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and -ß during Human Testicular Development and Disease
Dipartimento di Fisiopatologia Medica (S.B., S.M., M.A., G.S., L.G.) and Dipartimento di Medicina Sperimentale e Patologia (C.D., A.M.), Università di Roma "La Sapienza", Policlinico Umberto I, 00161 Rome, Italy; Dipartimento di Medicina Sperimentale, Università di L’Aquila (S.U., N.R., E.A.J.), 67100 L’Aquila, Italy; and Dipartimento di Medicina Interna, Cattedra di Endocrinologia, Università di Modena e Reggio Emilia, Policlinico di Modena (C.C.), 41100 Modena, Italy
Address all correspondence and requests for reprints to: Lucio Gnessi, M.D., Ph.D., Dipartimento di Fisiopatologia Medica, Policlinico Umberto I, Università di Roma "La Sapienza", 00161 Rome, Italy. E-mail: . lucio.gnessi{at}uniroma1.it
Abstract
Platelet-derived growth factor (PDGF) plays a critical role in regulating the development and functional control of various tissues and has been implicated in the pathogenesis of serious diseases, including cancer and atherosclerosis. Given the emerging role of PDGF in the development and function of male gonads, we compared the expression profiles of the mRNAs of the PDGF A- and B-chains and of the PDGF receptor (PDGFR) 
- and ß-subunits in fetal and adult human testis. The immunohistochemical localization of the corresponding proteins in fetal, adult, and diseased human testicular tissues was also analyzed. PDGFs and PDGFRs mRNAs were readily detected by both Northern analysis and RT-PCR. The transcript levels were higher during 16–20 wk gestation, significantly lower at 24–28 wk, and increased in the adult. An identical pattern of protein expression was confirmed by immunohistochemistry, although the cellular localization of the PDGF system changes during postnatal development, concomitantly with the progression of spermatogenesis. In the testicular samples from patients affected by either complete aplasia of germ cells or various grades of spermatogenic arrest, the immunohistochemical localization of PDGFs and PDGFRs was different from normal, confirming a close connection between germ cells and PDGF system distribution. These results indicate that PDGF, through complex interactions, could play a leading role in ontogenesis and testicular pathophysiology in humans. Finally, the expression of PDGF ligands and receptor proteins in Leydig cell tumors suggests a relationship of the PDGF system to tumorigenesis or tumor progression in this testicular neoplasm.
PLATELET-DERIVED GROWTH factors are homo- or heterodimers of A-chain (PDGF-A) and B-chain (PDGF-B) that exert their action via binding to and dimerization of two related receptor tyrosine kinases, -receptors (PDGFR-) and ß-receptors (PDGFR-ß) (1). Recently, however, PDGF-C and PDGF-D, two new protease-activated ligands for the PDGFR complexes, have been identified (2, 3, 4, 5, 6, 7). Due to the different ligand binding specificities of the PDGFRs it is now known that PDGFR- binds PDGF-AA, PDGF-BB, PDGF-AB, and PDGF-CC; PDGFR-ßß binds PDGF-BB and PDGF-DD; whereas PDGFR-ß binds PDGF-AB, PDGF-BB, PDGF-CC, and PDGF-DD (8). PDGF was originally isolated from blood platelets as a growth factor for cells of mesenchymal origin, but the PDGF genes are also expressed by a variety of cell types in developing and adult vertebrates. In vitro, a selective list of PDGF actions on target cells includes migration, proliferation, contraction, and alteration of cellular metabolic activities, including matrix synthesis, cytokine production, and lipoprotein uptake (1).
Genetic analysis using gene-targeting approaches has provided important information on the physiological functions of the PDGFs, including functions during embryonic development (8). The phenotypical appearance of PDGF knockout mice has revealed generic features of PDGF-A and PDGF-B function. Three types of smooth muscle cells/myofibroblasts show an obligatory requirement for different PDGF isoforms during development. Alveolar smooth muscle cells depend on PDGF-A (9), whereas microvascular pericytes (10) and kidney glomerular mesangial cells depend on PDGF-B (11). Furthermore, PDGF-A and its cognate receptor are required for oligodendrocyte development (12, 13, 14), development of mesenchymal components of the hair follicle (15), gastrointestinal villus morphogenesis (16), and adult Leydig cell recruitment (17). Thus, the data available suggest that PDGFs provide selective signals during development by acting on the PDGFR-carrying progenitor cells, allowing for their proliferation and spreading on PDGF-producing endothelial or epithelial layers. It has been suggested that this process involves proliferation and migration only, processes known to be regulated by PDGF in vitro, but it may involve other functions as well, such as cell survival or differentiation. Overexpression of PDGF has been observed in several pathological conditions, including malignancies, atherosclerosis, and fibroproliferative diseases (18).
The testis is one of the organs in which the PDGF system has been identified. mRNAs for PDGFs and PDGFRs are expressed in prenatal and postnatal rat testis, with maximal expression in the early postnatal period (19, 20, 21). In vitro experiments in the rat have shown that PDGF enhances T production by Leydig cells (20, 22), is chemotactic and mytotic for peritubular myoid cells (PMC) (19, 23, 24), stimulates extracellular matrix deposition (23) and contraction by PMC (25), and can be involved in spermatogenic cell differentiation (24) and gonocyte proliferation (26, 27). The expression and in vitro actions of PDGF in the male rat gonad as well as the lack of the adult Leydig cell population in PDGF-A knockout mice (17) suggest a central role for PDGF in the development and functional control of the testis (28). However, to date, PDGF expression in human testicular tissue has not been characterized.
Here we describe, for the first time, the expression pattern of PDGF-A, PDGF-B, PDGFR-, and PDGFR-ß genes and the immunohistochemical distribution of the corresponding proteins in fetal and adult human testis. The immunohistochemical localization of PDGFs and PDGFRs in testes affected by complete aplasia of germ cells [the so-called Sertoli cell-only syndrome (SCOS)], various degree of spermatogenetic arrests, and Leydig cells tumor is also reported. Our findings demonstrate that testicular expression of the PDGF system in man is developmentally controlled through a spatiotemporal pattern of expression and provide some insights into the possible role of this growth factor during intrauterine testicular development, adult life and in some diseases of the testis.
Materials and Methods
Tissue preparation
Normal fetal testes were obtained from second and third trimester pregnancies (16 wk, three cases; 20 wk, four cases; 24 wk, three cases; 28 wk, three cases) after induced or spontaneous abortions. Stillborn fetuses did not show any sign of developmental disorders on autopsy. The gestational age of the fetuses was calculated as the duration of the gestation in weeks minus 2 wk (from the date of the last menstrual bleeding). Normal adult testicular samples were from testicular biopsies of infertile patients with genital tract obstructions (four cases, aged 35–45 yr), that had not received hormonal medication, had endocrine diseases, or suffered for cryptorchidism. The tissues were either fixed in Bouin’s solution, processed, and embedded in paraffin according to conventional techniques for immunohistochemistry or immediately stored in liquid nitrogen for RNA extraction. Testicular biopsies, performed for diagnostic purposes during andrological work-up of infertility, were from patients affected by complete aplasia of germ cells (three cases), and various grades of spermatogenic arrest (two spermatogonial arrested samples and three spermatocytic arrested samples). None of the patients suffered from endocrinological diseases or past history of cryptorchidism. Tissue samples of Leydig cell tumors were from the tissue archive of the Department of Internal Medicine, University of Modena (four cases). The ethics committee of the University of Rome "La Sapienza" approved the study. Written informed consent was obtained from the subjects and, where appropriate, their parents.
Northern blot analysis
Frozen fetal testicular tissues were homogenized, and poly(A)+ mRNA was prepared using a commercial kit (MicroFastTrak, Invitrogen, San Diego, CA). A pool of 8 µg poly(A)+ mRNA from fetal testis (16–28 wk) was separated on 1% formaldehyde-agarose gel and blotted on Nytran (Schleicher \|[amp ]\| Schuell, Inc., Keene, NH). A commercial human multiple tissue Northern blot (CLONTECH Laboratories, Inc., Palo Alto, CA) containing 2 µg testicular poly(A)+ mRNA was used to analyze the mRNA content in normal adult testis. The filters were sequentially hybridized with different random primer 32P-labeled cDNAs at high stringency using ExpressHyb hybridization solution (CLONTECH Laboratories, Inc.). The following cDNA probes were used: a 650-bp SacII-StuI fragment of the human PDGF-A cDNA and the 462-bp SacII-PvuII fragment of the human PDGF-B cDNA excised from plasmids pMMTPDGFA and pMMTPDGFB, respectively (provided by Dr. Stuart Aaronson, National Cancer Institute, NIH, Bethesda, MD). The full-length coding region of the rat PDGFR - and ß-subunit cDNAs, which share 86% homology with human cDNA sequences, was a gift from Dr. Michael Peck (Hoffman-LaRoche Inc., Basel, Switzerland). All blots were rehybridized with a 32P-labeled human ß-actin cDNA probe.
Semiquantitative RT-PCR
One microgram of mRNA from fetal and adult testicular samples was reverse transcribed at 37 C for 1 h in a 25-µl reaction volume containing 250 mM Tris-HCl, 375 mM KCl, 15 mM MgCl2, 50 mM dithiothreitol, 0.5 mM dNTPs, 0.5 µg random hexamer oligonucleotide, 200 U Moloney murine leukemia virus reverse transcriptase, 26 U ribonuclease inhibitor (Promega Corp., Madison, WI). ß-Actin was used as a constitutively expressed gene product for comparison of PDGF and PDGFR mRNA abundance between samples. RT products (0.5 µl) were amplified with 2.5 U Taq DNA polymerase (Promega Corp.) and 20 µM of each human ß-actin primer (Table 1
) in 50 µl reaction mix containing 50 mM KCl, 10 mM Tris-HCl, 1.5 mM MgCl2, and 10 mM each of dNTPs as follows: 94 C for 30 sec, 50 C for 40 sec, and 72 C for 60 sec. Reactions were temporarily halted at 20, 23, 25, 28, and 30 cycles, and 10 µl PCR products were withdrawn from each tube. All products were then analyzed by 1.5% agarose gel electrophoresis. Quantitation of the signals was performed by densitometric analysis using densitometry computer software (Kodak Digital Science 1D Image Analysis software, Eastman Kodak Co., Rochester, NY). PCR signals revealed a strong linear relationship, and 25 cycles were chosen for further analysis. Dilutions of RT products were made where necessary, and the amplification procedure was repeated until all samples were standardized for ß-actin content. Two microliters of appropriately diluted RT products were then amplified using 2.5 U Taq DNA polymerase and 20 µM of each human PDGF and PDGFR primer (Table 1) in 50 µl of the reaction mix, as follows: initial denaturation for 3 min at 94 C, 30 cycles of amplification because levels of PCR products increased in a linear fashion for up to 40 cycles for all the genes analyzed, 1 min of denaturation at 94 C, different annealing temperatures for each pair of primers (PDGF-A, 62 C; PDGF-B, 60 C; PDGFR-, 60 C; PDGFR-ß, 58 C), 1 min of extension at 72 C, and a final elongation of 5 min at 72 C. Parallel RT-PCR reactions without reverse transcriptase were performed for each sample to confirm that the PCR products resulted from cDNA rather than from genomic DNA. All products were then analyzed after 1.5% agarose gel electrophoresis and ethidium bromide staining, the resultant bands were densitometrically scanned. Relative mRNA abundance was estimated by calculating the ratio of intensities of PDGFs and PDGFRs to corresponding ß-actin bands.
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Immunohistochemistry was carried out on 5-µm-thick sections of the fixed testes by the streptavidin-biotin immunoperoxidase method using a commercial kit (Zymed Laboratories, Inc., San Francisco, CA). The deparaffinized sections were incubated overnight in a moist chamber at 4 C with a 1:100 dilution of the primary antibodies. The following antisera were used: affinity-purified polyclonal rabbit antihuman PDGF-AA and PDGF-BB antibodies, monoclonal mouse antihuman PDGFR ß-subunit antibody (Genzyme Corp., Cambridge, MA), and affinity-purified polyclonal goat antihuman PDGFR -subunit antibody (R\|[amp ]\|D Systems, Inc., Minneapolis, MN). Parallel serial sections from each specimen incubated with a dilution buffer substituted for the primary antibodies were used as negative controls. Slides were developed using amino-ethylcarbazole as chromogenic substrate, which is converted by peroxidase into a red to brownish-red precipitate at the sites of antigen localization in the tissue. The preparations were lightly counterstained with hematoxylin and mounted.
Results
Northern analysis and RT-PCR of PDGF ligands and receptors mRNAs in fetal and adult human testes
Northern blot on mRNA extracted from fetal (Fig. 1A) and adult testes (Fig. 1B) showed transcripts for both the PDGF A- and B-chains and PDGFR - and ß-subunit genes. The band lengths corresponded to the expected sizes of mRNA transcripts. According to a previous report (29), PDGF-A chain mRNA occurs as three transcripts.
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, C and D). The overall trend in PDGF and PDGFR mRNA expression throughout fetal development showed a progressive reduction of transcript levels, which were higher in 16- to 20-wk-old fetal testis and exhibited a significant decrease at 24–28 wk gestation. In the adult tissue gene expression increased, reaching levels comparable to the highest levels observed during the period of fetal development. Immunohistochemical localization of PDGF ligands and receptors in fetal and adult human testicular tissue
Figure 2 and Table 2 show the immunohistochemical distribution of PDGFs and PDGFRs in a 20-wk-old fetal testis and in adult testis. In the 20-wk-old fetal testis the PDGF A-chain immunoreactivity was localized in the cytoplasm of Sertoli cells, whereas the gonocytes were negative (Fig. 2A). A more intense signal was observed in Leydig cells. The flattened PMCs surrounding the testicular cords and the mesenchymal cells scattered within the interstitium were negative (Fig. 2A). Leydig cells and PMCs were PDGF-B positive (Fig. 2C). PDGFR -subunit immunoreactivity was observed in all cellular somatic components of the tissue; the germ cells within the testicular cords were negative (Fig. 2E). Finally, PDGFR ß-subunit immunoreactivity was present in Leydig cells and in some gonocytes; in Sertoli cells immunostaining was very weak (Fig. 2G). The immunohistochemical localization and the staining intensity in 16-wk-old fetal testis were almost superimposable to those described for the 20-wk-old fetal testis (data not shown). In 24- and 28-wk-old fetal testes the cellular distribution of staining was similar to that described in the younger testicular samples, but in agreement with the lower expression of the corresponding genes, the intensity of the signal substantially declined (data not shown).
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). PDGF-B immunoreactivity was visible in PMCs and Leydig cells (Fig. 2D). PDGFR -subunit immunostaining was localized in Leydig and Sertoli cell cytoplasm (Fig. 2F). The Leydig cells were the only PDGFR ß-subunit-positive cells (Fig. 2H). The germ cells lineage was constantly negative for PDGF-B, PDGFR -subunit, and PDGFR ß-subunit immunostaining. Negative controls were always negative (data not shown). Immunolocalization of PDGF ligands and receptors in human testes affected by germ cell aplasia, spermatogonial arrest, spermatocytic arrest, and Leydig cell tumor
Figure 3 and Table 2 show the immunohistochemical localization of PDGF ligands and receptors in human testes affected by germ cell aplasia and various grades of spermatogenic arrest.
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) and PDGFR- (Fig. 3G) were localized in Sertoli cells, with a stronger staining intensity in their basal extension. PDGF-B was predominantly localized in PMCs and Leydig cells; light immunostaining, mainly basal, was consistently observed in Sertoli cells (Fig. 3D). Strong PDGFR ß-subunit expression was observed in Sertoli and Leydig cells (Fig. 3J).
In both spermatogonial and spermatocytic-arrested testicular samples PDGF-A (Fig. 3, B and C) and PDGFR- (Fig. 3, H and I) were expressed by Sertoli and Leydig cells. In testicular tissue affected by spermatocytic arrest the spermatocytes were PDGF-A positive (Fig. 3C). PDGF-B and PDGFR-ß localization showed significant differences between spermatogonial (Fig. 3, E and K) and spermatocytic (Fig. 3, F and L) arrests. In both cases PDGF-B was evident in PMCs; however, although in spermatocytic arrest the Leydig cells were positive (Fig. 3F), in the spermatogonial arrested testis they were negative (Fig. 3E). Concerning PDGFR-ß, the spermatogonia-arrested samples showed positive immunostaining in Leydig cells and faint staining in Sertoli cells (Fig. 3K), whereas in spermatocytic arrest only Leydig cells were PDGFR-ß positive (Fig. 3L). Negative controls were constantly negative (data not shown).
In all Leydig cell tumors studied, neoplastic cells showed an intense staining for PDGF A- and B-chains and PDGFR - and ß-subunits (Table 2 and Fig. 4), and the signal was clearly stronger than that in Leydig cells of healthy tissue.
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Our results indicate that the human testis expresses PDGF ligands and PDGFRs during fetal life and in adulthood, and that mRNA expression and cellular protein distribution are ontogenetically regulated. We also demonstrate that testicular localization of the PDGF system is altered in subjects with abnormalities of spermatogenesis and that PDGF and PDGFR proteins are abundantly expressed in Leydig cell tumors.
Northern analysis of fetal and adult testicular samples revealed transcripts corresponding to the expected size, confirming previous data demonstrating the presence of full-length PDGF-A, PDGF-B, and PDGFR- mRNAs in the adult human testis (2, 4, 30, 31).
Through semiquantitative RT-PCR we found that PDGF ligand and receptor mRNA show a characteristic expression pattern. In 16- to 20-wk-old fetal testis there were significantly higher levels of PDGF-A, PDGF-B, PDGFR-, and PDGFR-ß mRNAs than in 24- to 28-wk-old fetal testis. In the adult, gene expression reached levels comparable to those observed in 16- to 20-wk-old fetal testis.
The high expression of PDGF at wk 16–20 corresponds with the dramatically accelerated fetal growth that occurs during the midtrimester of human pregnancy and is coincident with a number of key morphogenetic events occurring in the male gonad at this time. Between 14 and 18 wk the interstitial fetal Leydig cells undergo rapid proliferation (32). At wk 18 the myoid cells reach the peritubulum, and through cooperation with Sertoli cells, the basement membrane is formed (33). The gonocytes actively proliferate and cease mitotic division between the end of the second and the beginning of the third trimester of gestation (34, 35). Although we do not know the expression pattern of PDGFs and PDGFRs in infancy and puberty, we speculate that the increase in their mRNA levels in the adult testis could be related to the initiation of spermatogenesis and reinitiation of steroidogenesis.
The strong Sertoli cell localization of PDGF-A and PDGFR- in both the fetus and the adult; the presence of PDGF-A, PDGF-B, PDGFR-, and PDGFR-ß in Leydig cells and PMCs; and the appearance of PDGF-A-positive staining in spermatocytes and spermatids of the mature testis are of particular note, suggesting that through a finely tuned distribution of ligands and receptors various cellular components of the testis may be either a target for PDGF or a source of the growth factor for action on the neighboring cells. For example, in coincidence with the time of the morphogenetic cascade resulting in seminiferous tubule formation, rat PMCs express PDGFRs (19), and PDGF stimulates PMC proliferation and extracellular matrix component secretion (23). Moreover, PDGF and PDGF-like substances produced by Sertoli cells in culture are strong chemoattractants for PMCs (19). This evidence supports the view that in the rat the Sertoli cell might direct the development of neighboring precursors of mature PMCs and seminiferous tubule formation via PDGF (28). We observed a similar reciprocal expression of PDGFR by PMCs and of PDGF by Sertoli cells in the human fetus. The correspondence between the spatiotemporal expression of PDGFs and their receptors and the full establishment of peritubular disposition of myoid cells and basal lamina production in man (33) suggests that involvement of PDGF in formation of the wall of the seminiferous tubule could also apply to humans. Interestingly, in the adult rat PMCs cease to express PDGFRs (19, 36). This phenomenon has been attributed to the shift from a synthetic to a contractile phenotype of PMCs, which occurs concomitantly with the morphological and functional maturation of the testis (37, 38), and is analogous to what described for the perivascular smooth muscle cells (39). The observed loss of PDGFR expression by PMCs in mature human testis suggests that a similar behavior could take place in man.
Human Leydig cells expressed both ligands and receptors of the PDGF system. These data agree with previous findings in animals (17, 19, 20, 40, 41) and suggest that the ontogeny of this cell type could be profoundly influenced by PDGF. Indeed, in earlier studies we observed the lack of development of adult-type Leydig cells in PDGF-A-null mice (17). Although we do not know the exact localization of PDGF-A and PDGFR- in the human testis at puberty, which is the time of development of adult-type Leydig cells in man, the prerequisite for an effect of PDGF on adult Leydig cell recruitment could be present in the human testis; in fact, in both prenatal and postnatal testes the Sertoli cell positively stains for PDGF-A, and the interstitial cells are PDGFR- positive. Additional evidence for Leydig cell regulation via PDGF was suggested by in vitro experiments. For example, PDGF-B exerts a discrete stimulatory effect on basal and LH-stimulated T production by rat Leydig cells (20, 22) and inhibits 5-reductase and 
5-3ß- hydroxysteroid dehydrogenase activities in cultured immature rat Leydig cells (42). The colocalization of PDGF-B and PDGFR-ß in Leydig cells and the recognition of PDGF-B in PMCs of both fetal and adult human testis suggest that a PDGF-B-mediated autocrine/paracrine regulation of Leydig cell steroidogenesis could be effective in man.
No direct effect of PDGF on Sertoli cells has been reported, except for the ability to increase activator protein-1-mediated gene transcription (43). However, Sertoli cell localization of PDGFR- in both fetal and adult testes confirms previous data in the rat (41), indicating that this cell type could be a target for PDGF action. Furthermore, there are many pieces of information that suggest directly or indirectly the presence of multiple, complex interactions between germ cells and Sertoli cells. One mechanism through which germ cells influence Sertoli cell function involves the secretion of humoral factors that act on the Sertoli cell, presumably via a receptor system. We have detected PDGF-A protein localization in spermatocytes and spermatids. This evidence coupled with the presence of PDGFR- in the Sertoli cell points to a possible intervention of PDGF-A in the modulation of Sertoli cell function by the germ cells. Furthermore, the demonstration that PDGF stimulates aromatase (44), a key enzyme of estrogen biosynthesis, mainly localized in Sertoli cells, would offer an additional possible site of regulation of the Sertoli cell by PDGF.
In the midtrimester human fetal testis some gonocytes were PDGFR-ß positive. This result is in line with what reported in the rat, in which the PDGFR-ß is present in some, but not all, gonocytes (26). In addition, PDGF is able to activate the proliferation of isolated rat and mouse gonocytes in a dose-dependent manner (26, 27). Whether this proliferative effect corresponds to a functioning regulatory system in the human fetal testis is not predictable. However, PDGFR-ß expression by gonocytes in second trimester fetal testis and its subsequent decrease parallel the human gonocyte proliferation pattern (34, 35), suggesting that a PDGF-mediated mechanism could be involved in fetal germ cell development in man. Although we have not found a cellular source of PDGF-B within the testicular cords of the fetal samples, its expression in 12-wk-old human fetal testis has been reported (21). Moreover, the recent discovery of PDGF-D (4, 5), a new ligand for PDGFR-ß that has been found in human testis (4), opens the question of whether PDGFR-ß-expressing gonocytes could be a target for locally produced PDGF-D.
Interesting results of this study were obtained from investigation of subjects with germ cell aplasia and complete or partial failure of meiosis. The complete absence of germ cells in SCOS was accompanied by profound differences in PDGF and PDGFR immunostaining compared with normal. Although in normal testis Sertoli cells were devoid of PDGF-B and PDGFR-ß immunoreactivity, in SCOS they showed a clear positivity, which was particularly intense for PDGFR-ß. On the contrary, the strong Leydig cell PDGF-A and PDGFR- immunostaining as well as the PMC PDGF-A positivity of normal adult tissue were absent in SCOS. In the spermatogonial and spermatocytic arrests, there was a tendency to restore the distribution pattern of normal tissue. Indeed, in testicular samples affected by spermatocytic arrest, immunohistochemical localization of PDGFs and PDGFRs was superimposable on that observed in the normal adult tissue, with the obvious exception of spermatids. These data suggest that germ cells, in particular spermatocytes, might have important functions in directing cellular localization of the PDGF system in the testis.
The identification of early genes essential for gonad development (45) and the nature of the genes regulated by them have opened new areas of investigation in revealing the mechanisms involved in the ontogenesis of the male gonad and possibly in the development of pathological processes. Notably, the potential significance of PDGF in testicular development and function is reinforced by evidence indicating that PDGF and PDGFR genes may be modulated by early genes.
For example, the pattern of PDGF-A expression in early and differentiated Sertoli cells is coincident to what was reported for the Wilms’ tumor gene product (WT1) in developing and adult human testes (46, 47, 48). WT1 is a transcriptional factor that regulates genes essential for gonadogenesis (49, 50); it is expressed in the genital ridge before the formation of the gonad, persists throughout the entire testis during the embryonic period, and starting from the eighth week of gestation, localizes away from the interstitium, being restricted to the Sertoli cells in the fetus and adult (47). WT1 mutations are implicated in childhood tumors of the kidney (51) and are also associated with three pediatric syndromes: WAGR (Wilms’ tumor, aniridia, genitourinary abnormalities, and mental retardation), Denys-Drash syndrome, and Frasier syndrome (52). One prominent feature shared by these distinct syndromes is a high incidence of urogenital defects, including ambiguous genitalia and male pseudohermaphroditism (52, 53). Interestingly, the WT1 protein has been involved in posttranscriptional processing within Sertoli cells (54) and interacts with the PDGF-A promoter region, acting as a transcriptional activator as well as a repressor (55, 56, 57). This difference in behavior occurs by RNA editing, developmentally regulated at several different levels (54, 58, 59). The recognition that WT1 mutations found in Denys-Drash syndrome may adversely affect the promoter activity of PDGF-A (60), the expression of PDGF-A in Wilms’ tumor (61), and the coexpression of WT1 and PDGF-A in glomerular epithelial cells of fetal and mature human kidney (61) suggest a close functional relationship of these molecules. Thus, the ability of WT1 to modulate PDGF A-chain gene expression coupled with our data on the testicular expression pattern of PDGF-A, indicate that WT1 could be involved in the control of PDGF-A in the human testis. Moreover, it is worth mentioning that the lack of adult Leydig cell development, the normal appearance of Sertoli cells, and the spermatogenic arrest observed in PDGF-A-null mice (17) closely resemble forms of male pseudohermaphroditism, which is the most common genital abnormality in XY individuals with WT1 mutations (53, 62).
GATA-4, a member of the GATA transcription factor family (63), which is a key regulator of Sertoli cell-specific gene expression (64, 65, 66, 67), is another example of a gonadal-specific transcription factor involved in regulation of the PDGF system. In the human testis GATA-4 is expressed in the Sertoli cells from early fetal development to adulthood, with a peak at 19–22 wk gestation (68); in the Leydig cells it is expressed during the fetal period and after puberty, coinciding with the periods of active androgen synthesis (68). The recognition that GATA-4 may be a trans-acting factor in regulating the PDGFR- gene (69), the temporal relationship between GATA-4 and PDGFR- testicular expression, and their colocalization in Sertoli and Leydig cells suggest that the testicular effects of GATA-4 may be mediated in part by PDGF.
Enhanced or persistent expression of PDGF/PDGFR has been reported in nongonadal tumors, including glioblastomas, sarcomas, meningiomas, prostatic cancer, gastric cancer, and lung cancer (18). Although these studies have to be interpreted with some caution, a supporting role for this growth factor in the progression of certain neoplasms has been suggested (18). Hyperexpression and aberrant expression of PDGFR- have also been reported in testicular tumors (30, 31). Being limited in number, these observations need additional investigation, particularly given the recent development of various types of PDGF antagonists whose potential clinical utility is currently being evaluated (70, 71, 72). We found an intense expression of PDGFs and PDGFRs in Leydig cell tumors. This observation proposes that PDGF might influence the growth of this somatic cell-derived gonadal neoplasm. In line with this hypothesis is our finding of intense PDGF and PDGFR expression during the proliferative phase of Leydig cells in the fetal period. Interestingly, the reported abundant expression of GATA-4, which is involved in PDGFR- gene regulation (69), and of WT1, which regulates PDGF-A gene expression (55, 56, 57), in Leydig cell tumors (68, 73) further support an involvement of PDGF in the progression of this cancer.
Footnotes
This work was supported by grants from the Italian Ministero dell’Istruzione and dell’Università e della Ricerca, Progetti di Ricerca di Ateneo (ex quota 60%).
S.B. and S.M. are recipients of a postdoctoral fellowship from University of Rome "La Sapienza".
Abbreviations: PDGF, Platelet-derived growth factor; PDGFR, PDGF receptor; PMC, peritubular myoid cells; SCOS, Sertoli cell-only syndrome.
Received October 22, 2001.
Accepted January 28, 2002.
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 S. Basciani, M. Brama, S. Mariani, G. De Luca, M. Arizzi, L. Vesci, C. Pisano, S. Dolci, G. Spera, and L. Gnessi Imatinib Mesylate Inhibits Leydig Cell Tumor Growth: Evidence for In vitro and In vivo Activity Cancer Res., March 1, 2005; 65(5): 1897 - 1903. [Abstract] [Full Text] [PDF]
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R. Thuillier, Y. Wang, and M. Culty Prenatal Exposure to Estrogenic Compounds Alters the Expression Pattern of Platelet-Derived Growth Factor Receptors {alpha} and {beta} in Neonatal Rat Testis: Identification of Gonocytes as Targets of Estrogen Exposure Biol Reprod, March 1, 2003; 68(3): 867 - 880. [Abstract] [Full Text] [PDF]
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, PDGF-A; •, PDGF-B; 
, PDGFR-
, PDGFR-ß.
