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RESEARCH |
School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, UK, 1 MRC Epidemiology Resource Centre, University of Southampton, Southampton General Hospital, Southampton SO16 6YD, UK and 2 Developmental Origins of Health and Disease Division, School of Medicine, University of Southampton, Princess Anne Hospital, Southampton SO16 5YA, UK
Correspondence should be addressed to W Y Kwong; Email: wyk{at}soton.ac.uk
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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The DOHaD concept may be extended by the identification of developmental ‘windows’ of plasticity when nutrient availability may act decisively to alter the pattern of future development. One such potential window is the preimplantation embryo when embryonic and extra-embryonic lineages are first established and segregated, and which is known to be sensitive to the environmental conditions with lasting consequences (reviewed in Khosla et al. 2001a, Thompson et al. 2001, Fleming et al. 2004a,b). Thus, we have shown that feeding pregnant rats with low protein diet (9 vs 18% casein) exclusively during the preimplantation period led to male offspring with altered postnatal growth rate and organ sizes and elevated systolic blood pressure, with less severe changes evidence in female offspring (Kwong et al. 2000).
In vitro culture conditions for preimplantation embryos have also been shown to alter incipient gene expression (Niemann & Wrenzycki 2000), fetal development rate after transfer (Khosla et al. 2001b, Thompson et al. 2001, Lane & Gardner 2003) and postnatal physiology (Young et al. 2001). In sheep and cattle, early embryo culture conditions can give a rise to large offspring syndrome (LOS) after transfer, associated with increased perinatal malformations and mortality rate (Walker et al. 1996). In addition, mouse offspring derived from cultured embryos have been shown to display altered behaviour with respect to anxiety and spatial memory compared with in vivo-derived counterparts (Ecker et al. 2004, Fernández-Gonzalez et al. 2004). Although a variety of mechanisms may associate preimplantation environment with future developmental changes, the aberrant expression of imprinted genes has been proposed to play a significant role (Young & Fairburn 2000, Fleming et al. 2004a,b).
Imprinted genes are only expressed from either maternal or paternal alleles due to epigenetic modifications such as DNA methylation to CpG islands in regulatory domains (Delaval & Feil 2004, Dean et al. 2005). Several studies have identified the changes in imprinted gene expression throughout the development associated with culture composition during the pre-implantation period when, through zygotic genome activation mechanisms, the DNA methylation pattern may be sensitive to environmental conditions (Doherty et al. 2000, Thompson et al. 2001, Jaenisch & Bird 2003). Thus, exposure of sheep embryos to serum leading to LOS induced the decreased fetal expression of imprinted insulin-like growth factor 2 receptor (IGF2R) transcript and the loss of methylation on the normally active maternal allele (Young et al. 2001). In mouse, fetuses produced from blastocysts cultured in the presence of fetal calf serum were lighter at embryonic day 14 and exhibited decreased insulin-like growth factor 2 (Igf2), H19 and Grb7 imprinted gene expression and an increase in Grb10 expression (Khosla et al. 2001b). Mouse preimplantation embryos cultured in Whitten’s medium displayed the aberrant expression of H19 from the normally silent paternal allele due to altered methylation pattern (Doherty et al. 2000). This aberrant H19 expression persisted in placental tissues in mid-gestation (Mann et al. 2004). Furthermore, the culturing of mouse zygotes to the blastocyst stage in the presence of ammonium increased the level of H19 gene transcription (Lane & Gardner 2003).
Fetal growth is largely controlled by the complex IGF system. Several imprinted genes are shown to be related to expression/function of IGF2, which is paternally expressed in several tissues. The deletion of maternal H19 allele gives rise to pups that are 27% heavier than their wild type littermate (Leighton et al. 1995a). The level of IGF2 protein can also be regulated by IGF2R (Lau et al. 1994). Previously, studies have shown that H19 is very sensitive to preimplantation culture condition (Doherty et al. 2000, Lane & Gardner 2003, Mann et al. 2004) and maternal dietary restriction altered H19 and IGF2 gene expression in fetal sheep (Brameld et al. 2000, Naimeh et al. 2001). The culturing of sheep embryos to blastocyst stage led to reduction in IGF2R transcript and protein in late gestation (Young et al. 2001). However, it is unknown whether embryo environment in vivo, through maternal LPD treatment, may similarly alter the pattern of embryonic imprinted gene expression with lasting consequences. Therefore, we examined the effect of maternal LPD fed solely during preimplantation period (switched diet) or throughout gestation (LPD) on fetal growth and the pattern of embryonic and fetal expression on these imprinted genes. For the first time, we show that maternal LPD alters blastocyst imprinted gene expression with reduced H19 mRNA evident in male embryos. A reduced expression level for H19 and Igf2 transcripts is also evident in day 20 male fetal liver. These changes in gene expression precede alteration in growth which is not evident until the postnatal period.
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Materials and Methods |
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Embryo collection
Embryos at different stages of development were collected from oviducts or uteri between day 2 and 4 by flushing with H6 medium containing BSA (4 mg/ml, Sigma; Sheth et al. 1997). Freshly collected embryos were rapidly washed once in H6+BSA, three times with H6+ polyvinyl pyrrolidone (6 mg/ml, Sigma) medium (Sheth et al. 1997) and three times with PBS. Single embryos in minimal volume (2–5 µl) of PBS were then transferred to siliconised tubes, snap frozen in liquid nitrogen and stored at –80 °C until used. Inner cell masses (ICMs) were isolated by immunosurgery from blastocysts as described (Eckert et al. 2004) prior to snap frozen in liquid nitrogen and stored at –80 °C until used.
Isolation of Poly A+ RNA in preimplantation embryos
Poly A+ RNA was isolated from single embryos using Dynabeads Oligo (dT)25 (Dynabeads mRNA DIRECT kit, Dynal Biotech, Wirral, UK). All reagents were provided in the Dynabeads mRNA DIRECT kit unless otherwise stated. Prior to isolation, 1 pg luciferase mRNA (1 pg/µl, Promega) was added to each tube containing single embryos to serve as an external standard. Single frozen-stored embryos were lysed by adding 150 µl lysis-binding buffer (100 mM Tris–HCl, pH 8.0, 500 mM LiCl, 10 mM EDTA, 1% (w/v) lithium dodecylsulfate (LiDS), 5 mM dithiothreitol) and incubated at room temperature for 10 min before addition of 10 µl washed Dynabeads Oligo (dT)25 in lysis-binding buffer and incubation at room temperature for 10 min on a roller. The beads with bound poly A+ RNA were separated employing a Dynal MPC-P-12 magnet (Dynal Biotech). The supernatant was kept for DNA extraction (see below) in order to determine the gender of the blastocyst. After two washes with 100 µl wash buffer A (10 mM Tris–HCl, pH 8.0, 0.15 M LiCl, 1 mM EDTA, 0.1% (w/v) LiDS) and three washes with 100 µl wash buffer B (10 mM Tris–HCl, pH 8.0, 0.15 M LiCl, 1 mM EDTA), the poly A+ RNA was eluted from Oligo (dT)25 beads in 10 µl nuclease-free water (Anachem, Luton, UK) by incubating at 65 °C for 2 min. RT was carried out using 80% of the eluted poly A+RNA in 20 µl reaction comprising 1xRT buffer, Sensiscript Reverse Transcriptase (Qiagen), 20 U RNase inhibitor (Roche), 0.19 µM random hexamers (Promega) and 500 µM of each dNTP (Invitrogen) according to the manufacturer’s instructions. To ensure the absence of DNA contamination, the remaining 20% of eluted poly A+ RNA was used in the same reaction except that Sensiscript enzyme was omitted.
Embryo gene expression
Different proportions of single embryo cDNA were used for parallel PCR to detect target gene expression using specific primers (Table 1
). PCR was performed in a total volume of 50 µl containing 1x PCR buffer (20 mM Tris–HCl, pH 8.4, 50 mM KCl), 2 mM MgCl2 (Invitrogen), 200 µM of each dNTP (Invitrogen) and 40 pmol of each primer. A hot start was used to ensure the specific amplification of the target gene. The PCR cycles involved one cycle of 94 °C for 3 min, 72 °C for 30 s prior to addition of 2.5 U of Taq DNA polymerase (Invitrogen). PCR was performed for varying numbers of cycles with the denaturing step at 94 °C for 30 s, different annealing temperatures (Table 1) for 30 s and extension step at 72°C for 45 s. After the last cycle, samples were kept at 72 °C for 10 min. PCR products (10 µl) were separated on 1% agarose gel in the presence of 1 µg/ml ethidium bromide and integrated density values (IDV) generated using a digitised camera system (Alpha Imager 1220) accomplished with AlphaEase software (Alpha Innotech). After subtracting the background intensity for each band, the relative abundance of a given transcript was determined as the ratio of the IDV of that transcript to IDV value of luciferase standard (Miller et al. 2003).
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). Then, samples were heated at 94 °C for 8 min, 60 °C for 2 min for three cycles before the addition of 2.5 U Taq DNA polymerase. PCR was performed for 30 cycles consisting of 94 °C for 1 min, 60 °C for 2.5 min and 72 °C for 2.5 min. After the last cycle, the samples were kept at 72 °C for another 10 min. The first stage product (2 µl) was transferred into two tubes each containing 47.5 µl master mix (as above) either with 40 pmol of each Hprt and Zfy inner primers or with Sry inner primers (Table 2). After the addition of 2.5 U Taq DNA polymerase, PCR was performed at the same conditions as described above. After the second stage PCR, 15 µl product was separated in 1.8% agarose gel and DNA bands were visualised under UV illumination.
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Preparation of biotinylated riboprobes
The PCR products of H19, Igf2, ß-actin and 28S rRNA from fetal liver or placenta were cloned into TOPO-2.1 vector according to manufacturer’s instructions (Invitrogen). In vitro transcription was performed in a final volume of 20 µl containing 1 µg of linearised plasmid, 1x reaction buffer (40 mM Tris, pH 7.9, 6 mM MgCl2, 2 mM spermidine and 10 mM NaCl, Promega), 0.01 M DTT, 2 mM each ATP, GTP and UTP, 0.5 mM CTP, 1 µl RNase inhibitor (10 U/µl, Promega), 0.5 mM Biotin-CTP (Amersham) and 1 µl T7 RNA polymerase (10 U/µl, Promega). Reaction mix was incubated at 37 °C for 1.5 h before treatment with RNase-free DNase I (Promega) at 37 °C for 15 min and the reaction terminated by adding 1 µl 0.5 M EDTA. In vitro-transcribed product was separated from unincorporated nucleotides in a 7 M urea, 6% polyacrylamide gel (Invitrogen) at 180 V for 50 min before visualising on Fluor-coated thin layer chromatography plate (Ambion, Huntingdon, UK) at 254 nm. Relevant bands were excised and eluted in probe elution buffer (Ambion).
RNase protection assay
RNase protection assay was carried out using RPA III kit (Ambion). Briefly, 5 µg total RNA from fetal liver or placenta was hybridised with biotinylated riboprobes (6 fmol for ß-actin, H19 and Igf2 and 10.65 pmol for 28S rRNA) at 42 °C overnight in hybridisation III buffer as described by the manufacturer. After hybridisation, a mixture of 2.5 U/ml RNase A and 100 U/ml RNase T1 was added and incubated at 37 °C for 30 min. Protected hybrids were purified and separated on 7 M urea, 6% polyacrylamide gel (Invitrogen) at 180 V. A ‘no RNase’ (undigested probes) and ‘no target’ (probes hybridised with yeast RNA treated with RNase enzyme mix) controls were included in every assay to check probe integrity and the completion of enzyme digestion. Then, bands were transferred to Biodyne B membrane (Pierce, Cramlington, UK) at 30 V for 2 h. Membranes were then air-dried and bands were detected using Supersignal RPA III Chemiluminescent Detection kit (Pierce) as described by the manufacturer. Band intensity was quantified using VersaDoc Imaging System and Quantity One software (Bio-Rad).
17ß-estradiol and progesterone analysis in maternal serum
Maternal blood samples were taken by cardiac puncture on day 4 of pregnancy at the same time as blastocyst collection. The serum was prepared by centrifuging the blood at 4 °C, 1000 g for 10 min. The 17ß-estradiol was measured by the Estradiol Maia kit (Serono) and progesterone by the Amerlex-M progesterone kit (Amersham). Control sera gave inter-assay coefficients of variation for 17ß-estradiol and progesterone of 9.9 and 9.4% respectively.
Bisulphite mutagenesis and sequencing
DNA from day 20 fetal liver was extracted using DNeasy Tissue kit (Qiagen) according to the manufacturer’s instruction. DNA samples (4 µg) were bisulphite treated as described by Warnecke et al.(1998), and bisulphite-treated DNA was dissolved in 20 µl water and stored at –20 °C until used. PCR was performed using 8 µl bisulphite-treated DNA in a reaction volume of 50 µl containing 2.5 U Taq polymerase, 1x reaction buffer (Invitrogen), 2 mM MgCl2, 200 µM of each dNTPs and 40 pmol of outer primers for H19 differentially methylated region (GenBank AF043428
[GenBank]
) for 30 cycles at 94 °C for 1 min, 50 °C for 1 min and 72 °C for 1 min. Then, 2 µl of first stage PCR product was amplified for another 30 cycles with the same conditions as the first stage reaction. Outer primers: (4207–4233) 5' GGTTTTTATGGTTTTTG-GATTTTTAAA 3' and (4873–4845) 5' AAAAACCATTCCA-TAAATCCAAATACCTA 3'. Inner primers: (4237–4270) 5' TTAGTGTGGTTTATTATTAGAAGATGTAGAAGT 3' and (4844–4802) 5' TAAACCTAAATAACTTAAAACTTTATCA-CAAAC 3'. PCR products were then gel purified by QIAquick Gel Extraction kit (Qiagen) and subjected to a sequencing using ABI PRISM Big-Dye v1.1 sequencing ready reaction mix (Applied Biosystems, Warrington, UK).
Statistical analysis
Data from blastocysts were not normally distributed and were normalised by ln(1+x) transformation. The gene expression data sets included either multiple embryos or at least two fetal and placental samples from the same dam. Because of the hierarchical nature of these data, a random effects regression analysis was employed (Stata program, Stata Corporation, College Station, TX, USA), which accounts for the variance between and within dams. Additional parameters such as gender, number of fetuses in the litter and their location in individual horns may also exert an effect on gene expression and were also accounted for estimating the effect of diet on gene expression (Kwong et al. 2004, Osmond et al. 2005). Thus, data presented (mean±S.E.M.) as significantly different (P<0.05) are independent of these parameters. Conceptus weight data were similarly analysed by random effects regression analysis.
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H19 transcripts were first detected at the morula stage (~16–32 cells) with increased band intensity apparent at the blastocyst stage (Fig. 1A). This finding is consistent with those reported for mouse embryos employing RT-PCR in which a major increase in H19 mRNA was observed between eight-cell and blastocyst stages (Doherty et al. 2000). Igf2 expression was first detected at the compacted eight-cell stage (Fig. 1A).
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), indicating trophectoderm specificity. Furthermore, another reported trophectoderm-specific transcript, urokinase plasminogen activator (uPA) (Harvey et al. 1995), gave an equivalent result (Fig. 1B). In contrast, the amplification of Igf2 transcript was detected in both isolated ICMs and whole blastocysts (Fig. 1B). In conclusion, the results indicate that H19 and Igf2 exhibit distinct temporal and spatial expression profiles in rat preimplantation embryos.
Gene expression analysis in blastocysts by semi-quantitative RT-PCR amplification
Although the Dynabead method for poly A+ RNA extraction and semi-quantitation has been used in single bovine embryo analyses (Miller et al. 2003), this is the first report of its use in single rat embryos. This method was selected in our study because it provided a fast and efficient way to extract and separate poly A+ RNA from genomic DNA so that expression and gender analysis could be performed co-ordinately.
In order to compare the relative abundance of gene expression in different samples, it is important to ensure that the PCR amplification is in the linear range. cDNA generated from each individual blastocyst was separated into appropriate amounts according to that required for each of the multiple target genes analysed. PCR was then performed for each target using different cycle numbers and also repeated in two to three different RT-PCR runs to ensure that there was no day-to-day variation. cDNA amplified for 30–38 cycles allowed the detection of individual transcripts without reaching the plateau phase of amplification. Typical semi-log plots of the amount of H19 and luciferase PCR products in relation to cycle number are shown in Fig. 2A and B. Cycle numbers within the linear range of amplification were selected for individual transcripts. A representative gel photograph of a RT-PCR assay of the transcripts is shown in Fig. 2C.
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). In addition, male DNA showed a band corresponding to the Sry fragment in a separate PCR amplification, whereas no product was found in female DNA (Fig. 3B). Hence, rat blastocysts showing Hprt/Zfy and Sry bands were assigned as male embryos (Fig. 3 lanes 3–5) and blastocysts only with a Hprt band were assigned as female embryos (Fig. 3 lane 6).
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). The gender of these blastocysts was also determined (Fig. 3A and B; 18%, dams=11, n=42; 9%, dams=13, n=47).
The relative abundance of H19 transcript was reduced (30%, P<0.05; 18%, dams=11, n=30; 9%, dams=11, n=28) in male blastocysts derived from LPD mothers (Fig. 4A). This reduction was not observed in female blastocysts (Fig. 4B; 18%, dams=7, n=12; 9%, dams=10, n=19). In both genders, the relative expression levels of Igf2, Igf2R and ß-actin transcripts were not altered by maternal LPD.
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Effect of maternal LPD on gene expression in day 20 fetal liver and placenta
Since H19 gene expression is reduced in male blastocysts from LPD dams, we analysed whether such changes persisted into later development. Hence, experimental dams either remained on LPD until day 20 or were switched back to control diet after preimplantation development (day 4.25). Total RNA was extracted from day 20 fetal liver and placenta, and the expression of H19, Igf2, ß-actin and 28S rRNA genes analysed in the same samples by multiplex RNase protection assay (Fig. 5A). The gender of these fetuses was also determined by PCR. A reduction (P<0.05) in relative levels of H19 (9.4%) and Igf2 (10.9%) transcripts in male fetal liver from the switched diet group was observed compared with controls (Fig. 5B; 18%, dams=10, n=11; 9%, dams=10, n=14; switched diet, dams=12, n=16). In contrast, no effect on the levels of these imprinted genes was observed when LPD was maintained throughout gestation. Neither feeding regimen altered the relative expression of ß-actin (Fig. 5B). No difference in gene expression was detected in female fetal liver in response to dietary manipulation (Fig. 5C; 18%, dams=11, n=12; 9%, dams=6, n=7; switched diet, dams=7, n=7). To investigate whether the reduction in H19 transcript correlated with the alteration in methylation status of H19 gene, bisulphite sequencing of differentially methylated region (DMR) (Manoharan et al. 2004) located upstream of the promoter was performed (Fig. 6A). By using the bisulphite treatment condition described by Warnecke et al.(1998), we found that the conversion of C in non-CpG to T is 100% (Fig. 6B). Preliminary experiments indicated that the maternal diet did not alter the methylation status of this region (Fig. 6B; 18%, n=3; 9%, n=3; switched diet, n=6). The expression of H19 and Igf2 mRNA was also analysed in placenta from the same conceptuses. No differences were observed in the relative level of these genes in placenta in either sex (Fig. 7A and B; male, 18%, dams=12, n=24; 9%, dams=11, n=24; switched diet, dams=12, n=26; female, 18%, dams=11, n=23; 9%, dams=11, n=18; switched diet, dams=11, n=19).
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). To test this possibility, a correlation analysis was performed on male fetal weight and the relative levels of H19 and Igf2 in the switched diet group. The analysis showed that there was no correlation between H19 or Igf2 gene expression and fetal weight (H19, P=0.56; Igf2, P=0.92) or combined H19/Igf2 and fetal weight (P=0.60). |
Discussion |
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H19 belongs to a subgroup of imprinted genes that code for untranslated RNA (Brannan et al. 1990). In rat embryos, H19 transcript is first detected at the morula stage and only in trophectoderm cells of blastocysts. The reduction in H19 transcript level in male blastocysts likely reflects a specific response to maternal dietary treatment rather than a global effect on blastomere proliferation rate. Thus, rat blastocysts used in this study were collected on day 4.0 for gene expression analysis, and we have shown previously that the number of trophectoderm cells was unaffected by dietary treatment at this stage. In addition, the relative expression of ß-actin, Igf2 and Igf2R transcripts did not show any changes in the same blastocyst. The more pronounced effect of maternal LPD on H19 compared with other genes suggests that it is more susceptible to environmental conditions. This relative sensitivity is in agreement with other reports (Doherty et al. 2000, Lane & Gardner 2003). As H19 detection is present in trophectoderm (progenitor of most extraembryonic tissues) but not the ICM (progenitor of embryo proper) at this early stage of development, it is proposed that it plays an important role in extraembryonic tissue proliferation to support nutrient transfer and the survival of the fetus after implantation (Rossant 1986).
The analysis of H19 and Igf2 expression at day 20 of gestation revealed that the level of H19 in placenta is unaffected by dietary treatment, coinciding with no effect of maternal diet on placental weight. However, we cannot exclude the possibility that the transport function of placenta is affected at this stage or at other stages of development. Interestingly, reduction in both H19 and Igf2 transcript levels was detected in fetal liver from the switched diet group. Although it is unclear when the reduced level of these genes is initiated, it does indicate that even a brief exposure to LPD, i.e. only during the preimplantation period, may have a long-term impact on fetal phenotype, particular on H19 since its expression in the embryonic tissues is not initiated until day 8.5 of gestation (Poirier et al. 1991). The effect of maternal LPD during the preimplantation period appeared to be gender specific, with both male blastocysts and fetal livers being affected. Such gender-related programming effects have been observed in several studies (Kwong et al. 2000, McMullen & Langley-Evans 2005). Although the precise mechanism is unknown, male sensitivity may reflect a faster rate of male embryo development, thus making them more vulnerable to suboptimal environmental conditions (Erickson 1997).
Several lines of evidence have suggested that DNA methylation status can be altered by maternal diet. For example, pregnant mice fed with methyl-supplemented diet containing methionine, betaine, folic acid and vitamin B12 at conception increased DNA methylation in the intra-cisternal A particle in the Avy allele of agouti mice offspring (Cooney et al. 2002). In another study, Rees et al.(2000) have shown that feeding pregnant rats with LPD, which contained excess methionine relative to other amino acids, led to hypermethylation of DNA in fetal liver. However, our results in the present study indicate that DNA methylation in this particular H19 DMR was not altered by maternal diet. Thus, the aberrant expression of H19 in fetal livers may result from other regulatory mechanisms such as histone acetylation status rather than alteration in DNA methylation in this region. However, we cannot exclude the possibility that the methylation status of other regions in H19 gene may be affected by diet.
Expression of H19 and Igf2 transcripts is also subjected to regulation by hormones or amino acids. In mouse uterus, it has been shown that 17ß-estradiol enhances H19 expression, whereas progesterone represses it through their effect on the H19 promoter (Adriaenssens et al. 1999). In the present study, however, neither 17ß-estradiol nor progesterone levels in serum from pregnant dams was affected by LPD. It is possible that other estrogenic compounds or their receptors may play a role in H19 expression in blastocysts. Glucocortiocoid has also been shown to suppress H19 and Igf2 transcripts levels in rat neonatal liver (Senior et al. 1996) and IGF2 in fetal sheep (Li et al. 1993). Given that maternal undernutrition can lead to alteration in steroid hormone levels (Gonzalez et al. 1997, Fernandez-Twinn et al. 2003), this may in turn affect H19 and/or Igf2 gene expression. Amino acids have also been shown to affect the expression of genes such as Igf2, Igf2R and Igf1 in preimplantation embryos (Ho et al. 1995). Moreover, alteration in maternal serum amino acid concentrations in response to LPD (Kwong et al. 2000, Petrie et al. 2002) may influence the expression of H19 (Doherty et al. 2000) and Igf2 expression (Straus & Takemoto 1988).
H19 and Igf2 genes are thought to be regulated co-ordinately and reciprocally as described by the ‘enhancer competition model’ (Bartolomei et al. 1993). Based on this model, the reduction in H19 in both male blastocysts and fetal livers may be expected to associate with an increase in Igf2 expression. Unexpectedly, a reduction in both H19 and Igf2 transcript levels in blastocysts and livers was observed. Such a non-reciprocal expression of H19 and Igf2 has also been reported in other studies. Fetuses from mouse embryos cultured in the presence of serum had 31 and 15% reduction in H19 and Igf2 respectively compared with in vivo counterparts (Khosla et al. 2001b). In addition, fetal liver from pregnant ewes fasted for 48 h showed a 61% reduction in H19 without any alteration in IGF2 expression (Naimeh et al. 2001). It is possible that the expression of H19 and Igf2 are regulated independently under certain circumstances. In fact, the transcription of Igf2 can be modulated by the degree of methylation in DMR within the Igf2 gene (Feil et al. 1994). In addition, several studies have shown that H19 and Igf2 transcripts can be regulated posttranscriptionally (Straus & Takemoto 1988, Jouvenot et al. 1999).
H19 encodes an untranslated RNA and its function remains unsolved. Some experiments suggest that it plays a growth control role (possibly via controlling IGF2 expression) during embryogenesis. Thus, deleting maternal H19 allele in mice led to 27% increased in birth weight compared with their wild type littermates. These animals also showed an elevated Igf2 transcript level in their tissues compared with wild type (Leighton et al. 1995a). In the present study, reduction in hepatic H19 and Igf2 gene expression in the switched diet group did not show any observed effect on fetal and organ weights nor did it influence the organ-to-body weight ratio. Statistical analysis also showed that there was no correlation between the decrease in expression of these genes and individual fetal growth. This may suggest that the level of reduction is insufficient to impede fetal growth. Alternatively, alteration in the availability of insulin-like growth factor binding proteins may account for the lack of effect on fetal growth (El-Khattabi et al. 2003). However, the reduction in Igf2 (and possibly protein level) and H19 may have a bigger impact on growth during the last 2 days of gestation. During this time, the weights of fetuses were nearly doubled (Langley-Evans et al. 1996). It is possible that the reduction in expression of these genes may lead to reduce weight gain or altered liver development during this rapid phase of growth. Such a possibility may explain the reduced birth weight, altered postnatal growth trajectory and reduced male offspring liver growth observed in the switched diet treatment, we have reported previously (Kwong et al. 2000). In relation to this speculation, mice with targeted disruption of Igf2 gene in liver, kidney and gut exhibit a 20% reduction in birth weight (Leighton et al. 1995b).
The effect of maternal LPD on imprinted gene expression was only seen in male fetal liver from the switched diet group but not in those fed with LPD for 20 day of gestation. The present findings are consistent with those reported by El-Khattabi et al.(2003) in which LPD fed to pregnant rats for 21.5 day of gestation did not alter Igf2 mRNA in LPD fetal liver. Although the mechanism by which maternal LPD at the preimplantation embryo stage can perpetuate into later gestation or postnatal life is unclear, it may indicate that a ‘predictive adaptive response’ can be initiated in the preimplantation embryo (Gluckman et al. 2005). By this concept, embryos may be able to control the growth rate or pattern of metabolism to maximise survival opportunity during future development relative to nutrient availability. However, the mismatch between preimplantation and postimplantation diets may distort such mechanisms. Consistent with this view, we have shown that programming of male hypertensive offspring can be induced irreversibly by maternal LPD fed only at the preimplanation period of development (Kwong et al. 2000).
In conclusion, our data show that maternal LPD for just the first 4 days of gestation can alter the pattern of expression of growth regulating imprinted genes from the blastocyst stage onwards. This early and maintained response to maternal LPD is gender specific and may contribute to changes in growth detected previously during the postnatal life (Kwong et al. 2000).
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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