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Mol. Hum. Reprod. Advance Access originally published online on March 7, 2008
Molecular Human Reproduction 2008 14(4):199-206; doi:10.1093/molehr/gan011
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© The Author 2008. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Platelet-derived growth factors (PDGF-A and -B) and their receptors in human fetal and adult ovaries

H. Pinkas1,2,3, B. Fisch1,2,3, G. Rozansky4, C. Felz1,2,3, G. Kessler-Icekson2,5, H. Krissi2,3, S. Nitke2,3, A. Ao4,6 and R. Abir1,2,3,7

1 IVF Research Laboratory, Infertility and IVF Unit, Helen Schneider Hospital for Women, Rabin Medical Center, Beilinson Campus, Petach Tikva 49100, Israel 2 Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel 3 Helen Schneider Hospital for Women, Rabin Medical Center, Beilinson Campus, Petach Tikva 49100, Israel 4Department of Obstetrics and Gynecology, Royal Victoria Hospital, McGill University, Montreal, Quebec, Canada H3A 1A1 5 Felsenstein Medical Research Center, Beilinson Campus, Petach Tikva 49100, Israel 6Department of Human Genetics, McGill University, Montreal, Quebec, Canada H3A 1A1

7 Correspondence address. Fax: +972 3 9240533; E mail: ronita{at}clalit.org.il


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
There is no information regarding the presence of platelet-derived growth factors (PDGFs) and their receptors in human ovaries. The expression of PDGF-A, -B and their two receptors, PDGFR-{alpha} and -β, was investigated in ovarian samples from women/girls and from human fetuses, at the protein and mRNA levels. The samples were prepared for immunohistochemical staining for PDGF-A and -B and their two receptors and in situ hybridization for the detection of the mRNA transcripts of the receptors. Total RNA was extracted from frozen ovarian samples, and the expression of PDGF-A and -B was investigated by reverse transcription–polymerase chain reaction. The proteins for PDGF-A and -B were detected in oocytes, and in granulosa cells (GC) of 50% of the follicles from women/girls. The proteins and mRNA transcripts for the two receptors were detected in oocytes (mRNA for PDGFR-β only in 25% of the oocytes). PDGFR-{alpha} mRNA was expressed in GC of a minority of the samples from women/girls, whereas PDGFR-β protein and mRNA were identified in over 50% of the GC from this source. PDGF-A and -B transcripts were identified in all the extracts. The presence of the receptors in GC suggests that PDGFs might be involved in the activation of primordial follicles.

Key words: primordial follicles/PDGFs and receptors/immunohistochemistry/in situ hybridization/RT–PCR


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Folliculogenesis in humans commences during the fourth–fifth month of pregnancy, when a single flat layer of ovarian granulosa cells (GC) surrounds the oocyte, thereby forming ‘primordial follicles’ (follicular diameter: 30–50 µm) (Gosden, 1995). The primordial follicles are activated when their GC become cuboidal (Gosden, 1995; Gougeon, 1996), and they are then termed primary follicles (50 µm–0.1 mm in diameter). Thereafter, the GC continue to proliferate until they are lined up in multiple layers around the oocyte, forming ‘secondary follicles’ with a theca cell layer (follicular diameter: 0.1–0.2 mm). ‘Antral follicles’ containing a fluid filled cavity, represent the final developmental stage. In this manuscript, the group of follicles (primordial, primary and secondary) preceding the antral stage are collectively termed ‘pre-antral follicles’ (Gougeon, 1996; Abir et al., 2006).

Most follicles in human ovaries remain quiescent primordial follicles (Gougeon, 1996; Fortune et al., 2000). It is well established that follicle-stimulating hormone (FSH) regulates folliculogenesis from the secondary stage onwards (Gougeon, 1996). However, the signals that trigger growth of unilaminar follicles are still unknown. Several growth factors may be involved in this early stage of folliculogenesis (Abir et al., 2006), including platelet-derived growth factors (PDGFs) (Nilsson et al., 2006; Yoon et al., 2006; Sleer and Taylor, 2007). Identifying these signals will promote the development of a potentially new therapeutic tool in assisted reproduction technology: in vitro maturation of primordial follicles from cryopreserved ovarian tissue (Abir et al., 1998, 2006). The method will restore fertility to former cancer patients without the risk of reseeding the disease.

PDGFs are a family of disulfide-bonded homo- or heterodimers of 30 kDa that include the classical and abundant A- or B-polymer chains (Heldin, 1993; Claesson-Welsh, 1996), in addition to the more recently discovered C- and D-polypeptide chains (Li et al., 2000; Bergsten et al., 2001; LaRochelle et al., 2001), each encoded by a different gene (Dalla-Favera et al., 1982; Swan et al., 1982; Betsholtz et al., 1986; Uutela et al., 2001). The chains form five dimeric isoforms: PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC and PDGF-DD. The dimerization-involved domains, which are structurally similar in all PDGFs, are responsible for receptor binding. The mature forms of the A- and B-chains are composed of ~100 amino acid residues with ~50% amino acid sequence identity, containing eight cysteine residues (Heldin and Westermark, 1999; Fredriksson and Eriksson, 2004).

The PDGF isoforms bind to structurally similar transmembrane glycoproteins, {alpha}- and β-protein tyrosine kinase receptors (PDGFR-{alpha} and -β); which are made up of 1089 and 1106 amino acid residues, respectively (Claesson-Welsh, 1994, 1996). These receptors contain an extracellular domain, a transmembrane domain, an intracellular juxtamembrane tyrosine kinase domain and a carboxyl terminal domain, and they undergo dimerization upon binding to the various PDGF isoforms: PDGF-AA creates {alpha}–{alpha} homodimers, PDGF-AB creates {alpha}–{alpha} homodimers as well as {alpha}–β heterodimers, and PDGF-BB creates {alpha}–{alpha}, β–β homodimers as well as {alpha}–β heterodimers.

PDGFs are major cellular mitogens, stimulating growth, differentiation and chemotaxis (Heldin and Westermark, 1999). In addition, recent studies have identified a connection with pre-antral follicles in rodents (Nilsson et al., 2006; Yoon et al., 2006; Sleer and Taylor, 2007). Specifically, PDGF isoforms and receptors were detected in murine (Yoon et al., 2006) and rat (Nilsson et al., 2006; Sleer and Taylor, 2007) follicles from primordial stages onwards. Furthermore, in vitro treatment of rat primordial (Nilsson et al., 2006) and secondary follicles (Sleer and Taylor, 2007) with various PDGF-A and -B isoforms stimulated their growth (Nilsson et al., 2006; Sleer and Taylor, 2007). However, there is no information regarding the expression of PDGFs and their receptors in human pre-antral follicles.

The aim of the present study was to fill this gap by investigating the expression of PDGF-A and -B and their receptors on the protein level using immunohistochemistry (IMH) and mRNA level using in situ hybridization (ISH) and reversed transcriptase–polymerase chain reaction (RT–PCR) in human ovaries from women/girls and second-to-third trimester fetuses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
Human ovaries from girls, women and fetuses
Ovarian samples were obtained from 17 aborted human fetuses aged 22–35 gestational weeks (GW) (Table I). The abortions were induced by prostaglandins. Thirteen of the fetuses had anatomical abnormalities, three had chromosomal aberrations and one was normal.


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  		Table I. Characteristics of fetuses from which ovaries were derived.

 
In addition to the fetal ovarian samples, small ovarian biopsy samples were donated by 20 women/girls aged 6–38 years or by their guardians (Table II). All had undergone gynecological laparoscopies. Eight patients had various forms of cancer, and their operation was performed for cryopreservation of ovarian tissue before the commencement of chemotherapy (Abir et al., 1998, 2006).


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  		Table II. Characteristics of women/girls from whom ovaries were derived.

 
The institutional ethics committee of Rabin Medical Center approved the study protocol, and every woman or minor’s parents signed an informed consent form. The samples were cut into uniform size (~2 mm x 2 mm) and fixed immediately in Bouin’s solution (components purchased from Sigma, St Louis, MO, USA) for IMH and ISH studies (Abir et al., 2004a, 2007a,b; Harel et al., 2006). The remaining sample material was frozen for subsequent RNA extraction.

Cryopreservation of ovarian tissue
Five to seven tissue slices measuring 1–2 mm were placed in cryogenic vials (Nalge Nunc International, Roskilde, Denmark) filled with a solution of 1.5 M dimethylsulfoxide (DMSO) (Sigma) and 0.1 M sucrose (Sigma) (Newton et al., 1998). Prior to freezing, the samples were kept on ice for 30 min. All samples were frozen gradually in a programmable freezer (Kryo 360-1/7, Planer Biomed, Sunbury on Thames, UK) as follows: –2°C/min from 15°C to –7°C, manual seeding at –7°C, –0.3°C/min to –35°C, and –10°C/min to –140°C. On completion of the automatic freezing cycle, the vials were immediately placed in liquid nitrogen. The slices were cryopreserved-stored for three months to 2 years until RNA extraction.

Histological preparation
Our histological preparation method has been described in detail elsewhere (Abir et al., 2004a,b, 2005, 2007a,b; Harel et al., 2006), and is therefore reviewed only briefly herein. The fixed specimens were rehydrated in a graded series of ethanol followed by paraffin embedding and sectioning (5 µm). Unstained sections were placed on OptiPlus positive charged microscope slides (BioGenex Laboratories, San Ramon, CA, USA) for IMH and ISH.

IMH for PDGFs and PDGF-Rs
Our IMH procedure has been described in detail elsewhere (Abir et al., 2004a,b, 2005, 2007a,b; Harel et al., 2006), and is therefore reviewed only briefly herein. Two sections per sample were utilized for the identification of each individual protein (PDGF-A, -B and PDGFR-{alpha}, -β). To enhance antigen retrieval, all the slides were microwaved with citrate buffer at pH 6.0 (CheMate buffer, DAKOCytomation, Glostrup, Denmark), and to block endogenous peroxidase activity, the slides were quenched in 3% hydrogen peroxide (H2O2, Vitamed, Binyamina, Israel).

All the primary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA): we included only those proven suitable for IMH by the manufacturer. Unfortunately, to the best of our knowledge, as noted also in our previous studies (Abir et al., 2004a,b, 2005, 2007a,b; Harel et al., 2006), there are no commercially available IMH positive controls for all four ligands. The samples were incubated with the primary antibodies (diluted: 1: 30, 1: 50) as follows: rabbit polyclonal antibodies against PDGF-A (Santa Cruz Biotechnology, catalog number: sc-128), PDGF-B (Santa Cruz Biotechnology, catalog number: sc-7878), PDGFR-{alpha} (Santa Cruz Biotechnology, catalog number: sc-338) and PDGFR-β (Santa Cruz Biotechnology, catalog number: sc-339). Negative control solutions were prepared by the absorption of the primary antibodies for PDGF-A, -B and PDGFR-{alpha}, -β with the corresponding blocking peptides (Santa Cruz Biotechnology, catalog numbers: sc-128P, sc-7878P, sc-338P and sc-339P, respectively).

Thereafter, the samples were incubated with solutions from an LSAB+System, horseradish peroxidase (HRP) kit (DAKOCytomation), which included biotinylated anti-rabbit, anti-mouse and anti-goat immunoglobulins and streptavidin conjugated to HRP (Harel et al., 2006). Finally, the sections were incubated with 3-amino-9-ethylcarbazole (AEC) substrate-chromogen (DAKOCytomation) containing H2O2 (red–brown staining, antigen detection), and counterstained with Mayer’s hematoxylin (Pioneer Research Chemicals Ltd., Colchester Essex, UK) (blue staining).

Non-radioactive ISH for PDGF receptors
Our ISH protocol has been described in detail previously (Abir et al., 2007a,b) and was used in the present study for detection of the mRNA transcripts for PDGFR-{alpha} and -β. All antisense oligonucleotide probes were 30 bases in length and custom-designed by Biognostik (Biognostik, Gottingen, Germany). The antisense phosphodiester DNA oligonucleotide probes were provided by the company with specific TriSeq Custom Design Hybriprobe kits (1 pmol/l in dionized sterile water, Biognostik), and were fluorescently labeled at the 5’ and 3’ ends with fluorescein-iso-thiocyanate. Specific antisense probes were designed for PDGFR-{alpha} (www.ncbi.nml.nih.gov/, accession number M21574 [GenBank] and M22734 [GenBank] ) and for PDGFR-β (www.ncbi.nml.nih.gov/, accession number NM_002609 [GenBank] and M21616 [GenBank] ) human genes. Negative controls consisted of random sequences of oligonucleotides (control HybriProbes) of comparable length to the targeted genes. Three positive control probes (polydeoxytymidine tissue control and two housekeeping genes: β-actin and {alpha}-tubulin) were also provided by the company and used in all experiments.

On the first day, the probes were applied on the sections, with negative controls being placed on adjacent sections. In addition, 10 separate sections were utilized for each of the three positive controls. All the probes were diluted in Hybribuffer (Biognostik, 40 units/ml = 120 µl/ml), and the slides were incubated overnight at 30°C in a humidified ThermoBrite Slide Hybridization/Denaturation System (StatSpin Inc., Norwood, MA, USA).

Hybridization was terminated by stringency rinses in a humidified ThermoBrite Slide Hybridization/Denaturation System (StatSpin Inc.): first with saline sodium citrate (SSC) buffer (diluted in distilled water from a 20x SSC buffer solution; Sigma) at room temperature, followed by 0.1% SSC buffer (diluted in distilled water from a 20x SSC buffer solution, Sigma) at 39°C. Thereafter, the slides were incubated with 0.5% Tris-blocking buffer (TNB, Perkin Elmer, Boston, MA, USA). The sections were further incubated with an anti-fluorescein-HRP conjugate (Perkin Elmer) (diluted 1: 25 with TNB), and then exposed to tyramide signal amplification -plus fluorescein (Perkin Elmer). Finally, the sections were rinsed and incubated overnight with AEC (red–brown staining, presence of PDGFR-{alpha} and -β). On the third day, the sections were counterstained with Mayer’s hematoxylin (Pioneer Research Chemicals) (blue staining).

Follicular counts
The number of follicles in every IMH and ISH stained section was counted with an image analyzer (analySIS, Soft Imaging System, Digital Solutions for Imaging and Microscopy, System GmbH, Munster, Germany), and the follicles were classified according to Gougeon (1996) as primordial, primary and secondary and antral (see Introduction for definitions).

RNA extraction
The frozen ovarian fragments were partially thawed at 37°C, rapidly removed from the semi-frozen DMSO (Sigma) solution, and placed in TRizol Reagent (Pioneer Research Chemicals) at room temperature and homogenized (Abir et al., 2004a,b; Harel et al., 2006). As the amount of ovarian tissue per individual fetus was very limited, in some cases, samples from fetuses of the same gestational age were pooled together. To obtain RNA fractions (supernatants) from these homogenates, chloroform (Biolab, Jerusalem, Israel) was added first and then a solution of phenol-chloroform-isoamyl alcohol (Sigma), followed by centrifugation at 4°C. To obtain RNA pellets, the RNA fractions were mixed with isopropanol (Biolab) and kept overnight at –20°C, followed by centrifugation at 4°C. Finally, ethanol (75%) was added in order to stabilize the pellet. The samples were kept at –80°C until RT–PCR was performed.

Reverse transcription–polymerase chain reaction
The primers for RT–PCR were designed to target the sequence corresponding to positions 2011 to 2241 of human PDGF-A and positions 1097 to 1481 of human PDGF-B mRNA sequences, published by the gene bank (www.ncbi.nml.nih.gov/).

The primers for PDGF-A were: forward, 5'-CACACCTCCTCGCTGTAGTATTTA-3', and reverse, 5'-GTTATCGGTGTAAATGTCATCCAA-3'; the primers for PDGF-B were forward, 5'-TCCCGAGGAGCTTTATGAGA-3' and reverse, 5'-ACTGCACGTTGCGGTTGT-3'; and the primers for control β-actin gene were forward, 5'-TCGTGCGTGACATTAAGGAG-3' and reverse, 5'-AGCACTGTGTTGGCGTACAG-3'.

Frozen total RNA samples of adult and fetal ovaries were centrifuged, and the pellet containing RNA was then resuspended in RNAse-free water. The concentration of each sample was measured by a spectrophotometer, and samples were stored at –80°C. The final reaction mixture of each sample contained 0.5 µg RNA, 2.5 mM magnesium acetate, 0.1 mM dNTPs, 0.4 nM forward primer, 0.4 nM reverse primer, 2 units of Taq polymerase (QIAGEN, Ontario, Canada) 1x buffer and molecular biology grade water, for a total volume of 40 ml. The amplification of the cDNA was performed by a single step RT–PCR procedure. The cycling conditions for PDGF-A were as follows: 95°C for 30 s, 53°C for 30 s, and 72°C for 1 min for 45 cycles, and a last extension cycle of 72°C for 8 min. Similar cycling conditions were used for PDGF-B and β-actin, except for annealing temperature, which was 54.5°C for PDGF-B and 56°C for β-actin. All primers were designed with the Primer 3, Version 4.0 program (http://frodo.wi.mit.edu/). Amplified products were electrophoresed on a 2% agarose gel along with a 100 bp DNA ladder as a reference for fragment size and stained with ethidium bromide. The expected product size for PDGF-A were 232 and 385 bp for PDGF-B. β-Actin gene (274 bp) was used as a positive control for the RT–PCR assay.

Statistical analysis
As the present study, similarly to our previous reports investigating expression of other growth factors and their receptors (Abir et al., 2004a,b, 2005, 2007a,b; Harel et al., 2006), was purely descriptive and qualitative, statistical analysis could not be applied to the IMH and ISH staining patterns or the RT–PCR results.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
IMH detection of PDGF-A and -B and their receptors
Figures 1A and B and 2A and B show the characteristic cellular localization for PDGF-A, -B and their {alpha} and β receptors, by IMH, in the human ovarian tissue from fetuses and women/girls. Full cytoplasmic oocyte staining for all four proteins was identified in all fetal samples. Partial cytoplasmic staining for PDGF-A was identified in samples from women/girls as well as in a portion of the sections stained for PDGFR-{alpha}. Partial and weak GC staining for PDGF-A was detected mainly in the samples from women/girls, and GC staining for PDGFR-β, only in samples from women/girls, often in a portion of the GC of individual follicles. Faint staining (compared with oocytes and GC) for all four proteins was identified in the theca layer. Stroma cells from both ovarian sources (fetuses and women/girls) expressed all four proteins. In addition, all four proteins were expressed in erythrocytes’ membranes with faint staining also in vascular endothelial cells from both ovarian tissue sources. There were no further correlations of the results by ovarian source, fetal abnormality, age or follicular class. The IMH (Figs 1C and D and 2C and D) negative controls did not stain positively (blue).


Figure 1
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  		Figure 1: IMH photographs of PDGF expression.

(A) PDGF-A expression. IMH staining of an ovary from an 18-year-old girl with acute lymphoblastic leukemia. Note the primordial (long black arrows) and primary (short black arrows) follicles, the large secondary follicle (black arrow head) and with red–brown staining in the oocytes (o) (as partial cytoplasmic staining with nuclear staining). Stroma cells (red arrow heads) are stained, some GC (g) of the secondary follicle are also stained (black arrow head), the theca (t) layer is faintly stained, endothelial cells of a blood vessel and erythrocytes within the blood vessel are also positively stained (red arrow) indicating PDGF-A protein expression. Original magnification x400. (B) PDGF-B expression. Section of human ovary from 33-GW-old fetus with achondroplasia. Note the primary (short black arrows) and primordial follicles (long black arrows), with the red–brown staining indicating PDGF-B protein expression in the oocytes (o) (as full cytoplasmic staining without nuclear staining), in some stroma cells (red arrow heads) and in erythrocytes’ membranes (red arrow). Original magnification x400. (C) Negative control (for PDGF-A). Section of human ovary from 24-GW-old fetus with oligohydramnion and growth retardation. Note the primordial follicles (long black arrows), the overall blue staining and the lack of red–brown staining. Original magnification x400. (D) Negative control (for PDGF-B). Section of human ovary from a 19-year-old woman with osteosarcoma. Note the secondary follicles (black arrow heads), the overall blue staining and the lack of red–brown staining. Original magnification x400.

 

Figure 2
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  		Figure 2: IMH photographs of PDGFR expression.

(A) PDGFR-{alpha} expression. Section of human ovary of 28-GW-old fetus with cardiac abnormalities. Note the primary (short black arrows) and primordial follicles (long black arrows) with brown staining indicating PDGFR-{alpha} protein expression in the oocytes (o) (as full cytoplasmic staining, without nuclear staining), stroma cells (red arrow heads) and in erythrocytes’ membranes (red arrow). Original magnification x400.

(B) PDGFR-β expression. Section of human ovary from a 38-year-old woman with familial breast cancer. Note the secondary follicle (black arrow head) with red–brown staining indicating PDGFR-β protein expression in the oocytes (o) (full cytoplasmic staining, without nuclear staining), in the GC (g), faint staining in the theca (t) layer and positive staining in stroma cells (red arrow heads) and in endothelial cells of blood vessels (red arrow). Original magnification x400. (C) Negative control (for PDGFR-{alpha}). Section of human ovary from a 17-year-old girl with Hodgkin lymphoma. Note the secondary follicle (black arrow head), the primordial follicles (long black arrows), the overall blue staining and the lack of red–brown staining. Original magnification x400. (D) Negative control (for PDGFR-β). Section of the same ovary as in Fig. 2B. Note the secondary follicle (black arrow head), the overall blue staining and the lack of red–brown staining. Original magnification x400.

 
ISH detection of PDGF receptors {alpha} and β
Figure 3A and B shows the characteristic mRNA cellular localization of the {alpha} and β receptors by ISH, in the human ovarian tissue from fetuses and women/girls. Cytoplasmic oocyte staining, without nuclear staining, was identified in all the fetal samples, and in a portion of the specimens from women/girls. Only a portion of the GC from women/girls stained positively. Weak positive theca cell staining was detected for PDGFR-{alpha} but not for PDGFR-β. Some of the stroma cells in all the samples were also stained. In addition, the mRNA transcripts for the two receptors were expressed in erythrocytes’ membranes with faint staining also in vascular endothelial cells from both ovarian sources. There were no further correlations of the results by ovarian source, fetal abnormality, age or follicular class. The ISH (Fig. 3C and D) negative controls did not stain positively (blue). The ISH positive controls stained in all the ovarian cells (Fig. 4). It is noteworthy that protein expression was strong compared with weak mRNA expression.


Figure 3
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  		Figure 3: ISH photographs of PDGFRs expression.

(A) PDGFR-{alpha} expression. Section of human ovary from 27-GW-old fetus with cardiac and pulmonary abnormalities. Note the red–brown staining indicating PDGFR-{alpha} mRNA expression in the oocytes (o) (as full cytoplasmic staining without nuclear staining) of primordial follicles (black arrows). Original magnification x400. (B) PDGFR-β expression. Section of human ovary from 33-GW-old fetus with achondroplasia. Note the primordial follicles (black arrows) with the red–brown staining indicating PDGFR-β mRNA expression in the oocytes (o) (full cytoplasmic staining, without nuclear staining), stroma cells (red arrow heads) and in erythrocytes’ membranes (red arrow). Original magnification x400. (C) Negative control (for PDGFR-{alpha}). Section of human ovary from 31-year-old woman with Hodgkin lymphoma. Note the primary follicle (short black arrow), the overall blue staining and the lack of red–brown staining. Original magnification x400. (D) Negative control (for PDGFR-β). Section of human ovary from 31-GW-old fetus with brain abnormalities. Note the primary (short black arrows) and primordial follicles (long black arrows), the overall blue staining, and the lack of red–brown staining. Original magnification x400.

 

Figure 4
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  		Figure 4: ISH positive control.

ISH positive control of the same ovary as in Fig. 3B. Note the primordial follicles (black arrows), with the red–brown staining indicating {alpha}-tubulin expression in the oocytes (o) (full cytoplasmic staining with nuclear staining) and in the GC (g), stroma cells (blue arrow heads), in endothelial cells of blood vessels and in erythrocytes’ membranes (blue arrows). Original magnification x400.

 
Follicular counts
Table III describes the number of follicles counted in the IMH and ISH stained samples from human fetuses and women/girls, distributed by follicular class. In the samples from women and girls, 441 follicles were counted: 373 were primordial (84.6%); 8 secondary (1.8%) and one antral. In samples from fetuses, 3324 follicles were counted: 3156 were primordial (94.9%), and only five were secondary (0.2%).


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  		Table III. Number and classification of the follicles examined.

 
Detection of PDGF-A and -B transcripts by RT–PCR
Ovarian RNA samples from eight human fetuses aged 22–35 GW and 10 women aged 21–38 years were tested for the presence of PDGF-A and -B transcripts by single-step RT–PCR assay. All samples yielded the expected fragment sizes (Fig. 5), with no difference in gene pattern or expression by sample source. None of the negative controls processed without reverse transcriptase (RT–) yielded an amplification product.


Figure 5
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  		Figure 5: Representative RT–PCR gel illustrating PDGF-A and PDGF-B gene expression in fetal and adult ovaries.

(A) PDGF-A (231 bp) and β-actin (274 bp). (B) PDGF-B (385 bp) and β-actin (274 bp). All intermediate lanes are the control (β-actin). Lane 1, ovary from 35-GW-old fetus with mosaic deletion of chromosome 9 (short limb bones); Lane 2, ovary from 24-GW-old fetus after intra uterine fetal death; Lane 3, pooled ovarian tissue from two 25-GW-old fetuses: one with hypoplastic left heart and the second with brain abnormalities; Lane 4, pooled ovarian tissue from three 22-23-GW-old fetuses: one with mosaic 46XX/47XX+marker non-satellite chromosome, the second with cardiac abnormalities and the third with renal abnormalities and intra uterine growth restriction; Lane 5, ovary from a 32-year-old women with an ovarian cyst.

 
Table IV summarizes the IMH, ISH and RT–PCR expression for PDGF-A and -B and their two receptors {alpha} and β in ovarian cells from fetuses and women/girls.


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  		Table IV. Proteins (IMH) and mRNA transcripts (ISH and RT–PCR) for PDGF-A, -B and their receptors (R-{alpha} and R-β) in human ovaries from women/girls and fetuses.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
In this study, we report for the first time the detection of the proteins and mRNA transcripts for PDGF-A and -B and their receptors, PDGFR-{alpha} and -β, in human ovaries from fetuses and women/girls. All four proteins were expressed in oocytes, theca cells and ovarian stroma cells from both sources. GC from the ovaries of women/girls expressed PDGF-A, -B and PDGFR-β. Transcripts of both receptors were identified in oocytes and stroma cells from both ovarian tissue sources, but only in GC from women/girls, whereas PDGFR-{alpha} mRNA was detected in theca cells from both sources. The mRNA transcripts of the two receptors were detected at a lower intensity by ISH than their corresponding proteins. The mRNA transcripts of PDGF-A and -B were identified by RT–PCR in ovarian extracts from both sources.

The availability of human fetal ovaries for research is extremely limited. This is especially true at the late gestational ages, when most abortions are performed because of fetal abnormalities. Although all but one of the fetal ovaries in our series were derived from abnormal fetuses, the spectrum of abnormalities was very wide and only three fetuses had chromosomal aberrations. It is, therefore, very unlikely that all the fetal samples were associated with ovarian developmental defects. Moreover, there were no differences in the general detection pattern of the four ligands among the fetal ovaries. Accordingly, in earlier studies, we used similar human fetal ovaries, including a minority from normal fetuses, to investigate the presence of other growth factors and their receptors, and no differences were noted in the detection pattern between ovaries from normal and abnormal fetuses (Abir et al., 2004a,b, 2007a; Harel et al., 2006). In addition, seven of the fetal ovarian specimens included in the present investigation were also used in a previous study, and the follicles survived in culture for 4 weeks, with an increase in 17β estradiol secretion (Biron-Shental et al., 2004), further supporting their viability.

The absence of proteins and transcripts in fetal GC may represent aspects of natural processes of follicular maturation and differentiation. The identification of PDGFR-β but not PDGFR-{alpha} in GC from women/girls suggests that binding of the β-receptor with its ligand may be important for the activation of primordial follicles. We detected the mRNA for PDGFR-{alpha} in some of the GC from women/girls, but not the corresponding protein, probably because of the low number of unstable transcripts that could not support protein synthesis (Alberts et al., 2002). Similar to our previous experiences (Abir et al., 2007a,b), IMH staining yielded higher intensities than ISH. We assume that the methodological sensitivity of ISH with DNA probes on paraffin sections to detect low mRNA transcript levels is poorer than the sensitivity of IMH to detect protein levels.

In a study of mouse ovarian follicles, protein expression of all PDGF isoforms and their receptors was evaluated by IMH (Yoon et al., 2006). PDGF-A and -B were expressed in the oocyte nucleus of primordial and secondary follicles. Protein expression of receptor {alpha} was observed in GC from primordial stages onwards and with low intensity in the oocyte cytoplasm. Receptor β was detected mainly in the oocyte cytoplasm and in proliferating GC.

The protein (Nilsson et al., 2006; Sleer and Taylor, 2007) and mRNA (Sleer and Taylor, 2007) expressions of all PDGF isoforms and of their receptors have been studied in the rat ovary, from the neonatal period (Nilsson et al., 2006; Sleer and Taylor, 2007) to adulthood, by IMH, real-time PCR and ISH (Sleer and Taylor, 2007). Protein expression of the PDGF isoforms was identified in oocytes of primordial, primary and developing follicles (Nilsson et al., 2006; Sleer and Taylor, 2007); PDGF-A was expressed in GC of young adult rats from secondary stages onwards (Days 20–24) (Sleer and Taylor, 2007). Protein expression of PDGFR-{alpha} and -β was identified in oocytes of primordial and primary follicles; PDGFR-{alpha} was also expressed in GC from secondary stages onwards in young adults, and PDGFR-β was expressed in the stroma cells surrounding the oocytes. Transcripts of PDGF-A and -B decreased from Day 2 onwards. Transcripts of the two receptors were detected from birth to early adulthood (Day 28), peaking in the neonatal period (Days 4–8). Contrary to the findings in rodents (Yoon et al., 2006; Sleer and Taylor, 2007), in the present study, PDGFR-{alpha} protein was not detected in human GC, but PDGFR-β protein was expressed in GC from women/girls. The discrepancies between the results can be attributed to differences between species.

Nilsson et al. (2006) cultured rat primordial follicles for 14 days and examined the localization of PDGF proteins at different time points. PDGF expression was noted in the oocytes of both primordial and growing follicles. Treatment with PDGF-AB resulted in a significant increase in the proportion of developing follicles with a concomitant decrease in the number of primordial follicles, whereas treatment with an anti-PDGF antibody reversed these actions. In addition, real-time PCR revealed that PDGF-AB induced an augmentation in KIT ligand (stem cell factor) mRNA expression. In another study, culturing rat secondary follicles with PDGF-AA or PDGF-AB or PDGF-BB led to a significant increase in follicular diameter (Sleer and Taylor, 2007).

It is very unlikely that only a single factor promotes the activation of primordial follicles (Abir et al., 2006). We assume that combinations of certain growth factors, perhaps species specific, are responsible for the transformation. Although interactions between PDGFs and KIT ligand stimulated growth of rat primordial follicles (Nilsson et al., 2006), the effects of KIT ligand itself differs among species, and even among rodents (Parrot and Skinner, 1999; Wang and Roy, 2004; Hutt et al., 2006). KIT ligand promoted the survival and proliferation of germ cells in rodents (Parrot and Skinner, 1999; Wang and Roy, 2004; Hutt et al., 2006), and the activation of primordial follicles in rats (Parrot and Skinner, 1999) and mice (Hutt et al., 2006). It stimulated only oocyte growth in rabbits, induced follicular assembly in hamsters (Wang and Roy, 2004), and prevented ovarian apoptosis in the sheep (Tissdall et al., 1999). In cultures of human tissue, KIT ligand only promoted survival of primordial follicles (Carlsson et al., 2006), despite its known presence in human fetal and adult oocytes (Abir et al., 2004b) as well as in human fetal GC (Høyer et al., 2005).

Our detection of the protein for PDGFR-β specifically in the GC of human primordial follicles from adults/girls suggests that PDGFs can interact with their protein receptor on GC of primordial follicles. This finding together with the results in rats (Nilsson et al., 2006; Sleer and Taylor, 2007) suggests that like in rats, PDGFs in humans might be involved in activation of primordial follicles. To further elucidate its role, PDGFs should be added to the culture medium of human primordial follicles. Additional studies in humans are still needed to clarify the place of PDGFs, other growth factors and synergistic interactions in early follicular development. Given that our present study as well as previous ones (Abir et al., 2006) revealed more receptors for growth factors in GC of human adults than of fetuses, it might be easier to first establish a successful culture system for human primordial follicles from women rather than from fetuses.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
The authors are grateful to Dr Dewen Kong from the Department of Obstetrics and Gynecology, Royal Victoria Hospital, McGill University, Montreal, for his technical help with the RT–PCR procedure. The authors are indebted to Ms G. Ganzach from the Editorial Board for the English editing, to the staff at the Gynecology Ward for their help in locating suitable patients and to the Ultrasound Unit for identifying fetal gender (all from Rabin Medical Center, Beilinson Campus).


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Abir R, Fisch B, Raz Ah, Nitke S, Ben Rafael Z. Preservation of fertility in women undergoing chemotherapy—current approach and future prospects. J Assist Reprod Genet (1998) 15:469–478.[CrossRef][Web of Science][Medline]

Abir R, Fisch B, Jin S, Barnnet M, Freimann S, Van den Hurk R, Feldberg D, Nitke S, Krissi H, Ao A. Immunocytochemical detection and reverse transcription polymerase chain reaction expression of leukemia inhibitory factor (LIF) and its receptor in human fetal and adult ovaries. Mol Hum Reprod (2004) a 10:313–319.[Abstract/Free Full Text]

Abir R, Fisch B, Jin S, Barnnet M, Kessler-Icekson G, Ao A. Expression of stem cell factor in human fetal and adult ovaries. Fertil Steril (2004) b 82:1235–1243.[CrossRef][Web of Science][Medline]

Abir R, Fisch B, Jin S, Barnnet M, Ben-Haroush A, Felz C, Kessler-Icekson G, Feldberg D, Nitke S, Ao A. Presence of NGF and its receptors in ovaries from human fetuses and adults. Mol Hum Reprod (2005) 11:229–236.[Abstract/Free Full Text]

Abir R, Nitke S, Ben-Haroush A, Fisch B. In vitro maturation of human primordial ovarian follicles- Clinical significance, progress and methods for growth evaluation. Histol Histopathol (2006) 21:887–898.[Medline]

Abir R, Ben-Haroush A, Melamed N, Felz C, Nitke S, Krissi H, Fisch B. The Expression of bone morphogenetic proteins 4 and 7 and their receptors IA, IB and II in Human Ovaries from Fetuses and Adults. Fertil Steril (2007) a.

Abir R, Garor R, Felz C, Nitke S, Krissi H, Fisch B. Growth Hormone and Its Receptor in Human Ovaries from Fetuses and Adults. Fertil Steril (2007) b.

Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P. Basic genetic mechanism, Chapter 7. In: Molecular Biology of the Cell—Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P, eds. (2002) 4th edn. New York, NY: Garland Science.

Bergsten E, Uutela M, Li X, Pietras K, Östman A, Heldin CH, Alitalo K, Eriksson U. PDGF-D is a specific, protease-activated ligand for the PDGF β-receptor. Nat Cell Biol (2001) 3:512–516.[CrossRef][Web of Science][Medline]

Betsholtz C, Johonsson A, Heldin CH, Westermark B, Lind P, Urdea MS, Eddy R, Shows TB, Philpott K, Mellor AL, et al. cDNA sequence and chromosomal localization of human platelet-derived growth factor A-chain and its expression in tumour cell lines. Nature (1986) 320:695–699.[CrossRef][Medline]

Biron-Shental T, Fisch B, Van Den Hurk R, Felz C, Feldberg D, Abir R. Survival of frozen-thawed human ovarian fetal follicles in long-term organ culture. Fertil Steril (2004) 81:716–719.[CrossRef][Web of Science][Medline]

Carlsson IB, Laitinen MPE, Scott JE, Louhio H, Velentzis L, Tuuri T, Aaltonen J, Ritvos O, Winston RML, Hovatta O. Kit ligand and c-Kit are expressed during early human ovarian follicular development and their interaction is required for the survival of follicles in long term culture. Reproduction (2006) 131:641–649.[Abstract/Free Full Text]

Claesson-Welsh L. Platelet-derived growth factor receptor signals. J Biol Chem (1994) 269:32023–32026.[Free Full Text]

Claesson-Welsh L. Mechanism of action of platelet-derived growth factor. Int J Biochem Cell Biol (1996) 28:373–385.[CrossRef][Web of Science][Medline]

Dalla-Favera R, Gallo RC, Giallongo A, Croce CM. Chromosomal localization of the human homolog (c–sis) of the simian sarcoma virus onc gene. Science (1982) 2:686–688.

Fortune JE, Cushman RA, Kito WS. The primordial to primary follicle transition. Mol Cell Endocrinol (2000) 163:53–60.[CrossRef][Web of Science][Medline]

Fredriksson L, Li H, Eriksson U. The PDGF family: four gene products from five dimeric isoforms. Cytokine Growth Factor Rev (2004) 15:197–204.[CrossRef][Web of Science][Medline]

Gosden RG. Oocyte development throughout life. In: Gametes—The Oocyte—Grudzinshes JG, Yovici JL, eds. (1995) Cambridge, UK: Cambridge University Press. 119–149.

Gougeon A. Regulations of ovarian follicular development in primates: facts and hypotheses. Endocrine Rev (1996) 17:121–154.[Abstract/Free Full Text]

Harel S, Jin S, Fisch B, Feldberg D, Krissi H, Freimann S, Tan SL, Ao A, Abir R. Tyrosine kinase B receptor and its activated neurotrophins in ovaries from human fetuses and adults. Mol Hum Rerod (2006) 12:357–365.[CrossRef]

Heldin CH. Purification and structure of PDGF. In: Biology of Platelet-Derived Growth Factor Cytokines—Westermark B, Sorge C, eds. (1993) Vol. 5. Basel: S. Karger AG. 1–10.

Heldin CH, Westermark B. Mechanism of action and in vivo role of platelet-derived growth factor. Physiol Rev (1999) 79:1283–1316.[Abstract/Free Full Text]

Høyer PE, Byskov AG, Møllgård K. Stem cell factor and c-Kit in human primordial germ cells and fetal ovaries. Mol Cell Endocrinol (2005) 234:1–10.[CrossRef][Web of Science][Medline]

Hutt KJ, Mclaughlin EA, Holland MK. Kit/Kit ligand in mammalian oogenesis and folliculogenesis: roles in rabbit and murine ovarian follicle activation and oocyte growth. Biol Reprod (2006) 75:421–433.[Abstract/Free Full Text]

LaRochelle WJ, Jeffers M, McDonald WF, Chillakuru RA, Giese NA, Lokker NA, Sullivan C, Boldog FL, Yang M, Vernet C, et al. PDGF-D, a new protease-activated growth factor. Nat Cell Biol (2001) 3:517–521.[CrossRef][Web of Science][Medline]

Li X, Pontén A, Aase K, Karlesson L, Abramsson A, Uutela M, Backstrom G, Hellstrom M, Bostrom H, Li H, et al. PDGF- C is a new protease-activated ligand for the PDGF-{alpha} receptor. Nat Cell Biol (2000) 2:302–309.[CrossRef][Web of Science][Medline]

Newton H, Aubard Y, Rutherford A, Sharma V, Gosden R. Low temperature storage and grafting of human ovarian tissue. Hum Reprod (1996) 11:1487–1491.[Abstract/Free Full Text]

Nilsson EE, Detzel C, Skinner MK. Platelet-derived growth factor modulates the primordial to primary follicle transition. Reproduction (2006) 131:1007–1015.[Abstract/Free Full Text]

Parrot JA, Skinner MK. Kit-ligand/stem cell factor induces primordial follicle development and initiates folliculogenesis. Endocrinology (1999) 140:4262–4271.[Abstract/Free Full Text]

Sleer LS, Taylor CC. Cell-type localization of platelet-derived growth factors and receptors in the postnatal rat ovary and follicle. Biol Reprod (2007) 76:379–390.[Abstract/Free Full Text]

Swan DC, McBride OW, Robbins KC, Keithley DA, Reddy EP, Aaronson SA. Chromosomal mapping of the simian sarcoma virus onc gene analogue in human cells. Proc Natl Acad Sci USA (1982) 79:4691–4695.[Abstract/Free Full Text]

Tissdal DJ, Filder AE, Smith P, Quirke LD, Stent VC, Heath DA, McNatty KP. Stem cell factor and c-kit gene expression and protein in the sheep ovary during fetal development. J Reprod Fertil (1999) 116:277–291.[Abstract/Free Full Text]

Uutela M, Lauren J, Bergsten E, Li X, Horelli-Kuitunen N, Eriksson U, Alitalo K. Chromosomal location, exon structure, and vascular expression patterns of the human PDGFC genes. Circulation (2001) 103:2242–2247.[Abstract/Free Full Text]

Wang J, Roy SK. Growth differentiation factor-9 and stem cell factor promote primordial follicle formation in the hamster: modulation by follicle stimulating hormone. Biol Reprod (2004) 70:577–585.[Abstract/Free Full Text]

Yoon SJ, Kim KH, Chung HM, Choi DH, Lee WS, Cha KY, Lee KA. Gene expression profiling of early follicular development in primordial, primary, and secondary follicles. Fertil Steril (2006) 85:193–203.[CrossRef][Web of Science][Medline]

Submitted on January 8, 2008; resubmitted on February 25, 2008; accepted on February 26, 2008.


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