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Carcinogenesis Advance Access originally published online on December 12, 2005
Carcinogenesis 2006 27(5):903-915; doi:10.1093/carcin/bgi305
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Aberrant expression of PDGF ligands and receptors in the tumor prone ovary of follitropin receptor knockout (FORKO) mouse

Xinlei Chen, Jayaprakash Aravindakshan, Yinzhi Yang, Rashmi Tiwari-Pandey and M.Ram Sairam *

Molecular Reproduction Research Laboratory, Clinical Research Institute of Montreal, 110 Pine Avenue West, Montreal, Quebec, Canada H2W 1R7

* To whom correspondence should be addressed. E-mail: sairamm{at}ircm.qc.ca


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Although PDGF family members play a vital role in cell proliferation, motility and chemotaxis via activation of structurally similar {alpha}- and ß-receptors, little is known of their function in ovarian regulation and induction of tumorigenesis. Microarray analyses of ovaries from young follitropin receptor knockout (FORKO) mice that are prone to late ovarian tumors upon aging have revealed significant imbalances in PDGF ligands and receptors. We hypothesized that FSH/FSH-R signaling may exert effects partly by regulation of PDGF the family. To further understand their implications for ovarian tumorigenesis, we studied FORKO ovaries and hormonal regulation of the PDGF family members in normal mice, by using RT–PCR, Q-PCR, immunohistochemistry and western blotting. While PDGF-C and PDGFR-{alpha} increased, PDGFR-ß mRNA and protein decreased significantly in absence of FSH-R signaling. In the normal ovary, PDGFR-{alpha} was not affected by gonadotropin (eCG) stimulation but PDGF-C and PDGFR-ß decreased. Administration of estradiol decreased PDGF and their receptors. To further probe the differential regulation of PDGF family members by eCG and estradiol, we co-administered eCG with estrogen antagonist, ICI 182780. Increase in PDGFR-{alpha} in the absence of estradiol suggests direct effects of FSH signaling. During the estrous cycle in mice PDGF-C, PDGF-D and PDGFR-{alpha} mRNA levels were higher at the proestrous. By IHC, we report for the first time the localization of PDGF-C, PDGFR-{alpha} and PDGFR-ß protein in mouse ovarian compartments including the surface epithelium that is also altered in mutants. Immunostaining of PDGFRs increased as the follicle developed to preantral stage and declined thereafter. Thus, FSH modulates PDGF family members, partly via E2, suggesting that loss of FSH-R signaling causes an imbalance of PDGF family members predisposing the abnormal ovarian follicular environment for inducing tumorigenesis in aging FORKO mice.

Abbreviations: E2, estradiol 17 ß; eCG, equine chorionic gonadotropin (pregnant mare serum gonadotropin-PMSG); FORKO, follitropin receptor knockout; FSH, follitropin or follicle-stimulating hormone; OSE, ovarian surface epithelium


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
To date only some growth factor systems such as insulin-like growth factors I and II (IGF-I and IGF-II) (1), the members of TGF beta superfamily (2) and EGF(3) are well characterized in the ovary, a tissue in which aggressive tumors occur during post-menopausal life in women (4). Other regulators like PDGF which have marked effects on cellular migration, proliferation and differentiation, survival and metabolism of the extracellular matrix (5–8), may also play an important role in the ovary, as PDGF-A and PDGF-B, and PDGFRs are coexpressed in the ovary (9). PDGFs are also known to stimulate thecal cell mitosis in vitro (10). In addition, PDGF-B and the corresponding receptors were found to be expressed in autonomous human ovarian cancer (11), and PDGFR tyrosine kinase inhibitors are noted as potential drugs for treating ovarian cancer (12,13).

The PDGF family of growth factors is composed of four different polypeptide chains encoded by four different genes, which combine to create five dimeric isoforms (PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC and PDGF-DD) linked by disulfide bonds. The classical PDGF chains, PDGF-A and PDGF-B, were discovered more than two decades ago, and recently PDGF-C and PDGF-D chains were added to the list (5,14–16).

The PDGF isoforms exert their biological actions via binding to their cell surface structurally related tyrosine kinase receptors (PDGFRs). Two types of distinct subunits, {alpha} and ß, dimerize to form three types of high affinity PDGF receptors ({alpha}{alpha}–receptor, {alpha}ß-receptor and ßß-receptor) (17). The five dimeric isoforms of PDGF display distinct abilities to bind and activate the two PDGFRs. The four dimeric isoforms, PDGF-AA, PDGF-AB, PDGF-BB and PDGF-CC can bind to and activate PDGFR-{alpha}, while PDGF-BB and PDGF-DD can specifically bind to and activate PDGFR-ß. PDGF-AB, PDGF-BB and PDGF-CC can also stimulate heterodimeric PDGFR{alpha}/ß complexes. Different biological responses may be mediated by different receptor dimmers (8). Upon ligand binding, PDGF receptors dimerize and autophosphorylate several tyrosine residues, leading to the recruitment and activation of various downstream signaling kinases, such as phosphatidylinositol-3-kinase (PI3-kinase) (18,19) and Src tyrosine kinase (20), as well as recruitment of docking proteins, such as Shc and Grb2, leading to activation of the Ras-Raf-MEK-extracellular signal-regulated kinase (ERK) pathway (21,22). Activation of these pathways increases cell growth and motility. Thus, promotion of cell survival and induction of chemoresistance in mouse epithelial cell lines with constitutive expression of Src tyrosine kinase (23) suggests an important potential link between PDGFs and ovarian tumor progression.

The ovary is composed of several specialized functional units and is amongst the most dynamic and plastic tissues in the body, involved in complex combinations of cell proliferation, migration and differentiation. These processes rest on intercellular communication, rather than relying entirely on intracellular molecular interactions and hormonal control. Follicle-stimulating hormone (FSH) and receptor signaling are required for optimal gametogenesis, growth and differentiation of somatic cells, and biosyntheses of steroid hormones (24–28). The endocrine actions of FSH are mediated by its receptor(s); follicle-stimulating hormone receptor (FSH-R), a transmembrane receptor of G-protein coupled signaling system as well as other signaling pathways. Although virtually nothing is known about gonadotropin regulation of the PDGF family, estrogen secreted by ovary, reportedly inhibited PDGFR-{alpha} mRNA and protein, while down-regulating PDGF-B mRNA (29). Considering the paucity of available information in the ovary, we became interested in exploring (i) which PDGF family members are expressed in the mouse ovary, including the novel members, PDGF-C and PDGF-D; (ii) how hormones such as FSH and estradiol (E2) regulate the expression of PDGF family members; and (iii) dynamics of changes in PDGF family members during the estrous cycle in the ovary. These questions assumed prominence because of the aberrations we found for the PDGF ligands and receptors during microarray analysis of ovaries from young follitropin receptor knockout mutants (30) long before the appearance of ovarian tumors in most animals (31). Our recent evaluations have also revealed early changes in the surface epithelium components that intensify with aging in FSH-R mutant mice (32). As majority of ovarian tumors in women are ascribed to the surface epithelium (4), understanding prominent growth factor–receptor relationships in normal and altered states would be desirable.

In the present studies, we have systematically examined the expression of all known PDGF ligands and receptors in young follitropin receptor knockout (FORKO) mice long before tumors appear in their ovaries upon aging and in normal immature mice that do not develop tumors. The results reveal that PDGF ligands and their receptors {alpha} and ß are all expressed in the mouse ovary, PDGF-C and PDGFR-{alpha} being predominant. Both FSH and estrogen could modulate the expression of PDGF ligands and their receptors. The use of estrogen antagonist in treatment combinations has allowed us to distinguish the effects of equine chorionic gonadotropin (eCG) and estradiol. Examination of normal females has provided data on variations during the estrous cycle. Having found early changes in FSH-R null mutant ovaries, we propose that the resulting aberrations in PDGFs and receptors could contribute to late onset of ovarian tumors in these mice (31,32).


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Animals
All experiments were performed according to accepted standards and with the approval of an institutional ethics committee. For all experiments, wild-type and virgin FORKO mice (24,31) in the 129T2 svEmsJ background were used. Littermate mutants and wild-type mice derived from heterozygous mothers were compared in each experiment. Genotyping by polymerase chain reaction (PCR) was performed on DNA isolated from tails or toes (33). The mice were housed (5 mice per cage) under controlled lighting (12D:12L) and were provided with standard commercial food pellets and tap water ad libitum. After CO2 asphyxiation, the ovaries were removed, cleared of adherent connective tissue and either placed in appropriate fixatives for histology and immunohistochemistry or immediately frozen in liquid nitrogen or RNA later (Ambion) for RNA extraction and purification. Because the ovary from young FORKO mice is very small, dissected ovaries (4–6 ovaries) from two or three mice were pooled for western blot and RNA analysis.

Experiment 1
To compare the gene profiles between FORKO and age-matched WT mice, 26 day-old FORKO and WT mice were killed. In subsequent experiments several treatment options were employed.

Experiment 2
This group of experiments was designed to test the hypothesis that FSH might be the regulatory factor for the expression of PDGF family members. Immature (21–24 days old) female mice were treated with a single injection of eCG, a well established surrogate for pituitary FSH used in experimental studies. In addition, to check if FSH might work indirectly through estrogen, 21–24-day-old WT and FORKO mice were treated eCG 5 U i.p and ±50 µg ICI182,780 (E2 antagonist; Tocris Bioscience, Ellisville, MO) s.c. per mouse. Groups of immature mice were also treated with estradiol 100 ng per mouse s.c., and ovaries were collected at 12 and 24 h later.

Experiment 3
This experiment was designed to follow up the results from the study of eCG and E2 regulated expression of PDGF family members. To understand cyclical variations, ovaries in different stages (proestrous, estrous, metestrous and diestrous) of the cycle were collected from 4-month-old WT mice. Female mice were acclimated for 1 week before the start of daily vaginal smears examined to determine the stage of the cycle. The mice underwent at least two complete estrous cycles before they were used.

Microarray analysis
Details of microarray analysis of dissected ovaries will be reported in a separate communication (Aravindakshan et al., in preparation). In brief, double-spotted array containing 15 264 mouse ESTs from the National Institute of Aging (NIA) were obtained from the Ontario Cancer Institute, Ontario, Canada. RNA was extracted from the tissue by using TRIZOL reagent. Twenty micrograms of RNA from 26 day-old FORKO and wild-type ovaries was reverse transcribed, and their corresponding cDNAs were independently labeled by incorporation of either Cyanine (Cy3)-dCTP or (Cy5)-dCTP according to manufacturer's specifications. The two labeled samples to be compared after purification were combined, and hybridized to the same microarray. Hybridization was done overnight at 65°C for 14–16 h. After washing in 2x SSC and 0.1% SDS for 5 min, with 1x SSC for 5 min, and with 0.1x SSC for 5 min at 55°C, slides were scanned using a GenePix scanner (Axon Instruments, Union City, CA), and intensities were analyzed with GeneSpring software (Silicon Genetics, Redwood City, CA). Statistical test was performed by using a parametric test with variances not assumed equal (Welch's t-test) and a P-value of <0.05.

Affymetrix gene chip mRNA expression analyses
Affymetrix 39 K GeneChip® Mouse Genome 430 2.0 Array has over 45 000 probe sets which analyses over 39 000 transcripts and variants from over 34 000 mouse genes. A total of 1 µg of double-stranded cDNA was transcribed in vitro using RNA Transcript Labeling Kit (Enzo, Farmingdale, NY) using biotinylated CTP and UTP (Enzo, Farmingdale, NY). Following 5 h incubation at 37°C, the resultant biotin-labeled cRNA was purified with RNAeasy column (Qiagen) and eluted in 40 µl of RNAse-free water. Target cRNAs corresponding to either wild-type ovary or FORKO ovarian tissue were hybridized to an individual GeneChip from an identical lot of Affymetrix 39 K GeneChip® Mouse Genome 430 2.0 array for 16 h. GeneChip arrays were washed and stained using antibody-mediated signal amplification and the Affymetrix Fluidics Station's standard Eukaryotic GE Wash 2 protocol, using Affymetrix equipment and protocols (Affymetrix, Santa Clara, CA). Data analysis was performed using GeneSpring software (Silicon Genetics, Redwood City, CA).

RT–PCR, real-time PCR
Expression of PDGF family members was determined in relation to ß-actin. The sequences of the specific PCR primers for RT–PCR and Q-PCR are shown in Table I. The primers of PDGF-A, PDGF-B, PDGFR{alpha} and PDGFRß are based on a recent report (34). RT–PCR parameters are ß-actin, 20 cycles; PDGF-A, PDGF-B, PDGF-D and PDGFR-ß, 27–29 cycles; PDGF-C and PDGFR-{alpha}, 24 cycles, and during PCR process, 94°C (denaturing temperature) for 45 s, 55°C for 40 s (annealing temperature) and 72°C for 45 s (extension temperature).


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  		Table I. The primers of PDGF family members for RT–PCR and Q-PCR

 
The real-time PCR (Q-PCR) assay was conducted following the manufacturer's procedures (Stratagene) using the fluorescent dye SYBR Green. All reactions were done in duplicate. Q-PCR parameters were 95°C for 15 min, and 40 cycles at 95°C for 30 s and at 55°C for 30 s, and 72°C for 30 s. No amplification of fragments occurred in control samples without reverse transcriptase. Quantity of mRNA was calculated using the {Delta}{Delta}Ct method. For each Q-PCR, the threshold cycle (Ct) was determined, being defined as the cycle at which the fluorescence exceeds 10 times the standard deviation of the mean baseline emission for cycles 3–10. Expression levels were normalized to the housekeeping gene ß-actin according to the following formula: Formula. Subsequently, the respective cytokine mRNA levels were calculated using the {Delta}{Delta}Ct method, that is, {Delta}{Delta}Ct = {Delta}Ct (treated) – {Delta}Ct (control). The relative mRNA level was calculated as 2–{Delta}{Delta}Ct based on the results of control experiments, where an efficiency of the PCR reactions of ~100% was determined.

Immunohistochemistry
Immunohistochemical staining was performed on 5-µm paraffin-embedded sections using the ImmunoCruz Staining System (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Briefly, sections were deparaffinized in xylene and rehydrated in a decreasing gradient of ethanol in water and then rehydrated in PBS buffer (pH 7.4) for 20 min. Antigen retrieval steps for PDGF-C included incubation in a Triton X-100 solution (0.5% in PBS) for 10 min at 37°C and washing twice for 5 min in PBS. For PDGFR-{alpha} and PDGFR-ß antigen retrieval, sections were subjected to a micro waving in 0.01 M citrate buffer, pH 6.0, for 20 min. Hydrogen peroxide (0.3%) was applied to quench the endogenous peroxidase activity. The slides were then incubated in protein blocking agent to reduce non-specific binding. Primary antibodies toward PDGF-C were diluted 1:15 in PBS containing 1% BSA and incubated on the slides overnight at 4°C in a humid chamber. Different dilutions of the primary antibodies were assessed, with positive staining observed at 1:20–1:5 for antibodies. The sections were incubated with primary antibodies (for PDGFR-{alpha} and PDGFR-ß diluted in blocking serum solution in 1:300 and 1:200, respectively) for 1 h. In negative controls, normal serum was substituted for primary antibody. The tissue sections were washed and incubated in biotinylated secondary antibody for 30 min at room temperature, followed by washing with PBS and incubation in avidin-biotin-horseradish peroxidase for 30 min. Reactions were visualized with 3,3'-diaminobenzidine tetrahydrochloride dehydrate and weakly counterstained with hematoxylin.

Western blotting
For immunoblotting, ovarian protein was extracted by lysis buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS in 1x PBS, pH 7.4) and concentration was determined in high speed supernatant using the Bio-Rad reagent. Samples were heated to 95°C for 5 min and then subjected to SDS-PAGE. Forty µg of protein were electrophoresed under reducing conditions on a 7.5% sodium dodecyl sulfate gel. Following electrotransfer to polyvinylidene difluoride membranes, they were blocked with TBST-5% milk [10 mM Tris–HCl (pH 8.0), 150 mM NaCl, 0.05–0.1% Tween-20, and 5% non-fat dry milk] for 1 h at room temperature before incubation with an antibody specific for the protein in question (e.g. PDGFR-{alpha}, 1:2000; PDGFR-ß, 1:800; PDGF-C, 1:100 dilution) in TBST-5% milk for 1 h or at 4°C overnight. Membranes were washed extensively with TBST, incubated with the appropriate peroxidase-conjugated secondary antibody in TBST-5% milk for 1 h, and then washed with TBST. Proteins were visualized by enhanced chemiluminescence, ECL plus.

The following antibodies were used in immunohistochemistry and western blotting: affinity-purified polyclonal goat anti-PDGF-CC (Santa Cruz Biologicals, CA), rabbit polyclonal antisera to the PDGF receptor {alpha}- and ß-subunit (kindly provided by Dr Carl-Henrik Heldin, Ludwig Institute for Cancer Research, Uppsala, Sweden).

Statistical analysis
Each experiment was performed three times. The significance of the results was determined by using the one-way ANOVA, following by Student's-test. Statistical differences were considered significant at P < 0.05. Data are presented as the mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Evidence of early changes in ovaries by microarray analysis
To survey early changes in the expression of ovarian mRNAs in the complete absence of FSH receptor(s), samples of ovarian mRNA from 26-day-old wild-type and FORKO mice were studied by cDNA microarray. To confirm the results of our cDNA microarray studies and to identify additional changes in ovarian genes, we also prepared complex biotin-labeled riboprobes from total ovarian RNA pooled from 26 day-old, wild-type and FORKO mice and hybridized them to the Affymetrix 39 K murine Gene chips of the Mouse Genome 430 2.0 array. Setting a conservative 2-fold difference for analyzing our data, all 64-up regulated genes and 53 down-regulated genes revealed by cDNA microarray were also represented among the 219 up-regulated and 72 down-regulated genes found by Affymetrix analysis. Full details of these changes will be reported elsewhere (Aravindakshan et al., in preparation). These analyses provided the first clues for differential expression of PDGF members in the mutants. PDGF-C and PDGF receptor {alpha} increased significantly in FORKO mice (~3-fold), while expression of PDGF receptor ß decreased to one-third.

Confirmation of differences by RT–PCR and Q-PCR
To extend findings from the microarray and study systematically all PDGF family members, we evaluated the expression of PDGF ligands and their receptors in 21–24-day-old immature FORKO and WT mouse ovaries by RT–PCR and Q-PCR by normalization of the amount of PCR products against that for ß-actin. Selecting immature mice permitted the study of external hormonal influences prior to the onset of the action of endogenous hormones. In order to rely on ß-actin for comparison, we evaluated the Q-PCR results of ß-actin among different groups treated with/without eCG and/or E2, and ascertained that its expression was not significantly altered by either treatment of WT mice (data not shown).

These evaluations shown in Figure 1A indicated that in the young FORKO and WT mouse ovaries PDGF-C is the predominant ligand among the four members and PDGFR-{alpha} is expressed several-fold more than PDGFR-ß. Relative to PDGF-D, the expression of ligand C in the wild-type ovary is 4-fold higher. This disparity increases to 15x in the FORKO ovary. It is also of interest to note that in the FORKO ovary PDGF-C was preferentially up-regulated over other members. Of the two receptors, PDGFR-{alpha} was about 5x that of PDGFR-ß. In the FORKO ovary, PDGFR-{alpha} expression was 2.5x that of WT. The reduction of PDGFR-ß expression and concomitant increase in PDGFR-{alpha} exaggerated the imbalance greatly in favor of the latter.


Figure 1
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  		Fig. 1. Expression of PDGFs and PDGFRs in WT and FORKO ovaries. (A) Q-PCR of PDGF-A, PDGF-B, PDGF-C, PDGF-D, PDGFR-{alpha} and PDGFR-ß mRNA expression in WT and FORKO mouse ovaries. Histogram shows relative amount of the different genes expressed as a ratio between the gene being analyzed and the constitutive gene ß-actin by Q-PCR analysis. Each value represents the mean ± SE of at least three different samples, each run in triplicate. *P < 0.05; **P < 0.01. (B). Western blot analysis of PDGF-C, PDGFR-{alpha} and PDGFR-ß subunits, demonstrating the increase of PDGF-C and PDGFR-{alpha} and decreased the expression of PDGFR-ß in the FORKO ovary compared with that in the WT ovary.

 
These mRNA expression data correlated generally well with western blotting (Figure 1B). Western blot analysis on total extracts of WT and FORKO ovaries showed bands of expected molecular mass for PDGF-C, PDGFR-{alpha} PDGFR-ß and confirming the PDGF system protein expression in the tissue (Figure 1B). A less intense PDGFR-{alpha} immunoreactive band of WT ovary, compared with the band of the FORKO ovary, was observed. PDGFR-ß protein decreased significantly as shown in Figure 1B. Of the two different PDGF-C antibodies (polyclonal anti-goat PDGF-C antibody, Santa Cruz; monoclonal anti-mouse PDGF-C antibody, R & D) that we tried, only the former proved satisfactory in providing indications of a probable increase in the FORKO ovary (Figure 1B).

Hormonal regulation by eCG
To further investigate hormonal modulation of the expression of PDGF family members, we treated 21–24 day-old FORKO and WT females by eCG. RT–PCR and Q-PCR analysis were used to study the time-dependent effects of a single treatment with eCG (5 U/per mouse) on the steady-state mRNA levels for all the PDGFs and their receptor {alpha}- and ß-subunits. Due to lack of FSH-Rs, the FORKO ovary is not responsive to eCG. Distinctive mRNA expression profiles of the PDGF ligands and their receptor genes were evident for the WT ovary in response to eCG. Figure 2A and B show that eCG tends to increase the level of PDGF-A within 12 h after treatment. The induction of PDGF-A chain mRNA was transient and fell to a level lower than that of control, within 24 h post-treatment followed by a rise above control level by 48 h. eCG treatment decreased the levels of PDGF-B, PDGF-C and PDGFR-ß within 24 h after treatment (P < 0.05), and the reduction of PDGF-B returned to control level while the reduction of PDGF-C remained consistently low 48 h post-treatment. In contrast to the expression of above three members, PDGF-D expression that remained unaltered at 24 h and increased 2-fold after 48 h of hormone treatment. While PDGFR-{alpha} in the wild-type ovary was not affected after administration of eCG, PDGFR-ß was down-regulated.


Figure 2
Figure 2
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  		Fig. 2. Time-dependent regulation of PDGFs and PDGFRs in the mouse ovary upon a single treatment of immature SV-129 mice with eCG. –/– C represents FORKO control mouse ovary; –/– 24 h represents FORKO mouse ovary treated by eCG for 24 h; +/+ C represents WT control mouse ovary; +/+ P12 h represents WT mouse ovary treated by eCG for 12 h; +/+ P24 h WT mouse ovary treated by eCG for 24 h; +/+ P48 h WT mouse ovary treated by eCG for 48 h. (A) RT–PCR of PDGFs and PDGFRs mRNA expression in WT and FORKO mouse ovaries after eCG treatment. Because there is no change in FORKO ovary after treatment, –/– C represents pool of all results. (B) Q-PCR of PDGF-A, PDGF-B, PDGF-C, PDGF-D, PDGFR-{alpha} and PDGFR-ß mRNA expression in WT and FORKO mouse ovaries after being treated by eCG. Histogram shows relative amount of the different genes expressed as a ratio relative to ß-actin. Each value represents the mean ± SE of at least three different samples, each run in triplicate. *P < 0.05; **P <0.01. (C) Western blot analysis of PDGF-C, PDGFR-{alpha} and PDGFR-ß subunits, demonstrating decreased expression of PDGF-C and PDGFR-ß, while PDGFR-{alpha} did not change in the treated WT ovary compared with age matched WT control.

 
Consistent with our RT–PCR and Q-PCR findings on eCG regulation of PDGF ligands and their receptor mRNAs, western blot demonstrated that within 24 h after eCG treatment of WT mice, a significant reduction of PDGF-C and PDGFR-ß occurred in the treated ovaries compared with control WT ovaries. On the contrary, PDGFR-{alpha} protein did not change much (Figure 2C). The expression of PDGFs and PDGFRs mRNA and protein of PDGF-C, PDGFR-{alpha} and PDGFR-ß were relatively constant without significant variations after eCG treatment in FORKO ovaries (Figure 2A, B and C).

Effect of estrogen on the expression of the PDGFs and receptors
To determine whether estrogen, a steroid hormone that is normally produced in response to eCG also modifies the expression of PDGFs and their receptors, we treated young WT and FORKO females with this hormone. Uterine weight, a measure of estrogen action in vivo (Table 2) indicates that treatment was effective in FORKO mice. By 24 h, equivalent stimulation (150% increase) was achieved in mice of both genotypes. The RT–PCR and Q-PCR results shown in Figure 3A and B reveal that E2 decreased the expression of all PDGFs and their receptors of WT ovaries 24 h after treatment. The effect on PDGF-D was most prominent as shown by ~50% decrease compared with matched control. In FORKO mice, ovarian PDGF-C, PDGF-D and PDGFR-{alpha} expression were decreased within 24 h of treatment by estradiol, at a time when they were not influenced by the tropic action of eCG.


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  		Table II. Uterine weight of different groups of mice

 

Figure 3
Figure 3
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  		Fig. 3. Time-dependent regulation of PDGFs and PDGFRs in the mouse ovary following single treatment of immature SV-129 mice with E2 (100ng). –/– C represents FORKO control mouse ovary; –/– 12 h represents FORKO mouse ovary treated by E2 for 12 h; –/– 24 h or –/–E24 FORKO mouse ovary treated by E2 for 24 h; +/+ C represents WT control mouse ovary; +/+E12h WT mouse ovary treated by E2 for 12 h; +/+24 h or +/+E24 WT mouse ovary treated by E2 for 24 h. (A) RT–PCR of PDGFs and PDGFRs mRNA expression in WT and FORKO mouse ovaries after E2 treatment. (B) Q-PCR of PDGF-A, PDGF-B, PDGF-C, PDGF-D, PDGFR-{alpha} and PDGFR-ß mRNA expression in the WT and FORKO mouse ovaries after E2 treatment. Histogram shows relative amount of the different genes expressed as a ratio between the gene being analyzed and the constitutive gene ß-actin by Q-PCR analysis. Each value represents the mean ± SE of three different samples, each run in triplicate. *P < 0.05; **P < 0.01. (C) Western blot analysis of PDGF-C, PDGFR-{alpha} and PDGFR-ß subunits, demonstrating the expression of PDGF-C, PDGFR-{alpha} and PDGFR-ß decreased in the WT and FORKO ovary compared with that in matched control.

 
Consistent with above findings from the RT–PCR and Q-PCR studies, western blot (Figure 3C) demonstrated that within 24 h after E2 treatment of both WT and FORKO mice, a pronounced reduction of PDGF-C, PDGFR-{alpha} and PDGFR-ß occurred in the treated ovaries compared with respective control ovaries.

FSH modulation of mRNA expression of the PDGFs and their receptors is partly mediated by estrogen
As there were some differences in the modulation of PDGFs by eCG and E2 in the WT ovary, we wanted to test the hypothesis that eCG (FSH) modulates the expression directly and not only through estrogen. To evaluate this, the WT mice were treated with eCG along with the estrogen antagonist, ICI 182780 using a dose that completely blocked the effect of estrogen on the uterus. The effects of eCG with estrogen antagonist on the steady-state mRNA levels for the PDGF ligands and their receptors are included in Figure 4A. The time point chosen to analyze the effects of ICI 182780 was 24 h after treatment, as shown by the previous time-course study (Figures 2 and 3). PDGF-A and PDGFR-{alpha} expression were increased (P < 0.05), while both PDGF-B and PDGFR-ß were reduced within 24 h after these treatments (P < 0.05 and P < 0.01, respectively). Western blot results (Figure 4B) confirmed Q-PCR data. More intense immunoreactive bands for PDGFR-{alpha} were observed and PDGFR-ß protein was decreased within 24 h treatment with eCG and estrogen antagonist. Although the result of probing for PDGF-C is not ideal, a decreasing trend after antagonist treatment was evident. From these results, we conclude that expression of PDGFs and PDGFRs was regulated by both FSH/E2, and furthermore, FSH modulated expression of the PDGFs and their receptors were also partly mediated by estrogen in the ovary.


Figure 4
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  		Fig. 4. Regulation of PDGFs and PDGFRs in the mouse ovary upon a single treatment of immature SV-129 mice with eCG and co-administration of ICI 182780, E2 antagonist. –/– C represents FORKO control ovary; +/+ C represents WT control ovary; +/+ ICI or P+ICI represents WT mouse treated by eCG co-administration with ICI 182780 (50 µg per mouse) for 24 h. (A) Q-PCR of PDGF-A, PDGF-B, PDGF-C, PDGF-D, PDGFR-{alpha} and PDGFR-ß mRNA expression in WT mouse ovaries after eCG and co-administration with ICI 182780, E2 antagonist. Histogram shows relative amount of the different genes expressed as a ratio between the gene being analyzed and the constitutive gene ß-actin by Q-PCR analysis (+/+ C being set as 1). Each value represents the mean ± SE of at least three different samples, each run in triplicate. *P < 0.05; **P < 0.01. (B) Western blot analysis of PDGF-C, PDGFR-{alpha} and PDGFR-ß subunits, demonstrating the expression of PDGFR-{alpha} increased and PDGFR-ß decreased while expression of PDGF-C did not change in the WT ovary compared with matched control.

 
Changes in expression of ovarian PDGFs and their receptors during the estrous cycle
Examining changes during the reproductive cycle, we found that PDGF-C, PDGF-D, PDGFR-{alpha} and PDGFR-ß subunit mRNA levels are at the highest at the proestrous stage, although there are no statistically significant differences due to wide variations. The trend of these fluctuations could indicate hormonal influences on ovarian compartments. The expression of PDGF-C and PDGFR-{alpha} in the FORKO ovary remained consistently higher than that in WT (P < 0.05) (Figure 5A) in most stages except perhaps at proestrous. Western blotting results were somewhat different from data obtained by Q-PCR. During estrous cycle, the expression of PDGF-C, PDGFR-{alpha} and PDGFR-ß was the lowest level at the proestrous stage, although there was no significant change among the four stages except PDGFR-ß (Figure 5B).


Figure 5
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  		Fig. 5. Expression of PDGFs and PDGFRs in the 4-month-old SV-129 mouse ovary during estrous cycle. –/– C represents age-matched FORKO mice; P, E, M and D represent the proestrous, estrous, metestrous and diestrous stages, respectively. (A) Q-PCR of PDGF-C, PDGF-D, PDGFR-{alpha} and PDGFR-ß mRNA expression in mouse ovaries during estrous cycle and age-matched FORKO group. Histogram shows relative amount of the different genes expressed as a ratio in relation to ß-actin. The results show that there was no dramatic change of the expression of PDGF-C, PDGF-D, PDGFR-{alpha} and PDGFR-ß mRNA during the estrous cycle except perhaps at the proestrous. (B) Western blot analysis of PDGF-C, PDGFR-{alpha} and PDGFR-ß subunits, demonstrating differences in protein and mRNA from Q-PCR. During estrous cycle, the expression of PDGF-C, PDGFR-{alpha} and PDGFR-ß was the lowest level at the proestrous stage, although there was no significant change among the four stages except PDGFR-ß. Each value represents the mean ± SE of at least three different samples, each run in triplicate. *P < 0.05; **P < 0.01.

 
Immunohistochemistry
The RT–PCR, Q-PCR as well as western blotting analyses data presented above clearly demonstrated that FSH and E2 regulated the steady-state mRNA and protein levels for the PDGF ligands and their receptors in a temporal fashion in the ovary of the prepubescent mouse. To better understand and appreciate the physiological role of the PDGFs in hormonal actions, we sought to identify the cell types within the ovary that expressed the PDGF and receptor genes. Cell-specific expression of protein for the PDGF ligands and their receptors was determined by immunohistochemistry. Consistent with our findings above, immunohistochemistry demonstrated positive immunoreactivity of PDGFR-{alpha} in granulosa cells, oocyte and ovarian surface epithelial cells in immature WT ovary, with stronger staining in immature FORKO ovaries. PDGFR-ß was present in the granulosa cell and oocyte of follicles and ovarian surface epithelial (OSE) cells, and less intense staining in the immature FORKO ovary. Immunostaining for PDGF-C (Figure 6A) revealed prominent expression in OSE cells and theca cells as well as the oocyte. In the 3–4 month-old cycling wild-type mouse ovaries, positive staining immunostaining for PDGF-C (Figure 6B), revealed expression in corpus luteum, theca cells as well as the oocyte, including ovarian surface epithelial cells. In the acyclic FORKO female, there was weaker expression in granulosa cells but stronger staining in the theca/stroma as well as OSE. Strong positive immunoreactivity to PDGFR-{alpha} was detected in the corpus luteum, and granulosa cells, oocyte and OSE; PDGFR-ß was present in the granulosa cell and oocyte of follicles and OSE cells as well as corpus luteum cells, with less intense staining in the FORKO ovary.


Figure 6
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  		Fig. 6. Immunohistochemical localization of PDGF-C, PDGFR-{alpha} and PDGFR-ß in mouse ovaries. (a) The immunohistochemical result of 21–24-day-old mouse ovaries were shown. (A), (C) and (E) represent WT ovaries; (B), (D) and (F) represent FORKO ovaries. (A) and (B) represent immunostaining of PDGF-C of WT and FORKO ovaries, respectively; (C) and (D) represent immunostaining of PDGFR-{alpha} of WT and FORKO ovaries, respectively; (E) and (F) represent immunostaining of PDGFR-ß of WT and FORKO ovaries. Immunostaining for PDGF-C revealed that prominent expression was found in OSE cells and theca cells as well as the oocyte in WT ovary, with stronger intense staining in FORKO ovaries. Immunohistochemistry demonstrated that a positive immunoreactivity to PDGFR-{alpha} was detected in granulosa cells, oocyte and OSE cells in the WT ovary, with stronger intense staining in FORKO ovaries, whereas PDGFR-ß was present in the granulosa cell and oocyte of follicles and OSE, and less intense staining in FORKO ovary. Arrow head shows that some part of OSE has been lost. (b) The immunostaining of PDGF-C, PDGFR-{alpha} and PDGFR-ß in 3–4 month-old adult mouse ovaries are shown. (A), (C) and (E) represent WT ovaries; (B), (D) and (F) represent FORKO ovaries. (A) and (B) represent immunostaining of PDGF-C of WT and FORKO ovaries, respectively; (C) and (D) represent immunostaining of PDGFR-{alpha} of WT and FORKO ovaries, respectively; (E) and (F) represent immunostaining of PDGFR-ß of WT and FORKO ovaries, respectively. Immunostaining for PDGF-C revealed that positive staining was found in the corpus luteum, theca cells as well as the oocyte in the WT ovary but not in OSE cells, but stronger intense staining in FORKO ovaries including OSE and weaker staining in the granulosa cells. Strong positive immunoreactivity to PDGFR-{alpha} was detected in the granulosa cells, oocyte and OSE cells, and less staining in corpus luteum cells in the WT ovary, but stronger intense staining in FORKO ovaries. (E and F) PDGFR-ß was present in the granulosa cell and oocyte of follicles and OSE cells as well as corpus luteum cells, and less intense staining in the FORKO ovary. The insets in each section are their lower magnifications, for comparison of the ovaries. OSE, ovarian surface epithelial cell; GC, granulosa cell; TC, thecal cell; OC, oocyte; and CL, corpus luteum.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Most ovarian cancers in women detected in menopausal years after cessation of reproductive functions are of the surface epithelium (4). Although it has been hypothesized that the trauma of incessant ovulation (35) occurring at monthly intervals in reproductive years could predispose the OSE to structural and functional alterations, we discovered a variety of tumorigenic changes (31) including those of the OSE (32) in ovaries of FORKO mice that never ovulate (26,33). This presented us an opportunity to explore and establish early molecular changes in the ovary that might be linked to cell proliferation and tumor induction. Based on data presented herein, we postulate that the early imbalance of growth factors such as PDGF family members could play a part in initiating and propagating this disease at later ages (31). The expression of PDGF family members (e.g. PDGF-A, PDGF-B and PDGFR-{alpha} and PDGFR-ß) has been associated with the development of tissues and male and female reproductive systems (9,10,36–43). To our knowledge, studies examining PDGF expression in the mouse ovary especially that of the novel ligand PDGF-C, have not been investigated. Here, we report that members of the PDGF signaling, PDGF-A, PDGF-B, PDGF-C, PDGF-D and their two receptors {alpha} and ß are expressed in the mouse ovary, and regulated by at least two hormones (FSH and E2). This does not preclude the action of other hormones such as progesterone, androgen and LH that are also part of hormonal aberrations in our mutants (33) and during menopause. Based on the protective role of progesterone (i.e. lower in the FORKO mutants) in ovarian cancer (33) and the risks posed by androgens (elevated in FORKO mice) (45) it is indeed likely that these steroids might also be involved in modulation of growth factor systems in mutant ovaries.

Our results on the localization of PDGF-C, PDGFR-{alpha} and PDGFR-ß family are different from the previous reports in other species (43,46). This might be a true reflection of species differences in expression or age related, as our studies were performed on either immature mice or young adult mice of 3–4 months. For example, our data showed PDGFR-{alpha} localized strongly to oocyte and granulosa cells in the mouse ovary, whereas PDGFR-{alpha} localized only to vascular endothelial cells and oocyte in the porcine ovary (46) and no PDGFR-{alpha} is localized to normal ovarian cells in the human (37,40,47). PDGFR-ß immunostaining was detected in granulosa cells and OSE cells in the mouse ovary (Figure 6), while granulosa cells and OSE were devoid of any immunoreactive PDGFR-{alpha} or PDGFR-ß in the porcine ovary (43) and in the human, no granulosa staining of PDGFR-ß was found in normal human ovaries (37,40,47). Here, we also report for the first time that PDGF-C, PDGFR-{alpha} and PDGFR-ß protein localize in corpus luteum, an important source of progesterone during the cycle and pregnancy.

The localization of PDGF-C, PDGFR-{alpha} and PDGFR-ß suggests that potential autocrine/paracrine functions for these growth factors in regulation of the ovarian development. This could be one of the explanations for our previous observation of thicker epithelial layer, faster follicle recruitment (25) and cellular markers (32) in young FORKO ovaries. Our data suggests that FSH, in the normal mouse ovary could exert its effects through regulation of PDGFs and PDGFRs. This work extends and supports other investigations, demonstrating the changes of expression of PDGF family members in the rat ovary by microarray in the eCG-treated immature female rat (48) and down-regulation of immunoreactivity for both PDGF-AA and PDGF-BB within the rat testis tubules of organ cultures after 24 h of treatment with eCG. However, no changes in the staining for the PDGFR-{alpha} and PDGFR-ß subunit after treatment with FSH were observed (48). These results are partly in agreement with our findings showing down-regulation of PDGF members by FSH signaling. When the FSH-R gene is ablated in FORKO mice opposite effects prevail.

Our time-course study of mRNA levels, reveal differential gene regulation of the PDGFs and their receptors in the ovary in response to estrogen. The use of specific antibodies demonstrates that protein expression mirrors variations in mRNA revealed by Q-PCR analysis. While these data are in agreement with findings showing down-regulation of mRNA and/or proteins for one or more of the PDGFs or receptors in response to estrogen, there are some gender-dependent discrepancies of effects on the PDGF family. While Gray et al. (39) reported estrogen increased expression of mRNA for the PDGF-A, PDGF-B and PDGFR-{alpha}, PDGFR-ß in mouse uterus and vagina, another study found that estrogen up-regulated PDGF receptors in rat testis (49). These data demonstrate that PDGF for autocrine action can be up-regulated or down-regulated by estrogen to switch on or switch off cellular growth. In the tumor suppressor gene tuberous sclerosis complex 2 (TSC2)-expressing VSMCs, growth inhibition by E2 was associated with down-regulation of PDGF, PDGF receptor (PDGFR), and limited activation of extracellular signal-regulated kinase (ERK) (50). In contrast, the growth-promoting effect of E2 in TSC2-null ELT-3 cells was associated with induction of PDGF and robust phosphorylation of PDGFR (50). Thus, the expression of Tsc2 protein in mouse ovary (51) and the estrogenic regulation of PDGF members and PDGF family members in the ovary suggests the existence of potential links.

Our results using treatments with ICI 187200, a specific estrogen receptor antagonist, co-administered with eCG could increase the expression of PDGFR-{alpha} suggest that FSH action on PDGF is in part mediated by E2. This may account for the significant decrease in expression of PDGFR-{alpha} after treatment by E2, while there is no obvious change after treatment with eCG, suggesting that FSH signaling could increase PDGFR-{alpha} mRNA only in the absence of estradiol. The effects of E2 in FORKO mice also indicate a direct effect of this steroid in the ovary in absence of the FSH receptors.

Because FSH/E2 could modulate the expression of PDGF family, we hypothesized that expression of PDGF family members is likely to change during estrous cycle, the expression of PDGF-C, PDGF-D and PDGFR-{alpha} mRNA appeared to increase at the proestrous when FSH and E2 levels are the lowest among the four stages. Other reports also found increased immunoreactivity in high estrogen states of early pregnancy in the pig and mouse (1,39,41). A working model relating FSH-R signaling (or loss) and relation of PDGF family in ovarian compartmental regulation is depicted in Figure 7 as a guide to studying mechanisms. Further studies are also required to evaluate distribution and regulation in ovarian tumors of FORKO mice.


Figure 7
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  		Fig. 7. A working model depicting interactions that are likely to be involved in PDGF regulation and potential effects in wild-type and FORKO ovary. The pathways of FSH-R and E2 signaling in the normal ovary (left) maintaining the balance of PDGF members are undefined. Altered steroid milieu in the FORKO ovary (right) in the absence of FSH-R signaling is seen as a contributing factor in causing PDGF imbalances.

 
In conclusion, we report that certain PDGF family members localize to mouse ovarian cells including the OSE. In the young ovary, PDGF-C and the PDGFR-{alpha} appear to be constitutively expressed while ligands B, C and D as well as receptor ß are likely to be regulated. As a consequence, lack of FSH-R signaling and hormonal aberrations lead to imbalances in the PDGF family affecting the local milieu of paracrine–autocrine regulation initiating tumorigenesis. Additional studies will be necessary to establish cause–effect relationships.


   Acknowledgments
 
We are very grateful to Dr Carl-Henrik Heldin, Ludwig Institute for Cancer Research, Uppsala, Sweden, for providing the rabbit polyclonal antisera to PDGF receptor {alpha}- and ß-subunit. This investigation was supported by the Canadian Cancer Society & CIHR (Canadian Institutes of Health Research).

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received August 17, 2005; revised November 27, 2005; accepted December 6, 2005.


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