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RESEARCH |
Institute of Clinical Research, Centre for Reproduction and Early Life, University of Nottingham, Nottingham NG7 2UH, UK
Correspondence should be addressed to M E Symonds at Academic Division of Child Health, School of Human Development, University Hospital, Nottingham NG7 2UH, UK; Email: michael.symonds{at}nottingham.ac.uk
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Introduction |
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UCP2 has the most widespread tissue abundance and is present in the uterus of mice (Pecquer et al. 2001, Rousset et al. 2003). Its function remains a subject of intense debate (Stuart et al. 2001), but the abundance of UCP2 mRNA and/or protein has been shown to be developmentally regulated in both the lung and adipose tissue of sheep where one postulated role includes the regulation of apoptosis (Voehringer et al. 2000). In both these tissues, the abundance of UCP2 mRNA and protein is strongly influenced by the current and the past nutritional states (Mostyn et al. 2003, Gnanalingham et al. 2005a, 2005c). It is currently not known whether UCP2 is present in the placenta nor whether it is developmentally controlled or nutritionally responsive in utero. Therefore, an extension of our study was to determine whether UCP2 is present in the ovine placenta and if it is developmentally regulated. Other important mitochondrial proteins whose function is related to UCP2 include voltage-dependent anion channel (VDAC; Voehringer et al. 2000) located in the outer mitochondrial membrane (Colombini 1979). VDAC along with UCP2 (Voehringer et al. 2000) could be responsible for the release of cytochrome c from the intermembrane space, a process that has been implicated in the chain of events culminating in apoptosis (Crompton 1999). Placenta of clinically compromised pregnancies shows increased rates of apoptosis (Huppertz & Herrler 2005), but the extent to which apoptosis is enhanced by maternal NR has not been examined. We therefore aimed to determine not only the effect of maternal food intake on apoptosis but also the cellular location of VDAC and cytochrome c in the placenta.
Maternal dietary manipulation in early pregnancy results in a number of endocrine adaptations that may potentially impact on placental function. These include a reduction in the plasma concentrations of a range of maternal metabolic hormones including cortisol, thyroid hormones, insulin-like growth factor (IGF)-I, and insulin (Bispham et al. 2003, Symonds et al. 2007). A reduction in placental IGF type-I receptor (IGF-IR) is associated with intrauterine growth retardation (Reid et al. 2002, Laviola et al. 2005). The extent to which the same effect on the IGF-IR may result from changes in maternal nutrition is unknown and was a further aim of the present study. In order to achieve the aims of the study, placenta was sampled from control and nutritionally manipulated singleton-bearing sheep at:
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UCP2, GR, and 11ßHSD2 mRNA were all detected in the fetal placentome at 80 and 140 days gestation (Fig. 1A–C
). UCP2 and GR mRNA abundance increased (P<0.01) with gestational age and were further raised, compared with controls (P<0.01), by early to mid-gestational NR (Fig. 1A and B). Interestingly, 11ßHSD2 mRNA abundance was lower (P<0.01) in the placenta of NR compared with control mothers at 80 days gestation (Fig. 1C). It then only decreased with gestational age in controls with the result that by 140 days gestation 11ßHSD2 mRNA abundance was significantly greater in placenta sampled from NR mothers compared with controls. Finally, irrespective of maternal diet and gestational age UCP2 and GR mRNA abundance were positively correlated (Fig. 2). IGF-IR mRNA abundance was decreased at 80 days gestation by NR, although by 140 days gestation this effect was negated by refeeding (Fig. 3).
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). PCNA expression was, however, markedly decreased in the placenta of nutrient-restricted mothers in which it was weakly expressed in all samples compared with moderate or strong expression in all control animals. Apoptosis, as assessed by either TUNEL staining or the detection of the cleaved form of caspase 3, was unaffected by NR (C 3.6 ± 0.2; NR 3.5 ± 0.3 positive cells per 200x magnification field). Both VDAC and cytochrome c were located within the maternal uterine syncytium region of the placenta (see representative example for cytochrome c in Fig. 5). The abundance of neither mitochondrial protein was influenced by NR or age and mRNA expression of VDAC was similarly unaffected (data not shown).
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Discussion |
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It is known that for adipose tissue during the period of exponential growth there is a strong correlation between total fat mass and both local glucocorticoid action and UCP2 expression (Gnanalingham et al. 2005a). In the present study on the ovine placenta, we found a positive relationship between UCP2 and GR mRNA with gestational age that is clearly not related to placental mass per se. The exact mechanisms by which both GR and UCP2 mRNA are up-regulated in the placenta remain to be established. Endocrine regulation of UCP2 during development has been most widely studied in fetal adipose tissue in which cortisol and the biologically active thyroid hormone, triiodothyronine (T3), both appear to be involved (Gnanalingham et al. 2005d). These hormones can affect local glucocorticoid action by specifically influencing the expression of GR and 11ßHSD isoforms. Indeed, regulation of fetal UCP2 mRNA by cortisol is in accord with the effects of umbilical cord occlusion which results in a precocious rise in UCP2 mRNA in the fetal lung and adipose tissue (Gnanalingham et al. 2005e). Under these adverse hypoxic conditions, however, it is cortisol, rather than T3, which regulates UCP2 abundance. In the present study, however, the endocrine mechanisms may be maternally driven as we have previously established that both maternal plasma cortisol and thyroid hormones are decreased over the period of NR (Bispham et al. 2003). It remains to be established whether these directly impact on placental glucocorticoid action and in particular increase its sensitivity to cortisol.
Interestingly, the increase in mitochondrial mRNA abundance for UCP2 was not accompanied by any change in VDAC or cytochrome c protein. This contrasts with adipose tissue in which nutritional programming of all of these proteins is observed (Mostyn et al. 2003) and emphasizes their tissue-specific regulation (Gnanalingham et al. 2005b, 2006). Unfortunately, we were unable to confirm whether UCP2 protein was similarly affected because of the current unavailability of specific antibodies for ovine UCP2 (Gnanalingham et al. 2005f). The functional consequence of an increase in placental UCP2 remains to be established. One putative role for UCP2 is in apoptosis (Voehringer et al. 2000), which, in the placenta, is determined in part by glucocorticoid sensitivity (Waddell et al. 2000). Apoptosis was, however, unaffected by NR in our study. Interestingly, we have shown for the first time that both VDAC and cytochrome c are located within the syncytial region of the placenta, which is the major site of glucose transport across the placenta (Dandrea et al. 2001). This process obviously requires appreciable amounts of energy, which will need to be met within the mitochondria, and does not appear to be impaired by reduced maternal food intake and the reduction in placental cell proliferation.
The persistent increase in placental GR mRNA abundance with maternal NR was not accompanied by a similar change in 11ßHSD2 mRNA, which was transiently decreased at 80 days, but then increased near to term. These data extend previous findings in which no effect of early to mid-gestational NR was found on 11ßHSD1 mRNA at term and a reduction in 11ßHSD2 was only present in mid-gestation (Whorwood et al. 2001). Unlike the present study that utilized RT-PCR, previous work may have been unable to detect this gene in term placenta because the less sensitive technique of Northern blotting was used (Whorwood et al. 2001). By term, the placentae of NR mothers demonstrate a marked up-regulation in 11ßHSD2 mRNA that is likely to be paralleled by an increase in 11ßHSD2 enzyme activity (Whorwood et al. 2001). The time course of this adaptation corresponds to the period in which maternal plasma cortisol adapts from being reduced during the period of NR to subsequently rising as the maternal diet is restored to the same level as controls (Bispham et al. 2003). Taken together, these findings indicate that it is not only the prevailing maternal cortisol that influences placental and, thus, fetal exposure to cortisol but it is the magnitude of change in the maternal circulation throughout pregnancy. As such, a transient rise in maternal cortisol in late gestation has no effect on fetal cortisol (Edwards & McMillen 2001). Indeed, the increase in placental 11ßHSD2 activity with gestation that only occurred in the nutrient-restricted group would be predicted to reduce fetal cortisol exposure (Langley-Evans et al. 1996). This may therefore be the mechanism by which fetal sensitivity is raised in these offspring, which is in direct contrast to the response of adipose tissue in offspring born to mothers nutrient restricted in late gestation (Gnanalingham et al. 2005a).
In contrast to the GR, mRNA abundance for the IGF-IR was decreased in placenta of NR mothers at 80, but not 140, days gestation. This coincided with the stage at which mean placentome mass was reduced and is in accord with findings in rats and humans in which intrauterine growth retardation is accompanied with reduced placental IGF-IR (Reid et al. 2002, Laviola et al. 2005). It should be noted, however, that the placenta is markedly different between sheep, rats, and humans which have discoid, hemochorial placenta, whereas in sheep placenta are cotyledonary synepitheliochorial that may represent an evolutionary development and can limit the transport of some molecules from the mother to fetus (Carter & Mess 2007). Therefore, in the sheep, restoration of the maternal diet together with the concomitant rise in maternal plasma IGF-I (Bispham et al. 2003) acts to restore placental responsiveness. At the same time, this adaptation is accompanied by a maintenance of placental mass, increased fetal length at term, and resetting of the relationship between fetal plasma IGF-I and body dimensions (Heasman et al. 2000).
In conclusion, we have shown that maternal NR targeted between the time of conceptus implantation throughout peak placental growth results in a pronounced change in local glucocorticoid action within the placenta, one consequence of which is increased UCP2 mRNA abundance. These adaptations occur in conjunction with a reduction in placental cell proliferation but in the absence of any affect on apoptosis. Changes in glucocorticoid action could then contribute to changes in cortisol exposure of the fetus, thereby, causing both immediate (Whorwood et al. 2001) and long-term changes in local glucocorticoid action and UCP2 abundance within specific offspring tissues (Gnanalingham et al. 2005a, 2005c).
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Materials and Methods |
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In order to determine the effect of early to mid-gestational maternal NR on fetal placentome development, five sheep within each nutrition group were randomized to tissue sampling at either 80 or 140 days gestation. Each animal was humanely euthanized following i.v. administration of 200 mg/kg pentobarbital sodium (Euthatal: RMB Animal Health, Dagenham, UK). The entire uterus was removed from each animal and a number of randomly chosen A type placentomes as determined by their visual appearance (Vatnick et al. 1991) were sampled. These represent the majority of placentomes in the sheep (Clarke et al. 1998, Heasman et al. 1998) and were immediately dissected and either put into 10% (v/v) formalin and embedded in paraffin wax for subsequent histological analysis, or separated into maternal and fetal components and immediately placed in liquid nitrogen and stored at –80 °C for later analysis. In addition, total placental and fetal weights were recorded together with all major organs. All operative procedures and experimental protocols had the required Home Office approval as designated by the Animals (Scientific Procedures) Act (1986).
Laboratory analyses
mRNA detection
Total RNA was isolated from fetal placentome tissue using Tri-Reagent (Sigma) and the expression of UCP2, GR (type 2), 11ßHSD2, and IGF-IR mRNA determined by reverse transcriptase-PCR (RT-PCR) as described previously (Bispham et al. 2003, Gnanalingham et al. 2005c). The analysis used oligonucleotide cDNA primers to UCP2, GR (type 2), 11ßHSD2, and IGF-IR genes as published previously (Bispham et al. 2003, Gnanalingham et al. 2005c), whilst VDAC expression was determined using the primers of Cesar & Wilson (2004), all generating specific intron-spanning products. Standard curves for each gene were initially established in order to optimize the amount of cDNA required for each subsequent analysis. Agarose gel electrophoresis and ethidium bromide staining confirmed the presence of both the product and the ribosomal 18S, and densitometric analysis was performed using a Fujifilm LAS-1000 cooled charge-coupled device (CCD) camera. Consistency of lane loading for each sample was verified and all results expressed as a ratio of a reference sample to r18S abundance. All analyses and gels were conducted in duplicate.
Protein detection
Mitochondria and plasma membranes were prepared from ~1 g of fetal placentome (Budge et al. 2000) and protein content determined by the Lowry method (Lowry et al. 1951). Western blotting was utilized to measure the abundance of VDAC and cytochrome c mitochondrial proteins (Mostyn et al. 2003). Identical amounts of placental protein were loaded (i.e. 10 µg) onto each gel for each sample. Following electroblotting of the polyacrylamide gel onto a nitrocellulose membrane, Ponceau red staining was used to visually confirm that similar amounts of protein had been transferred before subjecting the membranes to immunodetection (Mostyn et al. 2003). Abundance of cytochrome c was determined using a specific antibody (sc-7159; Santa Cruz, Biotechnology Inc., Santa Cruz, USA) at a dilution of 1 in 1000. VDAC abundance was determined using an antibody raised in rabbits to ovine VDAC1, purified from the kidney of a newborn sheep (Mostyn et al. 2003) at a dilution of 1 in 2000. Densitometric analysis was performed using AIDA software (Aida version 2.0; raytest Isotopenmeßgeräte GmBH Straubenhardt, Germany) on each membrane following image detection using a Fujifilm LAS-1000 cooled CCD camera (Fuji Photo Film Co. Ltd, Tokyo, Japan). All values were expressed in densitometric units. Specificity of detection was confirmed using nonimmune rabbit serum. All gels were run in duplicate and a reference sample (placental mitochondria from a control sheep sampled at 140 days gestation) was included on each to allow comparison between gels.
In addition, further Western blots were performed to confirm the effects of maternal nutrition on GR and IGF-IR protein abundance. This was undertaken using polyclonal antibodies for each protein purchased from Santa Cruz (catalogue numbers SC 8992 and 713 respectively). Each antibody was tested at a range of dilutions from 1:150 to 1:1000 under both mild reducing and nonreducing conditions using up to 80 µg protein. Unfortunately, neither antibody yielded a concise signal (in any of the animals) that was in accord with its predicted molecular mass (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 positive reference sample (plasma membranes isolated from 1-day-old sheep).
Immunohistochemistry and assessment of apoptosis
To establish the cellular location of VDAC and cytochrome c within the placenta, immunohistochemistry was performed using the antibodies described above at dilutions ranging from 1:100 to 1:1000 (Dandrea et al. 2001). Following incubation with enzyme-conjugated second antibody and chromogen substrate, sections were examined by light microscopy. The specificity of the procedure was confirmed by the absence of binding when adjacent sections were incubated with rabbit serum from an unimmunized rabbit in place of rabbit anti-ovine primary antibody. It was not possible to perform the same analyses with respect to the location of UCP2 due to the lack of appropriately validated antibodies (Gnanalingham et al. 2005f). The same analyses were undertaken for the GR and IGF-IR, but in accord with our failure to detect protein at the correct molecular weight by Western blotting neither antibody showed specificity of binding to the placenta. This procedure was undertaken on 20–30 randomly selected sections from each nutritional group.
Measurement of cell proliferation within the placenta was also undertaken by determining the amount of immunoreactive proliferating cell nuclear antigen (PCNA) expression (Waseem & Lane 1990). This was undertaken using a mouse MAB to PCNA (PC10) (Alexis Biochemicals, Bingham, Nottingham) at a 1:200 dilution. Relative staining intensity for immunoreactive nuclear PCNA expression was visually assessed in a blinded manner and scored as either absent (–; no nuclear staining), weak ( +), moderate ( + +), or strong ( + + +) (Taylor et al. 2000). Immunohistochemistry was performed on a computerized Bond automated immunohistochemistry system (Vision Biosystems Limited, Newcastle-upon-Tyne, UK) using a bond polymer refine detection kit. A negative control was performed for each test section by omitting incubation in the primary antibody. Tonsil, in which PCNA staining is pronounced, was used as the positive control (Waseem & Lane 1990). Sections were dehydrated in ascending concentrations of alcohol and xylene before coverslips were mounted.
These sections were also used to assess the relative incidence of apoptosis by the TUNEL assay (Roche Diagnostics; Gavrieli et al. 1992). Sections were stored at 4 °C in the dark and the magnitude of staining analyzed within 48 h. Appropriate negative controls were included to assess both nonspecific binding and the extent of autofluorescence. Positive controls were treated with DNase type I to produce fragmentation of chromosomal DNA and washed separately to prevent any residual DNase activity affecting other sections. All analyses were performed using the same assessor who was blinded to nutritional grouping using reference slides to check consistency of grading assessment. To assess the relative incidence of apoptosis between groups, slides were analyzed using fluorescence microscopy with FITC and u.v. filters to visualize green (fragmented DNA) and a semi-quantitative apoptosis index (0–4; Lepault et al. 2005) used. This procedure was undertaken on 30 randomly selected sections from each nutritional group at a magnification of 200x . In addition, as the TUNEL reaction is unable to discriminate apoptotic from necrotic cells, caspase-3, a marker of early apoptosis (Gown & Willingham 2002) was localized using a rabbit anti-caspase 3 polyclonal antibody (Abcam plc, Cambridge, UK) diluted 1:50. Immunohistochemistry was performed using the Bond automated system with a bond polymer refine detection kit (Vision Biosystems Limited). A negative control was performed for each test section by omitting incubation in the primary antibody. Tonsil, in which apoptosis is pronounced, was used as the positive control (Krajewska et al. 1997). For each section, the number of positive cells in five randomly selected fields was used to calculate the mean positive cell count ± S.E.M per 200x field.
Statistical analysis
All data are presented as the means ± S.E.M. Significant differences (P<0.05) between gestational ages and nutritional groups were analyzed by ANOVA. Significant correlations between molecular parameters were assessed by Spearman’s Rank Order Test (SPSS v11.0; SPSS Inc., Chicago, Illinois, USA).
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Footnotes |
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References |
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