The liver is a major metabolic and endocrine organ in growing neonates, but the extent to which its hormone receptor (R) sensitivity is potentially determined by maternal parity and the mother's nutritional environment is unknown. This was therefore investigated by sampling livers from postnatal sheep born to nulliparous or multiparous mothers. Offspring were sampled 1 or 30 days after birth from mothers consuming either 100 or 50% [i.e., nutrient-restricted (NR) group] of total metabolizable energy requirements from 110 days gestation to term (∼147 days). Regardless of maternal diet, offspring of nulliparous mothers were lighter at birth and had smaller livers. By 1 mo of age, they exhibited catch-up growth, an adaptation not seen when mothers were NR, but they retained their lighter livers. At both sampling ages, livers from offspring born to nulliparous mothers exhibited increased mRNA abundance for growth hormone (GH) receptor, IGF-IR, plus hepatocyte growth factor (HGF); and at day 1 only IGF-I, but not IGF-IIR mRNA was decreased. In addition, mRNA for IGF-II, the HGFR, c-Met, and Bax were persistently reduced in these offspring. Effects of parity were largely unaffected by maternal nutrient restriction. Maternal parity therefore has a substantial effect on liver size during postnatal development and its receptor population that is not dependent on maternal diet. First-born offspring appear to exhibit a resetting of the endocrine control of hepatic growth within the HGF and GH-IGF axis, which could have later consequences after their growth has caught up.
- birth weight
- insulin-like growth factor
- hepatocyte growth factor
an increased understanding of the prenatal factors that can determine birth weight is of major importance with regard to both neonatal (10) and longer-term (22) health consequences. Two major maternal factors determining birth weight are parity and the mother's diet in late gestation (16). In humans, for example, maternal parity not only influences birth weight but also postnatal growth and morbidity (2). First-born infants are more likely to be growth restricted in utero and exhibit compensatory growth after birth (38). The mechanisms behind these responses have received very little investigation, although recently the mother's genotype and cord blood IGF-II have been implicated (39). Several studies in a variety of species indicate that the smaller offspring born to nulliparous mothers (i.e., no previous pregnancies resulting in viable offspring) are not dependent on maternal age or body composition (30, 52, 55, 58). In both humans (30) and sheep (52), the increase in birth weight with number of pregnancies is greatest between first and second pregnancy (16) and occurs even when there is no change in maternal body weight and/or when the pregnancy is twin bearing (52). Furthermore, in sheep (16), as with humans (42), maternal nutrition during late gestation encompasses a critical period of fetal development that has the greatest impact on birth weight and can also determine both individual organ growth and endocrine sensitivity in the short and long term (19, 21). However, the extent to which late gestational maternal nutrient restriction may elicit differential effects depending on maternal parity is unknown.
Regulation of fetal development is likely to differ between first and subsequent pregnancies because of changes in the maternal metabolic and hormonal environment. For example, plasma IGF-I is higher in nulliparous compared with multiparous cows (56). While in humans, first-born offspring have significantly lower cord IGF-I and IGF binding protein-3 concentration in the presence of increased growth hormone (GH) compared with offspring of multiparous mothers, whereas cord IGF-II levels are unaffected (18). Other growth factors important in regulating liver growth include the hepatocyte growth factor (HGF) (24) and its receptor c-Met (7). In the adult, HGF acting through c-Met regulates liver mass by acting as a mitogen and survival factor, as well as inducing apoptosis (11, 45), primarily by affecting the abundance of the proapoptotic gene Bax (34). It is not known whether HGF may be influenced by maternal parity and prenatal nutrition and what effect this has upon neonatal liver growth. Maternal nutrient restriction during late gestation has a marked influence on tissue glucocorticoid sensitivity, causing an increase in the lung (19) but a reduction in adipose tissue (21). The extent to which glucocorticoid receptor (GR) mRNA abundance in the liver may be affected by maternal nutrition or parity remains to be established and was a further aim of this study.
Maternal nutrition and parity, independently, can have critical roles in determining weight at birth in a number of species (16, 58). However, no study to date has directly investigated the effects of both maternal parity and nutrient restriction in weight-matched mothers upon the neonatal liver, a key endocrine organ in the regulation of growth and metabolic homeostasis and later health. The present study was designed to examine liver growth in the resulting offspring with respect to mRNA abundance of hepatic growth factors critical in setting the hepatic GH-IGF axis. Neonatal development as determined by body size, organ weight, and hepatic receptor mRNA abundance was examined at 1 and 30 days of postnatal life. This represents the period over which the newborn effectively adapts to the extrauterine environment and concomitantly resets its metabolic response to feeding (50, 51). As such, the liver has a critical role in enabling the newborn to effectively adapt to the dramatic increase in metabolic demands at birth (31).
Twenty-four twin-bearing (14 nulliparous and 10 multiparous), Border Leicester cross Swaledale sheep of similar body weight [nulliparous, 79.76 ± 1.71 (n = 14); multiparous, 80.50 ± 3.45 kg (n = 10)], body condition score [nulliparous, 2.8 ± 0.6 (n = 14); multiparous, 2.5 ± 0.9 arbitrary units (a.u.) (n = 10)], and of known mating date were entered into the study. All nulliparous sheep were 2 yr old and had never been previously mated. The multiparous sheep were aged 3 to 4 yr and had all experienced two previous successful pregnancies. This is important because there is little increase in birth weight after a second pregnancy (16). Despite this difference in age, all sheep were adult and reproductively mature. To enable us to examine the offspring at 1 and 30 days of age, twin-bearing sheep were used (19). It is known that these are smaller at birth than singletons (16), but their postnatal growth and body composition is comparable when reared as a singleton (17). Following conception, animals were group housed and fed daily a diet sufficient to fully meet their total metabolic requirements. At 110 days gestation, i.e., 1 mo prior to predicted date of birth, all animals were individually housed and randomly assigned to one of two nutrition groups and fed according to current body weight. Six nulliparous and five multiparous mothers were allocated to the control group and were fed and consumed 100% of total metabolizable energy (ME) requirements for maternal body weight and stage of pregnancy as previously published (33). The remaining sheep were nutrient restricted (NR; n = 13); consuming 50% of total ME requirements until term. The diet comprised chopped hay and concentrate and was provided in a 3-to-1 weight ratio (33) with all animals having access to a mineral block and fresh water. At term, all offspring were delivered naturally at the expected gestational age i.e., 147 ± 2 days. There was no difference in gestational length between groups, and no mother required any birth intervention. Birth weight and body conformation (i.e., crown-rump length and chest girth) were then recorded. Within 6 h of birth, one twin was randomly selected from each mother to be tissue sampled following humane euthanasia by using an intravenous injection of barbiturate (200 mg/kg pentobarbital sodium; Euthatal; RMB Animal Health, UK). All mothers were then housed with their remaining offspring and fed a diet of hay ad libitum together with a fixed amount of concentrate that was sufficient to fully meet each mother's metabolizable requirements plus that required for maintaining lactation. The remaining twin was therefore reared with its mother and thus raised as a singleton until 30 days postnatal age when it, too, was tissue sampled. Again, each animal was euthanized, the liver rapidly dissected and weighed, and a representative portion (i.e., 20 g from the same position of the right lobe from each animal) was placed in liquid nitrogen and stored at −80°C until further analysis. All procedures were performed with the necessary institutional ethical approval as designated under the United Kingdom Animals (Scientific Procedures) Act, 1986.
Total RNA was isolated from a central region of the right lobe (see above) using Tri-Reagent (Sigma, Poole, UK). To maximize sensitivity, a two-tube approach to RT was adopted as previously described for mRNA detection in the ovine liver (23). The conditions used to generate first-strand cDNA RT were: 70°C (5 min), 4°C (2 min), 25°C (5 min), 4°C (2 min), 25°C (10 min), 42°C (1 h), 72°C (10 min), 4°C (5 min). The RT reaction (final volume 20 μl) contained: 1× RT buffer (250 mM Tris·HCl, 40 mM MgCl2, 150 mM KCl, 5 mM dithioerythritol, pH 8.5), 2 mM dNTPs, 1× hexanucleotide mix, 10 units RNase inhibitor, 10 units Moloney murine leukemia virus RT, and 1 μg total RNA. All of these commercially available products were purchased from Roche Diagnostics (Lewes, UK).
The expression of each gene was determined by RT-PCR, as previously described (5, 19). The analysis used oligonucleotide cDNA primers to each gene of interest by generating specific exon-intron spanning products (see Table 1). Briefly, the PCR program consisted of an initial denaturation [95°C (15 min)], amplification [stage 1, 94°C (30 s); stage 2, annealing temperature (30 s); stage 3, 72°C (60 s)], and final extension [72°C (7 min); 8°C “hold”]. The PCR mixture (final volume, 20 μl) contained 7 μl diethyl pyrocarbonate H2O, 10 μl Thermo-Start PCR Master Mix (50 μl contains 1.25 units Thermo-Start DNA polymerase, 1× Thermo-Start reaction buffer, 1.5 mM MgCl2, and 0.2 mM each of dATP, dCTP, dGTP, and dTTP; catalog no. AB-0938-DC-15; ABgene), 500 nM forward primer, 500 nM reverse primer, and 1 μl RT (cDNA; 2 ng) product. The annealing temperature and cycle numbers of all primers were optimized so as to be in the same linear range as the internal control, that is, 18S rRNA (see Table 1) as previously described (23). There were no developmental or nutritional effects on the mRNA abundance of hepatic 18S rRNA.
Agarose gel electrophoresis (2.0–2.5%) and ethidium bromide staining confirmed the presence of both the product of interest and 18S rRNA at the expected sizes. Densitometric analysis was performed on each gel by image detection using a Fuji film LAS-1000 cooled charge-coupled device camera and mRNA abundance determined for each gene. Consistency of lane loading for each sample was verified from the measurement of 18S rRNA. All results were calculated as a ratio of its own 18S rRNA abundance and expressed as a percentage of a reference sample (hepatic RNA extracted from a 1-day-old control sheep) run on all gels. Each analysis was performed in duplicate with appropriate positive (same as reference sample) and negative (RT: no RNA, no RT; PCR: no cDNA, no taq polymerase) controls and a full range of molecular weight markers. The resultant PCR product was extracted (catalog no. 28704; gel extraction kit, QIAquick) and sequenced, and results were cross referenced against the GenBank web site to determine specificity of the target gene. In addition, we undertook analyses for the antiapoptotic factor Bcl-2, and although it was highly abundant in the positive control i.e., adult spleen, it was undetectable in the liver of all animals (data not shown).
Statistical analysis was performed using SPSS software package (version 11.0). Data was first subjected to a Kolmogorov-Smirnov normality test to confirm normal distribution, thereby allowing parametric statistical tests to be performed. Mean body weight, dimensions, and organ weights, as well as gene abundance results were then analyzed by using a factorial (2 × 2) General Linear Model test to assess the main effects of parity and diet i.e., late gestational nutrient restriction and potential parity times diet interactions. Potential relationships between specific variables were investigated by using a parametric Pearson's correlation coefficient test. Data is expressed as means ± SE.
Day 1 of Postnatal Life
Birth weight, body composition, and liver weight.
At birth, both combined and individual twin body weights together with chest girth, but not crown-to-rump length, were all lower in offspring of nulliparous mothers, regardless of maternal nutrition (Table 2). These offspring had smaller livers (but not when expressed per kilogram body weight) and a lower liver-to-brain weight ratio, while relative perirenal adipose tissue mass [constituting 80% of fat in the newborn sheep (1)] was enhanced. The weights of all other major organs (i.e., brain, heart, kidney, lungs, and spleen) were reduced in offspring of nulliparous mothers and were in proportion to the reduction in total body weight (data not shown).
Hepatic mRNA abundance.
At 1 day of age, offspring of nulliparous mothers had the highest mRNA abundance for the GHR, which was reduced in NR offspring (Fig. 1). The offspring of nulliparous mothers also exhibited lower hepatic IGF-I and IGF-II mRNA abundance. Hepatic IGF-IR was, however, significantly increased in these offspring (Fig. 2). Neither maternal parity nor diet had a significant effect on IGF-IIR or GR mRNA abundance. Late gestational maternal nutrition had no effect upon IGF ligand or receptor mRNA. HGF mRNA abundance was upregulated in offspring of nulliparous mothers. This adaptation was accompanied by a downregulation in mRNA for both c-Met and Bax (Fig. 3). In addition, mRNA abundance for c-Met and Bax were positively correlated regardless of maternal parity and diet (r = 0.37; P < 0.01). Finally, mRNA abundance for both c-Met and Bax were upregulated by nutrient restriction. In summary, the primary factor determining mRNA abundance for the majority of genes examined in the newborn liver was parity. With increasing parity, and thus larger birth weight and liver mass, there was a decrease in mRNA abundance for GHR, IGF-IR, and HGF, whereas mRNA for IGF-I, IGF-II, and c-Met were all increased.
1 Mo of Postnatal Age
Growth rate, body composition, and organ weights.
There was no significant difference in mean birth weights between offspring that were sampled at 1 or 30 days of age, as all sheep produced twins that were within a 20% birth weight of each other. Despite being born of similar birth weight, NR offspring grew more slowly over the first month of life [nulliparous control: 406.3 ± 40.0 g/day (n = 6); nulliparous NR: 361.5 ± 21.1 g/day (n = 7); multiparous control: 404.7 ± 24.8 g/day (n = 5); multiparous NR 352.0 ± 17.1 g/day (n = 5)] and so weighed less than controls at 1 mo of age, regardless of maternal parity (Table 3). In contrast, offspring born to control nulliparous mothers experience relative “catch-up” growth over the first month of age and were of a similar weight to their multiparous counterparts. At the same time, offspring of nulliparous mothers maintained their smaller girth, having proportionately smaller livers and lower liver-to-brain weight ratio, while a greater proportion of body mass was made up of perirenal adipose tissue. In addition to the effects of parity, NR offspring had a significantly reduced girth, a difference not seen at birth.
Hepatic mRNA abundance.
As at 1 day of age, hepatic mRNA abundance for GHR was higher in offspring born to nulliparous compared with multiparous mothers and decreased with nutrient restriction, an adaptation that was reversed when offspring were born to multiparous mothers. That is, late gestational nutrient restriction of multiparous sheep increased hepatic GHR mRNA in the resultant offspring at 1 mo of age (Fig. 4). Neither maternal parity nor diet had any effect on GR mRNA abundance. IGF-II mRNA abundance remained reduced in offspring of nulliparous mothers, whereas IGF-IR was significantly increased a response that interacted with maternal diet (Fig. 5). In addition, the mRNA abundance for IGF-IR and IGF-IIR were both reduced by maternal nutrient restriction, whereas IGF-I mRNA was raised. As at 1 day of age, the mRNA abundance for HGF and c-Met plus Bax were up- and downregulated, respectively, in offspring of nulliparous mothers (Fig. 6). There was no effect of maternal nutrient restriction on these genes at this age. The mRNA abundance for c-Met was also negatively correlated with HGF (r = 0.26; P < 0.01) and Bax (r = 0.33; P < 0.01), with the latter relationship being dependent on maternal diet. In summary, the smaller livers of newborn offspring born to nulliparous mothers are maintained up to 1 mo of age in conjunction with persistently increased mRNA abundance for GHR, IGF-IR, and HGF, whereas mRNA for IGF-II and c-Met, were reduced. These longer-term adaptations are dependent, in part, on the prenatal maternal diet.
The main finding of our study was that maternal parity and late gestation nutrition have different effects upon body size, conformation, liver weight, and the hepatic GH-IGF axis in the resulting offspring over the first month of life. In particular, the reduction in both body and liver weight at birth in offspring born to nulliparous mothers was accompanied by a resetting of the hepatic GH-IGF axis together with changes in HGF responsiveness and potential action. Interestingly, these responses were largely unaffected by maternal nutrient restriction, and a majority of these adaptations persisted up to 1 mo of postnatal age. We have focused our attention on the neonatal liver as it provides a substantial endogenous energy reserve that can be rapidly mobilized and is known to be responsive to changes in maternal diet (9). Our analysis was therefore directed toward hepatic gene expression, as this critical stage of development is coincident with the maximal expression of a substantial number of genes that ensure tissue function meets the pronounced metabolic and endocrine changes occurring around the time of birth (15, 20, 49, 53).
Differential Effects of Maternal Parity and Late Gestation Nutrition on Weight at Birth and Body Conformation Over the First Month of Life
We have shown for the first time in the neonatal liver pronounced differences in mRNA abundance for specific hormones and their receptors that are mediated primarily by maternal parity rather than food intake. Importantly, our results in the sheep may be applicable to the human situation (38) given the marked similarities in reduction of birth weight and later catch-up growth in offspring of nulliparous mothers. There can be appreciable competition between the mother and her fetus for available nutrients during periods of maternal undernutrition and in her first pregnancy (25, 57). Consequently, the fetuses' supply and demand for nutrients are met by reducing overall growth rate at different stages of pregnancy (61). Our finding that the offspring born to nulliparous mothers were lighter at birth is in accord with a number of species (52, 55, 58), including humans (38). This effect may be mediated by decreased uterine vascularity (40) plus reduced placental efficiency (58). Offspring of nulliparous mothers were also asymmetrically smaller with reduced liver-to-brain weight ratios but possessing disproportionately more perirenal adipose tissue at both sampling ages. By 1 mo of age, these offspring had exhibited catch-up growth that is associated with later obesity (37). These results indicate that the underlying mechanisms behind this outcome may be in place prior to birth. It is noteworthy that catch-up growth did not occur when the mother was NR during late gestation. Both body weight and girth were reduced in NR offspring at 1 mo of age, which may be indicative of a negative effect on either mammary development and/or milk production, although this remains to be confirmed. The absence of any nutritional effect on weight at birth is in accord with previous studies that have used comparable numbers of pregnant sheep (8, 54).
Effect of Parity on Hepatic Growth Factor and c-Met
The liver, in sheep as in humans, forms early in gestation and then continues to grow up to term. Hepatocytes constitute ∼80% of liver tissue and do not express the IGF-IR (26). However, nonparenchymal cells do express IGF-IR (26), although by adulthood expression are greatly reduced (12). As a consequence, hepatocyte proliferation is largely regulated by other growth factors, such as HGF signaling through its receptor c-Met, which can have mitogenic, morphogenic, motogenic, and anti-apoptotic effects (4, 62). In the present study, we found that mRNA abundance for c-Met was significantly decreased in offspring born to nulliparous mothers, suggesting their livers are partly resistant to HGF. The regulation and role of HGF in the fetus has not been widely studied. HGF is critical to fetal development, with mice lacking HGF failing to develop normally as their liver is reduced in size with extensive loss of parenchymal cells (24, 47, 59). These HGF knockout mice also experience problems with placental development leading to severe intrauterine growth retardation and in utero death during late gestation.
Hepatic HGF mRNA abundance is nutritionally programmed and decreased in young adult sheep whose mothers were NR from early-to-mid gestation which was accompanied by smaller livers (23). However, livers from offspring born to nulliparous mothers exhibited raised hepatic HGF mRNA abundance despite reduced liver growth, suggesting that it is more likely to have an endocrine rather than paracrine role (27), possibly increasing cell proliferation in other tissues. To this end, HGF has recently been identified as an adipokine (44), with its synthesis and secretion elevated in obese patients (43). This is of particular interest with regard to the present study, as the one tissue whose mass was increased in offspring born to nulliparous mothers was perirenal fat. Enhanced hepatic HGF secretory capacity could influence the rate of apoptosis in liver cells by decreasing Bax expression and inhibiting its translocation from the cytosol to mitochondrial membrane (34). Interestingly, hepatic mRNA abundance for Bax and c-Met were positively correlated in 1-day-old but not 1-mo-old offspring, suggesting the immediate effects of reduced c-Met in the newborn are mediated through changes in Bax.
Differential Effects of Maternal Parity and Nutrition on the Hepatic GH-IGF Axis
A persistent increase in hepatic GHR mRNA abundance was apparent in offspring born to nulliparous mothers regardless of maternal food intake. This change in potential GH responsiveness was accompanied by a lower mRNA abundance for IGF-I at 1 day but not 30 days of age, whereas IGF-IR mRNA was upregulated in offspring born to nulliparous mothers at both ages. The increased GHR is likely to be in response to increased plasma GH (60), which is increased in individuals born to both first-time (18) and undernourished mothers in late gestation (3) although IGF-I remains low (3, 32, 36) but remains to be established in the present model. Further evidence of differences in GH responsiveness depending on maternal parity is that this had the opposite effect on GHR mRNA abundance compared with maternal nutrient restriction. In adults, fasting or nutrient restriction causes a transient state of GH resistance in which GH rises further following receptor downregulation (35, 46). During perinatal development, the GHR undergoes a major transition at birth coincident with the appearance of the adult liver-specific form of the receptor (29). This adaptation is coincident with a surge in fetal plasma cortisol around the time of birth and concomitant increase in GR (15). In the present study, we found no differences in hepatic GR mRNA abundance between maternal groups with all offspring born at ∼147 days, indicating that there was no major difference in maturation of the hypothalamic-pituitary-adrenal axis. Further factors implicated in fetal development of the GHR are thyroid hormones (48), which also respond to changes in maternal energy balance (6, 41). In cows, maternal plasma thyroid hormone concentrations increase with parity, a difference maintained through lactation (55), and could therefore be one factor important in mediating differential effects of maternal parity and nutrition.
At 1 day, but not 30 days of age, the only maternal factor influencing IGF mRNA abundance was parity. In this regard IGF-I and IGF-II mRNA were both reduced in livers of offspring born to nulliparous mothers with the lower IGF-II persisting up to 1 mo of age. This provides evidence at the tissue level for an involvement of IGF-II in the comparative fetal growth restriction in nulliparous mothers (39). The reduction in IGF-I, but not IGF-II, was accompanied by an upregulation in receptor mRNA that persisted up to 1 mo of age. It is therefore possible that the livers from offspring born to nulliparous mothers exhibit increased sensitivity to IGF-I and are therefore unaffected by any reduction in hepatic IGF secretory potential. A decrease in IGF-II compared with raised GHR mRNA abundance is in accord with the known effects of both cortisol and thyroid hormones on these genes in the late gestation fetus (14, 28). In the present study, however, mRNA abundance for the GR was unaffected. Clearly further investigation on the impact of maternal parity on fetal endocrine status, particularly the hypothalamic-pituitary-thyroid-adrenal axis is warranted. This may be particularly important with regard to fetal cortisol responses to maternal nutrient restriction. As to date this has been shown to be unresponsive even when maternal plasma cortisol is transiently raised (13).
In conclusion, maternal parity has a substantial effect on liver size at birth and mRNA abundance for its hormone receptor population that is only partly dependent on the maternal diet. Growth-restricted offspring of nulliparous mothers therefore exhibit a compensatory resetting of the endocrine control of hepatic growth particularly within the HGF and GH-IGF axis. These adaptations are not, however, amplified by maternal nutrient restriction in late gestation and offspring, born to nulliparous mothers, showing increased perirenal fat mass over the first month of life. The extent to which such changes in hepatic function may subsequently contribute to metabolic disturbances in these offspring as they show catch-up growth remains an important area for future research.
This work was supported by the University of Nottingham Children's Brain Tumour Research Fund.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2007 the American Physiological Society