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Center for Reproduction and Early Life, Institute of Clinical Research (M.A.H., H.B., T.S., M.E.S.), and Children’s Brain Tumor Research Center (M.A.H., D.W.), The University of Nottingham, Notingham NG7 2UH, United Kingdom
Address all correspondence and requests for reprints to: Michael E. Symonds, Academic Division of Child Health, School of Human Development, Queen’s Medical Center, University Hospital, Nottingham NG7 2UH, United Kingdom. E-mail: michael.symonds{at}nottingham.ac.uk.
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Abstract |
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Introduction |
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It is established that hepatic mRNA abundance for a number of hormones and their receptors, which have a critical role in metabolism and tissue growth, increase near to term in parallel with the prepartum surge in glucocorticoids (1, 8). These include GH receptor (GHR) (9) and prolactin receptor (PRLR) (10), together with IGF-I (11), whereas, in contrast, IGF-II is negatively regulated (12). The extent to which such adaptations are confined to the period immediately before birth, or whether they may also change between midgestation and late gestation, or soon after birth, has yet to be examined. Determining the fetal and postnatal ontogeny of the aforementioned genes is not only important in understanding the hepatic adaptations involved in the transition form intrauterine to extrauterine life, but will also enhance our ability to interpret the immediate and long-term effects of maternal nutritional manipulation at different stages of gestation (13, 14). In this regard, maternal nutrient restriction between early and midgestation results in adult offspring in which liver mass is reduced in conjunction with decreased gene expression for a number of important metabolic regulators including the GHR, PRLR, and IGF-II receptor (IGF-IIR) (15). The extent to which the mRNA abundance for both these and related [i.e. IGF-I, IGF-II, and IGF-I receptor (IGF-IR)] genes that are unaffected in the adult may be reset in utero in response to a shorter period of maternal nutrient restriction is currently not known. In contrast, maternal nutrient restriction in late gestation has little impact on hepatic gene expression in the neonate (16).
Therefore, the aim of our study was to examine the ontogenic changes in gene expression of hepatic GHR, PRLR, glucocorticoid receptor (GR), IGF-I and -II and their receptors between midgestation through to term, after birth, and then up to 6 months of juvenile life. This was accompanied by an examination of the effects of maternal nutrient restriction between early and midgestation, coincident with the period of maximal placental growth plus early hepatic growth, on later liver development. In this second part of the study, the fetal liver was examined both at the end of the period of nutrient restriction, i.e. 80 d gestation as well as 60 d later, i.e. near to term and, subsequently, in the resulting juvenile offspring.
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Materials and Methods |
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148 d) and sheep after birth at 1, 30, and 180 d (6 months) of postnatal age (n = 6 at each sampling point, 30 sheep in total) after euthanasia with an overdose of barbiturate (100 mg/kg pentobarbital sodium; Euthatal; RMB Animal Health, Dagenham, Essex, UK). All mothers were fed 100% of their total metabolizable energy (ME) requirements [taking into account requirements for both maternal maintenance and growth of the conceptus to produce a 4.5-kg newborn at term (18)]. Sheep randomized to postnatal sampling were born normally at term. The liver was rapidly dissected and weighed and a representative portion (i.e. 2 g from an 80-d fetus and 20 g at all other ages) from the same position in the right hepatic lobe from each animal was placed in liquid nitrogen and stored at –80 C until molecular analysis.
Maternal nutritional manipulation of hepatic gene expression
This study was designed to examine the effects of early to midgestational nutrient restriction, coinciding with the period of maximal placental growth, on the fetus and offspring and thus used singleton-bearing sheep. Thirty-six singleton-bearing Welsh Mountain sheep of similar age (median 3 yr) and weight [36.1 ± 0.9 kg (mean ± SEM)] were entered into the study and individually housed at 28 d gestation. Animals were allocated to one of two nutritional groups using stratified randomization by body weight. They consumed either 60% (i.e. nutrient restricted) or 150% (i.e. fed to appetite) of their calculated ME requirements (Agricultural Research Council, 1980). Feed intakes were measured daily. Food consumption between 28–80 d gestation was 3.2–3.8 MJ/d of ME in the nutrient-restricted group (60% of ME requirements) or 8.7–9.9 MJ/d of ME in the group fed to appetite (150% of ME requirements) (19). The amount of feed given to each mother was increased at 43 and 61 d gestation to meet the higher energy requirements associated with growth of the conceptus (Agricultural Research Council, 1980). The diet comprised chopped hay that had an estimated ME content of 7.91 MJ/kg dry matter and a crude protein content (nitrogen x 6.25) of 69 g/kg dry matter and barley-based concentrate that had an estimated ME content of 11.6 MJ/kg dry matter and a crude protein content of 162 g/kg dry matter. The proportion of hay to concentrate fed was approximately 3:1, with respect to dry weight. All diets contained adequate minerals and vitamins. These were added separately to the diet with equal amounts provided to all sheep and, thus, were sufficient to fully meet their requirements. After 80 d gestation, all sheep were offered sufficient feed to meet 100% of the ME requirements. They consumed between 6.5 and 7.5 MJ/d of ME with the amount of feed provided being increased at 100 and 120 d gestation to meet the increased ME requirements that accompany the increase in fetal weight with gestation. In those pregnant sheep allowed to go to term, all gave birth normally and the offspring were weaned at 3 months of age. Throughout lactation, all mothers were fed to requirements with hay provided ad libitum together with 1 kg concentrate.
To determine the effect of early to midgestational maternal nutrient restriction on fetal liver development, six sheep within each nutrition group were randomized for tissue sampling at either 80 or 140 d gestation. Each animal was humanely euthanized after iv administration of 100 mg/kg pentobarbital sodium. The fetus was rapidly dissected and weighed and a representative portion of the liver was placed in liquid nitrogen and stored at –80 C until further analysis. The remaining offspring (n = 6 per nutritional group) were sampled at 180 d (6 months) after birth.
All operative procedures and experimental protocols had the required Home Office approval as designated by the Animals (Scientific Procedures) Act (1986) and were approved by the animal experimentation ethics committee at the University of Nottingham.
Laboratory procedures
Messenger RNA detection.
Total RNA was isolated from a central region of the right hepatic lobe 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 (15). 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 optimized RT reaction (final volume 20 µl) contained: 1x reverse transcriptase buffer (250 mM Tris-HCl, 40 mM MgCl2, 150 mM KCl, 5 mM dithioerythritol pH 8.5), 2 mM dNTPs, 1x hexanucleotide mix, 10 U RNase inhibitor, 10 U Moloney murine leukemia virus reverse transcriptase, and 1 µg total RNA. All these commercially available products were purchased from Roche Diagnostics Ltd. (Lewes, UK).
The expression of each gene was determined by RT-PCR using ovine-specific oligonucleotide primers, as previously described for the liver (15, 16). Briefly, the PCR program consisted of an initial denaturation [95 C (15 min)], amplification [stage I, 94 C (30 sec); stage II, annealing temperature (30 sec); stage III, 72 C (60 sec)] 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 U Thermo-Start DNA Polymerase, 1x 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, Epsom, UK), 500 nM forward primer, 500 nM reverse primer, and 1 µl RT (cDNA; 2 ng) product. The amount of cDNA used for PCR, 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 as previously described (15, 16). 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 at the expected sizes. Densitometric analysis was performed on each gel by image detection using a Fujifilm LAS-1000 cooled charge-coupled device camera and mRNA abundance determined for each gene. Consistency of lane loading for each randomly loaded 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-d-old control sheep) ran on all gels. Each analysis was performed in duplicate with appropriate positive (same as reference sample) and negative (RT: 1) no RNA, 2) no reverse transcriptase; PCR: 1) no cDNA, 2) no Taq polymerase) controls and a full range of molecular weight markers. The resultant PCR product was extracted (QIAquick gel extraction kit; catalog no. 28704) and sequenced, and results were cross-referenced against the GenBank database to determine specificity of the target gene. For those genes in which we have previously validated their detection in sheep samples using real-time PCR (20), e.g. the GR, representative samples were reanalyzed and in each case confirmed the RT-PCR results with regard to molecular responses.
Protein detection.
Plasma membrane, mitochondria, and whole cell lysate were prepared from liver tissue sampled from each fetus and sheep (21), and their protein contents were determined using the Lowry protocol (22). The abundance of PRLR protein was determined on 10 µg plasma membrane protein by Western blot analysis using the R120 polyclonal antibody (23). The intraassay and interassay coefficient of variation for immunodetection of PRLR was 2.7 and 5.9%, respectively. Further Western blots were performed to confirm GHR, IGF-IIR, IGF-IR, and GR mRNA findings. Antibodies used included: 1) two GHR antibodies: a mouse antirabbit polyclonal antibody (RDI-GHRabm-5; Research Diagnostics Inc., Concord, MA) and a polyclonal antimouse GHR (gift from G. Thordarson, University of California, Santa Cruz, CA), 2) an IGF-IIR polyclonal antibody [gift from P. Lobel, Rutgers University, Camden, NJ as previously described (24)] 3) an IGF-IR polyclonal antirabbit antibody: Santa Cruz Biotechnology (Santa Cruz, CA; catalog no. SC 713), and 4) a GR polyclonal antirabbit antibody (Santa Cruz Biotechnology; catalog no. SC 8992). Each antibody was tested at a range of dilutions from 1:250–1:1000 under both mild reducing and nonreducing conditions on up to 80 µg of relevant protein preparations. Unfortunately, none of these antibodies yielded a signal (in any of the animals) that was in accord with its predicted molecular weight (i.e. signals were nonspecific). Nonspecificity of detected bands was confirmed through regression analysis of molecular weight markers to determine exact size and through incubation with nonimmune rabbit serum. All Western blots were run in duplicate and included a range of molecular weight markers and a reference sample (appropriate protein preparation from a 1-d-old sheep) to allow for comparisons between gels.
Statistical analyses
Statistical analysis was performed using SPSS software package (version 15.0). Data first were subjected to a Kolmogorov-Smirnov normality test to confirm normal distribution. Where appropriate, data were log10 transformed to achieve normality, thereby allowing parametric statistical tests to be performed. Liver weight and mRNA abundances obtained in the ontogeny study were compared by a restricted maximum likelihood (REML) analysis through SPSS univariate general linear model test followed by Bonferroni post hoc correction. The terms fitted to the model were developmental age, genotype, and gender. There were no differences in mean values obtained from male and female offspring within each age group. Therefore, data were pooled from these animals. Mean values for organ weight and abundance of target genes between control and nutrient-restricted offspring within each age group were compared using a Student’s unpaired t test. For consistency, all data are presented as untransformed means and their SE. For all statistical comparisons, significance was accepted when P < 0.05.
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Results |
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). A similar pattern of changes was observed for the PRLR with the modification that mRNA and protein abundance peaked at 140 d gestation rather than after birth. These adaptations were unrelated to changes in liver mass, which increased between 80–140 d gestation, remained unchanged at birth, and then rose up to 6 months of age (Table 1).
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). In contrast, mRNA abundance for the IGF-IR increased between late gestation and 1 d after birth to then decline up to 6 months of age. The ontogeny of gene expression for IGF-II and its receptor was very different from that of IGF-I in that mRNA abundance decreased between 80–140 d gestation only to then increase after birth and remain unchanged thereafter. In contrast, there was no effect of developmental age on IGF-IIR mRNA abundance.
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). In marked contrast, PRLR mRNA and protein was greater in the livers of nutrient-restricted fetuses at 80 d gestation but substantially lower near to term as a consequence of a failure to exhibit any developmental increase with gestational age. By 6 months of age, however, there was no difference between nutritional groups.
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). A similar pattern of developmental changes, as seen for IGF-I, was also observed for IGF-II. In contrast, hepatic IGF-IIR was significantly reduced in nutrient-restricted fetuses, an adaptation that did not persist into juvenile life.
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Discussion |
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Taken together, a number of aspects of our study are novel. First, gene expression of both GHR and PRLR remained low near to term in previously nutrient-restricted fetuses. This is surprising as the developmental increase in both GHR and PRLR are primarily mediated by the prepartum surge in cortisol (9, 10), and there is currently no evidence that plasma cortisol is different between control and previously nutrient-restricted offspring (19, 26). Indeed, glucocorticoid action is increased in the livers, as well as adipose tissue, kidneys, and lungs of neonates born to mothers nutrient restricted between early and midgestation (27). Secondly, ontogenic and nutritional manipulation of hepatic PRLR is similar in both its gene and protein expression, which lends support to mRNA data for the other genes we examined, but we are currently unable to confirm protein expression due to the lack of credible antibodies. Furthermore, we find no differences in GHR, PRLR, or GR mRNA abundance in juvenile offspring born to nutrient-restricted mothers. This may reflect a transitional period in metabolic adaptation in these animals before their attaining mature body weight. Indeed, at this age, only hepatic IGF-I and -II mRNA abundance was significantly higher and may, thus, be the dominant early hepatic growth factors before declining through to adulthood (28), in conjunction with loss of mRNA for the IGF-IR, but maintained IGF-IIR. Taken together, these adaptations would limit liver growth in the adult (15).
It is unlikely that the potential mechanisms by which hepatic gene expression was reset in nutrient-restricted fetuses reflects a prolonged change in plasma cortisol, because, as discussed above, there is no evidence to date that this is increased in either nutrient-restricted animals either during fetal (26) or later (29) life. Indeed, the fact that hepatic GHR and PRLR were significantly decreased near to term, but not at the time when maternal nutrient intake was reduced, suggests that the endocrine sensitivity to the prevailing plasma cortisol concentration was reset. In this regard, an increase in potential glucocorticoid action in the liver at 80 d of gestation (27) would potentially reduce hepatic sensitivity to the prepartum surge in cortisol, thus explaining why mRNA abundance for potentially glucocorticoid-sensitive genes was markedly reduced in late gestation. An adaptation of this type was most apparent for the PRLR with maternal nutrient restriction causing a precocious increase in mRNA and protein abundance at 80 d gestation with no further increase up to 140 d gestation.
A markedly different adaptation was observed for the IGF-IIR in which mRNA abundance was lower at 80 d gestation and remained so up to 140 d gestation. However, the IGF-IIR gene exhibited a much smaller increase with gestational age compared with the PRLR indicating very different transcriptional regulation and, thereby, potentially explaining differential responses between genes. It may also appear surprising that the reduction in IGF-IIR mRNA was not accompanied by any change in liver growth as, under the artificial conditions of in vitro fertilization, reduced IGF-IIR increases IGF-II availability and promotes fetal organ growth (24). The same type of adaptation does not appear to be apparent in the nutritionally manipulated fetus or the resulting offspring and suggests very different IGF-II regulation between in vitro fertilized and naturally conceived pregnancies. The lack of any nutritional effect on either liver weight in the fetus or the offspring is in accord with previous studies in which fetal number has been kept constant between nutritional groups (27), but not when a mix of singletons and twins are used (30). Thus, it appears that the reduced liver mass seen in adults born to mothers nutrient restricted between early gestation to midgestation is an adult adaptation that is mediated by both a reduction in mitogenic stimulation of the liver together with enhanced apoptosis (15).
Of the genes that were not nutritionally responsive in utero (i.e. IGF-I, IGF-II, and IGF-IR), all appear to show a different ontogeny compared with those that were sensitive to the maternal nutritional environment (i.e. GHR, PRLR, GR, and IGF-IIR). Hepatic IGF-II mRNA undergoes a pronounced decrease in gene expression before birth which reflects the negative influence of cortisol (12). The increase in IGF-II gene expression that then occurs between birth and 1 month of age may be mediated by the pronounced decrease in plasma cortisol over this period (31). In contrast, hepatic IGF-I and IGF-IR mRNA levels were very low at 80 d of gestation and, as such, could be predicted to be nutritionally insensitive in the fetus at this time. These two genes then exhibit differing developmental changes up to 1 d of postnatal age with hepatic IGF-IR mRNA showing an exponential increase. In contrast, IGF-I mRNA abundance rose up to 140 d gestation but remained unchanged thereafter up to 6 months of age. However, expression of IGF-IR mRNA rapidly decreased after birth and is no longer detectable in the adult liver (15). Taken together, these differences reflect the very different regulation of gene expression during a time when rapid liver growth occurs.
In conclusion, we have shown for the first time the ontogeny of the primary metabolic regulators of liver growth from the midgestational fetus up to juvenile life. This has enabled us to dissect the differential effects of maternal nutrient restriction over the period of early liver growth in the fetus. As such, the very different responses between the genes examined can be explained by the relative mRNA abundance at the time of this nutritional intervention, or by the subsequent change in gene expression up to the time of birth. At this time, the majority of genes exhibit maximal abundance necessary for ensuring the onset of liver function very soon after birth.
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Footnotes |
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Disclosure Statement: The authors have nothing to disclose.
First Published Online July 19, 2007
Abbreviations: GHR, GH receptor; GR, glucocorticoid receptor; IGF-IR, IGF-I receptor; IGF-IIR, IGF-II receptor; ME, metabolizable energy; PRLR, prolactin receptor; REML, restricted maximum likelihood.
Received March 5, 2007.
Accepted for publication July 6, 2007.
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