This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wang, Y. Articles by Culty, M. Search for Related Content PubMed PubMed Citation Articles by Wang, Y. Articles by Culty, M.

Identification and Distribution of a Novel Platelet-Derived Growth Factor Receptor ß Variant: Effect of Retinoic Acid and Involvement in Cell Differentiation

Department of Biochemistry and Molecular and Cellular Biology, Georgetown University Medical Center, Washington, D.C. 20057

Address all correspondence and requests for reprints to: Martine Culty, Ph.D., Department of Biochemistry and Molecular and Cellular Biology, Georgetown University Medical Center, Basic Science Building, 3900 Reservoir Road NW, Washington, D.C. 20057. E-mail: cultym{at}georgetown.edu.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have shown previously that neonatal testicular gonocytes express platelet-derived growth factor receptors (PDGFR) and ß. We report the expression of a novel PDGFRß (V1-PDGFRß) transcript in gonocytes of 3-d-old rat testes. V1-PDGFRß nucleotide sequence spans from intron 6 to exon 23 of the PDGFRß gene, and is predicted to encode a protein lacking part of the extracellular domain. V1-PDGFRß transcripts are expressed preferentially in developing gonads. The embryonic teratocarcinoma F9 cells, in which differentiation is driven by retinoic acid (RA), express V1-PDGFRß, but not wild-type PDGFRß. Green fluorescent protein-tagged V1-PDGFRß localized mainly in cytosol of F9, MA-10, and COS-1 cells. FLAG and green fluorescent protein-tagged V1-PDGFRß displayed tyrosine kinase activities and contain phosphotyrosine residues, suggesting that V1-PDGFRß is a cytosolic tyrosine kinase. Treatment of F9 cells with RA induced V1-PDGFRß gene expression, concomitant with changes in morphology and increased mRNA expression of collagen IV and laminin B1, suggesting that V1-PFGRß is involved in cell differentiation. Similarly, treatment of postnatal d 3 rat gonocytes with RA induced a dose-dependent increase in V1-PDGFRß expression together with an increase in c-kit and Stra8, markers of more differentiated germ cells and a concomitant decrease in GFR1, a marker of spermatogonial stem cells. However, an excess of V1-PDGFRß inhibited RA-mediated collagen IV and laminin B1 expression and altered both RA-dependent and RA-independent morphological changes in F9 cells, while increasing cell survival. These results suggest that the expression of V1-PDGFRß is tightly regulated during differentiation and that it may play an active role in germ cell differentiation.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
SPERMATOGONIA, THE FIRST cell type of the germ cell lineage in adult males that includes a subset of cells with stem cell properties, arise from the differentiation of their fetal/neonatal precursors, the gonocytes. Gonocytes arise themselves from the primordial germ cells and go through a complex succession of developmental events, including two mitotic phases, one in the embryo and the other at neonatal d 3 and 4, at which point, gonocytes migrate from the center to the periphery of the seminiferous cords and further differentiate into type A spermatogonia (1, 2, 3). Alteration of the developmental program of these cells can eventually lead to infertility or testicular germ cell cancer (4). Thus, understanding the cellular and molecular mechanisms regulating these developmental events could provide explanation and pinpoint at potential therapeutic targets in relation with germ cell pathologies.

Among the molecules that have been reported to affect gonocytes development, fibroblast growth factor 2 and leukemia inhibitory factor act as survival factors (5, 6); TGFß decreases gonocytes number in both fetal and neonatal periods (7); and the cKit ligand stem cell factor controls gonocyte migration toward the basement membrane (8). We have shown previously that PDGF induces neonatal gonocyte proliferation and that the expression of platelet-derived growth factor receptor (PDGFR) ß is up-regulated in neonatal gonocytes after fetal exposure to estrogenic compounds (9, 10). The common biological functions of PDGF signaling pathway include the regulation of cell proliferation and survival, angiogenesis, cell migration, membrane ruffles, and cytoskeletal rearrangements (11). The importance of the PDGF pathway during development is emphasized by the fact that both PDGFR and ß knockout mice do not survive past birth, with PDGFR knockout fetuses presenting abnormalities in the development of mesenchymal cell types of several organs and dying between gestation day (GD)8 and 16, and PDGFRß knockout mice dying just before birth and exhibiting cardiovascular, renal, and hematological defects due to abnormal development of smooth muscle cells (12). Several studies have suggested that PDGF signaling pathway is involved in the regulation of testis development. An early study has demonstrated that PDGFR was detected mostly in early postnatal peritubular myoid cells, where it was believed to regulate cell migration in response to PDGF secreted by Sertoli cells (13). PDGF-A was further shown to be critical for Leydig cell development (14). More recently, human fetal Leydig cells were found to express both PDGFR and ß, whereas subsets of gonocytes expressed PDGFRß (15). Our findings that neonatal testicular gonocytes express PDGFRs (9, 10) and proliferate in response to PDGF (10) further suggested that PDGF signaling may directly regulate germ cell development. Thus, the PDGF pathway appears to play a role in the development of both germ and somatic testicular cells and to be a key factor in the overall development of fetal/neonatal testis.

In previous studies, we demonstrated that at least two forms of PDGFRß proteins were present in neonatal gonocytes; a protein of a size similar to the full-length receptor and a truncated form (9). Interestingly, several variant forms of PDGFR and ß have been described in other models, including PDGFR mutants with strong tyrosine kinase activity in gastrointestinal stromal tumors (16), PDGFR variants in human testicular germ cell tumors and carcinoma in situ (17, 18), and a 4.2-kb transcript of PDGFRß in embryonic carcinoma (EC) and embryonic stem (ES) cells (19). Although none of these studies demonstrated a clear function for the variant PDGFRs, the study on the 4.2-kb transcript of PDGFRß reported that its expression appeared to decrease simultaneously to an increase of the wild-type receptor in cells stimulated to differentiate, and proposed that the truncated PDGFRß form might be a stage-specific embryonic receptor (19).

In the present study, we identified a new PDGFRß variant (V1-PDGFRß) in postnatal day (PND) 3 gonocytes and in embryonic teratocarcinoma cells F9. Sequence analysis showed that its mRNA spans from intron 6 to exon 23 of the PDGFRß gene. A survey of its expression throughout development showed that it was more abundant in the developing gonads. We further used the embryonic F9 cell model, where differentiation is driven by all trans-retinoic acid (RA), to perform functional studies of V1-PDGFRß and examine its regulation. These studies showed that V1-PDGFRß localizes in the cytosol and has tyrosine kinase activity. Moreover, its expression was found to play an active role in RA-induced F9 cell differentiation. Based on the present results, we proposed that V1-PDGFRß plays a role in gonad development, more specifically in testicular germ cell differentiation. Further studies are in progress to clarify the sequence of events and exact function of V1-PDGFRß in cell differentiation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals
BALB/c adult male mice, Sprague Dawley rats, including pregnant mothers and newborn male pups, were purchased from Charles River Laboratories (Wilmington, MA). Male and female rat offspring at GD15, GD17, and GD19, and PND3, PND10, PND21, PND35, and PND90 were killed by CO₂ inhalation; Their brain, heart, liver, kidney, testes, and ovaries were collected and snap-frozen in liquid nitrogen, or used for cell isolation. Male mice were also killed by CO₂ inhalation; their testes were collected and used for cell isolation. The animals were handled according to a protocol that was reviewed and approved by the Georgetown University Animal Committee.

Cell isolation
Gonocytes were isolated from PND3 rat testes using 30–40 pups per preparation as previously described (9, 10). Briefly, gonocytes were isolated by sequential enzymatic tissue dissociation, filtration, differential plating, and separation of the nonadherent cells on a 2–4% BSA gradient. The fractions containing the most gonocytes (as judged by their morphology and large size) were pooled and centrifuged, and the cell suspension was either submitted to an additional step of purification or directly cultured in function of the experiment performed. For studies concerning the identification and characterization of V1-PDGFRß transcript where the highest possible cell purity was required, the cells were further sorted using a micromanipulation device (TransferMan NK micromanipulator from Eppendorf, Westbury, NY) as previously described, where individual cells were harvested using a 15-µm glass transfer pipet allowing 100% purity (9). The final gonocyte pools (3000–6000 cells per sorting) were frozen at –70 C and further used for RNA extraction or protein analysis. In experiments where gonocytes were cultured to examine the effects of RA, the cells from the final BSA pool (with a purity of 75–85%) were resuspended in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 2.5% fetal bovine serum (FBS) and penicillin/streptomycin, plated in 24-well plates at 10,000 cells per 500 µl final volume containing different concentrations of RA for 1 d in 3.5% CO₂ at 36 C. Each experimental point was done in triplicate and at least three independent experiments were performed for each condition examined. Enriched interstitial cells or Sertoli cell preparations that contained around 20% myoid cells were also collected during the process of cell purification to serve as a comparison.

Immature mouse germ cells were isolated from 40 PND6 mice using the same protocol as that used to isolate PND3 rat gonocytes and stopping after the BSA gradient as described above. Adult (PND60) mouse germ cells were isolated using a slightly different protocol. Briefly, testes from eight adult mice per experiment were digested with 0.625 mg/ml collagenase (Invitrogen) and 0.1 mg/ml DNase I (Sigma, St. Louis, MO) for 30 min in RPMI containing antibiotics (100 U/ml penicillin; 0.1 mg/ml streptomycin) at 37 C; and then with 0.25% trypsin-1 mM EDTA (Mediatech, Herndon, VA), 0.25 mg/ml DNaseI, 3.125 mg/ml collagenase, and 0.125 mg/ml hyaluronidase (SIGMA) three times for 20 min each. Trypsin digestion was stopped by adding FBS; the suspension was filtered through 47-µm nylon mesh, the cells were pelleted by centrifugation, and the different germ cell types were separated by sedimentation velocity at unit gravity on a 2–4% BSA gradient. The fractions containing more than 80% round spermatids (as judged by their morphology and size) were pooled and centrifuged, and cell pellets were frozen at –70 C for RNA extraction.

Cell culture and treatment
Mouse F9 EC cells, ES cells 129, BALB/3T3 fibroblasts, rat C62B glioma, monkey COS1, and human breast cancer MCF7 cells were maintained in DMEM medium (Mediatech) containing 4.5% glucose, L-glutamine and sodium pyruvate supplemented with 10% FBS in 6% CO₂. Mouse MA-10 tumor Leydig cells, generously donated by Dr. M. Ascoli (University of Iowa, Iowa City, IA), were maintained at 37 C in DMEM/F12 (Mediatech) medium supplemented with 5% heat-inactivated fetal calf serum and 2.5% horse serum in 3.5% CO₂. F9 cells were also cultured in knockout DMEM supplemented with 15% knockout serum replacement (Invitrogen) for specific purpose. All culture flasks or dishes for F9 cell culturing were precoated with 0.1% gelatin (Sigma). All trans-retinoic acid-RA (Sigma) was used for cells treatment. F9 cells were plated in 12-well plates at 5000 cells per well and cultured overnight before adding RA, and the cells were further cultured for 24, 48, and 72 h with or without RA treatment. Gonocytes were cultured in RPMI 1640 medium containing 2.5% FBS and penicillin/streptomycin in 3.5% CO₂ at 36 C as described above.

RNA extraction
Total RNA was extracted from rat tissues using TRIzol reagent (Invitrogen) and digested with DNase I (Invitrogen). Cellular RNA was isolated using PicoPure RNA Isolation Kit (ARCTURUS, Mountain View, CA) and digested with DNase I (QIAGEN, Santa Clarita, CA). cDNA was synthesized from 2–5 µg of total RNA using PowerScript Reverse Transcriptase (CLONTECH, Mountain View, CA) following the manufacturer’s instructions.

Northern blot analysis
Northern blot analysis was performed as previously described (20). The RNA blots were hybridized with a ³²P-labeled PDGFRß cDNA probe generated from random priming (Promega, Madison WI) that recognized the C terminus of rat wild-type PDGFRß (region 2816–3568; accession no, AY090783). The same blot was stripped and reprobed with a ³²P-labeled glyceraldehyde-3-phosphate dehydrogenase (G3PDH) cDNA probe as internal control.

Rapid amplification of cDNA ends (RACE)
To generate the full-length sequence of the truncated PDGFRß, RACE was performed using BD SMART RACE cDNA Amplification Kit (CLONTECH). Briefly, total RNA from 5000 gonocytes or 1 µg total RNA from F9 cells was used to generate 5'-ready cDNA, which was linked to known nucleotides as an adaptor to the 3' end, or to generate 3'-ready cDNA, which was also linked to the same adaptor before oligo (dT) at the 5' end. Using 5'-ready cDNA or 3'-ready cDNA as template, one primer was designed based on the adaptor sequence to serve as the universal primer, whereas a series of specific primers were designed based on sequences from the wild-type PDGFRß (accession no. for rat PDGFRß, AY090783; accession no. for mouse PDGFRß, NM_008809) that were used in PCR assays to obtain the 5' end or 3'end cDNAs. The amplicons obtained were inserted into the TA-TOPO cloning vector (Invitrogen) and sequenced using M13-forward and M13-reverse primers. The ABI PRISM dye terminator cycle sequencing ready reaction kit and sequencer (both from PE Applied Biosystems, Foster City, CA) were used for nucleotide sequencing at the Lombardi Cancer Center Sequencing Core Facility (Georgetown University). Successive sets of PDGFRß-specific primers were designed to fit the newly sequenced 5' end and 3' end of cDNA, and the PCR and sequencing steps were continued until generation of the full-length truncated PDGFRß sequence.

RT-PCR analysis
Expression of V1-PDGFRß and wild-type PDGFRß mRNA in different rat organs or mouse/rat cells were examined by RT-PCR analysis. To identify the V1-PDGFRß, the following primers were used: 1) rat primers: forward, 5'-GCCCGCCCCTGACTGTTGTACCTTCA-3' and reverse, 5'-GGGAGTTTCTGGCAGGATGGATGGGA-3'; 2) mouse primers: forward, 5'-TTGTACCTTCCGCAGAGAATGGCTACG-3' and reverse, 5'-AAGGCAACTGCACAGGGTCCACGTAGA-3'. The forward primer sequences for rat and mouse V1-PDGFRß were localized in intron 6, a position chosen to identify exclusively V1-PDGFRß transcripts, whereas the reverse primers were localized in exon 23 and 12 for rat and mouse, respectively. To identify the wild-type PDGFRß, the following primers were used: 1) rat primers: forward, 5'-GTGGACTCCGATACTTACTATG-3' and reverse, 5'-TTTGAAAGGCAAGGAGTG-3'; 2) mouse primers: forward, 5'-CATCTCCAGCACCTTTGTTCTGACC-3' and reverse, 5'-CACCAGCCGCCCACTCTTCAT-3'. The forward primers were localized in exon 4 and exon 3 for rat and mouse transcripts, respectively, allowing the measurement of the full-length transcripts only, because these sequences were absent from V1-PDGFRß. The reverse primers localized in exon 11 and 6 for rat and mouse full-length PDGFRß, respectively. Advantage 2 PCR tag (Clontech) was used, and the reaction was amplified using the iCycler thermal cycler from Bio-Rad (Hercules, CA). PCR cycle conditions for rat V1-PDGFRß were 94 C for 3 min; 5 cycles of 94 C for 30 sec, 70 C for 3 min; and 35 cycles of 94 C for 30 sec, 68 C for 3 min; followed by a 3-min extension at 68 C. PCR cycle conditions for other genes were 94 C for 2 min; 5 cycles of 94 C for 15 sec, 62 C for 20 sec, 72 C for 2 min; and 30 cycles of 94 C for 15 sec, 59 C for 20 sec, 72 C for 2 min; followed by a 7-min extension at 72 C. PCR products were then separated on a 1% agarose gel. PCR products were sequenced. Simultaneous runs of the samples were performed using G3PDH primers (forward, 5'-TGTTCCAGTATGACTCTACCCACGGC-3' and reverse, 5'-GGAAGAATGGGAGTTGCTGTTGAAGT-3' as a housekeeping gene to normalize the data.

Real-time quantitative PCR (Q-PCR)
Q-PCR was performed on a DNA Engine Opticon2 Continuous Fluorescence Detector using a SYBR Green PCR Master Mix kit (Applied Biosystems) and primers specific for the genes of interest (Table 1). In experiments using transfected V1-PDGFRß, a set of primers was designed to measure exclusively the endogenous V1-PDGFRß transcript by choosing a sequence localized in the intron 6 portion of the mRNA that was not included in the transfected construct, whereas the measure of the total (endogenous + exogenous) V1-PDGFRß transcripts was performed using a set of primers designed against sequences from exons that were present in both transcripts. The cycling conditions were an initial step at 50 C for 2 min and 10 min at 95 C, followed by 40 cycles at 95 C for 15 sec and 60 C for 1 min. Direct detection of PCR products was monitored by measuring the increase in fluorescence caused by the binding of SYBR Green dye to double-stranded DNA, and the comparative C_T method was used to analyze the data. The amounts of the various genes were normalized to the endogenous reference (18S rRNA) by calculating the value of 2^Ct, with Ct being the difference between the threshold cycle (Ct) of the gene of interest and that of 18S rRNA. The final data were expressed in a relative unit representing the mRNA levels of the gene of interest present in the samples. Assays were performed in triplicate. For each treatment, the mRNA levels were determined in samples from three independent experiments and the mean ± SEM are presented.

Transient transfection
COS-1, MA-10, or F9 cells were transfected using Lipofectamine2000 (Invitrogen) following the manufacturer’s specifications. The cells were plated in six-well plates (with or without coverslips) 1 d before transfection. The cells transfected with the construct of enhanced green fluorescent protein (EGFP) fusion with V1-PDGFRß were observed under living conditions with an Olympus Fluorescence Inverted microscope (Olympus, Center Valley, PA). Cells were used in further experiments 48 h after transfection.

Immunoprecipitation
COS-1 or F9 cells were seeded in six-well plates 1 d before transfection. Each well was transfected with pEGFP-V1-PDGFRß. At 48 h after transfection, the cells were incubated for 30 min at 4 C with lysis buffer (1% Triton X-100, 10 mM Tris-HCl, pH 7.4, 150 mM NaCl), supplemented with protease inhibitors (Sigma). Samples were precleared by incubating with protein-G Sepharose (KPL, Gaithersburg, MD) for 1 h at 4 C, spun for 30 sec at 14,000 rpm, and incubated with 3 µg anti-green fluorescent protein (GFP) (Abcam, Cambridge, MA) overnight at 4 C. Then samples were incubated with protein-G Sepharose for 2 h at 4 C. Precipitants were washed three times with lysis buffer, resuspended in reduced sample buffer, and spun for 30 sec at 14,000 rpm. Samples were then separated by electrophoresis as described below.

Western blot analysis
Aliquots of cell preparations were solubilized in Laemmli buffer, and the proteins were separated on 4–20% SDS-PAGE gels using a Tris/glycine buffer, transferred to polyvinylidene fluoride membrane (Bio-Rad). PDGFRß immunodetection was carried out by immunoblot analysis using antibodies raised against either GFP, various domains of PDGFRß or the anti-phosphotyrosine antibody PY20 (BD Biosciences, San Jose, CA) (1:800 dilution). To identify the endogenous PDGFRß proteins in cell extracts as well as tagged-V1-PDGFRß, an antibody targeting PDGFRß C-terminal from Santa Cruz Biotechnologies (Santa Cruz, CA) (antibody no. 1) was used. Other anti-PDGFRß antibodies used to characterize GFP-V1-PDGFRß fusion protein included two anti-PDGFRß C-terminal antibodies, antibody no. 2 from Cell Signaling Technology, Inc. (Beverly, MA), and antibody no. 4 from Upstate Biotechnologies, Millipore Bioscience (Charlottesville, VA), anti-PDGFRß N-terminal (antibody no. 5, from BD Biosciences), and anti-PDGFRß kinase domain k (antibody no. 3 from Upstate Biotechnologies). G3PDH was used as loading reference and the protein band was visualized using an anti-G3PDH antibody at 1:3000 dilution (TREVIGEN, Gaithersburg, MD). Signal detection was carried out with a chemiluminescence kit (ECL western blotting detection kit; Amersham Pharmacia Biotech, Piscataway, NJ) as previously described (10).

Confocal microscopy
After transfection with pEGFP-V1-PDGFRß for 48 h as previously described, COS-1, MA-10, or F9 cells cultured on coverslips were washed three times with PBS, and then fixed with 3.7% buffered-formaldehyde for 10 min. Slides were incubated for 5 min at room temperature with 4',6-diamidino-2-phenylindole (DAPI) nucleic acid stain (1:40,000) (BD Biosciences). The slides were then mounted with the Prolong Antifade Kit (Molecular Probes Inc., Eugene, OR) and observed under Olympus Fluoview laser scanning microscope.

Kinase assays
Tyrosine kinase activity of FLAG-V1-PDGFRß fusion protein was measured using an ELISA-based protein tyrosine kinase assay kit (SIGMA). Briefly, FLAG-V1-PDGFRß fusion protein from F9 cells transfected with PCMV-Tag2-V1-PDGFRß was immunoprecipitated by anti-FLAG (SIGMA), then diluted in tyrosine kinase buffer and incubated with the Poly-Glu-Tyr substrate that was coated onto the walls of microtiter plates. The phosphorylated polymer substrate was probed with a purified phosphotyrosine-specific monoclonal antibody conjugated to HRP, which in turn, reacted with a chromogenic substrate, and the color signal was quantified by spectrophotometry. A standard curve of tyrosine kinase activity was generated using epidermal growth factor receptor (EGFR) provided in the kit as enzyme reference.

Autophosphorylation of the GFP-V1-PDGFRß fusion protein was measured using a tyrosine kinase activity assay kit also based on an ELISA (Chemicon, Temecula, CA) in the absence of added exogenous substrate. Briefly, GFP-V1-PDGFRß was immunoprecipitated using an anti-biotinylated GFP antibody (Abcam), then diluted in tyrosine kinase buffer and added into the wells of streptavidin-coated plates. After rinsing the wells, the autophosphorylation activity of GFP-V1-PDGFRß fusion protein was measured using a purified phosphotyrosine-specific monoclonal antibody conjugated to HRP, which in turn, reacted with a HRP chromogenic substrate to give a colorimetric signal quantified by spectrophotometry. The standard curve was generated using a phosphopeptide provided in the kit.

Establishment of stable overexpressing V1-PDGFRß cell lines
F9 cells were transfected with pcDNA3.1 (–) plasmid containing either a V1-PDGFRß insert sequence starting at bp 1166 and ending at bp 3445 of the wild-type full-length mouse PDGFRß (NM-008809) [pcDNA3.1 (–)-V1-PDGFRß] or no insert [pcDNA3.1 (–)]. These vectors were selected by incubation with 600 µg/ml G418 (Life Technologies/Invitrogen) in DMEM containing 10% FBS for 10 d, changing the medium every 3 d. The cells were then collected and diluted to get one cell per well, and further cultured for 2 wk into 96-well plates. Clones growing as single clones in a well were digested with 0.25% trypsin-1 mM EDTA, transferred, and cultured for an additional week into 48-well plates. The genomic DNA of selected clones was extracted by DNAzol (Invitrogen) and their identity was verified by PCR. The pcDNA3.1 (–)-V1-PDGFRß-positive clones were determined using primers located at neo gene and V1-PDGFRß, as follows: forward, 5'-CCCAGAGCTGCCCATGAACGACCA-3' (located at V1-PDGFRß) and reverse, 5'-TTCGGCAAGCAGGCATCG-3' (located at neo), that was expected to amplify a 2.5-kb product. The pcDNA3.1 (–)-positive clones were determined using the following two primers located at neo gene: forward, 5'-GTGGAGAGGCTATTCGGCTATGA-3' (located at neo), and reverse, 5'-TTCGGCAAGCAGGCATCG-3' (located at neo). Several positive clones for each transfected cell line were found. One positive clone for pcDNA3.1 (–)-V1-PDGFRß, named vg1, and one positive clone for pcDNA3.1 (–), named C3, were used for further investigation. The expression level of V1-PDGFRß in both clones was determined by Q-PCR using specific primers (Table 1). One of the primers to detect endogenous V1-PDGFRß was located at intron 6 of the genomic PDGFRß (not located at the pcDNA3.1 (–)-V1-PDGFRß plasmid), a sequence that was not included in V1-PDGFRß generated by the pcDNA3.1 (–)-V1-PDGFRß plasmid. Another set of primers was selected to detect total V1-PDGFRß, by recognizing two V1-PDGFRß exonic sequences located in the insert on pcDNA3.1 (–)-V1-PDGFRß plasmid and present in both endogenous and overexpressed V1-PDGFRß.

Apoptotic cell death measurement
The levels of apoptosis occurring in vg1 and C3 cells grown with or without RA for 3 d were determined using the Cell Death Detection ELISA^plus kit (Roche Applied Science) that quantifies the amounts of mononucleosomes and oligonucleosomes present in cell cytosol as a measure of DNA fragmentation using a biotin-anti-histone antibody to trap nucleosomes on the plates and a peroxidase-coupled anti-DNA antibody to quantify them following the protocol recommended by the manufacturer. The data were expressed as differences between the absorbance at 405 nm and the absorbance at the reference wavelength of 490 nm for each sample. Three independent experiments were performed with each individual sample run in triplicates.

Statistical analysis
Statistical analysis was performed by unpaired two-tail t test using statistical analysis functions in GraphPad Prism 4.0 program (GraphPad Inc., San Diego, CA). All experiments were performed at least three independent times and a P value less than 0.05 was considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Identification of truncated PDGFRß in rat PND3 gonocytes and F9 cells
Our previous studies had identified at least two PDGFRß forms in protein extracts of isolated rat PND3 gonocytes examined by Western blot analysis (9), and shown that an antibody against the C-terminal region of PDGFRß recognized one single PDGFRß band around 70 kDa in these cells, although the same antibody reacted with the full-length receptor in Sertoli/myoid cell extracts. To confirm the existence and characterize further the variant PDGFRß form, Northern blot analysis of PND3 whole testis, Sertoli/myoid cell, and gonocyte RNA extracts were performed using a ³²P-labeled DNA probe recognizing the C terminus of rat PDGFRß (accession no. AY090783, region 2816–3568, exon 20-exon 23). As shown in Fig. 1A, PND3 testis expressed three transcripts of PDGFRß, the wild-type at 5.4 kb and two minor forms at 4 and 2 kb; Sertoli/myoid cells expressed two transcripts of PDGFRß, the wild-type at 5.4 kb, and a minor form at 2 kb; whereas gonocytes expressed three transcripts of PDGFRß at similar levels, the wild-type at 5.4 kb, and two shorter transcripts of 4 and 2 kb, respectively. These results confirmed the existence of different PDGFRß mRNA species in PND3 gonocytes, indicating that the variant proteins were due to pretranslational modifications, and that the variant transcripts included the C-terminal sequence of wild-type PDGFRß. We then focused our interest on the 4-kb mRNA form expressed in PND3 gonocytes, because this transcript should encode a protein at around 70 kDa, corresponding to the size of the variant form we previously reported in these cells (9).

View larger version (15K): [in this window] [in a new window]   FIG. 1. Identification of the PDGFRß variant transcript V1-PDGFRß in neonatal rat gonocytes and F9 cells. A, Northern blot analysis was performed on RNA extracts from neonatal d 3 (PND3) rat testis, isolated Sertoli/myoid cells, and purified gonocytes using a 32P-labeled cDNA probe against the C-terminal region (exons 20–23) of PDGFRß. The long arrow indicates the position of the 5.4-kb full-length PDGFRß transcript, whereas the short arrow shows a 4-kb variant transcript. Positions of standards are indicated on the left. B, Sequencing of V1-PDGFRß mRNA and comparison with full-length PDGFRß. V1-PDGFRß sequence was determined by performing 5' and 3' RACE on RNA extracts from rat PND3 gonocytes and mouse F9 cells. Both sequences started in intron 6 (intron sequences represented by underlined lowercase letters), and ended at exon 23 of their corresponding wild-type PDGFRßs. Exon sequences are represented by uppercase letters, and the predicted start codons are represented by bold uppercase letters. The dotted line indicates that mouse start codon is located in exon 7; the solid line shows that rat start codon is located in exon 8. The parts of the nucleotide sequences that are not shown were found to be identical to that of full-length PDGFRßs. A schematic representation of wild-type PDGFRß domains is aligned with a depiction of the exons present in full-length and variant PDGFRß. V1-PDGFRßs lack most of the extracellular domain (ED) of wild-type PDGFRß, but have retained the transmembrane (T), juxta-membrane (JM), tyrosine kinase 1 (TK1), kinase insert (KI), tyrosine kinase 2 (TK2), and C-terminal (CT) domains.

View larger version (29K): [in this window] [in a new window]   FIG. 2. Expression of V1-PDGFRß in PND3 testicular cells and cell lines. The mRNA expression of V1-PDGFRß and wild-type PDGFRß were determined by RT-PCR analysis on the indicated PND3 rat testicular cells (A); and in mouse EC F9 cells, mouse 129 ES cells, round spermatids isolated from adult mice and spermatogonia from PND6 mice (B). G3PDH mRNA was measured as loading reference. C, V1-PDGFRß and full-length PDGFRß protein expression were examined by Western blot analysis in the indicated PND3 primary testicular cells, and in several cell lines, including F9 cells, MA-10 mouse Leydig tumor cells, C6–2B rat glioma cells, BALB/3T3 mouse fibroblasts, and MCF7 human breast cancer cells. G3PDH was used as a loading reference. The positions of molecular weight standards are indicated on the left. A representative experiment is shown.

Differential tissue and developmental expression of V1-PDGFRß in rat organs
To determine the tissue distribution of V1-PDGFRß in different organs and at various ages, RT-PCR analysis was performed on RNA extracts of several organs collected at different fetal and postnatal ages. As shown in Fig. 3A, V1-PDGFRß transcript was expressed mainly in testes and ovaries at all ages examined, but also in GD17 brain and GD19 kidney, whereas it was conspicuously absent in heart and liver. By contrast, the full-length PDGFRß mRNA was found in all tissues examined and at all ages. These data suggested that V1-PDGFRß might be involved more specifically in gonad development and functions. Because V1-PDGFRß mRNA was expressed in testes and ovaries at all ages, we then quantified its expression in these tissues by Q-PCR. These experiments showed that V1-PDGFRß mRNA levels varied in testis and ovary throughout development, with the highest levels found at PND3 and the lowest levels in the adult for both tissues (Fig. 2, B and C).

View larger version (18K): [in this window] [in a new window]   FIG. 3. Tissue distribution of V1-PDGFRß transcript in rat organs at different ages. A, The expression of rat V1-PDGFRß and wild-type PDGFRß mRNAs were examined by RT-PCR in brain (Br), heart (H), liver (L), kidney (K), testis (T), and ovaries (O) in rats at GD15, GD17, GD19 and PND3, PND10, prepuberty (PND21), puberty (PND35), and adulthood (PND90). Representative results are shown. The quantification of V1-PDGFRß mRNA levels was performed by Q-PCR analysis in rat testes (B) and ovaries (C) dissected at the indicated ages. Results show the mean ± SEM of at least three independent experiments.

View larger version (22K): [in this window] [in a new window]   FIG. 4. Expression and subcellular location of GFP-tagged V1-PDGFRß (GFP-V1-PDGFRß) in cell lines. The pEGFP-V1-PDGFRß plasmid was constructed and transfected into F9 cells, Leydig tumor cells MA-10, and COS1 cells. Confocal microscopy was performed using an antibody against GFP and an immunofluorescent detection kit. DAPI was used to label the nuclei, and the images were merged to determine the subcellular location of V1-PDGFRß. Representative pictures are shown.

View larger version (11K): [in this window] [in a new window]   FIG. 5. Tyrosine kinase activity and autophosphorylation of FLAG/GFP-tagged V1-PDGFRß in F9 cells. A, PCMV-Tag2-V1-PDGFRß plasmid was transfected into F9 cells and the resulting protein was immunoprecipitated using an anti-FLAG antibody. FLAG-V1-PDGFRß protein was used to perform an ELISA-based tyrosine kinase activity assay using a Poly-Glu-Tyr exogenous substrate. B, pEGFP-V1-PDGFRß plasmid was constructed and transfected into F9 cells and the fusion EGFP-V1-PDGFRß protein was immunoprecipitated using a biotinylated anti-GFP antibody. Autophosphorylation of EGFP-V1-PDGFRß was determined by ELISA. C, Western blot analysis of the EGFP-V1-PDGFRß immunoprecipitates was performed using various anti-PDGFRß antibodies directed against either the C-terminal (# 1, 2, 4), N-terminal (# 5), kinase domain (# 3), PY20 antibody that identifies phosphotyrosine residues and an anti-GFP antibody to identify the band corresponding to the expressed protein.

To further confirm the identity of the tyrosine-phosphorylated protein, GFP-V1-PDGFRß fusion protein was immunoprecipitated with an anti-GFP antibody and the protein extract was submitted to Western blot analysis using anti-GFP, several anti-PDGFRß, and anti-PY20 antibodies. As shown in Fig. 5C, the band identified by anti-GFP antibody as GFP-V1-PDGFRß was also recognized by five different anti-PDGFRß antibodies raised against either the C terminus (antibodies 1, 2, 4) or kinase domain (antibody no. 3) of wild-type PDGFRß, but it did not react with an antibody raised against the N terminus of wild-type PDGFRß (antibody no. 5). This later result was consistent with the predicted V1-PDGFRß protein analysis indicating that V1-PDGFRß lacked the N-terminal region. Moreover, the band corresponding to GFP-V1-PDGFRß was also recognized by an anti-PY20 antibody, suggesting that the fusion protein can autophosphorylate. Taken together, these results demonstrated that FLAG/GFP-V1-PDGFRß fusion proteins contained tyrosine kinase activity and could also autophosphorylate its own tyrosine residues, thereby providing potential docking site for other SH2-domain containing proteins and influencing signal transduction pathways.

Regulation of V1-PDGFRß expression by RA in F9 cells
F9 cells are a well-established model to study RA-induced differentiation. As shown in Fig. 6A, after 72 h of treatment with 0.1 µM RA, the morphology of F9 cells changed from an appearance of compact clusters observed in control samples to a monolayer presenting more scattered and flattened cells and corresponding to a primitive endoderm-like cell morphology. Because increases in the expression of collagen IV and laminin B1 are considered hallmarks of RA-induced differentiation in F9 cells (22, 23), the mRNA levels of these two marker genes were quantified by Q-PCR after 24, 48, and 72 h of RA treatment. As expected, the mRNA expression of both genes were dramatically increased over time, with the collagen IV mRNA increasing by 6.3-, 16.0-, and 59.7-fold and laminin B1 mRNA increasing by 3.8-, 8.3-, and 31.5-fold of the untreated values after 24-, 48-, and 72-h RA treatments, respectively (Fig. 6, B and C). It should be noted that small increases of collagen IV and laminin B1 mRNAs were also occurring over time in the absence of RA, but that the amplitudes of these responses were much smaller than those observed with RA. Similar observations were made for V1-PDGFRß mRNA, which was increased in a time-dependent manner after RA treatment, as shown by RT-PCR analysis (Fig. 6D). This pattern of expression was similar to that observed for full-length PDGFR mRNA, except that the increases in V1-PDGFRß appeared delayed by 1 d and to be smaller than those observed for PDGFR (Fig. 6D). Further quantification by Q-PCR of V1-PDGFRß mRNA showed increases of 4.1-, 3.1-, and 4.2-fold of the corresponding untreated values after 24, 48, and 72 h of RA treatments, respectively (Fig. 6E). The amounts of V1-PDGFRß transcripts present in RA-treated cells were doubling from 1 d to the next, translating to a progressive increase over time as observed for collagen IV and laminin B1. Here again, small increases appear to occur over time in the absence of RA, but at much smaller levels than in the presence of RA. To further confirm that the effects observed on V1-PDGFRß expression were due only to RA stimulation and did not involve other factors from the serum, we performed experiments where the regular medium containing 10% FBS was substituted with a knockout medium containing 15% of serum-replacement reagent devoid of RA, so that the only source of RA would be the RA added separately in the cultures. As shown in Fig. 6F, addition of 0.1 µM RA in cells cultured in this special medium induced increases in V1-PDGFRß mRNA expression similar to those observed with regular culture medium, increasing by 3.8-, 2.4-, and 2.9-fold after 24, 48, and 72 h of RA treatments, respectively. Interestingly, the responses obtained for collagen IV and laminin B1 expression in these conditions were qualitatively similar to those observed with the regular medium, although smaller in amplitude (data not shown). These results demonstrated that V1-PDGFRß expression was changed by RA in a manner similar to the differentiation markers collagen IV and laminin B1, further suggesting the involvement of V1-PDGFRß in RA-induced F9 cell differentiation.

View larger version (26K): [in this window] [in a new window]   FIG. 6. Effects of RA on V1-PDGFRß expression and cell differentiation in F9 cells. F9 cells were plated at 5000 cells per well and cultured for 20 h in DMEM containing 10% FBS, then treated with or without 0.1 µM RA for up to 5 d. Cells without treatment were collected at d 1–5; cells with RA treatment were collected at d 1–3. A, Changes in the morphology of F9 cells after 3 d of RA treatment. The magnification of the pictures is indicated by the 200 µm scale in the top picture. The mRNA levels of two differentiation markers, collagen IV (B) and laminin B1 (C) were quantified by Q-PCR analysis. D, The mRNA expression of V1-PDGFRß and PDGFR were examined by RT-PCR, using G3PDH as a loading reference. E, The mRNA levels of V1-PDGFRß in F9 cells grown with normal FBS ± 0.1 µM RA were quantified by Q-PCR analysis using 18S rRNA as a reference. F, Q-PCR quantification of V1-PDGFRß mRNA in F9 cells (5000 cells per well) transferred to knockout DMEM with 15% serum replacement 20 h after plating, and treated with or without 0.1 µM RA for 3 d. Results in B, C, E, and F show the mean ± SEM of at least three independent experiments for each condition (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

View larger version (18K): [in this window] [in a new window]   FIG. 7. Effects of RA on V1-PDGFRß expression and cell differentiation in rat gonocytes. Gonocytes were isolated from PND3 rat testes and cultured at 10,000 cells per well in RPMI 1640 supplemented with 2.5% FBS and antibiotics with or without 0.1 and 1 µM RA for 1 d. The cells were then collected and the RNA extracted. The mRNA levels of V1-PDGFRß, c-kit, GFR1, Stra8, and Itg6 were determined by Q-PCR analysis. Results are the mean ± SEM of at least three independent experiments for each condition (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

View larger version (33K): [in this window] [in a new window]   FIG. 8. Effects of overexpressed V1-PDGFRß on RA-induced differentiation and survival of F9 cells. A, Two stable transfected F9 cell lines were established, a mock pcDNA3.1 (–) vector and pcDNA3.1 (–) vector containing a mouse V1-PDGFRß insert. The plasmids were transfected into F9 cells and neomycin was used to select stably transfected cells. In both conditions, several clones were selected and verified as stably transfected (left and center panels). The clone 1 for V1-PDGFRß construct (vg1) and the clone 3 for empty vector (C3) were used for further analysis. The mRNA levels of total V1-PDGFRß and endogenous V1-PDGFRß were quantified by Q-PCR analysis (right panel). B, vg1 and C3 cells were plated (5000 per well) and incubated with or without 0.1 µM RA for 3 d, and the morphology of the cells was observed to evaluate differentiation under a phase contrast microscope equipped with a digital camera. The magnification was the same as in Fig. 6. C, Q-PCR analysis of collagen IV mRNA expression used as differentiation marker, in the presence or absence of 0.1 µM RA for 3 d, in C3 and vg1 cells. D, Q-PCR analysis of Laminin B1 mRNA expression used as differentiation marker, in the presence or absence of 0.1 µM RA for 3 d, in C3 and vg1 cells. E, Apoptosis was examined by measuring DNA fragmentation in cell cytosol using a nucleosomal ELISA kit in vg1 or C3 cells (5000 per well) treated with or without 0.1 µM for 3 d. Results in C–E show the mean ± SEM of at least three independent experiments for each condition (*, P < 0.05; **, P < 0.01).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the current study, we identified and characterized a novel form of truncated PDGFRß (V1-PDGFRß) in rat neonatal testicular germ cells and in a murine embryonic teratocarcinoma cell line. The mRNA sequences of V1-PDGFRß included part of intron 6 and the exons 7–23 of the full-length PDGFRß, with start codons located in exon 7 and 8 in mouse and rat cells. The predicted protein sequences indicated that V1-PDGFRß contains a partial extracellular domain, the transmembrane domain, juxtamembrane domain, tyrosine kinase domain, and C-terminal tail. Using mammalian expression vectors, FLAG and GFP-tagged V1-PDGFRß fusion proteins were generated in which tyrosine kinase activity was maintained, demonstrating both the ability to phosphorylate exogenous substrates as well as an autophosphorylation activity. Moreover, when expressed in several mammalian cell lines, V1-PDGFRß was found to localize in the cytosol of the cells rather than at the plasma membrane where the full-length receptor resides. Together, these results identify V1-PDGFRß as a cytosolic tyrosine kinase that is no longer under the direct control of PDGF because it is missing the PDGF binding domain.

Interestingly, a number of alternative mRNA transcripts derived from tyrosine kinase receptor genes have been described, including variant transcripts of the EGFR (31), fibroblast growth factor receptor (32), c-kit (33), PDGFR (34, 35), and the fer gene (36). Some of these variant transcripts, such as that of EGFR (31), initiated transcription at the normal site and contained alternatively spliced exons. However, other variants, such as a truncated c-kit (tr-kit) found in round spermatids (33) presented the striking analogy with V1-PDGFRß to initiate transcription within intronic sequences and to be devoid of additional splicing in the exons localized beyond the initiation sequence. Tr-kit transcripts were found to have an open reading frame starting in intron 16 and to encode for a protein containing 12 hydrophobic amino acids exclusive to tr-kit and the amino acid sequences corresponding to the phosphotransferase domain and C-terminal tail of c-kit; but lacked the extracellular, transmembrane, ATP binding site, and interkinase domains of the wild-type receptor (33, 37). Other examples of retention of intronic sequences in mature mRNA have been reported, such as that observed in variant transcripts of the kallikrein gene resulting in the formation of truncated proteins in prostate (38) as well as truncated transcripts of PDGFR expressed in human teratocarcinoma cells and human germ cell tumors (34, 35). Interestingly, in this later case, initiation of transcription was also found to occur within an intron, further suggesting comparable mechanisms of transcript rearrangements for PDGFR and PDGFRß. Such findings of intronic retention have become more frequent with the availability of complete genomes, and have led to the concept that intronic retention might represent a rearrangement mechanism, together with exon splicing and fusion, by which variant transcripts are generated and contribute to the higher diversity observed in proteomes relative to their corresponding genomes.

A truncated 4.2-kb transcript of PDGFRß was identified some years ago by Northern blot analysis in the three ES cell lines 129 ES, C3H, and AKR, as well as in the EC cells PSA, together with the 5.3-kb full-length PDGFRß transcript (19). This study found that the truncated transcript was initiated within the genomic region immediately upstream of exon 6 of the full-length PDGFRß transcript and, therefore, lacked the first five exons, which encode much of the extracellular domain of the wild-type PDGFRß, but its exact sequence was not determined. This study also described the presence of the 4.2-kb truncated PDGFRß transcript in the mouse teratocarcinoma cells F9, which differed from the other cell lines by not expressing the full-length PDGFRß transcript. Moreover, in all the ES and EC cells tested except F9 cells, the 4.2-kb PDGFRß transcript was predominant. However, after differentiation induced by RA and dibutyryl cAMP, expression levels of the 5.3-kb PDGFRß increased dramatically and became the dominant form in these cell lines. Based on these observations, the authors concluded that during early embryogenesis, a stem cell-specific PDGFRß promoter was used in a stage- and cell type-specific manner to express a 4.2-kb PDGFRß. In the present study, we initially identified and determined the full sequence of V1-PDGFRß by 5',3' RACE and PCR in neonatal rat testicular germ cells. Because of our finding that V1-PDGFRß initiates from intron 6 and ends at exon 23 of the wild-type PDGFRß and the study mentioned above, we examined whether a homologous form of the rat V1-PDGFRß was expressed in F9 cells and 129 ES cells. Indeed, we identified and sequenced V1-PDGFRß transcripts in F9 and 129 ES cell lines, suggesting that the 4.2-kb PDGFRß transcript previously described in these cells is the murine homolog of rat V1-PDGFRß expressed in germ cells. We also confirmed that F9 cells do not express the full-length PDGFRß, making this cell line a good model to study V1-PDGFRß function.

Considering that neonatal rat gonocytes correspond in human to a germ cell population found in the fetal testis (39), whereas F9 teratocarcinoma correspond to pluripotent ES cells (22), we examine whether V1-PDGFRß expression was restricted to fetal-type cells or if it could be found at the mRNA and/or protein level in various tumor cell lines and rat organs at different ages. These experiments clearly showed that V1-PDGFRß was expressed not only in neonatal rat germ cells but also in more differentiated germ cell stages such as murine round spermatids, as well as in the mouse tumoral Leydig cell line MA-10 and the human breast cancer MCF7 cells. It should be noted that the antibody used in these experiments recognized full-length PDGFRß in several cell types but not in gonocytes. However, our previous studies using an antibody directed against the kinase domain of the receptor had demonstrated the presence of a PDGFRß band corresponding in size to the full-length receptor (9), although its failure to react with an antibody recognizing the C terminus of PDGFRß suggested that the receptor present in gonocytes carry some differences in its primary amino acid sequence or tertiary structure compared with the receptor expressed in somatic cells. When several organs from GD15 to PND90 rats were probed for V1-PDGFRß mRNA expression, we observed a strong and exclusive expression in testis and ovary at most ages examined, except at GD17 and 19, where it was also detected in brain and kidney, respectively. The restricted tissue distribution of V1-PDGFRß transcript, by contrast with the wild-type PDGFRß mRNA that was found in all tissues examined, suggested that the two PDGFRß forms might play distinct roles in development. Moreover, V1-PDGFRß differential expression throughout ages suggests a preferential role of the truncated form in the development of the gonads, but also in a narrow time frame of brain and kidney development. In testis, V1-PDGFRß mRNA expression culminated at PND3, at an age when we had found its expression limited to germ cells, and then decreased progressively over time. In ovary, V1-PDGFRß presented a different profile of expression, with the highest levels being at PND3 and 10. At present, we do not know what ovarian cell type is expressing V1-PDGFRß and, therefore, we cannot speculate on the potential role of V1-PDGFR in ovary, but its expression profile definitively suggests a preferential role in the gonads, two organs presenting homologies, especially in their early developmental phases and also at the levels of their steroidogenic somatic cells. Moreover, at PND3 in female the primary oocyte is in the diplotene stage of meiosis, whereas in male, the gonocyte is in mitotic phase, suggesting that, if V1-PDGFR was expressed in oocyte, then its role in male and female germ cell development must be different. All together, these observations suggest potential roles for V1-PDGFRß in gonadal function, more specifically germ cell development, as well as in cancer.

Such properties are reminiscent of those described for the c-kit tyrosine kinase receptor and its variant form tr-kit which were both found to play a role in germ cell maturation (37), whereas c-kit was found to be highly expressed in testicular germ cell cancers (40), and tr-kit was shown to be involved in neoplastic transformation of prostate cancer cells (41). Both PDGFR and c-kit belong to the type III family of receptor tyrosine kinases, which is defined by the presence of five extracellular immunoglobin-like domains, one transmembrane and one juxtamembrane domain, a cytoplasmic kinase domain that is interrupted by a kinase insert segment, and a C-terminal tail (11). Upon ligand binding, PDGFR/c-kit undergoes dimerization and autophosphorylation. Phosphorylated tyrosine in the receptors will bind to the SH2 domain-containing signal relay proteins [e.g. Src, phosphatidylinositol 3-kinase (PI3-kinase), phosphatase PTP), triggering the activation of these proteins and their downstream signaling targets (11, 36, 38). Interestingly, we found that V1-PDGFRß was strongly expressed in PND3 gonocytes and round spermatids, whereas full-length PDGFRß was present in GD21 and PND3 gonocytes but absent from spermatogonia in PND7 rats (10). By comparison, c-kit was shown to be expressed in primordial germ cells, the precursor cells of gonocytes (42), but not in mouse gonocytes prior and at birth (25). Although we could not detect c-kit protein in PND3 rat gonocytes (M. Culty, unpublished data), the present study found significant levels of c-kit transcripts in RNA extracts from isolated PND3 gonocytes. This is in agreement with a study where 30% of gonocytes isolated at birth and cocultured for 3 d with Sertoli cells showed a positive signal for c-kit mRNA by in situ hybridization, whereas the protein was clearly shown after 5 d in coculture (8). Indeed, it is now well established that c-kit is expressed in differentiated type A spermatogonia but not in the germline stem cells in mice (25, 43). Interestingly, this pattern of successive expression, disappearance, and reexpression at later stages is similar to the expression pattern of c-kit observed in human germ cells, where an initial c-kit-positive PGC population differentiates into a c-kit-negative population, then further gives rise to subpopulations of c-kit-positive cells (39, 40). By contrast, tr-kit was only expressed in specific postmeiotic stages in mice, including sperm and round spermatids, a stage we found to contain V1-PDGFRß, and is believed to be involved in spermatid maturation as well as egg activation by spermatozoa (31). Moreover, a PDGFRß truncated form lacking extracellular and transmembrane domains has been previously shown to retain the ability to dimerize and interact with downstream proteins (44), whereas tr-kit expression was found to result in the activation of Fyn and a Src-like kinase in mouse eggs (45), further demonstrating the ability of both types of truncated proteins to interact with downstream signaling molecules. Taken together, these and our results suggest that V1-PDGFRß, full-length PDGFRß, c-kit, and tr-kit either alternate in their expression or are coexpressed depending on the differentiation stages of germ cells, suggesting that they all play specific roles in spermatogenesis; and that the truncated variants of both receptors might represent physiological modulators of their full-length counterparts in this process.

Although the role of PDGFR in germ cells has been established only for PND3 gonocytes, c-kit gene has been shown to be critical for the survival, migration, and proliferation of primordial germ cells (37, 42), growth and maturation of oocytes (37, 46), and survival and proliferation of type A spermatogonia (31, 25). We previously showed that PDGF activates the proliferation of PND3 rat gonocytes (10, 47) and that fetal estrogen exposure increases PDGFRß mRNA and protein expression in neonatal gonocytes (9). Interestingly, one of the antibodies used to perform immunohistological analysis of PND3 rat testes exposed to estrogens that showed a strong increase in PDGFRß protein in estrogen-exposed gonocytes did not identify the full-length receptor by Western blot analysis of gonocytes, but only a 70-kDa truncated PDGFRß band (9) that we have identified as V1-PDGFRß in the present study. Taking together our earlier and present findings, this suggests that estrogens can modulate the expression of V1-PDGFRß in gonocytes. Moreover, PND3 gonocytes were also found to express a 120-kDa full-length PDGFRß using an antibody against the kinase domain of PDGFRß, as well as PDGFR (9), similarly to the EC and ES cells expressing either both or one type of full-length receptors together with the truncated PDGFRß (19). We have also found that PDGF proliferative action on neonatal gonocytes requires the concomitant activation of estrogen receptor in these cells (47), which express high levels of ERß (48). Such interactions between a growth factor receptor and nuclear receptor signaling pathways have been observed in other systems, such as the interaction of EGFR and ER pathways in human breast cancer cells (49), the induction of PDGFR by the estrogenic compound diethylstilbestrol in mouse vagina and uterus (50), or in the case of the increase of full-length PDGFR after RA-induced differentiation of F9 cells (19). Interestingly, neonatal gonocytes have been shown to express RA receptors, and in these cells, RA has been found to be involved in cell survival (51).

In search of V1-PDGFRß function, we investigated whether RA might regulate V1-PDGFRß expression concomitantly with cell differentiation in F9 cells. Our finding that RA treatment induced a progressive increase in V1-PDGFRß mRNA expression with time that paralleled the increases observed in collagen IV and laminin B1 expression and the morphological alterations corresponding to a differentiation of F9 cells into primitive endoderm-like cells suggested that V1-PDGFRß plays a role in the RA-dependent differentiation process of these cells. We also observed a progressive increase in PDGFR mRNA expression in RA-stimulated F9 cells, whereas in the absence of RA, the increase in PDGFR mRNA clearly preceded that of V1-PDGFRß, suggesting different kinetics and probably functions for the two forms of PDGFRs in F9 cells. Interestingly, an earlier study comparing PDGFR expression during the differentiation of several ES and EC cell lines had reported that the treatment of PSA-1 EC cells with a combination of RA and cAMP resulted in the increase of a large 5.3-kb PDGFR transcript, believed to be coded by the same gene as a 4.2-kb truncated form that was decreased in the same conditions (19). Moreover, this study described how the cotreatment with RA and cAMP of F9 cells induced a decrease in a truncated PDGFRß mRNA, whereas no full-length transcript was visible in these cells. Thus, our present data showing a RA-induced increase in a full-length PDGFR transcript is similar with the observations made on PSA-1 cells stimulated with RA and cAMP, whereas our results showing an increase in V1-PDGFRß in RA-stimulated F9 cells contrasts with the data reported earlier for F9 cells incubated with a combination of RA and cAMP. This further suggests that V1-PDGFRß might be affected differently by RA alone vs. RA combined with cAPM in F9 cells, and therefore, that it might play different roles depending of the differentiation path followed by the cells, reminiscent of the ability of RA-stimulated F9 cells to undergo either parietal or visceral endoderm differentiation in different conditions, including presence or absence of cAMP and aggregation (52).

However, when V1-PDGFRß was overexpressed in F9 cells, it clearly inhibited by more than 50% the increases of collagen IV and laminin B1 in RA-stimulated cells, but not in untreated cells, where it was instead augmenting the small increases occurring with time in collagen IV and laminin B1 transcripts in the absence of RA. Moreover, V1-PDGFRß overexpression in absence of RA appeared to totally block the spontaneous differentiation that took place in control cells after 3 d in culture, or at least to modify the type of differentiation occurring in these cells, because their morphology was reminiscent of a progression into visceral endoderm and their levels of collagen and laminin production remained much lower than the levels found in cells differentiating into primitive endoderm. By contrast, in the presence of RA, V1-PDGFRß overexpression appeared to delay but not totally block RA-induced differentiation, which is in agreement with the fact that it inhibited only partially the production of extracellular matrix proteins and decreased it to levels that remained much higher than those found in the absence of RA. Concomitant to these effects on differentiation, V1-PDGFRß overexpression was found to increase F9 cell survival by decreasing the level of apoptosis that occurred both in basal conditions and in the presence of RA, suggesting that V1-PDGFRß may also play a role in the survival of differentiating cells. These results clearly show that F9 cell differentiation is accompanied by changes in V1-PDGFRß expression and suggest that the time and levels of V1-PDGFRß expression might be critical events in the differentiation process and survival of these cells.

To verify whether the F9 model was relevant to the developmental process of gonocytes, we performed a series of experiments where enriched populations of PND3 gonocytes were incubated for 1 d with two concentrations of RA. RA induced a dose-dependent increase in V1-PDGFRß mRNA expression similar to that observed in F9 cells treated with RA, validating further the use of F9 cells as a model to examine the role of V1-PDGFRß in cell differentiation. Another important finding of the present study is that RA induced changes in PND3 gonocytes of genes that have been associated either with stemcellness or differentiation, suggesting that RA might play a role in germ cell differentiation as it has been shown for hematopoietic cells. Indeed, our results showed that incubation of isolated gonocytes with RA induced an increase in c-kit mRNA expression, which would be in agreement with a progression of the cells toward a more differentiated stage. Interestingly, the highest increase in gene expression was observed in Stra8, a protein originally found to be induced by RA in embryonal carcinoma F9 and P19 cells, as well as in premeiotic germ cells (27). More recently, Stra8 expression was found to be induced in embryonic ovaries and testes collected at GD11.5 and GD12.5, respectively, and cultured with RA for 2 d, suggesting the presence of a factor delaying the expression of Stra8 in the embryonic testis but not ovary (53). By contrast, the expression of glial cell line-derived neurotrophic factor receptor GFR1, a protein identified as SSC marker (54) was strongly decreased in gonocytes incubated with RA, suggesting that the cells were losing their stem cell phenotype while differentiating to become spermatogonia. The fact that a small level of GFR1 expression remained after RA treatment may indicate that a subpopulation of the gonocytes was refractory to the effects of RA and retained stem cell properties. This further suggests that the initial population of gonocytes is heterogeneous and comprises a subpopulation of stem cells that do not differentiate, implying that an asymmetrical division of the gonocytes might occur, where the parent cell keeps its stem cell properties while the daughter cell progress into the differentiation process to become a differentiated A spermatogonia. Simultaneously, the Int6 previously described as a SSC marker was not significantly affected by RA treatment, suggesting that it may remain expressed in differentiated spermatogonia. Alternatively, this could indicate that the paradigm of cultured isolated cells used in this study does not mimic perfectly all the parameters involved in the in vivo differentiation process, and that only portions of the differentiation process toward a spermatogonia phenotype occur, which would not be surprising considering that this system is devoid of Sertoli cells, which normally exert a constant interaction with germ cells. Despite the possible limitations of this system, this is the first time that RA is reported to change the expression of these genes in gonocytes.

Taken together, the findings that 1) V1-PDGFRß has retained its tyrosine kinase activity and ability to autophosphorylate, presumably allowing for the formation of functional phosphotyrosine sites involved in protein docking, 2) it lacks the ligand binding domain and may behave as a constitutively active enzyme, 3) it is localized in the cytosolic compartment, 4) it is expressed preferentially in the developing gonads and in neonatal testicular gonocytes, 5) its expression is induced by RA in differentiating F9 EC and in isolated gonocytes, 6) changes in V1-PDGFRß expression levels coincide with changes in the expression of genes suggestive of a progression from a stem-cell phenotype to a differentiated phenotype both for F9 cells and PND3 gonocytes, 7) V1-PDGFRß overexpression inhibits the expression of RA-induced proteins and alter the differentiation of F9 cells, suggesting that V1-PDGFRß acts as a regulatory molecule, possibly by being a dominant negative form of PDGFR that may compete with the full-length receptors for binding downstream signaling molecules or by activating distinct signaling pathways involved in cell differentiation. In this manner, V1-PDGFRß could modulate or even terminate responses to PDGF in specific time-frames and cell types during developmental processes such as those occurring in the gonads, in which several cell types appear to express simultaneously PDGFRs. Examples of truncated forms of receptors exerting negative regulations have been reported, including a recent report describing the inhibitory effect of ERß5 on the transcriptional activity of ER in ovarian cells (55). Interestingly, ERß5, which includes the exons 1 to 7 of ERß1 in its sequence, has been shown to present intronic retention similarly to V1-PDGFRß and tr-kit. Alternatively, the role of V1-PDGFRß could be to trigger downstream cascades distinct from those activated by the full-length receptors and also required by the differentiation processes occurring in the cells. The identity of the downstream elements of the signaling cascades of V1-PDGFRß and the full-length receptors in gonocytes and F9 cells remains to be determined. Although PND3 rat gonocytes have been shown to express PI3-kinase, PKC, and PKC (10), Raf1, MEK1/2, and ERK1/2 (47), the F9 cells have been found to contain PI3-kinase, Akt (55), and PLC- (56). Additional candidate targets of PDGFRs in germ cells include the full-length 94-kDa Fes-related protein Fer, a member of the nonreceptor tyrosine kinase subfamily Fps/Fes that is phosphorylated upon PDGF stimulation (57, 58), and its truncated 51-kDa Fer (Fert), both expressed in rat round spermatids (40), a cell type that was also shown to express also V1-PDGFRß and full-length PDGFRß in the present work. Moreover, the possibility that V1-PDGFRß might regulate cells functions that are known to depend on PDGF is further supported by an article published during the preparation of the present manuscript reporting the presence of a 70-kDa truncated form of PDGFRß in rat vascular smooth muscle cells that was tyrosine-phosphorylated and interacted with PI3-kinase in response to angiotensin II stimulation (59). Although the exact sequence of the vascular truncated PDGFRß was not determined, the provided partial amino acid sequence together with the fact that the protein was recognized by the same antibody as that used in the present study suggest that it might be homologous to V1-PDGFRß. Experiments designed to characterize the signaling cascade of V1-PDGFRß and its relationships with RA and PDGF stimulation of F9 cells and gonocytes are under progress that should clarify the exact role that V1-PDGFRß plays in the differentiation, survival and/or proliferation processes in these cells.

	Acknowledgments

 
We thank Drs. Vassilios Papadopoulos and Jun Liu for their fruitful comments and critical reading of the manuscript.

	Footnotes

 
This work was supported by National Institutes of Health Grant ES10366.

Disclosure Statement: The authors have nothing to disclose.

First Published Online February 15, 2007

Abbreviations: EC, Embryonic carcinoma; EGFP, enhanced green fluorescent protein; EGFR, epidermal growth factor receptor; ER, endoplasmic reticulum; ES, embryonic stem; FBS, fetal bovine serum; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; GD, gestation day; GFP, green fluorescent protein; Itg6, integrin ₆; PDGFR, platelet-derived growth factor receptor; PI3-kinase, phosphatidylinositol 3-kinase, PND, postnatal day; Q-PCR, real-time quantitative PCR; RA, retinoic acid; RACE, rapid amplification of cDNA end; SSC, spermatogonial stem cell; tr-kit, truncated c-kit.

Received September 5, 2006.

Accepted for publication February 2, 2007.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. McGuinness MP, Orth JM 1992 Reinitiation of gonocyte mitosis and movement of gonocytes to the basement membrane in testes of newborn rats in vivo and in vitro. Anat Rec 233:527–537[CrossRef][Medline]
2. Orth JM, Boehm R 1990 Functional coupling of neonatal rat Sertoli cells and gonocytes in coculture. Endocrinology 127:2812–2820[Abstract/Free Full Text]
3. McGuinness MP, Orth JM 1992 Gonocytes of male rats resume migratory activity postnatally. Eur J Cell Biol 59:196–210[Medline]
4. Giwercman A, Andrews PW, Jorgensen N, Muller J, Graem N, Skakkebaek NE 1993 Immunohistochemical expression of embryonal marker TRA-1–60 in carcinoma in situ and germ cell tumors of the testis. Cancer 72:1308–1314[CrossRef][Medline]
5. De Miguel MP, De Boer-Brouwer M, Paniagua R, van den Hurk R, De Rooij DG, Van Dissel-Emiliani FM 1996 Leukemia inhibitory factor and ciliary neurotropic factor promote the survival of Sertoli cells and gonocytes in coculture system. Endocrinology 137:1885–1893[Abstract]
6. Van Dissel-Emiliani FM, De Boer-Brouwer M, De Rooij DG 1996 Effect of fibroblast growth factor-2 on Sertoli cells and gonocytes in coculture during the perinatal period. Endocrinology 137:647–654[Abstract]
7. Olaso R, Pairault C, Boulogne B, Durand P, Habert R 1998 Transforming growth factor ß1 and ß2 reduce the number of gonocytes by increasing apoptosis. Endocrinology 139:733–740[Abstract/Free Full Text]
8. Orth JM, Qiu J, Jester Jr WF, Pilder S 1997 Expression of the c-kit gene is critical for migration of neonatal rat gonocytes in vitro. Biol Reprod 57:676–683[Abstract]
9. Thuillier R, Wang Y, Culty M 2003 Prenatal exposure to estrogenic compounds alters the expression pattern of platelet-derived growth factor receptors and ß in neonatal rat testis: identification of gonocytes as targets to estrogen exposure. Biol Reprod 68:867–880[Abstract/Free Full Text]
10. Li H, Papadopoulos V, Vidic B, Dym M, Culty M 1997 Regulation of rat testis gonocyte proliferation by platelet-derived growth factor and estradiol: identification of signaling mechanisms involved. Endocrinology 138:1289–1298[Abstract/Free Full Text]
11. Claesson-Welsh L 1994 Platelet-derived growth factor receptor signals. J Biol Chem 269:32023–32026[Free Full Text]
12. Tallquist M, Kazlauskas A 2004 PDGF signaling in cells and mice. Cytokine Growth Factor Rev 15:205–213[CrossRef][Medline]
13. Gnessi L, Emidi A, Jannini EA, Carosa E, Maroder M, Arizzi M, Ulisse S, Spera G 1995 Testicular development involves the spatiotemporal control of PDGFs and PDGF receptors gene expression and action. J Cell Biol 131:1105–1121[Abstract/Free Full Text]
14. Gnessi L, Basciani S, Mariani S, Arizzi M, Spera G, Wang C, Bondjers C, Karlsson L, Betsholtz C 2000 Leydig cell loss and spermatogenic arrest in platelet-derived growth factor (PDGF)-A-deficient mice. J Cell Biol 149:1019–1026[Abstract/Free Full Text]
15. Basciani S, Mariani S, Arizzi M, Ulisse S, Rucci N, Jannini EA, Della Rocca C, Manicone A, Carani C, Spera G, Gnessi L 2003 Expression of platelet-derived growth factor-A (PDGF-A), PDGF-B, and PDGF receptor- and -ß during human testicular development and disease. Clin Endocrinol Metab 87:2310–2319
16. Heinrich MC, Corless CL, Duensing A, McGreevey L, Chen CJ, Joseph N, Singer S, Griffith DJ, Haley A, Town A, Demetri GD, Fletcher CD, Fletcher JA 2003 PDGFRA activating mutations in gastrointestinal stromal tumors. Science 299:708–710[Abstract/Free Full Text]
17. Looijenga LH, Oosterhuis JW 2002 Pathobiology of testicular germ cell tumors: views and news. Anal Quant Cytol Histol 24:263–279[Medline]
18. Oosterhuis JW, Gillis AJ, van Roozendaal CE, van Zoelen EJ, Looijenga LH 1998 The platelet-derived growth factor -receptor 1.5 kb transcript: target for molecular detection of testicular germ cell tumours of adolescents and adults. APMIS 106:207–213[Medline]
19. Vu TH, Martin GR, Lee P, Mark D, Wang A, Williams LT 1989 Developmentally regulated use of alternative promoters creates a novel platelet-derived growth factor receptor transcript in mouse teratocarcinoma and embryonic stem cells. Mol Cell Biol 9:4563–4567[Abstract/Free Full Text]
20. Gazouli M, Yao ZX, Boujrad N, Corton JC, Culty M, Papadopoulos V 2002 Effect of peroxisome proliferators on Leydig cell peripheral-type benzodiazepine receptor gene expression, hormone-stimulated cholesterol transport, and steroidogenesis: role of the peroxisome proliferator-activator receptor . Endocrinology 143:2571–2583[Abstract/Free Full Text]
21. Shoelson SE, White MF, Kahn CR 1988 Tryptic activation of the insulin receptor. Proteolytic truncation of the -subunit releases the ß-subunit from inhibitory control. J Biol Chem 263:4852–4860[Abstract/Free Full Text]
22. Strickland S, Smith KK, Marotti KR 1980 Hormonal induction of differentiation in teratocarcinoma stem cells: generation of parietal endoderm by retinoic acid and dibutyryl cAMP. Cell 21:347–355[CrossRef][Medline]
23. Clifford J, Chiba H, Sobieszczuk D, Metzger D, Chambon P 1996 RXR-null F9 embryonal carcinoma cells are resistant to the differentiation, anti-proliferative and apoptotic effects of retinoids. EMBO J 15:4142–4155[Medline]
24. Livera G, Rouiller-Fabre V, Pairault C, Levacher C, Habert R 2002 Regulation and perturbation of testicular functions by vitamin A. Reproduction 124:173–180[Abstract]
25. Ohta H, Yomogida K, Dohmae K, Nishimune Y 2000 Regulation of proliferation and differentiation in spermatogonial stem cells: the role of c-kit and its ligand SCF. Development 127:2125–2131[Abstract]
26. Orwig KE, Shinohara T, Avarbock MR, Brinster RL 2002 Functional analysis of stem cells in the adult rat testis. Biol Reprod 6:944–949
27. Oulad-Abdelghani M, Bouillet P, Decimo D, Gansmuller A, Heyberger S, Dolle P, Bronner S, Lutz Y, Chambon P 1996 Characterization of a premeiotic germ cell-specific cytoplasmic protein encoded by Stra8, a novel retinoic acid-responsive gene. J Cell Biol 135:469–477[Abstract/Free Full Text]
28. Meng X, Lindahl M, Hyvonen ME, Parvinen M, de Rooij DG, Hess MW, Raatikainen-Ahokas A, Sainio K, Rauvala H, Lakso M, Pichel JG, Westphal H, Saarma M, Sariola H 2000 Regulation of cell fate decision of undifferentiated spermatogonia by GDNF. Science 287:1489–1493[Abstract/Free Full Text]
29. Shinohara T, Avarbock MR, Brinster RL 1999 ß1- and 6-integrin are surface markers on mouse spermatogonial stem cells. Proc Natl Acad Sci USA 96:5504–5509[Abstract/Free Full Text]
30. Rochette-Egly C, Chambon P 2001 F9 embryocarcinoma cells: a cell autonomous model to study the functional selectivity of RARs and RXRs in retinoid signaling. Histol Histopathol 16:909–922[Medline]
31. Petch LA, Harris J, Raymond VW, Blasband A, Lee DC, Earp HS 1990 A truncated, secreted form of the epidermal growth factor receptor is encoded by an alternatively spliced transcript in normal rat tissue. Mol Cell Biol 10:2973–2982[Abstract/Free Full Text]
32. Jaye M, Schlessinger J, Dionne CA 1992 Fibroblast growth factor receptor tyrosine kinases: molecular analysis and signal transduction. Biochim Biophys Acta 1135:185–199[Medline]
33. Rossi P, Marziali G, Albanesi C, Charlesworth A, Geremia R, Sorrentino V 1992 A novel c-kit transcript, potentially encoding a truncated receptor, originates within a kit gene intron in mouse spermatids. Dev Biol 152:203–207[CrossRef][Medline]
34. Mosselman S, Claesson-Welsh L, Kamphuis JS, van Zoelen EJ 1994 Developmentally regulated expression of two novel platelet-derived growth factor -receptor transcripts in human teratocarcinoma cells. Cancer Res 54:220–225[Abstract/Free Full Text]
35. Mosselman S, Looijenga LH, Gillis AJ, van Rooijen MA, Kraft HJ, van Zoelen EJ, Oosterhuis JW 1996 Aberrant platelet-derived growth factor -receptor transcript as a diagnostic marker for early human germ cell tumors of the adult testis. Proc Natl Acad Sci USA 93:2884–2888[Abstract/Free Full Text]
36. Greer P 2002 Closing in on the biological functions of Fps/Fes and Fer. Nat Rev Mol Cell Biol 3:278–289[CrossRef][Medline]
37. Sette C, Dolci S, Geremia R, Rossi P 2000 The role of stem cell factor and of alternative c-kit gene products in the establishment, maintenance and function of germ cells. Int J Dev Biol 44:599–608[Medline]
38. David A, Mabjeesh N, Azar I, Biton S, Engel S, Bernstein J, Romano J, Avidor Y, Waks T, Eshhar Z, Langer SZ, Lifschitz-Mercer B, Matzkin H, Rotman G, Toporik A, Savitsky K, Mintz L 2002 Unusual alternative splicing within the human kallikrein genes KLK2 and KLK3 gives rise to novel prostate-specific proteins. J Biol Chem 277:18084–18090[Abstract/Free Full Text]
39. Gaskell TL, Esnal A, Robinson LL, Anderson RA, Saunders PT 2004 Immunohistochemical profiling of germ cells within the human fetal testis: identification of three subpopulations. Biol Reprod 2004 71:2012–2021[Abstract/Free Full Text]
40. Devouassoux-Shisheboran M, Mauduit C, Tabone E, Droz JP, Benahmed M 2003 Growth regulatory factors and signalling proteins in testicular germ cell tumours. APMIS 111:212–224[CrossRef][Medline]
41. Paronetto MP, Farini D, Sammarco I, Maturo G, Vespasiani G, Geremia R, Rossi P, Sette C 2004 Expression of a truncated form of the c-Kit tyrosine kinase receptor and activation of Src kinase in human prostatic cancer. Am J Pathol 164:1243–1251[Abstract/Free Full Text]
42. Lacham-Kaplan O 2004 In vivo and in vitro differentiation of male germ cells in the mouse. Reproduction 128:147–152[Abstract/Free Full Text]
43. Sandlow JI, Feng HL, Sandra A 1997 Localization and expression of the c-kit receptor protein in human and rodent testis and sperm. Urology 49:494–500[CrossRef][Medline]
44. Zaman GJ, Vink PM, van den Doelen AA, Veeneman GH, Theunissen HJ 1999 Tyrosine kinase activity of purified recombinant cytoplasmic domain of platelet-derived growth factor ß-receptor (ß-PDGFR) and discovery of a novel inhibitor of receptor tyrosine kinases. Biochem Pharmacol 57:57–64[CrossRef][Medline]
45. Kierszenbaum AL 2006 Tyrosine protein kinases and spermatogenesis: truncation matters. Mol Reprod Dev 73:399–403[CrossRef][Medline]
46. Hutt KJ, McLaughlin EA, Holland MK 2006 Kit ligand and c-kit have diverse roles during mammalian oogenesis and folliculogenesis. Mol Hum Reprod 12:61–69[Abstract/Free Full Text]
47. Thuillier R, Wang Y, Culty M Mechanisms underlying PDGF- and estradiol-induced gonocyte proliferation in neonatal rat testis. Program of the Annual Meeting of the American Society of Andrology, Baltimore, MD, 2004
48. Wang Y, Thuillier R, Culty M 2004 Prenatal estrogen exposure differentially affects estrogen receptor-associated proteins in rat testis gonocytes. Biol Reprod 71:1652–1664[Abstract/Free Full Text]
49. Ignar-Trowbridge DM, Nelson KG, Bidwell MC, Curtis SW, Washburn TF, McLachlan JA, Korach KS 1992 Coupling of dual signaling pathways: epidermal growth factor action involves the estrogen receptor. Proc Natl Acad Sci USA 89:4658–4662[Abstract/Free Full Text]
50. Gray K, Eitzman B, Raszmann K, Steed T, Geboff A, McLachlan J, Bidwell M 1995 Coordinate regulation by diethylstilbestrol of the platelet-derived growth factor-A (PDGF-A) and -B chains and the PDGF receptor - and ß-subunits in the mouse uterus and vagina: potential mediators of estrogen action. Endocrinology 136:2325–2340[Abstract]
51. Livera G, Rouiller-Fabre V, Durand P, Habert R 2000 Multiple effects of retinoids on the development of Sertoli, germ, and Leydig cells of fetal and neonatal rat testis in culture. Biol Reprod 62:1303–1311[Abstract/Free Full Text]
52. Darrow AL, Rickles RJ, Pecorino LT, Strickland S 1990 Transcription factor Sp1 is important for retinoic acid-induced expression of the tissue plasminogen activator gene during F9 teratocarcinoma cell differentiation. Mol Cell Biol 10:5883–5893[Abstract/Free Full Text]
53. Koubova J, Menke DB, Zhou Q, Capel B, Griswold MD, Page DC 2006 Retinoic acid regulates sex-specific timing of meiotic initiation in mice. Proc Natl Acad Sci USA 103:2474–2479[Abstract/Free Full Text]
54. Poola I, Abraham J, Baldwin K, Saunders A, Bhatnagar R 2005 Estrogen receptors ß4 and ß5 are full length functionally distinct ERß isoforms: cloning from human ovary and functional characterization. Endocrine 27:227–238[CrossRef][Medline]
55. Bastien J, Plassat JL, Payrastre B, Rochette-Egly C 2006 The phosphoinositide 3-kinase/Akt pathway is essential for the retinoic acid-induced differentiation of F9 cells. Oncogene 25:2040–2047[CrossRef][Medline]
56. Lee YH, Lee HY, Ryu SH, Suh PG, Kim KW 1993 Reduced expression of PLC- during the differentiation of mouse F9 teratocarcinoma cells. Cancer Lett 68:237–242[CrossRef][Medline]
57. Craig AW, Zirngibl R, Williams K, Cole LA, Greer PA 2001 Mice devoid of fer protein-tyrosine kinase activity are viable and fertile but display reduced cortactin phosphorylation. Mol Cell Biol 21:603–613[Abstract/Free Full Text]
58. Kim L, Wong TW 1995 The cytoplasmic tyrosine kinase FER is associated with the catenin-like substrate pp120 and is activated by growth factors. Mol Cell Biol 15:4553–4561[Abstract]
59. Gao BB, Hansen H, Chen HC, Feener EP 2006 Angiotensin II stimulates phosphorylation of an ectodomain-truncated platelet-derived growth factor receptor ß and its binding to class IA PI3-kinase in vascular smooth muscle cells. Biochem J 397:337–344[CrossRef][Medline]

This article has been cited by other articles:

				S. Mithraprabhu and K. L Loveland Control of KIT signalling in male germ cells: what can we learn from other systems? Reproduction, November 1, 2009; 138(5): 743 - 757. [Abstract] [Full Text] [PDF]

				S. Basciani, G. De Luca, S. Dolci, M. Brama, M. Arizzi, S. Mariani, G. Rosano, G. Spera, and L. Gnessi Platelet-Derived Growth Factor Receptor {beta}-Subtype Regulates Proliferation and Migration of Gonocytes Endocrinology, December 1, 2008; 149(12): 6226 - 6235. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wang, Y. Articles by Culty, M. Search for Related Content PubMed PubMed Citation Articles by Wang, Y. Articles by Culty, M.