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Research Article |
School of Anatomy and Human Biology, The University of Western Australia, Perth, Western Australia 6009, Australia
ABSTRACT
Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors. Recent gene deletion studies indicate that PPARG and PPARD play critical roles in rodent development, including effects on placental vascularization. In this study we investigated the expression of the PPAR isoforms and their heterodimeric partner, RXRA, in the two functionally and morphologically distinct zones of the rat placenta during normal gestation and after glucocorticoid-induced fetal and placental growth restriction. Real-time reverse transcription-polymerase chain reaction and immunohistochemical analysis demonstrated markedly higher expression of Ppara, Pparg, and Rxra mRNA in labyrinth zone trophoblast as compared with basal zone near term. There was also a marked increase in Pparg (65%, P < 0.05) and Ppara (91%, P < 0.05) mRNA specifically in the labyrinth zone over the final third of pregnancy. In contrast, expression of Ppard mRNA fell (P < 0.001) in both placental zones over the same period. Maternal dexamethasone treatment (1 µg/ml in drinking water; Days 13–22, term = 23 days) reduced placental (44%) and fetal (31%) weights and resulted in a fall in Pparg (37%, P < 0.05) mRNA expression specifically in the labyrinth zone at Day 22. Placental expression of Ppara, Ppard, and Rxra was unaffected by dexamethasone treatment. These data suggest that PPARG:RXRA heterodimers play important roles in labyrinth zone growth late in pregnancy, possibly supporting vascular development. Moreover, glucocorticoid inhibition of placental growth appears to be mediated, in part, via a labyrinth-zone-specific suppression of PPARG.
developmental biology, glucocorticoid receptor, placenta, pregnancy, syncytiotrophoblast
INTRODUCTION
Intrauterine growth restriction (IUGR) is a major problem confronting obstetric and paediatric medicine, with low birth weight increasing the rate of neonatal morbidity and mortality [1]. IUGR associated with a compromised environment in utero can also increase the risk of adult onset diseases due to fetal programming [2]. The control of fetal growth involves a range of genetic and environmental factors mediated, in part, via the placenta [3, 4], and although a relationship between placental function and fetal well-being is well established [5–8], the complex regulation of placental growth and development remains poorly understood. Recent gene deletion studies [9, 10] suggest that the peroxisome proliferator-activated receptors (PPARs) may be key players in this regulation, together with downstream effectors that impact on placental growth and vascularity.
PPARs are a subclass of the nuclear hormone receptor superfamily of ligand-regulated transcription factors. Activation of the PPARs regulates a diverse range of biological processes, including cellular growth, development, differentiation, and homeostasis [11–13]. To date, three PPAR subtypes (PPARA, PPARD, and PPARG) encoded by separate genes have been described, each with distinct tissue distribution patterns and groups of target genes [13, 14]. PPARA is expressed predominantly in the liver, with a primary role in lipid metabolism [15]. PPARD is ubiquitously expressed, and recent studies indicate a possible role in fat burning and adaptive thermogenesis [11]. PPARG, although originally characterized for its role in adipocyte differentiation, has since been linked to various physiological processes, including insulin sensitivity and tumor growth [11, 16]. Ligand-activated PPARs form a functional heterodimer with another nuclear receptor, the retinoid X receptor (RXR) [17], of which three distinct RXR isoforms (RXRA, RXRB, and RXRG) have been described [18].
Gene deletion studies have shown that PPARG is critically important for placental development with Pparg-null mice dying by midgestation due to a failure in placental vascular development [9]. Consistent with this aspect of the Pparg-null phenotype, activation of PPARG has been shown to promote angiogenesis in other tissues [19–21]. Moreover, recent studies reveal that several aspects of human placental development have been linked to PPARG activation, including trophoblast differentiation and invasion [22–24]. Ppard-null mice also exhibit placental defects leading to embryonic death by midgestation in the majority of cases [10], and Rxra and/or Rxrb knockouts exhibit a phenotype similar to that seen in Pparg-null mice [25]. In addition to these vascular effects, it has also been proposed that PPARs may play important roles in relation to placental transport and metabolism of fatty acids [14].
Although each of the three PPAR isoforms is known to be expressed in the rat placenta [14, 26, 27], their temporal and zone-specific patterns of expression have not been quantified. The current study, therefore, investigated the expression of the three PPAR isoforms and RXRA (the predominant binding partner of the PPARs) over the final third of pregnancy, the period of maximal fetal and placental growth. Separate analyses were conducted for the two functionally and morphologically distinct zones of the rat placenta, the basal and labyrinth zones, because only the latter grows in association with fetal growth in the period of maximal fetal growth [28], consistent with its role in fetal-maternal exchange. The expression patterns of the PPARs and RXRA were also examined in a model of increased glucocorticoid exposure known to restrict fetal and placental growth [29] since previous reports indicate that glucocorticoids can modulate PPAR expression in various tissues [30–32].
MATERIALS AND METHODS
Animals
Nulliparous albino Wistar rats aged between 8 and 12 wk were obtained from Animal Resources Center (Murdoch, Australia) and maintained under controlled conditions as described previously [28]. Rats were mated overnight with the first day of pregnancy designated as the day in which spermatozoa were present in a vaginal smear. All procedures involving animals were conducted after approval by the Animal Ethics Committee of The University of Western Australia.
Glucocorticoid Manipulations
Increased fetal and placental glucocorticoid exposure was achieved by administration of dexamethasone acetate (1 µg/ml; Sigma, St. Louis, MO) in the drinking water from Days 13–22 of pregnancy (term = 23 days). We have previously shown that these treatments reduce fetal weight by approximately 30% at Day 22 [33].
Tissue Collection
For collection of placental zones, pregnant rats were anesthetized with halothane/nitrous oxide at either Day 16 or Day 22 of gestation, and three fetuses and placentas were removed from each mother and weighed. Placental zones were separated by blunt dissection, individually weighed, and either snap frozen in liquid nitrogen for real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) or fixed in Histochoice MB (Amresco, Solon, OH) for immunohistochemistry.
RNA Sample Preparation
Total RNA was isolated from placental zones at Days 16 and 22 of pregnancy using Tri-Reagent (Molecular Resources Center, Cincinnati, OH) as per the manufacturer's instructions. RNA integrity was assessed by ethidium bromide staining of the nucleic acids before agarose gel electrophoresis (data not shown). Total RNA was treated to remove contaminating genomic DNA using DNA-free reagent (Ambion, Austin TX). DNase-treated total RNA (1 µg) was used to synthesize cDNA using M-MLV Reverse Transcriptase RNase H Point Mutant and random hexamer primers (Promega, Madison, WI) as per the manufacturer's instructions. The resultant cDNAs were purified using the Ultraclean PCR Cleanup kit (MoBio Industries, Solana Beach, CA).
Real-Time RT-PCR
Analyses of expression levels for the three PPAR isoforms, Rxra, and L19 transcripts were performed by real-time RT-PCR on the Rotorgene 3000 (Corbett Industries, Sydney, Australia) using iQ SYBR Green Supermix (Bio-Rad, Hercules, CA). Primers for Ppard and total Pparg (Table 1) were adapted from Hoekstra et al. [34], whereas those for Ppara and Rxra were designed using Primer 3 software (MIT/Whitehead Institute; http://www-genome.wi.mit.edu) [35]. Each of the selected primer pairs were positioned to span introns to ensure that no product was amplified from genomic DNA, and the resulting amplicons were sequenced to confirm specificity (data not shown). All samples were standardized against L19 as previously described [36]. Standard curves for each product were generated from gel extracted (QIAEX II; Qiagen, Melbourne, Australia) PCR products amplifying 10-fold serial dilutions and the Rotorgene 3000 software.
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Immunohistochemistry
Sections were cut at 4 µm, deparaffinized, and rehydrated before incubation for 10 min in 3% H2O2 to block endogenous peroxidases. Mouse monoclonal primary antibody to PPARG (sc-7273) and rabbit polyclonal primary antibody to RXRA (sc-553) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and each used at a dilution of 1:100 overnight at 4°C. This was followed by a 30-min room-temperature incubation with biotinylated anti-mouse or anti-rabbit secondary at 1:200 (BA2001 and BA1000; Vector Laboratories, Burlingame, CA) and streptavidin-peroxidase complex at 1:50 (Vectastain ABC Kit; Vector Laboratories). Sections were visualized by application of diaminobenzidine substrate (Sigma) and counterstained with haematoxylin before dehydration in graded alcohols and mounting in DPX.
Statistical Analysis
Fetal, total placental, and placental zone weight values were determined for each mother as the average of the three estimates. All group values are expressed as means (±SEM) of gene expression standardized by L19. Two-way ANOVAs (GenStat7; Hemel Hempstead, UK) were used to assess variation in expression levels for each of the PPAR isoforms and Rxra, with gestational age and placental zone as sources of variance. Where the F-test for the ANOVA reached statistical significance (P < 0.05), differences were assessed by least significant difference (LSD) tests [37]. The effects of dexamethasone on fetal and placental weights were assessed by unpaired t-tests.
RESULTS
Gestational Changes in Fetal, Placental, and Placental Zone Weights
Fetal and placental weight both increased markedly from Days 16–22 of pregnancy (fetus: 18-fold increase, P < 0.001; placenta: 2-fold increase, P < 0.01). This increase in placental weight was attributable entirely to growth of the labyrinth zone (3-fold increase, P < 0.001), with no change evident basal zone weight (Fig. 1).
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Gestational Changes in Ppara, Ppard, Pparg, and Rxra Expression
The mRNAs for all PPAR isoforms and Rxra were readily detectable in both zones of the rat placenta (Fig. 2). Expression of Ppara and Pparg mRNA increased in the labyrinth zone (91%, P < 0.05; and 65%, P < 0.05, respectively) from Day 16 to 22 of pregnancy but remained unchanged in the basal zone over the same period. In contrast, expression of Ppard mRNA decreased in both the basal (76%, P < 0.001) and labyrinth zones (63%, P < 0.001) from Day 16 to 22. Expression of Rxra mRNA was higher (P < 0.05) in the labyrinth zone compared to the basal zone at both Day 16 (2.1-fold) and Day 22 (4.8-fold).
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Immunohistochemical analysis confirmed the overall pattern of PPARG and RXRA distribution between the two placental zones. Moreover, both PPARG and RXRA were localized to the nuclei of the labyrinth zone trophoblast, with no immunostaining present in fetal endothelial cells of the labyrinth zone or giant trophoblast cells of the basal zone (Fig. 3). No change in distribution of PPARG or RXRA was observed between Days 16 and 22.
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Fetal and Placental Weights after Dexamethasone Treatment
Dexamethasone treatment from Day 13 of pregnancy reduced fetal weight at Day 22 by 24% (P < 0.01) and placental weight by 32% (P < 0.01), with the latter due to comparable effects on both basal and labyrinth zone weights (Fig. 4).
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Effect of Dexamethasone on Expression of PPAR Isoforms and Rxra
Expression of all the PPAR isoforms and Rxra were assessed following treatment with dexamethasone. Expression of Pparg mRNA at Day 22 was reduced by 37% (P < 0.05) in the labyrinth zone after dexamethasone treatment compared with placentas from the untreated mothers (Fig. 5). In contrast, expression of the other PPAR isoforms and Rxra were all unaffected by dexamethasone treatment (data not shown). Immunohistochemical analysis revealed no change in the cellular distribution of either PPARG or RXRA following treatment with dexamethasone.
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DISCUSSION
This study investigated the expression of PPARA, PPARD, PPARG, and their heterodimeric partner, RXRA, in the basal and labyrinth zones of the rat placenta over the final third of pregnancy. The major findings were that Ppara, Pparg, and Rxra mRNA expression were all higher in the rapidly growing labyrinth zone near term and that Ppara and Pparg mRNA increased from Day 16 to 22 specifically in the labyrinth zone. In contrast, Ppard expression fell in both the basal and the labyrinth zones over the final third of pregnancy. Maternal dexamethasone treatment restricted fetal and placental growth and resulted in a marked reduction in Pparg mRNA expression specifically within the labyrinth zone but had no effect on the expression of Rxra or the other PPAR isoforms. These data are consistent with the previously reported obligatory role for PPARG in placental development and further suggest that PPARG is particularly important in the labyrinth zone during the period of maximal fetal and placental growth.
The higher expression of PPARG and RXRA in the labyrinth zone compared with the basal zone observed in this study is consistent with a role for PPARG in promoting placental growth, possibly through increased trophoblast differentiation and/or angiogenesis of fetal blood vessels. Accordingly, the increase observed in labyrinth zone expression of PPARG occurred in association with the major growth of this zone during the final third of rodent pregnancy, which includes a marked increase in placental vascularization [38]. Immunohistochemical localization of both PPARG and RXRA to the nuclei of labyrinth zone trophoblasts is consistent with the proposed role of these transcription factors acting as a heterodimer to alter gene transcription. In vitro human studies suggest that PPARG promotes trophoblast invasion [23] and differentiation [22], and thus PPARG expression may influence these key developmental events of the rat placenta. Gene deletion studies in the mouse have also established a role for PPARG in placental development. Barak et al. [9] demonstrated that Pparg knockout mice die by midgestation because of inadequate development of the placental vasculature. In particular, fetal vessels appeared not to invade the labyrinth of Pparg-null placentas. More recently, Asami-Miyagishi et al. [26] investigated the role of PPARG during midpregnancy by maternal administration of PPARG agonists between Days 9 and 11. This treatment decreased the rate of spontaneous fetal mortality by half, consistent with PPARG support of placental vascular development. Interestingly, in contrast to the marked changes in spatial or temporal expression of placental PPARs and RXRA observed in the present study, Wang et al. [14] previously reported consistent expression of all three PPARs between the basal and labyrinth zones and across the second half of rat pregnancy. In addition, the expression of Rxra mRNA was noted to be higher in the basal zone when compared to the labyrinth zone, increasing throughout the placenta from Day 13 to Day 19 [14]. These inconsistencies presumably reflect the far more accurate assessment of mRNA expression possible by real-time, quantitative RT-PCR as used in the present work.
Localization of PPARG to the labyrinth trophoblast (and its absence from fetal endothelium) suggests that activation of PPARG may result in trophoblastic secretion of angiogenic factors to stimulate the growth of fetal blood vessels. Consistent with this proposal, several studies have suggested that PPARG exerts a positive effect on angiogenesis by stimulating expression of vascular endothelial growth factor (VEGF) [19, 20, 39, 40]. Indeed, activation of PPARG by the endogenous PPARG ligand, 15-deoxy-
12, 14-prostoglandin J2 (15PGJ2), has demonstrated a role for 15PGJ2 in the PPARG-mediated upregulation of VEGF in a human macrophage cell line [40]. Interestingly, 15PGJ2 appears to be synthesized in the rat placenta [26, 41] and so may serve as the endogenous ligand for placental PPARG activation. Further studies are required to determine whether activation of PPARG in trophoblast cells influences VEGF expression and downstream effects on fetal vasculature.
The current study also shows that maternal dexamethasone treatment, which clearly restricted both fetal and placental growth, resulted in reduced expression of Pparg mRNA specifically in the labyrinth zone. These data suggest that dexamethasone-induced placental growth restriction may be due, at least in part, to a downregulation of PPARG expression specifically within trophoblast cells of the labyrinth zone. This reduction in PPARG expression may reduce the expression of secreted growth or angiogenic factors from labyrinthine trophoblasts, thus inhibiting fetal vascular development. In a previous report, total placental PPARG expression at Day 21 of rat pregnancy was found to be unaffected by maternal dexamethasone treatment (Days 15–21; 100–200 µg kg–1 day–1) [27]. In this case, however, placental expression of PPARG was measured in whole placentas rather than individual zones, thus highlighting the need for individual examination of the two distinct zones of the rodent placenta.
PPARD expression is also critical for placental development as shown by gene deletion studies, with limited survival of homozygous null pups [10]. However, unlike Pparg-null placentas, the labyrinth zone of Ppard-null mice displayed normal vascular development. Our data show that placental Ppard mRNA expression falls during the last third of rat pregnancy, possibly a reflection of reduced functional importance for PPARD at this time. Recent work in human trophoblast cells suggests that PPARD may suppress expression of 11ß-hydroxysteroid dehydrogenase-2 (11BHSD2), the enzyme responsible for metabolism of active glucocorticoids within the placenta [42]. Such a role appears unlikely in the rat, however, since we have previously shown that labyrinthine expression of 11BHSD2 falls between Days 16 and 22 of pregnancy [28, 43]. Alternatively, the reduction in PPARD expression near term may facilitate the actions of PPARG, since all the PPARs competitively bind RXRA as determined by their relative abundance and binding affinities. Therefore, lower expression of PPARD would be expected to enhance PPARG interaction with RXRA and thus facilitate its downstream effects on placental vascularization.
This study also confirmed that the rat placenta expresses PPARA, and as with PPARG, this expression increased specifically within the labyrinth zone toward term. Although the role of placental PPARA remains to be determined, it may impact on fatty acid transport and metabolism either alone or in combination with PPARG. Wang et al. [14] suggested that the presence of PPARA and PPARG late in pregnancy may facilitate the necessary increase in transplacental fatty acid transfer stimulated by increased fetal demand. Consistent with this hypothesis, the reduction in labyrinthine PPARG expression observed following glucocorticoid-induced placental growth restriction may reduce transfer of maternal fatty acids to the fetus and possibly contribute to the restricted fetal growth. In addition, placental PPARA may be important in relation to placental steroid production, since previous studies indicate that PPARA activation may promote steroidogeneisis in human trophoblasts by increasing the pool of available cholesterol through fatty acid oxidation [44]. Although the rodent placenta exhibits only limited steroidogenic capacity relative to other species [45], the rat placenta is a significant local source of progesterone within the intrauterine compartment [46].
In conclusion, our data suggest that, in addition to promoting early placental development, PPARG is important for placental growth and development late in pregnancy, particularly in the rapidly growing labyrinth zone. Moreover, inhibition of placental and fetal growth by dexamethasone treatment was associated with reduced PPARG expression specifically within the labyrinth zone, suggesting that placental vascular development may be impeded in this model of IUGR. Further ligand binding studies are required to assess whether PPARs may provide a therapeutic target in the treatment of IUGR.
FOOTNOTES
1 Supported by the National Health and Medical Research Council of Australia (Project Grant 254576). 
2 Correspondence: Brendan J. Waddell, School of Anatomy and Human Biology, The University of Western Australia, 35 Stirling Highway, Crawley, Western Australia 6009, Australia. FAX: 61 8 6488 1051; bwaddell{at}anhb.uwa.edu.au
Received: 22 July 2005.
First decision: 18 August 2005.
Accepted: 29 August 2005.
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