Biol Reprod Email Content Delivery
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print July 5, 2007.
Biol Reprod 2007, 10.1095/biolreprod.107.061929
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
77/5/829    most recent
biolreprod.107.061929v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Desmarais, J. A
Right arrow Articles by Murphy, B. D
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Desmarais, J. A
Right arrow Articles by Murphy, B. D
Agricola
Right arrow Articles by Desmarais, J. A
Right arrow Articles by Murphy, B. D
BIOLOGY OF REPRODUCTION 77, 829–839 (2007)
DOI: 10.1095/biolreprod.107.061929
© 2007 by the Society for the Study of Reproduction, Inc.

The Peroxisome Proliferator-Activated Receptor Gamma Regulates Trophoblast Cell Differentiation in Mink (Mustela vison)1

Joëlle A Desmarais 3, Flavia L Lopes 3, Hao Zhang 4, Sanjoy K Das 4, and Bruce D Murphy 2 3

Centre of Animal Reproduction Research,3 Université de Montréal, Québec, Canada J2S 7C6 Department of Pediatrics,4 The Division of Reproductive and Developmental Biology, Vanderbilt University, Nashville, Tennessee 37232

ABSTRACT

Nuclear receptors of the peroxisome proliferator-activated receptor (PPAR) family are implicated in implantation and early placental formation. In carnivores, the trophoblast invades to develop intimate contact with the endothelial cells of the maternal circulation, resulting in an endothelio-chorial form of placentation. Spatio-temporal investigation demonstrated that peroxisome proliferator-activated receptor gamma (PPARG) was strongly and specifically expressed in the mink trophoblast at the time of formation of the syncytiotrophoblast during early implantation, and in trophoblast of the placental labyrinth. The retinoid-X-receptor alpha (RXRA), the heterodimeric partner of PPARG in transcriptional regulation, is, with very few exceptions, co-expressed with PPARG in mink trophoblast. We used mink trophoblast cell lines together with a natural (15-deoxy-delta12,14-prostaglandin J2 ) or a synthetic (troglitazone) PPARG ligand to demonstrate that PPARG is an authentic regulator of gene expression in this tissue. Ligand-activated PPARG stimulated transcription of the PPRE-luc reporter gene transfected into these cell lines. The prostaglandin-induced morphologic changes were accompanied by attenuation in cell proliferation, an increase in PPARG mRNA and protein levels, and the appearance of enlarged and multinuclear cells. Furthermore, 15-deoxy-delta12,14-prostaglandin J2 stimulated the expression of invasion-related genes in trophoblast cells, namely, adipophilin and osteopontin. The results demonstrate that PPARG ligands attenuate proliferation and induce differentiation of mink trophoblast cells to the multlinuclear phenotype. The upregulation of differentiation-specific genes in the placenta under the influence of PPARG ligands provides a mechanism by which blastocyst and endometrial prostanoids regulate implantation, as well as the formation and maintenance of the placenta.

embryo, endotheliochorial placenta, implantation, placenta, PPARG, syncytiotrophoblast, trophoblast

INTRODUCTION

The formation of the diverse types of mammalian placenta is a function of the invasiveness of the trophoblast and its capacity to degrade and eliminate the tissue barriers between itself and the maternal vascular elements of the endometrium. In carnivores, the trophoblast invades to develop intimate contact with the endothelial cells of the maternal circulation, resulting in an endothelio-chorial form of placentation [1]. Morphological analysis of the early stages of implantation and placental formation in two mustelid carnivores, the ferret (Mustela putorius) [2] and the spotted skunk (Spilogale gracilis) [3], indicates that the trophoblast cells of the chorion proliferate and expand rapidly prior to direct association with the maternal epithelium. These changes are accompanied by enlargement of some trophoblast cells and the initial formation of structures known as syncytial plaques, with the result that, as implantation approaches, the trophoblast is bilaminar, comprising inner cytotrophoblast and outer syncytial trophoblast layers [2, 4]. These plaques attach to the endometrial epithelium and form the trophoblastic villi that penetrate into the epithelium via the necks of the endometrial glands, inducing formation of maternal symplasma from glandular epithelial cells [5]. From thence, as illustrated by the spotted skunk, the syncytial trophoblast surrounds the maternal capillary plexus, and fetal vessels form within the mesenchyme adjacent to the cytotrophoblast [4]. According to Enders [6], four distinct layers compose the mink placenta. The exchange function of the placenta occurs in the allantochorionic and the labyrinth zones. The third layer is the invasion front, comprising the maternal cell debris and given the name junctional zone, and the more distal maternal remains of the endometrium are referred as the glandular zone.

Recently, nuclear receptors of the peroxisome proliferator-activated receptor (PPAR) family have been implicated in the processes of implantation and early placental formation. These receptors serve as transcription factors, forming functional heterodimers with another nuclear receptor, the retinoid-X receptor-{alpha} (RXRA) [7]. Ablation of the PPARG gene in mice resulted in fetal loss at Embryonic Day 10.5 [8, 9], which is attributed to defects in formation of the placenta [8]. Phenotypic analysis of these null mice revealed that PPARG is essential for the proper development of the labyrinth layer of the placenta and for the establishment of proper maternal vascularity within the labyrinth [8]. Further, the disruption of the RXRA gene in mice resulted in abnormalities of the labyrinthine and spongiotrophoblast layers of the placenta, leading to embryonic lethality and consistent with the PPARG knockout phenotype [10]. PPARG plays an important role in cell differentiation in other tissues, best exemplified by its ability to induce adipogenesis and adipocyte differentiation [8, 11].

We have previously shown the presence of prostaglandin synthetic enzymes associated with embryo implantation in mink [12] and, more recently, demonstrated prostaglandin regulation of vascular endothelial growth factor transcription in mink uterine cells [13]. The prostaglandins can act through their G-coupled membrane-bound receptor or through nuclear receptors. The bioactive metabolite of prostaglandin D2, 15-deoxy-{Delta}12,14-prostaglandin J2 (15-d-PGJ2), is a natural ligand for PPARG [14, 15]. In addition, there are numerous synthetic ligands for PPARG, the most potent being the thiazolidinediones (TZDs), a family of antidiabetic drugs [15, 16]. As PPARG ligands attenuate cell proliferation and induce cell differentiation, the antioncogenic action of these ligands has attracted great interest [17].

The molecular pathways involved in trophoblast differentiation are not well elaborated for any of the mammalian placental subtypes, and virtually nothing is known about the endotheliochorial placenta. Further, the role of PPARG in trophoblast differentiation has not been extensively investigated in the context of implantation. Here we use the mink model to investigate PPARG function in the carnivore endotheliochorial type of placenta. We report the first evidence for an early expression of PPARG associated with trophoblast attachment and implantation. In addition, we will present data supporting a role for PPARG in syncytiotrophoblast differentiation.

MATERIALS AND METHODS

Sampling

All procedures involving live animals were approved by the Comité de déontologie de la Faculté de Médecine Vétérinaire, Université de Montréal, which is accredited by the Canadian Council on Animal Care. Ranch mink of the Dark and Pastel varieties were purchased from Armand Richard (St. Damase, QC, Canada) and were bred during the mating season to two fertile males according to the usual farming procedures. To terminate embryonic diapause and induce implantation, we used a treatment regimen that we have previously employed [18] in which animals were injected intramuscularly with ovine prolactin, 1 mg kg–1 day–1 (Sigma, St. Louis, MO), after 21 March, for a maximum of 11 days. Embryos were collected by uterine flushing with TC-199 medium (Gibco, Burlington, ON, Canada) containing 10% fetal bovine serum (Gibco). Uterine horns were dissected separately, and one was fixed in 4% paraformaldehyde (Sigma) overnight for immunohistochemistry, whereas the other was flash frozen in liquid nitrogen for subsequent RNA extraction or in situ hybridization.

RNA Probes and In Situ Hybridization

To generate the RNA antisense probes for in situ hybridization, RT-PCR using a mink uterine tissue was performed with the forward primer 5-CTT GAC AGG AAA GAC AAC AGA C-3 and the reverse primer 5-CAG CAA ACT CAA ACT TGG GTT C-3 to obtain a 705-bp PPARG fragment. To amplify the 450-bp RXRA fragment, the forward primer used was 5-TGA TCG ACA AGC GGC AGC GGA AC-3, and the reverse primer was 5-GGA GAA GGA GGC GAT GAG CAG C-3. PCR products were subcloned into a pDrive cloning vector (Qiagen, Mississauga, ON, Canada) and amplified in competent bacteria of XL1-Blue strain, displaying flanking Sp6 and T7 RNA polymerase recognition sites for the generation of 35S-CTP-labeled RNA probes.

In situ hybridization using PPARG, as well as RXRA antisense or sense (control) RNA probes was achieved as described [19]. Briefly, cryostat sections (11 µm), mounted on poly-lysine-coated slides, were postfixed in 4% paraformaldehyde and washed in PBS. Following a prehybridization step, hybridization of the antisense probe continued for 4 h at 45°C. Sections were washed and incubated with 20 µg ml–1 RNase A for 20 min at 37°C to eliminate excess probe. The positive signal was revealed by autoradiography using NTB-2 liquid emulsion (Eastman Kodak, Rochester, NY).

Immunohistochemistry

Fixed uterine tissues were embedded in paraffin and sectioned at 4 µm. Immunohistochemistry employed 1:50 PPARG (H-100) and 1:75 RXRA (D-20) rabbit polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) and biotinylated anti-rabbit IgG second antibody (1µg ml–1; Vector Laboratories, Burlingame, CA) following the protocol described in Lord et al. [20]. Briefly, paraffin sections were dehydrated in ethanol and washed in TBS, and the endogenous peroxidase was inactivated by incubation in 3% peroxide in methanol. Antigen was retrieved by brief incubation of the sections in boiling 0.1 M citrate buffer (pH 6.0). Nonspecific primary antibody binding activity was blocked with normal goat serum (10% in TBS; Jackson ImmunoResearch, West Grove, PA), and the tissues were incubated overnight with the first antibody at 4°C in a humid, dark slide chamber. After repeated washes, sections were incubated with biotinylated second antibody for 2 h at room temperature, and the Vectastain ABC kit (Vector Laboratories) with the Vector NovaRED substrate kit (Vector Laboratories) were used to detect the peroxidase activity. Negative control sections were submitted to the same procedures, except that the first antibody was replaced by 10% normal goat serum in TBS.

We performed single or double fluorescence immunolabeling on trophoblast cells in culture, as described [21]. The antiosteopontin monoclonal antibody MPIIIB101 was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa (Iowa City, IA). In brief, the cells were grown on a coverslip in a four-well dish, fixed with cold methanol (–20°C), and washed with PBS. Nonspecific antibody binding was blocked over 2 h of incubation with 0.1% Triton X-100 (Sigma) in PBS. Cells were incubated overnight with antiosteopontin antibody (1:50) and anti-PPARG antibody (1:100). Following a wash with 0.1% Triton in PBS for 2 h, the second antibodies were added at a concentration of 1:1000 for CY3-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch) and 1:25 for fluorescein (FITC)-conjugated goat anti-mouse IgG, (Jackson ImmunoResearch). The cells were incubated with diamidino-4'6-phenylindol-2 dichlorhydrate (DAPI) and washed in PBS before the coverslips were mounted on slides with DABCO antifade mounting medium (2.5% [w/v] 1,4-diazabicyclo(2,2,2) octane (Sigma), 50 mM Tris-HCl, pH 8.0; and 90% glycerol). For negative control, the primary antibody was replaced by the blocking buffer. All steps were carried out at 4°C. The signal was visualized by fluorescence microscopy (Leica ASLMD microscope; Leica Cambridge, Cambridge, UK) with a mercury lamp (HBO 103 W/2; LEJ, Jena, Germany). The micrographs were acquired with the Leica DC500 camera and a DFC Twain Software (Leica).

Trophoblast Cell Culture and Treatments

Trophoblast cell culture was established as previously described [22], with some modifications. The embryo was placed in culture with a mouse fetal fibroblast feeder monolayer and mouse trophoblast stem cell medium (TS) with 20% fetal bovine serum, containing 25 ng ml–1 fibroblast growth factor (FGF4; Sigma) and 1 µg ml–1 heparin (Sigma) as described [23]. When the blastocyst was attached to the monolayer and the trophoblast had begun to proliferate, the ICM was removed with a glass pipette. At confluence, cells were passaged to a gelatin- and poly-lysine-coated Petri dish or six-well plate that was free of the monolayer, and conditioned media was used thereafter for continuous culture as described [23]. Medium was changed at 2-day intervals, and cells were passaged at confluence (approximately 7-day intervals) by pipetting. A stock of the trophoblast cells was conserved at –80°C and thawed as needed.

To verify the trophoblastic provenence and nature of the cells, mink primers were designed using a multispecies comparative approach for a series of trophoblast marker genes [24, 25]. Subsequent RT-PCR analysis (Fig. 1) demonstrated the putative trophoblast cells to be positive for the mink orthologs of the trophoblast markers, caudal type homeobox transcription factor 2 (CDX2, GenBank AY460115), eomesodermin (EOMES, GenBank AY460116), fibroblast growth factor receptor 2 (FGFR2, GenBank AY460117), heart- and neural crest derivative-expressed protein (HAND1, GenBank AY460118), Achaete-scute homolog 2 (MASH2), and proliferation-related acidic leucine-rich protein (PAL31, GenBank AY460119). In general, the abundance of transcript for each increased with time in culture. Trophoblast cell lines were negative for the embryonic stem cell marker, the homeobox gene NANOG [25] (data not shown). In all cases, the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), served as a positive control for mRNA recovery and efficiency of reverse transcription (Fig. 1).


Figure 01
View larger version (44K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 1 Trophoblast cultures express marker genes indicative of chorionic provenance of the cells. Mink primers were designed using a comparative, multispecies approach for a series of putative marker genes in mink trophoblast cells at 2 and 6 days after initiation of culture, and PCR amplicons were resolved by electrophoresis. The markers are caudal type homeobox transcription factor 2 (CDX2), eomesodermin (EOMES), fibroblast growth factor receptor 2 (FGFR2), the basic helix-loop-helix transcription factor (HAND1), Achaete-scute homolog 2 (MASH2), and proliferation-related acidic leucine-rich protein (PAL31). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as positive control for mRNA recovery and reverse transcription.

To probe PPARG transcriptional activity, we treated the cells either with 10–25 µM 15-d-PGJ2 (Cayman Chemicals, Ann Arbor, MI), 1–25 µM troglitazone (Calbiochem, San Diego, CA), 10–20 µM rosiglitazone (Cayman Chemicals), 5 µM retinoic acid (RA; Sigma), and/or 1 µM GW 9662 (Cayman Chemicals), all diluted in dimethyl sulphoxide (DMSO; Sigma at 0.044% v/v). Experiments were conducted a minimum three times using freshly thawed trophoblast cells for each replicate experiment.

Proliferation Assay

To determine DNA synthesis and cell proliferation, mink trophoblast cells were incubated overnight in the presence of 100 µM bromodeoxyuridine 5'-triphosphate (BrdU; Sigma) 6 days after initiation of culture with DMSO (control) or 15-d-PGJ2. Cells were fixed in methanol and submitted to immunofluorescent analysis to detect incorporated BrdU using 10 µl anti-BrdU mouse monoclonal antibody (Amersham, Oakville, ON, Canada) containing 1 µg ml–1 DNase and a Cy3-labeled goat anti-mouse IgG (Sigma) at 1:1000 dilution.

Reporter Gene Assay

Trophoblast cells were grown to confluence, seeded equally onto 24-well plates, and grown for 48 h in TS-conditioned medium with FGF4 and heparin. Cells were transfected using Effectene Transfection Reagent (Qiagen) with 250 ng ml–1 PPREx3-tk-Luc construction vector (a generous gift from R. M. Evans, Salk Institute, La Jolla, CA) and 20 ng ml–1 simian virus-40 Renilla luciferase vector (pRLSV40; Promega Corp., Nepean, ON, Canada). All plasmids used for transfection were prepared with Qiagen HiSpeed Plasmid Midi kit. To prepare the tk-Luc control vector harboring no PPREs, the original plasmid was digested with the restriction enzymes HindIII and XbaI. After dose and time optimization, plasmids were transfected for 12 h in the presence of the Effectene reagent (according to manufacturer's specifications). Cells were then washed with 1x PBS, and transfection medium was replaced with fresh TS medium containing the treatments. Luciferase fluorescence was detected with the Dual Luciferase Assay System (Promega Corp.) using a Berthold 9501 luminometer. This experiment comprised three wells for each treatment and was repeated three times using three populations of freshly thawed cells.

Flow Cytometry

Mink trophoblast cells were grown to confluency and seeded equally onto six-well plates. At the time of plating, DMSO or 25 µM 15-d-PGJ2 was added to the TS culture medium. The medium and treatment were changed every 2 days, and the cells were allowed to grow for 6 days before trypsinization and fixation in 70% cold EtOH. The cells were stored at –20°C until flow cytometry analysis, at which time the fixed cells were washed once in 1x PBS and incubated for 30 min to 1 h at room temperature with a propidium iodide (PI) solution (1x PBS, 0.1 % [v/v] Triton X-100, 0.02 mg ml–1 PI (Sigma), and 2 mg RNAse A). Flow cytometry analysis was performed with a BD Bioscience FACSVantage SE, and the data were processed with Cell Quest Pro software (BD Bioscience, Oakville, ON, Canada).

RNA Isolation and Real-Time Polymerase Chain Reaction

Total RNA from uterine tissues and trophoblast cells in culture was isolated with the RNeasy mini kit (Qiagen) according to the manufacturer's protocol. Reverse transcription was achieved with an initial quantity of 250 µg total RNA and 200 IU of Superscript RNase H– reverse transcriptase (Invitrogen Life Technologies, Burlington, ON, Canada). The real-time relative quantification of cDNA from trophoblast cells in culture was achieved using a LightCycler (Roche Molecular Biochemicals, Laval, QC, Canada) with the QuantiTect SYBR Green PCR kit from Qiagen. An initial hot-start step was carried out at 95°C for 15 min. Each amplification cycle consisted of a denaturation step for 20 sec at 94°C, an annealing step according to the specific annealing temperature described in Table 1 for 20 sec, and an amplification step at 72°C for 20 sec. PCR quantification data were analyzed using the second derivative method with the LightCycler Software version 3.5. Standard curves were generated for each set of primers. Amplified products were verified on agarose gel electrophoresis and by sequencing. The primers (Table 1) used were designed with the Oligo Primer analysis software 4.0-s (National Biosciences, Plymouth, MN) and were based on mink-specific sequences or on homology between humans and at least another species (rat, mouse, dog, etc.), aligned with Gene Jockey 5.0 Software (Biosoft, Cambridge, UK).


View this table:
[in this window]
[in a new window]
[Download PPT slide]
  		TABLE 1 Primers used for real-time PCR.

Statistical Analyses

Treatment means were analyzed using least-square ANOVA and the general linear model procedures of SAS (Cary, NC). In the presence of a significant overall treatment effect, means were compared by Duncan multiple-range and Tukey tests. A probability level of P < 0.05 was considered statistically significant.

RESULTS

PPARG and RXRA Expression Are Upregulated at the Time of Implantation

The PPARG antisense probe in in situ hybridization revealed the PPARG mRNA signal first appearing as the embryo attaches to endometrium (designated Day 0 of implantation; Fig. 2a). The expression was specific to the trophoblast, and it was further found only in trophoblasts in contact with the endometrial epithelium. It was absent in uterine tissue and not detectable in either the preimplantation uterus or between implantation sites (Fig. 2a). At Day 4 following implantation, intense PPARG mRNA signal was detected in the placenta, principally in the region of the incipient labyrinth. There was prominent localization of RXRA mRNA, the heterodimerization partner of PPARG in the trophoblast, colocalizing with the PPARG mRNA manifestation at the implantation site on the day of attachment (Fig. 2b). The RXRA message was also distributed more widely, present in the uterine glands and endometrial stroma on the day of implantation (Fig. 2b). At Day 4 after implantation, in situ hybridization revealed that RXRA mRNA, while again more widespread, was present in the same regions that expressed the PPARG message (Fig. 2b).


Figure 02
View larger version (38K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 2 PPARG (a) and RXRA (b) mRNA localization by in situ hybridization in mink uteri. The samples for frozen sections were collected at preimplantation, 5 days after the escape of the embryo from diapause, at implantation (Day 0), or at early postimplantation (Day 4). The dark-field photomicrographs reveal the autoradiographic signal in orange. Note the expression of PPARG exclusive to trophoblastic tissue, as well as the broader distribution of RXRA mRNA in the trophoblastic tissue and in the epithelia and stroma of the uterus. The preimplantation uterus and the interimplantation site display no signal for PPARG. Bars = 1 mm.

Immunohistochemistry indicated that messages identified by in situ hybridization were accompanied by the occurrence of the cognate PPARG and RXRA proteins, in almost all cases indicating co-expression. As expected, both signals were restricted to the nuclei of trophoblast cells. On the day of implantation, PPARG protein localized to the nuclei of trophoblastic plaques (Fig. 3a). PPARG was likewise present in nuclei of syncytiotrophoblast cells of the incipient chorionic villi invading the luminal and the glandular epithelium of the uterus (Fig. 3, b and c). By Day 4 following implantation, PPARG protein was strongly expressed in the syncytiotrophoblast that surrounds the fetal villi and the maternal endothelium in the proximal labyrinth region (Fig. 3d), as well as in the syncytiotrophoblast surrounding the villi from the distal labyrinth region (Fig. 3e). On the day of implantation, RXRA protein localized to the cells of the trophoblastic plaques prior to and following contact with the endometrial epithelium (Fig. 3, f and g). It was most prominent in the enlarged cytotrophoblast associated with the invading villus (Fig. 3, h and i). RXRA was not evident in the syncytiotrophoblast at Day 4 after implantation (Fig. 3, h and i). As shown in Figure 3, j and k, the sections that were not incubated with first antibody are negative for the signal.


Figure 03
View larger version (104K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 3 Immunocytochemical localization of PPARG and RXRA proteins at implantation sites at Days 0, 1, and 4 after implantation. On the day of implantation, the red signal indicates that the PPARG and RXRA signals are present in trophoblastic plaques (arrowhead in a; long arrow in f), and RXRA is detected along with PPARG in the syncytial trophoblast intruding in the maternal epithelium (arrowheads, b and f). One day after implantation, PPARG and RXRA are expressed in the syncytiotrophoblast cells (arrowheads, c and g). At Day 4 after implantation, PPARG signal is evident in the syncytiotrophoblast cells, surrounding the fetal villi, and in the maternal capillaries in the labyrinth zone (d). Panel k shows that RXRA, while less pronounced, is present in the syncytiotrophoblast (arrowhead) and also in cytotrophoblast cells (arrow). In the distal labyrinth zone, PPARG is again found to be specific to the syncytiotrophoblast cells (e). RXRA is specific to the cytotrophoblast cells but was not evident in the syncytiotrophoblast (i). Panels j and k: The negative controls for the early implantation site and Day 4 implantation site in the labyrinth zone, respectively. The schematic drawing on the bottom right represents the placental areas described in each panel. le, luminal epithelium; ge, glandular epithelium; t, trophectoderm; me, maternal endothelium; fm, fetal mesenchyme of the villus; ms, maternal symplasma. The arrow indicates cytotrophoblast cells; the arrowheads, syncytotrophoblast cells. Bars = 100 µm.

The Prostanoid, 15-d-PGJ2, an Endogenous Ligand for PPARG, Modulates Cell-Cycle Progression of the Trophoblast Cells In Vitro

As in situ hybridization and immunohistochemistry revealed PPARG expression to be specific to differentiating trophoblast cells during implantation and placental development, we employed trophoblast cells derived from preimplantation mink blastocysts to further delineate the effects of PPARG activation. We previously showed both PPARG transcripts and protein to be present in these cells. To establish the occurrence of PPAR signaling, trophoblast cells were passaged and grown to approximately 80% confluence, then transfected with a luciferase reporter gene construct driven by three PPRE elements and a minimal promoter (PPREx3-tk-Luc). We tested two PPARG ligands, the antidiabetic thiazolidinedione, troglitazone, and a natural ligand, 15-d-PGJ2. Both ligands triggered PPARG transcriptional activity 2- to 3-fold over 36 h, with apparent dose dependence for 15-d-PGJ2 but not troglitazone, where the response was of equal magnitude with doses of 10 and 25 µmol/L (Fig. 4). The specific PPARG antagonist, GW 9662, inhibited the generation of the luciferase signal and DMSO, whereas at the molar concentration employed it had no distinguishable effect on basal PPRE-induced transcription (Fig. 4a). Retinoic acid mildly activated the construct and modestly potentiated the effects of 15-d-PGJ2 (data not shown). The control vector, the tk-Luc plasmid, which does not harbor PPREs, did not display enhanced luciferase activity in the presence of 25 µM 15-d-PGJ2 (Fig. 4). These results indicate that both ligands act specifically through the canonical PPAR transcriptional signaling pathway.


Figure 04
View larger version (12K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 4 The PPARG ligands 15-d-PGJ2 and troglitazone induce transcriptional activation of PPARG target genes. In mink trophoblast cells cultured in vitro, a PPREx3-tk Luc reporter construct was transfected along with the SV40-Renilla reporter gene (Luc/Ren) as a control for transfection efficiency. Following treatment for 36 h, 15-d-PGJ2 (25 µmol/L) promoted an optimal luciferase activity, which was reduced by the addition of the specific PPAR antagonist GW9662 (1 µmol/L) to the culture medium (upper panel). Troglitazone (Trog) enhanced luciferase activity significantly. At 10 µM concentration, and GW9662 limited the luciferase (middle panel). DMSO had a negligible effect on PPARG transcriptional activity (upper and middle panels). In the absence of the PPRE3x in the construct there was no enhancement of luciferase signal by PPARG ligands (bottom panel). Mean ± SEM promoter activity in terms of relative luciferase signal is presented. The asterisk indicates a mean value significantly different from the DMSO control (P > 0.05).

Based on these findings, we chose doses of 25 µM 15-d-PGJ2 and 10 µM troglitazone to further examine PPARG effects and mechanisms in trophoblast cells in vitro. Treatment of trophoblast cultures with 15-d-PGJ2 induced an evolution in cell morphology (Fig. 5a). In the control group treated with DMSO, after 48 h of culture, trophoblast cells proliferated in the form of well-delimited colonies of small cells, reaching confluence over the first 2 days of culture. At 96 h after plating, cells of the control group were substantially more abundant and appeared to proliferate actively and, while remaining loosely attached to the Petri surface, were strongly linked together. Between Days 4 and 8 following initiation of culture in the control group, cells detached gradually from the plate and formed blastocyst-like floating vesicles. The formation of these vesicles concurs with observations typical of mouse trophoblast cell culture, as described in Tanaka et al. [23]. In contrast to control cultures, the 15-d-PGJ2-treated cell group appeared to cease proliferation. This was confirmed by studies of BrdU incorporation into trophoblast cells, indicating extensive proliferation in control cells but none in 15-d-PGJ2-treated cells (Fig. 5b). In addition, prostanoid-treated cells displayed extensive expansion of the cytoplasm, accompanied by increased adherence to the surface of the plate (Fig. 5a). These radical changes in cell morphology were first observable at Day 2 following initiation of 15-d-PGJ2 treatment. Staining of cultures with the DAPI reagent to localize nuclei revealed substantial reductions in cell density and the presence of enlarged nuclei and multinuclear cells in cultures treated with 15-d-PGJ2 (Fig. 5a). The addition of both retinoic acid and 15-d-PGJ2 to the cells accelerated the rate of morphological differentiation (Fig. 5a). In contrast, the ligand troglitazone did not induce morphological changes as profound as those seen in 15-d-PGJ2-treated cultures (Fig. 4a). Troglitazone appeared to reduce cell proliferation at Days 2 and 4, whereas on subsequent days the cells underwent rapid proliferation, evoking the pattern observed in the control cultures.


Figure 05
View larger version (92K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 5 The PPARG ligand 15-d-PGJ2 induces (a) a change in trophoblast cell morphology and (b) a decrease in cell proliferation activity in vitro. a) Morphologic changes are evident at 2 days of culture with the ligand, where arrows indicate the colony delimitation; at Day 8, the cells display a morphology characteristic of the mouse TS cells. The cells are growing compactly and form floating vesicles. The cells treated with 15-d-PGJ2 are fewer in a same microscopic area, their expanded cytoplasm attached to the bottom of the Petri dish, and the cells frequently contain two nuclei, as seen with DAPI DNA coloration in the dark field microphotograph. In the presence of RA, 15-d-PGJ2 accelerated the rate of the morphologic changes of the trophoblast cells. In contrast, the synthetic ligand troglitazone (Trog) did not trigger a complete arrest in cell proliferation, as did 15-d-PGJ2 alone or with retinoic acid. b) BrdU (BrDU in figure) incorporation as detected by immunofluorescence in the control culture (DMSO) or in the 15-d-PGJ2-treated culture after 6 days of treatment (left panel). The DNA is revealed with DAPI staining (right panel). The cell culture in the negative control was submitted to immunofluorescence staining but was not previously incubated with BrdU. Bars = 200 µm.

To examine the apparent influence of 15-d-PGJ2 on the regulation of trophoblast cell cycle, cells were cultured as described above, treated with 15-d-PGJ2 for 8 days, and subjected to flow cytometric analysis for DNA content (Fig. 6). Large, multinuclear cells that did not enter the capillary of the apparatus were excluded from the analysis. The results revealed that 15-d-PGJ2 substantially altered the DNA content of the cell populations. The proportion of cells displaying the 2n complement of DNA was reduced from 68% to 39% by the higher dose of 15-d-PGJ2, whereas the proportion containing 4n or more increased from 13% to 41%. Troglitazone produced a similar, albeit muted, response as the increase in the frequency of the 4n or more complement approximated 3% (data not shown). This result was anticipated, since troglitazone treatment of the trophoblast cells in vitro did not induce the same changes in cell morphology as did 15-d-PGJ2 (Fig. 5a). The augmentation in the proportion of cells with two or more times the concentration of the G-phase complement of DNA under the influence of 15-d-PGJ2 is interpreted to indicate the presence of a polyploid cell population, as has been previously reported for whole-mink blastocysts [26]. These findings are interpreted as further indication that 15-d-PGJ2 directs trophoblast differentiation.


Figure 06
View larger version (14K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 6 Treatment of trophoblast cells with 15-d-PGJ2 promotes a change in DNA complement. Frequency analysis of the numbers of cells per channel as a function of relative fluorescence intensity. The peak at 200 fluorescence units represents cells with the 2n complement of DNA; the region from approximately 350–400 represents those with a 4n or greater complement, the region between represents cells in transition. Doses of 10 and 25 µM 15-d-PGJ2 increased the number of cells with greater than 2n complement of DNA.

Treatment with 15-d-PGJ2 Induces a PPARG-Positive and a Potentially Invasive Trophoblast Cell Phenotype In Vitro

We then undertook immunolocalization of PPARG to determine its association with the differentiated phenotype in trophoblast cells in culture. The results demonstrate a low level of PPARG nuclear signal in control cultures (Fig. 7a). For cultures treated with 15-d-PGJ2, both cells with large nuclei and multinucleated cells displayed an intense signal (Fig. 7a).


Figure 07
View larger version (34K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 7 Treatment with 15-d-PGJ2 increases PPARG protein and mRNA and promotion of an attachment-competent and potentially invasive trophoblast cell phenotype in the trophoblast cells under the influence of nuclear localization of PPARG in the control trophoblast cells or in the trophoblast cells cultured in the presence of 15-d-PGJ2 (a). The cells were fixed on a coverslip and processed for immunocytochemistry with the PPARG first antibody and a Cy3-coupled second antibody. Note that the large binucleated cells strongly express PPARG, whereas the nuclei of proliferating cells are essentially PPARG negative (Bar = 25 µm). b) Real-time PCR amplification and relative quantification of PPARG mRNA showing increases in transcript abundance in 15-d-PGJ2-treated cultures but not DMSO control cultures. Cyclophilin (PPIA) served as control (c). Concomitant immunolocalization of PPARG and osteopontin (SPP1) in the trophoblast cells treated with the control DMSO or with 15-d-PGJ2 for 6 days. The DNA was counterstained with DAPI, and signals were visualized by fluorescence microscopy. Treatment with 15-d-PGJ2 increased PPARG and SPP1 protein signals in trophoblast cultures at Day 2 of culture. Bars = 50 µm. In panels b and d, means bearing different superscripts are different at P < 0.05.

To determine whether PPARG expression was upregulated by its own ligand in the course of differentiation, real-time PCR amplification was employed. The results show that PPARG transcript abundance was significantly elevated in cultures subjected to 15-d-PGJ2 over 8 days, relative to the control, where the mRNA was substantially lower and remained stable throughout the time of culture, beginning as early as Day 2 (Fig. 7b). Overall, these data suggest that 15-d-PGJ2 acting through PPARG is involved in the differentiation of cytotrophoblast cells into enlarged trophoblast cells and multinucleated cells.

To further investigate the PPARG induction of mink trophoblast cell differentiation, real-time PCR measurement of the abundance of the genes related to trophoblast attachment and differentiation was conducted. Immunocytochemical analysis was then used to confirm the presence of the corresponding proteins. Osteopontin is a secreted glycoprotein involved in the integrin signaling pathway. It is expressed by invading trophoblasts [27], and there is evidence for osteopontin modulation by PPARG in other tissues [28]. We tested 15-d-PGJ2 regulation of osteopontin gene expression in mink trophoblast cells. Real-time RT-PCR revealed that osteopontin mRNA is upregulated 6-fold over 8 days of chronic 15-d-PGJ2 treatment (Fig. 7d). To establish a functional link between PPARG transcriptional activity and osteopontin protein expression, we examined the localization of the two proteins, osteopontin and PPARG, in the trophoblast cells in cultures. The results (Fig. 7c) demonstrate the virtual absence of nuclear PPARG and cytoplasmic osteopontin in DMSO-treated control cultures. In contrast, treatment with 15-d-PGJ2 induced development of both the large and multinuclear PPARG-positive cell phenotypes, with accompanying extensive cytoplasmic expression of osteopontin (Fig. 7c). We also noted an inexplicable nucleolar signal to the osteopontin antibody, which was present in DMSO-treated and 15-d-PGJ2-treated cultures.

The expression of the adipose differentiation-related protein adipophilin (also known as ADRP and ADPH) was evaluated in response to PPARG ligands. Adipophilin is ubiquitously expressed and is a known target gene of PPARG that is upregulated during adipocyte [29] and human trophoblast cell differentiation [30]. In the carnivore trophoblast line, the abundance of adipophilin mRNA was stably increased in cultures treated for 48 and 96 h with 15-d-PGJ2 (Fig. 8a). The activation of RXRA was expected to increase the transcriptional activity of the heterodimer formed by PPARG and RXRA. The results (Fig. 8a) demonstrate that a combination of retinoic acid and 15-d-PGJ2 provoked a marked increase in the expression of adipophilin at 96 h, providing additional evidence to suggest that PPARG mediates adipophilin gene expression in trophoblast cells. Using the same paradigm, we examined two synthetic PPARG ligands, troglitazone and rosiglitazone, which are two members of the TZD family. There was a trend (P = 0.07) toward modest increases in adipophilin transcript abundance induced by troglidizone at 48 h, but no differences were evident at 96 h (Fig. 8b). Rosiglitazone produced identical results (data not shown).


Figure 08
View larger version (10K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 8 Adipophilin (ADFP) is a PPARG target gene in trophoblast cells. a) Real-time PCR revealed that treatment with 15-d-PGJ2T for 48 or 96 h induced increases in adipophilin transcript abundance in cultured mink trophoblast cells at 48 and 96 h (mean ± SEM, means bearing different superscripts are different at P < 0.05). b) Treatment with troglitazone (Trog) produced a tendency toward increase at 48 h (P = 0.07), whereas no effect was seen at 96 h.

DISCUSSION

The information generated in this investigation demonstrates that transcriptional modulation by the nuclear receptor PPARG is involved in implantation and in the formation of the endothelio-chorial placenta in a carnivore model. The clear distribution of the PPARG signal in enlarged cytotrophoblast and syncytiotrophoblast nuclei, as shown by in situ hybridization and immunohistochemistry, suggests it may play an important role for PPARG in formation of the syncytiotrophoblast. Following adhesion to the mustelid uterus, the syncytial cells penetrate the uterine luminal epithelium and insinuate through the glandular epithelium, which degenerates with contact to trophoblast cells [2, 4]. PPARG mRNA and protein are strongly expressed in these invading cells, and a representative expression of RXRA mRNA and protein was shown in the same cell type. This early expression of PPARG and its transcriptional partner in trophoblastic plaques before and during attachment, as well as during the initial epithelial penetration has not been previously described in any species. Gene deletion studies in mice have shown PPARG to be essential to placental formation [8], and PPAR delta (PPARD) in trophoblast differentiation [31]. The present work is the first demonstration of a temporal expression of PPARG associated with early invasion of the trophoblast, and it is consistent with the well-known role of this nuclear receptor as a modulator of cell invasion in liposarcomas and colon, breast, and prostate carcinomas [32, 33]. It further suggests a new role for PPARG in organization of the implantation process.

We employed mink trophoblast cell lines to demonstrate that PPARG is an authentic regulator of gene expression in the trophoblast. PPARG is transcriptionally active in the presence of both natural (15-d-PGJ2) and synthetic (troglitazone) ligands, as evidenced by their robust stimulation of transcription of genes driven by PPRE in our in vitro culture system. Moreover, this stimulation of transcription was specific to the nuclear receptor, as GW 9662, a specific antagonist of PPARG, abrogated the transcriptional effects of both ligands. The response was dose dependent when 15-d-PGJ2 was used to stimulate PPRE-mediated transcription. A similar low-magnitude effect of PPARG ligands on luciferase signal was also reported by other authors, notably by Allred and Kilgore [34], in cell lines expressing PPARG.

As a principal effect of PPARG in adipose tissue [8, 11], colon cancer, and other cancers is the induction of terminal differentiation (reviewed in [17]), we examined the effects of the natural ligand on trophoblast cell differentiation. In these trials, 15-d-PGJ2 attenuated proliferation and induced differentiation of the cells over 8 days, with the first morphological signs present as early as 48 h. These morphological changes were accompanied by an increase in PPARG protein and mRNA and by formation of enlarged and multinucleated cells. The rapid induction of differentiation in the presence of PPARG ligands and the upregulation of PPARG during differentiation in the mink trophoblast cells are consistent with observations in other species. In the human BeWo choriocarcinoma cell line, troglitazone accelerates differentiation of cells into human chorionic gonadotropin (hCG)-producing syncytia [35]. In that study, the acquisition of the completely differentiated phenotype, as indicated by an increase in hCG secretion, occurs within 72 h of initiation of treatment of the human cell line. It is likewise marked by a strong increase in PPARG transcriptional activity [35]. In addition, PPARG is robustly expressed during differentiation in the murine trophoblast stem cells [36]. Furthermore, the PPARG promoter contains PPAR-regulatory sequences, suggesting that PPARG can autoregulate the transcription of its own gene [37, 38]. Together, these observations support the view that PPARG-activated transcription is an early event in mink trophoblast cell differentiation.

Phenotypic analysis of PPARG knockout mice revealed that the nuclear receptor is essential for development of the labyrinth layer of the placenta and for the proper maternal vessel establishment within the labyrinth [8]. Likewise, the RXRA knockout mouse displays abnormalities of the labyrinthine and spongiotrophoblast layers of the placenta, also leading to embryonic lethality [10]. The mouse placenta is hemochorial and monocotyledonary, and PPARG mRNA is present in both the labyrinth and spongiotrophoblast regions from Embryonic Day 8.5 onward [8, 39]. The situation is similar to that of the rat, where the syncytial cells of the spongiotrophoblast and the labyrinth regions express both PPARG and RXRA from Embryonic Day 11 [37, 40]. In humans, PPARG is expressed in the placenta during the second trimester in the villous and columnar cytotrophoblasts, whereas during the third trimester or in term placentas, PPARG expression appears also in the syncytiotrophoblast [35, 39]. Morphological evidence in the present investigation suggests a similar role for PPARG and RXRA in development of the fetal villi that form the surface of exchange in the mink placenta. We show PPARG and RXRA to be expressed in the trophoblast at the time of attachment and implantation. In contrast, in mice, rats, and humans, PPARG is not expressed at the time of implantation, suggesting a fundamental difference between the strategies of embryo implantation among these species.

Given the distribution of PPARG during placental development, we sought to determine by real-time PCR what genes might mark the process of differentiation, and thus might be targeted. Adipophilin (ADFP), the human homolog of the mouse adipose differentiation-related protein, is believed to be involved in cellular lipid uptake [41, 42]. In the mink, 15-d-PGJ2 triggered upregulation of ADFP in trophoblast cells by 48 h. This upregulation of ADFP has also been demonstrated in human trophoblasts following exposure to troglitazone for 4 h [30]. These data support the view that the transcriptional activation of PPARG target genes is an early event, occurring following initiation of trophoblast differentiation. Moreover, the consistently elevated levels of adipophilin in the cells under the influence of 15-d-PGJ2 for 8 days suggest that the syncytiotrophoblast cells have a lipid uptake requirement during the process of differentiation. In differentiated human trophoblasts, expression patterns of PPARG and the related nuclear receptor, PPARD, are similar [43]. It is therefore of interest that PPARD is necessary for trophoblast differentiation in the mouse and that it drives adipophilin expression [31]. Our in situ hybridization analysis of PPARD transcripts during implantation and placenta formation in mink revealed expression specific to uterine stroma and virtually absent in the trophoblast (data not shown). This further points to disparity between the implantation processes in the rodent hemochorial and the carnivore epitheliochorial placenta types.

Osteopontin is involved in cell-cell and cell-extracellular matrix contact through binding to the alpha-nu category of integrin receptors. In the rat, pig, and sheep, osteopontin is present in luminal and glandular epithelium throughout pregnancy [27]. The expression of osteopontin was shown to be regulated in human cytotrophoblasts and ovine uterine luminal epithelium by progesterone [44, 45]. When human cytotrophoblast cells are placed in culture, they rapidly aggregate, and osteopontin expression is downregulated [46]. When the aggregated cells undergo fusion, osteopontin expression increases [45]. These data demonstrate that in the human model, osteopontin is upregulated in syncytiotrophoblast cells. In the present study, osteopontin mRNA and protein were shown to be upregulated following 15-d-PGJ2-mediated PPARG activation. This provides additional evidence to suggest the involvement of PPARG in trophoblast cell attachment and syncytium formation in the trophoblast cells at the onset of embryo implantation.

In summary, we provide the first evidence to demonstrate that the nuclear receptor PPARG is present in the carnivore trophoblast during implantation and placenta formation. We further demonstrate by use of homologous trophoblast cells in vitro that PPARG ligands induce differentiation of the mink trophoblast, including induction of syncytiotrophoblast formation and escape from the cell cycle. The upregulation of differentiation-specific genes in the placenta under the influence of PPARG ligands provides a mechanism by which blastocyst and endometrial prostanoids regulate the formation and maintenance of the mink placenta through gestation.

ACKNOWLEDGMENTS

The authors acknowledge Mira Dobias-Goff for invaluable technical assistance; M. Armand Richard, Marie-Ève Charest, and Geneviève Charest for aid with experimental animals; Dr. Patrick Vincent for flow cytometry; and Simon-Pierre Demers for aid in preparation and revision of the manuscript.

FOOTNOTES

1Supported by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada to B.D.M.; J.A.D., F.L.L., H.Z, S.K.D., and B.D.M. have no real or potential conflicts of interest to declare with entities related to the material being published. Back

Correspondence: 2Bruce D. Murphy, 3200 Sicotte, St-Hyacinthe, QC, Canada J2S 7C6. FAX: 450 778 8103; e-mail: bruce.d.murphy{at}umontreal.ca

Received: 29 March 2007.

First decision: 13 April 2007.

Accepted: 26 June 2007.

REFERENCES

  1. Carter AM and Enders AC. Comparative aspects of trophoblast development and placentation Reprod Biol Endocrinol 2004 246[CrossRef][Medline]
  2. Enders AC and Schlafke S. Implantation in the ferret: epithelial penetration Am J Anat 1972 133291–315[CrossRef][Medline]
  3. Enders AC, Schlafke S, Hubbard NE, Mead RA. Morphological changes in the blastocyst of the western spotted skunk during activation from delayed implantation Biol Reprod 1986 34423–437[Abstract]
  4. Enders AC and Mead RA. Progression of trophoblast into the endometrium during implantation in the western spotted skunk Anat Rec 1996 244297–315[CrossRef][Medline]
  5. Gulamhusein AP and Beck F. Light and electron microscope observations at the pre- and early post-implantation stages in the ferret uterus J Anat 1973 115159–174[Medline]
  6. Enders AC. Histological observations on the chorio-allantoic placenta of the mink Anat Rec 1957 127231–245[CrossRef][Medline]
  7. Kliewer SA, Forman BM, Blumberg B, Ong ES, Borgmeyer U, Mangelsdorf DJ, Umesono K, Evans RM. Differential expression and activation of a family of murine peroxisome proliferator-activated receptors Proc Natl Acad Sci U S A 1994 917355–7359[Abstract/Free Full Text]
  8. Barak Y, Nelson MC, Ong ES, Jones YZ, Ruiz-Lozano P, Chien KR, Koder A, Evans RM. PPAR gamma is required for placental, cardiac, and adipose tissue development Mol Cell 1999 4585–595[CrossRef][Medline]
  9. Kubota N, Terauchi Y, Miki H, Tamemoto H, Yamauchi T, Komeda K, Satoh S, Nakano R, Ishii C, Sugiyama T, Eto K, Tsubamoto Y, et al. PPAR gamma mediates high-fat diet-induced adipocyte hypertrophy and insulin resistance Mol Cell 1999 4597–609[CrossRef][Medline]
  10. Sapin V, Dolle P, Hindelang C, Kastner P, Chambon P. Defects of the chorioallantoic placenta in mouse RXRalpha null fetuses Dev Biol 1997 19129–41[CrossRef][Medline]
  11. Tontonoz P, Hu E, Graves RA, Budavari AI, Spiegelman BM. mPPAR gamma 2: tissue-specific regulator of an adipocyte enhancer Genes Dev 1994 81224–1234[Abstract/Free Full Text]
  12. Song JH, Sirois J, Houde A, Murphy BD. Cloning, developmental expression, and immunohistochemistry of cyclooxygenase 2 in the endometrium during embryo implantation and gestation in the mink (Mustela vison) Endocrinology 1998 1393629–3636[Abstract/Free Full Text]
  13. Lopes FL, Desmarais J, Ledoux S, Gevry NY, Lefevre P, Murphy BD. Transcriptional regulation of uterine vascular endothelial growth factor during early gestation in a carnivore model, Mustela vison J Biol Chem 2006 28124602–24611[Abstract/Free Full Text]
  14. Forman BM, Umesono K, Chen J, Evans RM. Unique response pathways are established by allosteric interactions among nuclear hormone receptors Cell 1995 81541–550[CrossRef][Medline]
  15. Kliewer SA, Lenhard JM, Willson TM, Patel I, Morris DC, Lehmann JM. A prostaglandin J2 metabolite binds peroxisome proliferator-activated receptor gamma and promotes adipocyte differentiation Cell 1995 83813–819[CrossRef][Medline]
  16. Berger J, Bailey P, Biswas C, Cullinan CA, Doebber TW, Hayes NS, Saperstein R, Smith RG, Leibowitz MD. Thiazolidinediones produce a conformational change in peroxisomal proliferator-activated receptor-gamma: binding and activation correlate with antidiabetic actions in db/db mice Endocrinology 1996 1374189–4195[Abstract]
  17. Panigrahy D, Huang S, Kieran MW, Kaipainen A. PPARgamma as a therapeutic target for tumor angiogenesis and metastasis Cancer Biol Ther 2005 4687–693[Medline]
  18. Lopes FL, Desmarais J, Gevry NY, Ledoux S, Murphy BD. Expression of vascular endothelial growth factor isoforms and receptors Flt-1 and KDR during the peri-implantation period in the mink, Mustela vison Biol Reprod 2003 681926–1933[Abstract/Free Full Text]
  19. Das SK, Wang XN, Paria BC, Damm D, Abraham JA, Klagsbrun M, Andrews GK, Dey SK. Heparin-binding EGF-like growth factor gene is induced in the mouse uterus temporally by the blastocyst solely at the site of its apposition: a possible ligand for interaction with blastocyst EGF-receptor in implantation Development 1994 1201071–1083[Abstract]
  20. Lord E, Murphy BD, Desmarais JA, Ledoux S, Beaudry D, Palin MF. Modulation of peroxisome proliferator-activated receptor delta and gamma transcripts in swine endometrial tissue during early gestation Reproduction 2006 131929–942[Abstract/Free Full Text]
  21. Micchelli CA and Perrimon N. Evidence that stem cells reside in the adult Drosophila midgut epithelium Nature 2006 439475–479[CrossRef][Medline]
  22. Desmarais JA, Bordignon V, Lopes FL, Smith LC, Murphy BD. The escape of the mink embryo from obligate diapause Biol Reprod 2004 70662–670[Abstract/Free Full Text]
  23. Tanaka S, Kunath T, Hadjantonakis AK, Nagy A, Rossant J. Promotion of trophoblast stem cell proliferation by FGF4 Science 1998 2822072–2075[Abstract/Free Full Text]
  24. Cross JC. Genetic insights into trophoblast differentiation and placental morphogenesis Semin Cell Dev Biol 2000 11105–113[CrossRef][Medline]
  25. Simmons DG and Cross JC. Determinants of trophoblast lineage and cell subtype specification in the mouse placenta Dev Biol 2005 28412–24[CrossRef][Medline]
  26. Zybina EV and Zybina TG. Polytene chromosomes in mammalian cells Int Rev Cytol 1996 16553–119[Medline]
  27. Johnson GA, Burghardt RC, Bazer FW, Spencer TE. Osteopontin: roles in implantation and placentation Biol Reprod 2003 691458–1471[Abstract/Free Full Text]
  28. Oyama Y, Akuzawa N, Nagai R, Kurabayashi M. PPARgamma ligand inhibits osteopontin gene expression through interference with binding of nuclear factors to A/T-rich sequence in THP-1 cells Circ Res 2002 90348–355[Abstract/Free Full Text]
  29. Brasaemle DL, Barber T, Wolins NE, Serrero G, Blanchette-Mackie EJ, Londos C. Adipose differentiation-related protein is an ubiquitously expressed lipid storage droplet-associated protein J Lipid Res 1997 382249–2263[Abstract]
  30. Bildirici I, Roh CR, Schaiff WT, Lewkowski BM, Nelson DM, Sadovsky Y. The lipid droplet-associated protein adipophilin is expressed in human trophoblasts and is regulated by peroxisomal proliferator-activated receptor-gamma/retinoid X receptor J Clin Endocrinol Metab 2003 886056–6062[Abstract/Free Full Text]
  31. Nadra K, Anghel SI, Joye E, Tan NS, Basu-Modak S, Trono D, Wahli W, Desvergne B. Differentiation of trophoblast giant cells and their metabolic functions are dependent on peroxisome proliferator-activated receptor beta/delta Mol Cell Biol 2006 263266–3281[Abstract/Free Full Text]
  32. Tontonoz P, Singer S, Forman BM, Sarraf P, Fletcher JA, Fletcher CD, Brun RP, Mueller E, Altiok S, Oppenheim H, Evans RM, Spiegelman BM. Terminal differentiation of human liposarcoma cells induced by ligands for peroxisome proliferator-activated receptor gamma and the retinoid X receptor Proc Natl Acad Sci U S A 1997 94237–241[Abstract/Free Full Text]
  33. Kubota T, Koshizuka K, Williamson EA, Asou H, Said JW, Holden S, Miyoshi I, Koeffler HP. Ligand for peroxisome proliferator-activated receptor gamma (troglitazone) has potent antitumor effect against human prostate cancer both in vitro and in vivo Cancer Res 1998 583344–3352[Abstract/Free Full Text]
  34. Allred CD and Kilgore MW. Selective activation of PPARgamma in breast, colon, and lung cancer cell lines Mol Cell Endocrinol 2005 23521–29[CrossRef][Medline]
  35. Schaiff WT, Carlson MG, Smith SD, Levy R, Nelson DM, Sadovsky Y. Peroxisome proliferator-activated receptor-gamma modulates differentiation of human trophoblast in a ligand-specific manner J Clin Endocrinol Metab 2000 853874–3881[Abstract/Free Full Text]
  36. Shalom-Barak T, Nicholas JM, Wang Y, Zhang X, Ong ES, Young TH, Gendler SJ, Evans RM, Barak Y. Peroxisome proliferator-activated receptor gamma controls Muc1 transcription in trophoblasts Mol Cell Biol 2004 2410661–10669[Abstract/Free Full Text]
  37. Asami-Miyagishi R, Iseki S, Usui M, Uchida K, Kubo H, Morita I. Expression and function of PPARgamma in rat placental development Biochem Biophys Res Commun 2004 315497–501[CrossRef][Medline]
  38. Elbrecht A, Chen Y, Cullinan CA, Hayes N, Leibowitz M, Moller DE, Berger J. Molecular cloning, expression and characterization of human peroxisome proliferator activated receptors gamma 1 and gamma 2 Biochem Biophys Res Commun 1996 224431–437[CrossRef][Medline]
  39. Schaiff WT, Barak Y, Sadovsky Y. The pleiotropic function of PPAR gamma in the placenta Mol Cell Endocrinol 2006 24910–15[Medline]
  40. Wang J and Armant DR. Integrin-mediated adhesion and signaling during blastocyst implantation Cells Tissues Organs 2002 172190–201[CrossRef][Medline]
  41. Gao J and Serrero G. Adipose differentiation related protein (ADRP) expressed in transfected COS-7 cells selectively stimulates long chain fatty acid uptake J Biol Chem 1999 27416825–16830[Abstract/Free Full Text]
  42. Schaiff WT, Bildirici I, Cheong M, Chern PL, Nelson DM, Sadovsky Y. Peroxisome proliferator-activated receptor-gamma and retinoid X receptor signaling regulate fatty acid uptake by primary human placental trophoblasts J Clin Endocrinol Metab 2005 904267–4275[Abstract/Free Full Text]
  43. Rodie VA, Young A, Jordan F, Sattar N, Greer IA, Freeman DJ. Human placental peroxisome proliferator-activated receptor delta and gamma expression in healthy pregnancy and in preeclampsia and intrauterine growth restriction J Soc Gynecol Investig 2005 12320–329[CrossRef][Medline]
  44. Johnson GA, Bazer FW, Jaeger LA, Ka H, Garlow JE, Pfarrer C, Spencer TE, Burghardt RC. Muc-1, integrin, and osteopontin expression during the implantation cascade in sheep Biol Reprod 2001 65820–828[Abstract/Free Full Text]
  45. Omigbodun A, Ziolkiewicz P, Tessler C, Hoyer JR, Coutifaris C. Progesterone regulates osteopontin expression in human trophoblasts: a model of paracrine control in the placenta? Endocrinology 1997 1384308–4315[Abstract/Free Full Text]
  46. Daiter E, Omigbodun A, Wang S, Walinsky D, Strauss JF III, Hoyer JR, Coutifaris C. Cell differentiation and endogenous cyclic adenosine 3',5'-monophosphate regulate osteopontin expression in human trophoblasts Endocrinology 1996 1371785–1790[Abstract]



This article has been cited by other articles:


Home page
 Mol Hum ReprodHome page
E. Capobianco, V. White, R. Higa, N. Martinez, and A. Jawerbaum
Effects of natural ligands of PPAR{gamma} on lipid metabolism in placental tissues from healthy and diabetic rats
Mol. Hum. Reprod., August 1, 2008; 14(8): 491 - 499.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
77/5/829    most recent
biolreprod.107.061929v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Desmarais, J. A
Right arrow Articles by Murphy, B. D
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Desmarais, J. A
Right arrow Articles by Murphy, B. D
Agricola
Right arrow Articles by Desmarais, J. A
Right arrow Articles by Murphy, B. D

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS