to investigate intrauterine programming of adult disease.
The minimum recommended protein content of diets for pregnant and lactating mice is 18% total protein (Subcommittee on Laboratory Animal Nutrition, 1995). If the diet is made synthetically with the milk protein casein, the total protein recommendation equates to a casein content of 20% (Langley-Evans, 2000). Casein diets are frequently used in studying the nutritional programming of adult disease with low protein diets of 8–9% total protein compared to control diets which range in total protein content from 16% to 20% (Langley-Evans, 2000; Fernandez-Twinn et al. 2003; Warner & Ozanne, 2010). Therefore, the aim of this study was to address the hypothesis that even small changes in dietary composition have consequences for placental development by determining placental phenotype in mice fed casein diets containing 20%, 16% and 8% total protein supplemented with starch and sugar in a constant ratio to maintain an isocaloric energy content. Placental morphology and nutrient transport were measured at day 16, when placental growth is maximal, and at day 19, when the fetus is growing most rapidly in absolute terms. These characteristics were related to the placental expression of the principal placental glucose transporters (Slc2a1/GLUT1, Slc2a3/GLUT3), the System A family of amino acid transporters (Slc38a1/SNAT1, Slc38a2/SNAT2, Slc38a4/SNAT4) involved in the transfer of neutral amino acids essential for fetal growth and to key placental growth regulatory genes (Igf2, Grb10, H19) known to affect the transport phenotype of the mouse placenta (Constanciaet al. 2002; Angiolini et al. 2011).
Methods
Animals
Two hundred and thirty three virgin 6- to 8-week-old C57Bl/6J female mice (Harlan, UK) were mated with C57Bl/6J males (Harlan, UK). On the morning of the plug, females were group-housed and randomly assigned to one of three synthetic isocalorific diets (Dietex International, Witham, UK): 23% casein diet (C23, n = 66, 20% crude protein, 9.7% crude oil, 37% starch, 20% sugar; 16.07 MJ kg−1, cat no.: RB 23% casein SY (P) 829274), 18% casein diet (C18, n = 97, 16% crude protein, 9.6% crude oil, 40% starch, 22% sugar; 16.13 MJ kg−1, cat no.: RB 18% casein SY (P) 829252) or 9% casein diet (C9, n = 70, 8.2% crude protein, 9.5% crude oil, 46% starch, 25% sugar, 16.22 MJ kg−1, cat no.: RB 9% casein SY (P) 829253). All diets were supplemented with equal quantities of vitamins, minerals and d-methionine. Food intake per cage was recorded on a daily basis and divided by the number of occupants to provide an estimate of the daily food intake per mouse. All mice were housed under 12 h dark–12 h light conditions with free access to food and water. The presence of a copulatory plug was designated as day 1. All procedures were carried out in accordance with the UK Home Office regulations under the Animals (Scientific Procedures) Act 1986.
Experimental procedures
Placental transfer of non-metabolisable, radiolabelled substrates
Unidirectional materno-fetal clearance of non-metabolisable radioactive tracers (Perkin Elmer, Cambridge, U.K.) was measured in pregnant mice at D16 (C23, C18 and C9, n = 19 litters per diet group) and D19 (C23, n = 31 litters; C18, n = 29 litters; C9, n = 16 litters). Briefly, mice were anaesthetised and injected with either 100 μl of [14C]methyl-d-glucose (cat no.: NEN NEC-377; specific activity 2.1 GBq mmol−1) or [14C]methyl amino-isobutyric acid (MeAIB) (cat no.: NEN NEC-671; specific activity 1.86 GBq mmol−1) to provide a measure of transplacental transport by facilitated diffusion and active transport, respectively (further details in Coan et al. 2010). At specific times <5 min after tracer injection, a maternal blood sample was taken for the measurement of plasma counts and blood glucose concentration (day 19 only). The mean time between injection of isotope and collection of the maternal blood sample did not differ between the dietary groups or with gestational age. The mother was then killed by cervical dislocation. Conceptuses were dissected out, decapitated and weighed. The fetal to placental weight ratio was calculated as a measure of placental efficiency. Whole fetuses were minced, and lysed in Biosol (National Diagnostics, Hessle, U.K.), then aliquots of the fetal lysate were counted in a β counter (LKB Wallac 1216 Liquid Scintillation Counter, LKB Wallac, Turku, Finland) according to Coan et al. 2010. Fetally accumulated radioactivity and maternal plasma radioactivity were used to calculate clearance in μl min−1 (g placenta)−1 and fetal uptake in fetal dpm (g fetus)−1 (Sibley et al. 2004). The remaining mice were allowed to deliver spontaneously (C23, n = 8; C18, n = 7; C9, n = 7). The newborn pups were weighed, decapitated and a small blood sample taken to determine glucose concentration.
Stereological analysis of placental structure
The placenta closest to the mean placental weight within a litter was fixed in 4% paraformaldehyde in Pipes buffer. Following fixation, the fixed tissue was dehydrated, embedded in paraffin wax and sectioned completely at 7 μm. The Computer Assisted Stereological Toolbox (CAST v2.0, Olympus, Ballerup, Denmark) was employed to measure the gross structure of the placenta in a systematic random fashion using paraffin sections. Volume densities of placental compartments were determined by point counting and converted to absolute volumes according to the total number of sections, section thickness and shrinkage as described previously (Coan et al. 2004).
Biochemical analyses
Blood glucose concentrations
Blood glucose concentrations were measured in the mothers at day 19 and in the newborn pups using a hand held glucometer (One Touch, Ortho-Clinical Diagnostics, High Wycombe, UK).
Quantitative real-time PCR analysis of placental gene expression
Expression levels were analysed by quantitative real time PCR (7500 Fast Real-Time PCR System, Applied Biosystems) for the growth regulatory genes (Grb10, Igf2, H19), the placenta-specific transcript of Igf2 (Igf2P0), the placental glucose and System A amino acid transporter genes (Slc2a1, Slc2a3, Slc38a1, Slc38a2 and Slc38a4). Ribonucleic acids (RNAs) were isolated from whole placentas using Tri-reagent and a standard protocol (Sigma-Aldrich). RNAs were reverse transcribed to cDNAs using Multiscribe Reverse Transcriptase with random primers according to the manufacturer's protocol (Applied Biosystems). All cDNA samples were analysed in triplicate with cDNA derived from six placentas per diet group per gestational age and cDNAs derived from a pool of placentas used with 5-fold dilutions to create a standard curve. The primers and the PCR conditions used for gene expression analyses have been published previously (Coan et al. 2008, 2010). The relative standard curve method was used for quantifying levels of gene expression. In order to normalise gene expression levels, transcripts from Sdha, Actb, Tbp and Gapd were compared in order to find the optimal combination as follows: for each placental RNA sample, the expression of each housekeeping gene was determined by real-time PCR. The mean expression of combinations of two or more housekeeping genes was determined and the total mean and standard deviation for combined expression levels in all samples were calculated. The combination of housekeeping genes that yielded the lowest standard deviation was then used to normalise each gene of interest. Consequently, genes of interest were normalised to mean expression levels of Gapd and Tbp as described previously (Silver et al. 2006).
Statistical analyses
Values for all data are expressed as the mean (±SEM) of litter means. Litter size has been included as a co-variant in analysing differences with dietary treatment, where appropriate. Significant differences in clearance, structure and gene expression between diets were determined by ANOVA and Fisher's protected least significant difference post hoc test (StatView v. 5.0, SAS Institute Inc., Cary, North Carolina, U.S.A.). Significant differences between gestational ages within diet groups were assessed by Student's unpaired t test. Linear regression and simple correlation analysis and their significance were determined using GraphPad Prism 4.0 (GraphPad Software Inc., La Jolla, CA, USA).
Results
Food intake
Food intake and dietary consumption of protein, carbohydrate and energy were measured across three periods of gestation; days 2–9, before the haemochorial placenta is fully formed; days 10–16, the main period of placental growth; days 17–19, the period of maximal fetal growth. During the first two periods, C9 mice consumed significantly more food and energy than C18 mice with intermediate values in the C23 group (Table 1). In late gestation (days 17–19), there were no significant differences in food or energy intake with diet (Table 1). For the two periods of gestation up to day 17, protein intake was greatest on the C23 diet, followed by the C18 and C9 diets (Table 1). However, thereafter, the difference between C18 and C23 mice was not significant, whereas the C9 group ate less (Table 1). Conversely, carbohydrate intake was significantly greater on the C9 than C23 or C18 diets during the first two periods of gestation, but thereafter did not differ significantly with diet (Table 1). Blood glucose concentrations were measured at day 19, and were significantly greater on the C23 than C18 or C9 diets (C23, 11.6 ± 0.5 mmol l−1, n = 22 mice; C18, 7.3 ± 0.5 mmol l−1, n = 9 mice; C9, 6.3 ± 0.6 mmol l−1, n = 8 mice, P < 0.0001).
Table 1.
Estimated mean nutrient intake per mouse for 23%, 18% and 9% casein diets between days 2 and 9, days 10 and 16 and days 17 and 19
Gestation period | Diet | Food (g) | Protein (g) | Carbohydrate (g) | Energy (kJ) |
---|---|---|---|---|---|
C23 | 3.0 ± 0.1a | 0.62 ± 0.02a | 1.74 ± 0.06a | 49 ± 2a | |
Days 2–9 | C18 | 2.7 ± 0.1b | 0.44 ± 0.01b | 1.70 ± 0.06a | 44 ± 1b |
C9 | 3.2 ± 0.1a | 0.27 ± 0.01c | 2.28 ± 0.07b | 53 ± 2a | |
C23 | 3.2 ± 0.1a | 0.67 ± 0.03a | 1.82 ± 0.08a | 51 ± 2a | |
Days 10–16 | C18 | 3.0 ± 0.1a | 0.48 ± 0.01b | 1.87 ± 0.05a | 49 ± 1a |
C9 | 3.5 ± 0.1b | 0.29 ± 0.01c | 2.46 ± 0.06b | 57 ± 1b | |
C23 | 3.2 ± 0.2a | 0.66 ± 0.03a | 1.83 ± 0.09a | 52 ± 2a | |
Days 17–19 | C18 | 3.4 ± 0.4a | 0.54 ± 0.07a | 2.13 ± 0.26a | 55 ± 7a |
C9 | 3.2 ± 0.4a | 0.26 ± 0.03b | 2.26 ± 0.29a | 52 ± 7a |
Mean (±SEM) daily food constituent intake per cage, divided by the number of occupants to obtain estimate intake per mouse, where n = number of cages; C23, n = 6; C18, n = 13; C9, n = 8. Significant differences between diets at each gestational time period are represented by a, b, c, and assessed by ANOVA followed by Fisher's least significant difference post hoc test, P = 0.048 to P < 0.0001. Abbreviations of isocalorific diets C23, 23% casein; C18, 18% casein; C9, 9% casein.
Biometry
Maternal weight
At both ages, total maternal weight was greatest in the C18 group (Table 1). Carcass weight of the dams showed the same trend at day 16 but was lowest in the C9 mice at day 19. The weight of the gravid uterus did not differ with diet at either age but accounted for a significantly greater proportion of carcass weight in the C9 than C23 dams (Table 2).
Table 2.
Biometric data of mice fed 23%, 18% or 9% casein diets
Day 16 | Day 19 | |||||
---|---|---|---|---|---|---|
C23 | C18 | C9 | C23 | C18 | C9 | |
Maternal weight (g) | ||||||
Gravid | 28.7 ± 0.5a | 30.9 ± 0.7b | 29.5 ± 0.6ab | 31.5 ± 0.6a | 32.9 ± 0.4b | 30.9 ± 0.6a |
Carcass (minus uterus) | 22.8 ± 0.4a | 24.8 ± 0.7b | 23.0 ± 0.5a | 22.3 ± 1.2a | 22.3 ± 0.3a | 20.5 ± 0.4b |
Gravid uterus | 6.5 ± 0.3a | 6.6 ± 0.2a | 7.4 ± 0.8a | 10.0 ± 0.3a | 10.7 ± 0.3a | 10.4 ± 0.3a |
G:H% | 19 ± 7a | 27 ± 2ab | 30 ± 2b | 45 ± 2a | 49 ± 2b | 51 ± 2b |
Conceptus weight (mg) | ||||||
Fetus | 410 ± 5a | 408 ± 6a | 412 ± 7a | 1138 ± 11a | 1113 ± 11a | 1035 ± 23b |
Placenta | 93 ± 1a | 98 ± 1b | 90 ± 2a | 79 ± 1a | 83 ± 1b | 82 ± 2b |
F:P ratio | 4.5 ± 0.1a | 4.2 ± 0.1b | 4.5 ± 0.1a | 14.5 ± 0.2a | 13.7 ± 0.2b | 12.8 ± 0.3c |
Litter size | ||||||
6.9 ± 0.3a | 7.3 ± 0.2a | 6.9 ± 0.3a | 6.1 ± 0.2a | 6.7 ± 0.2b | 6.9 ± 0.3b |
Mean±SEM. a–c indicate homogenous groups assessed by ANOVA, with litter size as a covariate, and Fisher's least significant difference post hoc test: P < 0.05 to P < 0.0001. Abbreviations of isocalorific diets: C23, 23% casein; C18, 18% casein; C9, 9% casein. Day16: C23, n = 24; C18, n = 42; C9, n = 32; day 19: C23, n = 42; C18, n = 55; C9, n = 38 (n = litters); G:H% refers to the weight of the gravid uterus as a percentage of hysterectomised (carcass – uterus) weight.
Conceptus weights
At day 16, there was no significant difference in the mean weight of the fetuses per litter with diet (Table 2). In contrast, the C18 placenta weighed more than C9 or C23 placentas and produced fewer grams of fetus on a weight specific basis (Table 2). At day 19, C9 fetuses were growth restricted compared to the C18 and C23 fetuses (Table 2). The C9 and C18 placentas weighed more than the C23 placentas at day 19 and were less efficient at supporting fetal growth measured as gram fetus produced per gram placenta (Table 2). When data from all dietary groups were combined, mean fetal weight at day 19 was positively correlated to maternal protein intake between day 10 and day 16 (r = 0.64, n = 8 cages, P < 0.02) and between day 17 and day 19 (r = 0.74, n = 8 cages, P < 0.01), but not during the earlier period of gestation (P > 0.05). Positive correlations were also observed between the ratio of fetal to placental weight at day 19 and maternal dietary intake of protein between day 10 and day 16 (r = 0.59, n = 8 cages, P < 0.05) and between day 17 and day 19 (r = 0.89, n = 8 cages, P < 0.02), but not between day 2 and day 9 (P > 0.05). There was no relationship between mean fetal weight at day 19 and maternal carbohydrate intake at any period of gestation (P > 0.05, all cases). At birth, C9 neonates were significantly lighter than C18 and C23 pups (C9, 1160 ± 10 mg, n = 47 pups; C18, 1250 ± 13 mg, n = 55 pups; C23, 1256 ± 14 mg, n = 62 pups, P < 0.0001) and had significantly lower blood glucose concentrations (C9, 1.45 ± 0.01 mmol l−1, n = 47 pups; C18, 2.34 ± 0.12 mmol l−1, n = 32 pups; C23, 2.37 ± 0.11 mmol l−1, n = 55 pups, P < 0.0001).
Placental transport
The effects of dietary composition on unidirectional transplacental clearance of glucose (facilitated diffusion) and amino acid (active transport) depended on the mode of transport and gestational age. For [14C]methyl-d-glucose at day 16, unidirectional clearance per gram placenta was significantly less in C23 mice than in the other two dietary groups, which had similar clearances (Fig. 1A). The C23 mice also had the lowest glucose accumulation per gram fetus at D16 (Fig. 1B). At day 19, transplacental glucose clearance was greater on the C9 than C18 diet with intermediate values in the C23 group (Fig. 1A). Fetal accumulation of glucose was also significantly greater per gram C9 than C23 or C18 fetus at day 19 (Fig. 1B). Transplacental glucose clearance increased between day 16 and day 19 in both the C23 and C9 mice but not in the C18 group. Weight specific fetal accumulation of glucose was, therefore, maintained on the C23 and C9 diets but decreased per gram C18 fetuses between day 16 and day 19 (Fig. 1B).
Figure 1. Unidirectional transplacental transfer and fetal accumulation of [14C]glucose (A and B) and [14C]MeAIB (C and D) in mice fed 23% (black columns), 18% (grey columns) and 9% (open columns) casein diets.
Significant differences between groups were determined by ANOVA, with litter size as a covariate, followed by Fisher's least significant difference post hoc test, indicated by letters a and b. *Significant differences between gestational ages within diet groups were assessed by paired t test. Number of pregnant mice for glucose clearance and uptake at day 16: C23 = 8; C18 = 10; C9 = 10; day 19: C23 = 12; C18 = 13; C9 = 7. Number of pregnant mice for MeAIB clearance and uptake at day 16: C23 = 11; C18 = 9; C9 = 9; day 19: C23 = 19; C18 = 16; C9 = 9.
In contrast to glucose, dietary composition had no significant effect on the transplacental transfer of [14C]MeAIB per gram placenta at day 16 (Fig. 1C). However, fetal accumulation of MeAIB, like glucose, was significantly greater per gram C18 than C23 fetuses at day 16 with intermediate values in the C9 group (Fig. 1D). Placental MeAIB clearance increased significantly between day 16 and day 19 in all dietary groups but, at day 19 was significantly less in C18 than C23 mice with intermediate values in the C9 group (Fig. 1C). Weight specific fetal accumulation of MeAIB, therefore, fell between day 16 and day 19 in the C18 but not C23 or C9 mice (Fig. 1D). Thus, by day 19, fetal accumulation of MeAIB per gram fetus was similar in the three dietary groups (Fig. 1D). When data from all dietary groups were combined, there was a significant positive correlation between placental clearance of glucose and the body weight of individual fetuses at day 16 but not at day 19 (Fig. 2A and B). Conversely, placental clearance of MeAIB was positively correlated with fetal weight at day 19 but not day 16 (Fig. 2C and D).
Figure 2. Relationship between fetal weight and placental unidirectional clearance of glucose (A and B) and MeAIB (C and D) at day 16 (A and C) and day 19 (B and D) from litters exposed to 23% (black circles), 18% (grey circles) and 9% (open circles) casein diets.
Glucose: Day 16 (A): y = 275x− 0.02, r = 0.22, n = 207 conceptuses, P = 0.0016; day 19 (B): y = 17.1x− 3, r = 0.03, n = 194 conceptuses, P > 0.05; MeAIB: Day 16 (C): y = 183x− 0.21, r = 0.12, n = 195 conceptuses, P = 0.086; Day 19 (D): y = 161x+ 0.24, r = 0.33, n = 282 conceptuses, P < 0.0001.
Placental structure
The absolute and relative volumes of the different compartments of the placenta varied with diet. There were no significant differences in the absolute volume of the labyrinthine zone (Lz) responsible for nutrient transfer between the dietary groups at either day 16 or day 19 (Table 3). However, Lz volume expressed as a percentage of the total placental volume was less in mice fed the C18 diet than the other two diets at both ages (Table 3). The absolute volume of junctional zone (Jz) responsible for placental endocrine secretions was less in C9 than C18 or C23 placentas at day 16 and D19 (Table 3). The relative Jz volume was also lowest in C9 placentas at day 16 (Table 3). The C18 placentas had the greatest absolute and relative volume of decidua basalis (Db), followed by C9 then C23 placentas at both ages (Table 3). The percentage of total placental volume occupied by the Lz increased in all three dietary groups between day 16 and day 19 but the increment was greatest in C23 placentas followed by the C9, and then C18 groups (Table 3). Coincident with this, the absolute and relative volume of the Jz decreased significantly between day 16 and day 19; mean decrements in these volumes were similar in the three dietary groups (Table 3). At both gestational ages, the ratio of the Lz volume to the Jz volume was significantly lower in the C18 placentas than C23 and C9 placentas (Table 3). When data from all dietary groups were combined, there was a significant positive correlation between fetal weight and the Jz volume fraction (r = 0.550, n = 18, P < 0.02) and a significant inverse correlation between fetal weight and the Lz:Jz ratio (r = −0.574, n = 18, P < 0.02) at day 19. No significant relationship between fetal weight and these morphological parameters was observed at day 16 or between fetal weight and the Lz absolute volume or volume fraction at either age (P > 0.05, all cases).
Table 3.
Stereological analysis of placentas on day 16 and day 19 of gestation, from mice fed 23%, 18% or 9% casein diets
Day 16 | Day 19 | |||||
---|---|---|---|---|---|---|
C23 | C18 | C9 | C23 | C18 | C9 | |
Absolute volume (mm3) | ||||||
Placenta | 87 ± 4a | 97 ± 3a | 92 ± 3a | 79 ± 1a* | 86 ± 3a* | 81 ± 2a* |
Lz | 40 ± 2a | 39 ± 2a | 43 ± 1a | 47 ± 2a | 42 ± 3a | 45 ± 3a |
Jz | 34 ± 4ab | 37 ± 2a | 30 ± 2b | 22 ± 2ab* | 25 ± 2a* | 19 ± 1b* |
Db | 13 ± 1a | 18 ± 1b | 16 ± 1ab | 10 ± 1a* | 14 ± 1b* | 12 ± 1ab* |
Lz:Jz | 1.31 ± 0.13a | 1.10 ± 0.07b | 1.50 ± 0.09a | 2.28 ± 0.27a* | 1.75 ± 0.24b* | 2.40 ± 0.25a* |
Volume fraction (%) | ||||||
Lz | 43 ± 1a | 40 ± 1b | 43 ± 1a | 51 ± 2a* | 44 ± 2b* | 48 ± 1ab* |
Jz | 38 ± 2ab | 38 ± 1a | 34 ± 1b | 32 ± 1a* | 33 ± 2a* | 29 ± 1a* |
Db | 23 ± 2a | 26 ± 1a | 24 ± 1b | 20 ± 1a | 24 ± 1b | 22 ± 1ab |
Mean ± SEM. C23, 23% casein diets; C18, 18% casein diet; C9, 9% casein diet. The placenta closest the mean for each litter was analysed day 16: C23, 6 litters; C18, 9 litters; C9, 10 litters. Day 19: C23, C18 and C9, 6 litters for each. Significant differences between diets assessed by ANOVA and Fisher's least significant difference post hoc test and indicated with different lettered superscripts (P < 0.05). *Significant differences between gestational ages within the same diet assessed by unpaired t test (P < 0.05). Abbreviations: Db, decidua basalis; Jz, junctional zone; Lz, labyrinthine zone.
Placental gene expression
Altering dietary composition had no effect on expression of the placenta-specific transcript of Igf2, Igf2P0, at either day 16 or day 19 (Fig. 3). In contrast, expression of Igf2 from all promoters in the placenta was significantly elevated in C9 placentas to 180% of the values seen in C23 placentas at day 16 but was unaffected by diet at day 19 (Fig. 3). Both H19 and Grb10 were expressed in similar quantities by all placentas at the two ages (Fig. 3). At day 16, expression of the facilitated glucose transporter gene, Slc2a1, was greatest in C18 placentas, followed by C9 then C23 placentas (Fig. 3A). By day 19, placental Slc2a1 expression was similar amongst diets (Fig. 2B). Expression of the other placental glucose transporter, Slc2a3, was not affected by dietary manipulation at either age (Fig. 2). At day 16, expression of the Slc38a2 isoform of the System A amino acid transporters in C9 placentas was greater than C23 placentas, whereas placental expression of the other two isoforms of these transporters, Slc38a1 and Slc38a4, was similar in the three dietary groups (Fig. 3A). At day 19, expression of both Slc38a1 and Slc38a4 was significantly reduced in C9 and C18 placentas compared to C23 placentas, whereas placental Slc38a2 expression was similar for all diets (Fig. 3B).
Figure 3. Placental expression of selected growth and transporter genes, at day 16 (A) and day 19 (B), from mice fed 23% (black columns), 18% (grey columns) or 9% (open columns) casein diet.
Number of litters per diet per gestational age = 6. Significant differences between diets were determined by ANOVA followed by Fisher's least significant difference post hoc test, indicated by letters a and b.
Discussion
The results demonstrate that the mouse placenta can adapt(its phenotype to help maintain fetal growth when the protein content of the diet is reduced and replaced with carbohydrate. These adaptations were both morphological and functional, and involved changes in placental size and expression of growth regulatory and nutrient supply genes. They were also dependent on gestational age. Adaptive changes in placental phenotype were observed not only in response to the 9% casein diet commonly fed in studies of intrauterine programming but also when dietary protein content was reduced more modestly within the range found in many control diets used experimentally. During mid gestation, dams on the 9% casein diet ate approximately 10% more food per day than those fed higher protein diets. This affected the intake of energy, nutrients and essential minerals and vitamins, independently of the actual composition and calorific content of the diet. Placental phenotype is, therefore, responsive to relatively subtle changes in maternal dietary intake and adapts to favour the conceptuses in the balance of resource allocation between the mother and gravid uterus.
Fetal growth and maternal resource allocation
In common with previous findings in pregnant rats (Langley-Evans, 2000; Doherty et al. 2003; Fernandez-Twinn et al. 2003), varying the dietary content of total protein within the 8–20% range isocalorically had little effect on fetal growth until close to term in the mouse. Mean fetal weight was similar in the three dietary groups at day 16 but was reduced on the 9% compared to 18% or 23% casein diets by day 19, even when adjusted for the differences in litter size. However, the day 19 fetuses and the newborn pups of C9 dams were only 5–10% smaller than those of the other dietary groups, despite a 60% reduction in maternal protein intake throughout pregnancy. With a 30% restriction in maternal protein intake for most of gestation on the C18 diet, normal fetal weight was maintained right up to birth. This relative maintenance of intrauterine growth was due, in part, to the effects of diet on maternal nutrient partitioning and on placental size and morphology. On both the C18 and C9 diet, nutrients appeared to be partitioned preferentially to the conceptuses as hysterectomised carcass weight was significantly less in C9 dams than in the other two groups at day 19 and decreased between day 16 and day 19 in the C18 and C9 but not the C23 groups. Indeed, C18 dams appeared to gain more carcass weight initially despite the decreased food intake and then mobilised this reserve to sustain fetal growth in late gestation as the hysterectomised carcass weight of the C18 dams was higher than that of other groups at day 16 but similar to the C23 dams by day 19. The maternal strategy to supporting conceptus growth, therefore, appears to differ with diet. Certainly, in pregnant rats fed an isocaloric 4–10% protein diet, there are increases in the insulin resistance and glucogenic capacity of the dam, which, in turn, will increase nutrient availability for transplacental transfer to the fetus and intrauterine growth relative to control 20–23% total protein diets (Jimenez-Gancedo et al. 2004; Franko et al. 2009).
Morphological adaptations of the placenta
The re-distribution of maternal resources in favour of the fetus also appears to have a placental component. Placental weight was greater on the C9 and C18 diets than on the C23 diet at both gestational ages. Similar compensatory increases in placental growth near term have been observed in other species deprived of total or specific nutrients during the period of maximal placental growth (Lumey, 1998; Fowden et al. 2009). Increased placental weight at birth has also been observed in women who ate relatively more carbohydrate in early pregnancy and less protein closer to term (Godfrey et al. 1996). Despite a similar increase in placental weight on the C18 and C9 diets, growth of the different placental zones was affected differentially by the specific intake of protein and carbohydrate. On the C18 diet, the volume of the Lz responsible for nutrient exchange tended to be smaller while the volume of the endocrine Jz tended to be greater than seen on the C23 diet at both gestational ages. This resulted in a smaller Lz volume fraction at day 19 and a smaller Lz:Jz ratio at both ages relative to the C23 diet, which suggests preferential Jz growth on the C18 diet. This adaptive strategy is likely to have most influence on the endocrine functions of the C18 placenta with potential consequences for maternal metabolic adaptation to pregnancy, consistent with the greater changes in carcass weight observed in the C18 dams. In contrast, the Lz volume and Lz:Jz ratio were maintained at C23 values in the C9 group at both ages, at the expense of Jz growth. This reduction in the endocrine potential of the placenta may be an adaptive ‘trade off’ to ensure adequate Lz growth for nutrient exchange, similar to the Lz sparing effect seen in mice undernourished by restricting food intake for most of gestation (Coan et al. 2010). The reciprocal nature of the relationships between fetal weight and the Lz:Jz ratio and the former values and the Jz volume fraction across the dietary groups in the present study also suggests that the endocrine functions of the Jz zone may be sacrificed to help maintain the nutrient transfer functions of the Lz zone when fetal growth is compromised. Taken together, these and our earlier findings in normal and genetically modified mice indicate that the relative development of the different zones within the mouse placenta is determined by interaction between the fetal drive for growth and the maternal metabolic adaptations and availability of nutrients during late gestation (Coan et al. 2008, 2010).
Expression of placental growth regulatory genes
The increased placental weight, particularly in the C9 group, may be due, in part, to the enhanced placental expression of the Igf2 gene known to be important in stimulating placental growth (Constancia et al. 2002, 2005). Although the labyrinthine specific Igf2P0 transcript was unaffected by diet, total expression of Igf2 from all transcripts was increased in the C9 placentas at day 16, which may also explain, in part, the smaller weight loss of the C9 placenta between days 16 and 19 and counter any increased apoptotic activity associated with decreased protein intake (Gheorghe et al. 2009). In contrast, diet had no effect on placental expression of the H19 or Grb10 genes, which suppress growth of the mouse placenta (Charalambous et al. 2003; Coan et al. 2005). However, when protein availability is reduced during pregnancy by undernutrition in mice and other species, placental Igf2 gene expression is decreased in association with placental growth restriction (Roberts et al. 2001; Coan et al. 2010). Since carbohydrate availability is restricted during undernutrition but increased on the C9 diet, these observations suggest that placental Igf2 gene expression may be more responsive to changes in maternal carbohydrate than protein intake, consistent with the predominance of carbohydrate in the natural rodent diet. There may, therefore, be a reciprocal relationship between placental Igf2 gene expression, placental growth and the ability of the mother to allocate carbohydrate to fetal growth. Certainly, when placental Igf2 gene expression is up-regulated genetically by loss of imprinting due to deletion of the H19 gene, there is placental overgrowth and elevations in maternal glucose levels and insulin resistance at day 16, which will increase glucose availability for fetal growth (Petry et al. 2010). Placental Igf2 gene expression may, therefore, act to integrate maternal and fetal signals of nutrient availability and adapt placental development to optimise fetal growth with respect to the prevailing nutritional conditions. Imprinted genes, like Igf2, H19 and Grb10, which are expressed monoallelically in a parent-of-origin manner, have a disproportionately important role in placental development and are believed to be central in the conflict between maternal and paternal genomes in allocating maternal resources to fetal growth (Constancia & Reik, 2004). Since the Igf2 gene is paternally expressed, the potential action of Igf2 in integrating nutritional signals is in keeping with the role of imprinted genes in mediating the parental conflict in resource allocation and will lead to a better match between the paternal drive for fetal nutrient acquisition and the maternal ability to distribute nutrients to the gravid uterus in an individual pregnancy.
Adaptation in placental nutrient transport capacity
In addition to the morphological adaptations to the placenta, there were functional changes in placental nutrient transport with the different diets, which also contributed to the maintenance of fetal growth, particularly at day 16. When total dietary protein content was reduced below 20%, placental clearance of glucose was increased on a weight specific basis at day 16, in association with enhanced placental expression of Slc2a1/GLUT1, the glucose transporter localised to the blood facing sides of the trophoblast layers (Wooding & Burton, 2008). This led to increased glucose accumulation per gram of C9 and C18 fetuses relative to C23 pups at day 16. Upregulation of placental Slc2a1/GLUT1 expression and glucose clearance has also been observed in the naturally small placenta within a litter and when nutrient availability was reduced by undernutrition of pregnant mice (Coan et al. 2008, 2010). Whether upregulation in gene expression of the glucose transporters demonstrated here leads to increased protein expression has yet to be determined, although the changes in mRNA expression were associated with physiological changes in glucose transport at day 16. Increasing fetal glucose availability will stimulate growth of the fetal tissues both directly and indirectly as previous studies in fetal sheep have shown that glucose is used preferentially for oxidative metabolism, thereby sparing amino acids for tissue accretion in the fetus (Hay, 2006). Indeed, placental glucose clearance was positively correlated with fetal weight at day 16 in the present study. The increase in placental clearance and fetal accumulation of glucose was maintained in the C9 relative to the C18 group at day 19. This may reflect the greater Lz volume fraction in the C9 group as placental Slc2a1/GLUT1 expression was unaffected by diet at this age. Although there was no relationship between placental glucose clearance and fetal weight at day 19, these observations suggest that glucose may continue to be an important substrate for fetal growth and oxidative metabolism near term in the dams with the lowest dietary protein intake, despite their hypoglycaemia.
There were also changes in the placental clearance and fetal accumulation of MeAIB with dietary composition although these were not as pronounced as seen with glucose. They were also confined mainly to the C18 group with increased fetal MeAIB accumulation at day 16 and decreased placental MeAIB clearance at day 19 relative to the C23 group. At day 19, this change in MeAIB transfer across the C18 placenta was associated with decreased expression of Slc38a4. In addition, in C9 mice, there were increases in placental expression of the Slc38a2 isoform at day 16 and decreases in expression of both the Slc38a1 and Slc38a4 isoforms at day 19 relative to the C23 mice, but these did not result in significant changes in MeAIB clearance per gram C9 placenta. These observations suggest that changes in gene expression for the accumulative System A amino acid transporters do not coincide immediately with the availability of active transporter protein. Certainly, in rats fed a 4% protein diet placental MeAIB transport changed 5 days after alterations in placental Slc38a2 mRNA expression at a time when gene expression had normalised (Jansson et al. 2006). Alternatively, there may be dietary-induced changes in placental expression of other amino acid transporters, such as the facilitated transporters responsible for efflux of amino acids down the concentration gradient from placenta to fetus, which may influence transplacental MeAIB transport. Up-regulation of Slc38a2 and down-regulation of Slc38a4 expression has also been observed in placentas of undernourished mice (Coan et al. 2010). Since Slc38a4 appears to be localised predominantly to the Jz (G.D. Kelsey & M. Constancia unpublished observations), down-regulated expression of this isoform may restrict Jz growth, as seen in the C9 group, but, by limiting amino acid uptake into this zone, spare amino acid for transport to the fetus, particularly when dietary protein intake is low. The current findings that fetal weight was positively correlated to both placental MeAIB clearance and maternal protein intake, but not to placental glucose clearance at day 19, suggests that availability of amino acids may be more important than glucose for growth in late gestation, when the rate of fetal tissue accretion is at its greatest in absolute terms. Taken together, the morphological and functional changes in the placenta induced by dietary manipulation suggest that the placenta adapts to maximise nutrient transfer in the prevailing conditions although this may not always result in maintained fetal growth depending on the maternal and fetal response to the nutritional challenge. Placental efficiency measured as grams fetus produced per gram placenta may, therefore, decline as occurred in the C18 and C9 placentas, despite the improved ability of the placenta per se to deliver nutrients (Fowden et al. 2009).
Conclusion and perspectives
In summary, dietary composition altered the development and nutrient transport capacity of the mouse placenta during late gestation. These changes helped maintain fetal growth for much of gestation and altered maternal nutrient partitioning progressively in favour of the conceptuses as dietary protein content was reduced and replaced by carbohydrate. Changes in placental phenotype were observed in response to relatively minor changes in dietary composition within the range seen in control diets used to study the prenatal nutritional programming of adult disease. This may help to explain the variation in offspring phenotype observed between studies feeding diets with apparently similar low protein contents. Indeed, in the current study, the C9 and C18 mice adopted different strategies in adapting placental phenotype to help maintain growth in utero. The C18 mice increased placental weight at the expense of the Lz zone while the larger C9 placenta reduced Jz volume in favour of Lz growth. Both groups increased the placental capacity for glucose transport at day 16 but this was only sustained to term in the C9 relative to the C18 group. However, whether changes in placental phenotype are due to alterations in the protein or carbohydrate content of the diet remains unclear. Whatever the cause, these changes will alter the absolute and relative quantities of nutrients supplied to the fetus with consequences for the growth and functioning of tissues both before and after birth. Indeed, recently, placental Slc38a2 expression has been shown to correlate with postnatal growth rate of offspring from rat dams fed low protein diets during pregnancy (Strakovsky et al. 2010). Thus, placental phenotype and, more specifically, the expression pattern of key growth regulatory and nutrient supply genes may provide a good index of the conditions experienced during intrauterine development and allow more accurate prediction of the adult risk of developing diseases programmed in utero.
Acknowledgments
We would like to thank Nuala Daw, Julie Gautrey, Chris Cardinal and Wendy Cassidy for their technical assistance on the study. This study was funded by the BBSRC.
Glossary
Abbreviations
- C23, C18 and C9
diets with 23%, 18% and 9% casein content
- Jz
junctional (endocrine) zone of the placenta
- Lz
labyrinthine (transport) zone of the placenta
- MeAIB
methyl aminoisobutyric acid
Author contributions
P.M.C. and A.L.F.: conception and design of experiments, collection, analysis and interpretation of data, drafting the article and revising it critically for important intellectual content; G.J.B. and M.C.: conception and design of experiments, interpretation of data, and revising article critically for important intellectual content; V.O.R., J.M. and C.M.: collection, analysis and interpretation of data and revising the article critically for important intellectual content. The final version of the manuscript was approved by all authors. All experiments in this study were carried out at the Department of Physiology, Development & Neuroscience, University of Cambridge, U.K.
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