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73 Boney, C.M. et al. (1998) Modulation of insulinlike growth factor I mitogenic signaling in 3T3-L1 preadipocyte differentiation. Endocrinology 139, 1638–1644 74 Vaziri, C. and Faller, D.V. (1996) Down-regulation of platelet-derived growth factor receptor expression during terminal differentiation of 3T3-L1 pre-adipocyte fibroblasts. J. Biol. Chem. 271, 13642–13648 75 Serrero, G. et al. (1992) Paracrine regulation of adipose differentiation by arachidonate
metabolites: prostaglandin F2α inhibits early and late markers of differentiation in the adipogenic cell line 1246. Endocrinology 131, 2545–2551 76 Casimir, D.A. et al. (1996) Preadipocyte differentiation blocked by prostaglandin stimulation of prostanoid FP2 receptor in murine 3T3-L1 cells. Differentiation 60, 203–210 77 Borglum, J.D. et al. (1999) Differential expression of prostaglandin receptor mRNAs during adipose cell differentiation. Prostaglandins Other Lipid Mediat. 57, 305–317
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78 Reginato, M.J. et al. (1998) Prostaglandins promote and block adipogenesis through opposing effects on peroxisomal proliferator-activated receptor γ. J. Biol. Chem. 273, 1855–1858 79 Marques, B.G. et al. (1998) Association of fat cell size and paracrine growth factors in development of hyperplastic obesity. Am. J. Physiol. 275, R1898–R1908 80 Faust, I.M. et al. (1978) Diet-induced a dipocyte numbers increase in adult rats: a new model of obesity. Am. J. Physiol. 235, E279–E296
PDGF and the testis Stefania Mariani, Sabrina Basciani, Mario Arizzi, Giovanni Spera and Lucio Gnessi Testicular development is controlled by a complex hierarchy of gene regulatory proteins, growth factors, cell adhesion molecules, signaling molecules and hormones that interact, often acting within short time windows, via reciprocal control relationships. The identification in the testis of platelet-derived growth factor (PDGF), a key regulator of connective tissue cells in embryogenesis and pathogenesis, has focused attention on the role of this growth factor in testicular pathophysiology. This review summarizes recent advances in the study of the actions of PDGF in the male gonad, and attempts to incorporate complex in vitro and in vivo experimental data into a model that might clarify the role played by PDGF in the mammalian testis.
Testicular development and functional control involve a complex combination of cell proliferation, hypertrophy, migration, differentiation and apoptosis, which occur within strict temporal and anatomical constraints [1]. These highly coordinated processes, driven by the sequential activation of specific genes [2,3], require a precise temporal regulation of growth and differentiation of somatic and germ cell elements and imply several cell–cell interactions that are accomplished by locally produced growth and differentiation factors, hormones and cell adhesion molecules [4]. During the past decade, evidence has accumulated that shows that platelet-derived growth factor (PDGF) should be included as one of the locally produced growth factors that mediate testicular cell–cell interactions. This review focuses on the role of PDGF in the male gonad during prenatal and postnatal phases of development. Stefania Mariani Sabrina Basciani Mario Arizzi Giovanni Spera Lucio Gnessi* Dept Medical Physiopathology, Policlinico Umberto I, University of Rome ‘La Sapienza’, 00161 Rome, Italy. *e-mail: [email protected]
Structure and general functions of PDGFs and PDGF receptors PDGF isoforms
PDGFs are members of the PDGF-vascular endothelial growth factor (PDGF-VEGF) family of growth factors [5]. Their constituent polypeptide chains share a core motif of Cys residues with a characteristic spacing [6]. The three-dimensional structure of PDGFs is similar to that of VEGFs, but also bears some resemblance to the structures of http://tem.trends.com
glycoprotein hormones, of nerve growth factor and of the transforming growth factor β family of peptides, despite the fact that there is no amino acid sequence similarity among them [7]. All of these factors have dimeric configurations and show the characteristic Cys-knot motifs that are involved in the formation of inter- and intramolecular disulfide bonds [8]. For almost 20 years, only two PDGF polypeptides, PDGF-A and PDGF-B, were known. Recently, however, PDGF-C [9,10] and PDGF-D [11–13], two new PDGFs, were discovered. The biologically active PDGF molecules are either homodimers or heterodimers. The four homodimers, PDGF-AA, PDGF-BB, PDGF-CC and PDGF-DD, and the heterodimer, PDGF-AB, have all been shown to be endogenous cell products [14]. PDGF-C does not heterodimerize with PDGF-A or PDGF-B, probably because PDGF-C is rather distantly related to PDGF-A and PDGF-B in its core domain. By contrast, PDGF-C and PDGF-D are closely related structurally, but it remains to be established whether they can heterodimerize. PDGF-C and PDGF-D are as closely related to the VEGFs as they are to PDGF-A and PDGF-B, based on their primary sequence. However, they are characterized as novel PDGFs because of their PDGF receptor binding specificity. The A- and B-chains of PDGF are synthesized as precursor molecules that undergo proteolytic processing at the N-termini and, in the case of the B-chain, also intracellularly at the C-terminus [15]. Cells naturally producing both A- and B-chains contain all three PDGF isoforms, suggesting that the assembly of PDGF dimers could be a random process. PDGF bioavailability in vivo is also dependent on the association of the secreted growth factor with extracellular matrix molecules, which for PDGF-A and PDGF-B seems to be mediated by a C-terminal basic motif [16]. PDGF-C and PDGF-D possess an N-terminal domain (CUB domain; domain found in complement subcomponents C1r/C1s, urchin epidermal growth factor-like protein and bone
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and inhibited by PDGFR-α, and the receptor heterodimer appears to mediate a slightly stronger mitogenic signal than do either of the two receptor homodimers [6]. Functions of PDGF
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Fig. 1. Schematic representation of PDGF ligand and PDGF receptor isoforms and binding specificities. A- and B-chains of PDGF are synthesized as precursor molecules that undergo proteolytic processing and intracellular assembly before secretion. PDGF-C and PDGF-D possess an N-terminal domain, referred to as a CUB domain, which remains on the secreted PDGF-CC and PDGF-DD molecules. Extracellular proteolytic removal of the CUB domain from the secreted PDGF-CC and PDGF-DD dimers is a prerequisite for binding to PDGF receptors. The different PDGF isoforms bind to and dimerize α- and β-receptors into the configuration shown. The extracellular part of the receptors consist of five Ig-like domains. Ligands bind to the three outermost Ig-like domains, whereas the fourth domain is involved in direct receptor–receptor interactions. Intracellular parts of receptors contain tyrosine kinase domains. Abbreviations: CUB, domain found in complement subcomponents C1r/C1s, urchin epidermal growth factor-like protein and bone morphogenetic protein 1; Ig, immunoglobulin; PDGF, platelet-derived growth factor.
morphogenetic protein 1), which appears not to be removed obligatorily by intracellular proteolytic processing before secretion. Proteolytic removal of the CUB domain is a prerequisite for binding to PDGF receptors, but the proteases involved in the assumed extracellular processing are unknown. Thus, PDGF-CC and PDGF-DD are synthesized and secreted as latent growth factors.
In vitro, a list of the best-documented functions of PDGFs on target cells includes: proliferation, migration and actin reorganization, contraction, extracellular matrix production, differentiation and survival [6]. Gene-targeting approaches have provided important information about the physiological role of the PDGFs [14]. The phenotypical analysis of Pdgf-knockout mice has revealed generic features of the function of PDGF-A and PDGF-B. Three types of smooth muscle cells and/or myofibroblasts show an obligatory requirement for PDGF during development. Alveolar smooth muscle cells depend on PDGF-A [17], and microvascular pericytes and kidney glomerular mesangial cells depend on PDGF-B [18,19]. Furthermore, PDGF-A and its cognate receptor are required for oligodendrocyte development [20–22], development of mesenchymal components of the hair follicle [23], gastrointestinal villus morphogenesis [24] and adult Leydig cell recruitment [25]. No data are available on the in vivo functions of PDGF-C and PDGF-D. However, the phenotypic differences between mice lacking PDGFR-α and those lacking PDGF-A support the idea that the other PDGFR-α ligand, PDGF-C, might provide distinct signals for PDGFR-α-expressing cells. Comparisons of mice lacking PDGFR-β and those lacking PDGF-B have not revealed a phenotypic discrepancy, indicating that PDGF-D and PDGF-B might have partially redundant functions. Thus, PDGFs provide selective signals during development by acting on the PDGF receptor-carrying progenitor cells, enabling them to proliferate and spread on PDGF-producing endothelial and/or epithelial layers. Overexpression of the PDGF gene has been observed in several pathological conditions, including malignancies, atherosclerosis and fibroproliferative diseases [26].
PDGF receptors
Developmental production of PDGFs and PDGF receptors in the mammalian testis
PDGFs interact with two different but structurally related cell surface receptor tyrosine kinases (PDGFR-α and PDGFR-β), which dimerize upon ligand binding and can be expressed individually or together in cells [14]. The PDGF receptor dimers have different ligand-binding capacities: PDGFR-αα binds PDGF-AA, PDGF-BB, PDGF-AB and PDGF-CC; PDGFR-ββ binds PDGF-BB and PDGF-DD; and PDGFR-αβ binds PDGF-AB, PDGF-BB, PDGF-CC and PDGF-DD (Fig. 1). PDGF receptors have partially overlapping and partially distinct signaling capabilities, which could have specific biological consequences. For example, chemotaxis of fibroblasts and smooth muscle cells is stimulated by PDGFR-β
PDGFs and PDGF receptors are produced in the testis during prenatal and postnatal life in a timeand space-dependent manner. In the rat, transcripts from the Pdgfa, Pdgfb, Pdgfra and Pdfgrb genes are found on embryonic day 18 (E18), reach high levels by postnatal day 5 (P5) and then decline to lower levels in older animals [27]. These age-related changes were confirmed for Pdgfra – the highest levels of transcript were observed in the testes of five-day-old animals, followed by a decline in relative abundance with increasing age [28]. Localization studies have shown that, in the prenatal period, Sertoli cells are the primary source of PDGF-A and PDGF-B molecules, whereas the cells expressing PDGFR-α and PDGFR-β
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are scattered in the tissue between the testicular cords [29]. During the first week of postnatal life, the rat testis shows an intense staining for PDGF-A and PDGF-B in the cytoplasm of the Sertoli cells, and PDGFR-α- and -β-subunit immunoreactivity in the peritubular myoid cells (PMCs) [27,29]. Li and colleagues also reported PDGFR production in some but not all gonocytes during the first five days after birth [29]. In pubertal animals, only minimal positive staining is seen for PDGF-A and PDGF-B in Sertoli cells and for PDGFR-α- and -β-subunits in PMCs. Finally, in the adult, Leydig cells are the only cellular component showing a positive reaction for PDGF-A, PDGF-B, PDGFR-α and PDGFR-β [27,30]. These results confirmed those from studies on isolated cells. In fact, a small percentage of gonocytes purified from three-day-old rat testis produces PDGF receptors [29]; rat PMCs from prepubertal animals show specific high-affinity PDGF receptors mainly of the β-type [31]; Pdgfa, Pdgfb, Pdgfra and Pdgfrb mRNA was extracted from purified adult rat Leydig cells [27,32]; and cultured adult Leydig cells produce PDGF-like molecules and bind PDGF [27]. In the mouse, high amounts of Pdgfra and Pdgfrb mRNA are found from E12.5 to E17.5 in the mesenchyme cells between the testicular tubules [25,33,34], whereas PDGF-A is localized to the tubular epithelium. These results were confirmed for PDGFR-β by both immunoblot analysis and immunohistochemistry. Indeed, E12.5 and E13.5 mouse testes produce increasing amounts of PDGFR-β protein along with a progressive increase of PDGFR-β-positive mesenchymal cells (A. Puglianiello et al. *) More recently, expression of Pdgfc was found on the ectodermic surface of the mouse genital regions at E12.5 [35]. Only PDGF-A and PDGFR-α localization data are available for the postnatal mouse testis. In situ hybridization and immunohistochemistry have shown PDGFR-α-positive cells in the interstitium of pubertal and adult animals. PDGF-A is seen in the Sertoli cells of all the testicular tubules before puberty and in a subset of tubules after puberty. A weak PDGF-A immunohistochemical signal is also localized in some interstitial cells at this age [25]. In summary, the spatial and temporal expression patterns PDGFR-α and PDGFR-β suggest that, with the noticeable exception of gonocytes, mesenchymal PMCs and Leydig cells are the probable targets for an autocrine or paracrine action of PDGF in the testis. The cellular origin of the PDGF ligands depends on the developmental period and the animal species. Sertoli cells are the main source of PDGF ligands before birth until puberty in both the rat and mouse. In the adult rat, there is a shift in the production of PDGF-A and PDGF-B from the Sertoli cells to the *Puglianiello, A. et al. (2000) The PDGF/PDGFR system is involved in testis morphogenesis in the mouse embryo. 11th European Workshop on Molecular and Cellular Endocrinology of the Testis. Saint-Malo, France A2 (abstract). http://tem.trends.com
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Leydig cells. However, in the adult mouse, Sertoli cells continue to produce PDGF-A even when Leydig cells start to synthesize this ligand. Information on the expression of PDGF genes in the human testis is still incomplete. However, a strong PDGF-B and PDGFR-β immunoreactivity was seen in sex cords of the human fetus after 12 weeks of gestation [36], and northern analysis of adult testicular samples revealed the presence of full-length PDGFA [9], PDGFB [11], PDGFC [9,10,37], PDGFD [11] and PDGFRA [38,39] mRNAs, suggesting that PDGF might play a role in the local control of testicular function in humans. Testicular actions of PDGF in vitro and in vivo In vitro studies
The testicular localization of the PDGF receptors suggests that PMCs, Leydig cells and possibly gonocytes might be targets for the local action of PDGF, a hypothesis that has been confirmed by in vitro and in vivo studies. PDGF is a potent chemoattractant for PMCs in culture [27] and stimulates contractility [40,41], protein synthesis [41] and the production and proliferation of extracellular matrix components in PMCs [31]. The chemotactic response of PMCs to PDGF is rapid, and PDGF dimers show different potencies, with PDGF-BB being the most active. This behavior confirms that, similar to the classic target cells for PDGF (fibroblasts and smooth muscle cells), PMCs express both α- and β-receptors, although there are generally higher levels of PDGFR-β. Moreover, PMCs show a migratory response to early postnatal Sertoli cellconditioned medium that is inhibited by anti-PDGF antibodies [27], demonstrating that this effect is PDGF mediated. The strong mitogenic and attractive properties of PDGF-B indicate that its secretion by Sertoli cells could be ideally suited to chemotactically attracting and mitogenically stimulating PMCs in close proximity of the tubule, and that the production of PDGF-B by Sertoli cells could be crucial in seminiferous tubule formation through paracrine interactions with PMC precursors. The response of PMCs to PDGF is analogous to that which has been well documented for vascular smooth muscle cells, a cell type in which PDGF is regarded as a powerful chemotactic and proliferative signal involved in vascular pathological changes [42]. Unexpectedly, a hypertrophic rather than hyperplastic response of PMC primary cultures to PDGF-BB has also been reported [41]. However, the high concentration of cells used in these experiments could have masked the PDGF-mediated proliferative effect by contact inhibition. Interestingly, the reciprocal expression of PDGF receptors by PMCs and the production of PDGFs by Sertoli cells correlates, in temporal sequence, with the timing of recruitment of PMCs from the undifferentiated intertubular stromalfibroblast population and the migration of PMCs from the interstitium to the peritubulum [43]. The
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Sertoli cells might direct the development of their neighboring PMC precursors via PDGF. The ability of rat Leydig cells to produce both PDGFs and to express PDGF receptors in adulthood suggests an autocrine effect of the PDGF system on this cell type. Accordingly, PDGF-BB inhibits the activity of 5-α-reductase and ∆5-3β-hydroxysteroid dehydrogenase in cultured Leydig cells from 25-day-old rats [48] and increases luteinizing hormone-stimulated testosterone production in vitro [28,49]. These observations are consistent with a current working model where, during the early phases of postnatal development, PDGFR-β-positive PMC precursors relocate to the peritubulum and proliferate in response to PDGF-BB produced by Sertoli cells. As testicular development progresses, the shift of production of PDGFs and PDGF receptors towards Leydig cells could modulate testosterone production in an autocrine manner (Fig. 2). PDGF-BB was also shown to induce the proliferation of isolated neonatal rat and mouse gonocytes [29,50]. These data indicate that PDGF might have functional importance for cells of non-mesenchymal origin, such as gonocytes, within the testis. However, further studies are required to confirm this hypothesis because only a small percentage of gonocytes were PDGF receptor positive and PDGF responsive. In vivo studies
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Fig. 2. Schematic illustration of the proposed model for the role of PDGF during seminiferous tubule formation in the rat. (a) During the perinatal period, PMC progenitors, scattered in the interstitium, express PDGF receptors, mainly PDGFR-β, and Sertoli cells secrete PDGF ligands. (b) Under the influence of PDGF secreted by the Sertoli cells, and in conjunction with the maturation of the tubule, PDGFR-β-positive PMC progenitors are chemotactically attracted and spread to acquire positions surrounding the tubule, proliferate and, in association with Sertoli cells, form the basal membrane. At puberty, the secretion of PDGF by Sertoli cells drops, whereas the PMCs, as part of their differentiation program, lose the ability to express PDGF receptors on their surface and acquire the ability to contract. (c) In the adult testis, the mature Leydig cells start to secrete PDGF and express PDGF receptors, modulating testosterone production autocrinally. Abbreviations: PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor; PMC, peritubular myoid cell.
expression of the PDGF receptor by PMCs also correlates with the period of highest proliferative activity of these cells [44] and the ability of PMCs to secrete extracellular matrix components to form, in cooperation with Sertoli cells, the basal membrane of the tubule [45]. PMCs cease to express PDGF receptors as soon as they shift from a synthetic to a contractile phenotype, which corresponds with the morphological and functional maturation of PMCs and the development of the ability of the tubule to contract [46,47]. This evidence supports the view that http://tem.trends.com
The description of the testicular phenotype of the Pdgfa−/− mouse has provided important insights into the in vivo function of PDGF in the male gonad, attributing a crucial role to PDGF-A in adult Leydig cell development [25]. In the fetus, Leydig cells appear in the interstitium shortly after the differentiation of the testicular cords. This population of fetal Leydig cells produces androgens that are required for masculinization during fetal and neonatal life. They regress thereafter and are replaced during puberty by adult Leydig cells, which supply the testosterone necessary for the completion of spermatogenesis and maintenance of male reproductive function. Adult Leydig cells are not derived from preexisting fetal cells but from undifferentiated mesenchymal stem cells [51]. Testicular development before birth was found to be normal in Pdgfa−/− mice, and the fetal population of Leydig cells is present and functional as demonstrated by normal androgenization of the animals [25]. Postnatally, Pdgfa-deficient mice develop a progressive reduction in testicular size, loss of Leydig cells, reduced circulating testosterone and arrested spermatogenesis, despite normal plasma levels of luteinizing hormone [25]. The cellular mechanisms responsible for these effects of null mutation can be ascribed to the lack of recruitment of the adult Leydig cell population from undifferentiated PDGFR-α-positive interstitial mesenchymal cells, putative precursors of the adult Leydig cells [25] (Fig. 3). Lack of PDGF-A could lead to adult Leydig cell deficiency through proliferative arrest and progressive
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Fig. 3. Model of Leydig cell development. (a) Adult Leydig cell PDGFR-α-positive precursors originate from PDGFR-α-positive mesenchymal cells located in the interstitium together with the fetal population of Leydig cells. (b) In early puberty, fetal Leydig cells are progressively lost and a new generation of maturing Leydig cells, not derived from preexisting fetal Leydig cells, appears. (c) In the adult, no more fetal Leydig cells are present and the interstitium is filled with mature testosterone-producing adult Leydig cells. (d) In Pdgfa−/− testis, the substitution of the fetal Leydig cells with the adult generation of cells does not occur and consequently, (e) in the adult, there is a progressive reduction in testicular size, lack of Leydig cells and arrested spermatogenesis. Abbreviations: PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor; PMC, peritubular myoid cell.
depression of the Leydig cell precursors. Alternatively, PDGF-A could directly control Leydig cell differentiation. This evidence, coupled with the distribution of PDGF-A and PDGFR-α in the normal testis, suggests that this system is essential in epithelial–mesenchymal interactions that are crucial for testicular organogenesis. http://tem.trends.com
Conclusions and perspectives
The results described here provide clear evidence that PDGF has an important role during testicular development. PDGF-B–PDGFR-β interactions might be involved in the proliferation and migration of PMC precursors from the intertubular space to the peritubulum. PDGF-A–PDGFR-α interactions drive the commitment of the precursors of the adult Leydig cells to develop. In both cases, the expression pattern of the genes encoding ligands and receptors follows the overall consensus, with ligands found in the epithelium and receptors in the mesenchyme. Genetic analysis of Pdgfa-null mice has furnished the in vivo evidence for the crucial role of PDGF-A–PDGFR-α in Leydig cell development, although further studies are required for a better understanding of the intimate cellular mechanisms of Leydig cell loss in these mice. Pdgfb- or Pdgfrbknockout mice die during late gestation and no data are available on the reproductive system of these
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Acknowledgements This work was supported by grants from the Italian Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR) and progetti di ricerca di Ateneo (ex quota 60%). S.M. and S.B. are recipients of a postdoctoral fellowship of the University of Rome ‘La Sapienza’.
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animals [19,52]. The most conspicuous phenotype of PDGF-B and PDGFR-β mutants is the absence of vascular smooth muscle cells and/or pericytes and of mesangial cells of the kidney glomerulus caused by failure of these cell progenitors to locate to their appropriate final destination, expand and differentiate. The functional and structural analogy between vascular smooth muscle cells, kidney mesangial cells and testicular PMCs suggests analogous developmental roles of PDGF-B/PDGFR-β for these cell types. The significance of the PDGF–PDGF receptor system in testicular physiology is further suggested by studies identifying important transcription factors, implicated in the male sex determination pathway, testicular development and disease [3], as regulators of PDGF chains and PDGF receptor subunit synthesis. For instance, the pattern of synthesis of PDGF-A in early and differentiated testicular epithelial structures is identical to that reported for the Wilms’ tumor suppressor gene product (WT1) in developing and adult testis [53,54]. WT1, a transcriptional factor that regulates genes essential for gonadogenesis [2], can either repress or activate PDGFA [55–58], and has been suggested to be subject to post-transcriptional processing within the Sertoli cell [59]. Whether PDGFA is one of the transcriptional targets through which the profound effects of WT1 on testicular organogenesis and function could be exerted needs further investigation. However, it is worth mentioning that the lack of adult Leydig cell development, normal appearance of Sertoli cells and seminiferous tubules with spermatogenic arrest seen in Pdgfa-knockout mice [25] closely resembles forms of male pseudohermaphroditism, which is the most common genital abnormality in XY individuals with WT1 mutations [60,61]. GATA-4, a member of the GATA
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transcription factor family, is another example of a gonad-specific regulatory factor involved in the control of the PDGF system [62]. GATA-4 is found in Sertoli cells from early fetal development to adulthood, and in Leydig cells during the fetal period of life and after puberty [63]. GATA-dependent testicular promoters have been postulated, including the promoters for genes encoding inhibin α [64], Mullerian inhibiting substance [65], SF-1 [66] and the steroidogenic acute regulatory protein [67]. The temporal relationship between GATA-4 and PDGFR-α synthesis in the testis, their colocalization in Sertoli and Leydig cells and the recognition that the promoter element of the PDGFRA gene contains a consensus binding site for GATA-4 [68] suggest that the testicular effects of GATA-4 might be mediated in part by PDGF. Unfortunately, mice carrying a null mutation for the gene encoding GATA-4 exhibit early embryonic lethality [69], precluding their use in assessing the role of GATA-4 in the gonad. However, the use of tissue-specific knockout animals might shed light on our understanding of whether the testicular effects of GATA-4 could be exerted via modulation of PDGFRA. Hyperexpression and aberrant expression of PDGFRA have also been reported in testicular tumors [38,39,70]. Being limited in number, these observations need additional investigation, particularly with the recent development of various types of PDGF antagonists, the potential clinical utility of which is currently being evaluated [71–73]. Because of the high complexity of the PDGF system, and owing to the recent cloning of two genes encoding additional PDGF receptor ligands, PDGF-C and PDGF-D, both found in testicular tissue, further studies to define the physiopathological relevance of PDGF in the testis are needed.
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15 Östman, A. et al. (1992) PDGF-AA and PDGF-BB biosynthesis: proprotein processing in the Golgi complex and lysosomal degradation of PDGF-BB retained intracellularly. J. Cell Biol. 118, 509–519 16 Betsholtz, C. (1993) The PDGF genes and their regulation. In Biology of Platelet-derived Growth Factor (Westermark, B. and Sorg, C., eds), pp. 11–30, Basel-Karger 17 Boström, H. et al. (1996) PDGF-A signaling is a critical event in lung alveolar myofibroblast development and alveogenesis. Cell 85, 863–873 18 Lindahl, P. et al. (1997) Pericyte loss and microaneurysm formation in PDGF-B-deficient mice. Science 277, 242–245 19 Levéen, P. et al. (1994) Mice deficient for PDGF B show renal, cardiovascular, and hematological abnormalities. Genes Dev. 8, 1875–1887 20 Fruttiger, M. et al. (1999) Defective oligodendrocyte development and severe hypomyelination in PDGF-A knock out. Development 126, 457–467 21 Calver, A.R. et al. (1998) Oligodendrocyte population dynamics and the role of PDGF in vivo. Neuron 20, 869–882
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22 Soriano, P. (1997) The PDGF alpha receptor is required for neural crest cell development and for normal patterning of the somites. Development 124, 2691–2700 23 Karlsson, L. et al. (1999) Role for PDGF-A and sonic hedgehog in development of mesenchymal components of the hair follicle. Development 126, 2611–2621 24 Karlsson, L. et al. (2000) Abnormal gastrointestinal development in PDGF-A and PDGFR-α deficient mice implicates a novel mesenchymal structure with putative instructive properties in villus morphogenesis. Development 127, 3457–3466 25 Gnessi, L. et al. (2000) Leydig cell loss and spermatogenic arrest in platelet-derived growth factor (PDGF)-A-deficient mice. J. Cell Biol. 149, 1019–1025 26 Östman, A. and Heldin, C.H. (2001) Involvement of platelet-derived growth factor in disease: development of specific antagonists. Adv. Cancer Res. 80, 1–38 27 Gnessi, L. et al. (1995) Testicular development involves the spatio–temporal control of PDGFs and PDGF receptors gene expression and action. J. Cell Biol. 131, 1105–1121 28 Loveland, K. et al. (1993) Identification of receptor tyrosine kinases in the rat testis. Mol. Reprod. Dev. 36, 440–447 29 Li, H. et al. (1997) Regulation of rat testis gonocyte proliferation by platelet-derived growth factor and estradiol: identification of signaling mechanisms involved. Endocrinology 138, 1289–1298 30 Gnessi, L. et al. (1992) Rat Leydig cells bind platelet-derived growth factor through specific receptors and produce platelet-derived growth factor-like molecules. Endocrinology 130, 2219–2224 31 Gnessi, L. et al. (1993) Platelet derived growth factor effects on purified testicular peritubular myoid cells: binding, cytosolic Ca2+ increase, mitogenic activity, and extracellular matrix production enhancement. Endocrinology 133, 1880–1889 32 Loveland, K. et al. (1995) Platelet-derived growth factor ligand and receptor sub-unit mRNA in the Sertoli and Leydig cells of the rat testis. Mol. Cell. Endocrinol. 108, 155–159 33 Orr-Urtreger, A. et al. (1992) Developmental expression of the alpha receptor for plateletderived growth factor, which is deleted in the embryonic lethal Patch mutation. Development 115, 289–303 34 Shinbrot, E. et al. (1994) Expression of the platelet-derived growth factor β receptor during organogenesis and tissue differentiation in the mouse embryo. Dev. Dyn. 199, 169–175 35 Ding, H. et al. (2000) The mouse Pdgfc gene: dynamic expression in embryonic tissues during organogenesis. Mech. Dev. 96, 209–213 36 Burton, P.B. et al. (1993) Growth factor expression during rat development: a comparison of TGF-β3, TGF-α, bFGF, PDGF and PDGF-R. Int. J. Exp. Pathol. 74, 87–96 37 Gilbertson, D.G. et al. (2001) Platelet-derived growth factor C (PDGF-C), a novel growth factor that binds to PDGF α and β receptor. J. Biol. Chem. 276, 27406–27414 38 Peltomäki, P. et al. (1991) Oncogenes in human testicular cancer: DNA and RNA studies. Br. J. Cancer 63, 851–858 http://tem.trends.com
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39 Mosselman, S. et al. (1996) Aberrant plateletderived growth factor α-receptor transcript as a diagnostic marker for early human germ cell tumors of the adult testis. Proc. Natl. Acad. Sci. U. S. A. 93, 2884–2888 40 Tung, P.S. and Fritz, I.V. (1991) Transforming growth factor-β and platelet-derived growth factor synergistically stimulate contraction by testicular peritubular cells in culture in serum-free medium. J. Cell. Physiol. 146, 386–393 41 Chiarenza, C. et al. (2000) Platelet-derived growth factor-BB stimulates hypertrophy of peritubular smooth muscle cells from rat testis in primary cultures. Endocrinology 141, 2971–2981 42 Giese, N.A. et al. (1999) The role of alpha and beta platelet-derived growth factor receptor in the vascular response to injury in nonhuman primates. Arterioscler. Thromb. Vasc. Biol. 19, 900–909 43 Bressler, R.S. and Ross, M.H. (1972) Differentiation of peritubular myoid cells of the testis: effects of intratesticular implantation of newborn mouse testis into normal and hypophysectomized adult. Biol. Reprod. 6, 148–159 44 Palombi, F. et al. (1992) Development and cytodifferentiation of peritubular myoid cells in the rat testis. Anat. Rec. 233, 32–40 45 Pelliniemi, L.J. et al. (1984) Extracellular matrix in testicular differentiation. Ann. New York Acad. Sci. 438, 405–416 46 Kormano, M. and Hovatta, O. (1972) Contractility and histochemistry of the myoid cell layer during postnatal development. Z. Anat. Entwicklungsgesch. 137, 239–248 47 Worley, R.T.S. et al. (1984) Testicular oxytocin: an initiator of seminiferous tubule movement? In Recent Progress in Cellular Endocrinology of the Testis (Saez, J.M. et al., eds), pp. 205–212, INSERM 48 Murono, E.P. and Washburn, A.L. (1990) Platelet derived growth factor inhibits 5α-reductase and ∆5-3β-hydroxysteroid dehydrogenase activities in cultured immature Leydig cells. Biochem. Biophys. Res. Commun. 169, 1229–1234 49 Risbridger, G.P. et al. (1993) Discrete stimulatory effects of platelet-derived growth factor (PDGFBB) on Leydig cell steroidogenesis. Mol. Cell. Endocrinol. 97, 125–128 50 Hasthorpe, S. et al. (1999) Neonatal mouse gonocyte proliferation assayed by an in vitro clonogenic method. J. Reprod. Fertil. 116, 335–344 51 McLaren, A. (1998) Gonad development: assembling the mammalian testis. Curr. Biol. 8, R175–R177 52 Soriano, P. (1994) Abnormal kidney development and hematological disorders in PDGF β-receptor mutant mice. Genes Dev. 8, 1888–1896 53 Mundlos, S. et al. (1993) Nuclear localization of the protein encoded by the Wilms’ tumor gene WT1 in embryonic and adult tissues. Development 119, 1329–1341 54 de Santa Barbara, P. et al. (2000) Expression and subcellular localization of SF-1, SOX9, WT1, and AMH proteins during early human testicular development. Dev. Dyn. 217, 293–298 55 Wang, Z.Y. et al. (1992) The Wilms’ tumor gene product, WT1, represses transcription of the platelet-derived growth factor A-chain gene. J. Biol. Chem. 267, 21999–22002
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PDGF and the testis Stefania Mariani, Sabrina Basciani, Mario Arizzi, Giovanni Spera and Lucio Gnessi Testicular development is controlled by a complex hierarchy of gene regulatory proteins, growth factors, cell adhesion molecules, signaling molecules and hormones that interact, often acting within short time windows, via reciprocal control relationships. The identification in the testis of platelet-derived growth factor (PDGF), a key regulator of connective tissue cells in embryogenesis and pathogenesis, has focused attention on the role of this growth factor in testicular pathophysiology. This review summarizes recent advances in the study of the actions of PDGF in the male gonad, and attempts to incorporate complex in vitro and in vivo experimental data into a model that might clarify the role played by PDGF in the mammalian testis.
Testicular development and functional control involve a complex combination of cell proliferation, hypertrophy, migration, differentiation and apoptosis, which occur within strict temporal and anatomical constraints [1]. These highly coordinated processes, driven by the sequential activation of specific genes [2,3], require a precise temporal regulation of growth and differentiation of somatic and germ cell elements and imply several cell–cell interactions that are accomplished by locally produced growth and differentiation factors, hormones and cell adhesion molecules [4]. During the past decade, evidence has accumulated that shows that platelet-derived growth factor (PDGF) should be included as one of the locally produced growth factors that mediate testicular cell–cell interactions. This review focuses on the role of PDGF in the male gonad during prenatal and postnatal phases of development. Stefania Mariani Sabrina Basciani Mario Arizzi Giovanni Spera Lucio Gnessi* Dept Medical Physiopathology, Policlinico Umberto I, University of Rome ‘La Sapienza’, 00161 Rome, Italy. *e-mail: [email protected]
Structure and general functions of PDGFs and PDGF receptors PDGF isoforms
PDGFs are members of the PDGF-vascular endothelial growth factor (PDGF-VEGF) family of growth factors [5]. Their constituent polypeptide chains share a core motif of Cys residues with a characteristic spacing [6]. The three-dimensional structure of PDGFs is similar to that of VEGFs, but also bears some resemblance to the structures of http://tem.trends.com
glycoprotein hormones, of nerve growth factor and of the transforming growth factor β family of peptides, despite the fact that there is no amino acid sequence similarity among them [7]. All of these factors have dimeric configurations and show the characteristic Cys-knot motifs that are involved in the formation of inter- and intramolecular disulfide bonds [8]. For almost 20 years, only two PDGF polypeptides, PDGF-A and PDGF-B, were known. Recently, however, PDGF-C [9,10] and PDGF-D [11–13], two new PDGFs, were discovered. The biologically active PDGF molecules are either homodimers or heterodimers. The four homodimers, PDGF-AA, PDGF-BB, PDGF-CC and PDGF-DD, and the heterodimer, PDGF-AB, have all been shown to be endogenous cell products [14]. PDGF-C does not heterodimerize with PDGF-A or PDGF-B, probably because PDGF-C is rather distantly related to PDGF-A and PDGF-B in its core domain. By contrast, PDGF-C and PDGF-D are closely related structurally, but it remains to be established whether they can heterodimerize. PDGF-C and PDGF-D are as closely related to the VEGFs as they are to PDGF-A and PDGF-B, based on their primary sequence. However, they are characterized as novel PDGFs because of their PDGF receptor binding specificity. The A- and B-chains of PDGF are synthesized as precursor molecules that undergo proteolytic processing at the N-termini and, in the case of the B-chain, also intracellularly at the C-terminus [15]. Cells naturally producing both A- and B-chains contain all three PDGF isoforms, suggesting that the assembly of PDGF dimers could be a random process. PDGF bioavailability in vivo is also dependent on the association of the secreted growth factor with extracellular matrix molecules, which for PDGF-A and PDGF-B seems to be mediated by a C-terminal basic motif [16]. PDGF-C and PDGF-D possess an N-terminal domain (CUB domain; domain found in complement subcomponents C1r/C1s, urchin epidermal growth factor-like protein and bone
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and inhibited by PDGFR-α, and the receptor heterodimer appears to mediate a slightly stronger mitogenic signal than do either of the two receptor homodimers [6]. Functions of PDGF
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Fig. 1. Schematic representation of PDGF ligand and PDGF receptor isoforms and binding specificities. A- and B-chains of PDGF are synthesized as precursor molecules that undergo proteolytic processing and intracellular assembly before secretion. PDGF-C and PDGF-D possess an N-terminal domain, referred to as a CUB domain, which remains on the secreted PDGF-CC and PDGF-DD molecules. Extracellular proteolytic removal of the CUB domain from the secreted PDGF-CC and PDGF-DD dimers is a prerequisite for binding to PDGF receptors. The different PDGF isoforms bind to and dimerize α- and β-receptors into the configuration shown. The extracellular part of the receptors consist of five Ig-like domains. Ligands bind to the three outermost Ig-like domains, whereas the fourth domain is involved in direct receptor–receptor interactions. Intracellular parts of receptors contain tyrosine kinase domains. Abbreviations: CUB, domain found in complement subcomponents C1r/C1s, urchin epidermal growth factor-like protein and bone morphogenetic protein 1; Ig, immunoglobulin; PDGF, platelet-derived growth factor.
morphogenetic protein 1), which appears not to be removed obligatorily by intracellular proteolytic processing before secretion. Proteolytic removal of the CUB domain is a prerequisite for binding to PDGF receptors, but the proteases involved in the assumed extracellular processing are unknown. Thus, PDGF-CC and PDGF-DD are synthesized and secreted as latent growth factors.
In vitro, a list of the best-documented functions of PDGFs on target cells includes: proliferation, migration and actin reorganization, contraction, extracellular matrix production, differentiation and survival [6]. Gene-targeting approaches have provided important information about the physiological role of the PDGFs [14]. The phenotypical analysis of Pdgf-knockout mice has revealed generic features of the function of PDGF-A and PDGF-B. Three types of smooth muscle cells and/or myofibroblasts show an obligatory requirement for PDGF during development. Alveolar smooth muscle cells depend on PDGF-A [17], and microvascular pericytes and kidney glomerular mesangial cells depend on PDGF-B [18,19]. Furthermore, PDGF-A and its cognate receptor are required for oligodendrocyte development [20–22], development of mesenchymal components of the hair follicle [23], gastrointestinal villus morphogenesis [24] and adult Leydig cell recruitment [25]. No data are available on the in vivo functions of PDGF-C and PDGF-D. However, the phenotypic differences between mice lacking PDGFR-α and those lacking PDGF-A support the idea that the other PDGFR-α ligand, PDGF-C, might provide distinct signals for PDGFR-α-expressing cells. Comparisons of mice lacking PDGFR-β and those lacking PDGF-B have not revealed a phenotypic discrepancy, indicating that PDGF-D and PDGF-B might have partially redundant functions. Thus, PDGFs provide selective signals during development by acting on the PDGF receptor-carrying progenitor cells, enabling them to proliferate and spread on PDGF-producing endothelial and/or epithelial layers. Overexpression of the PDGF gene has been observed in several pathological conditions, including malignancies, atherosclerosis and fibroproliferative diseases [26].
PDGF receptors
Developmental production of PDGFs and PDGF receptors in the mammalian testis
PDGFs interact with two different but structurally related cell surface receptor tyrosine kinases (PDGFR-α and PDGFR-β), which dimerize upon ligand binding and can be expressed individually or together in cells [14]. The PDGF receptor dimers have different ligand-binding capacities: PDGFR-αα binds PDGF-AA, PDGF-BB, PDGF-AB and PDGF-CC; PDGFR-ββ binds PDGF-BB and PDGF-DD; and PDGFR-αβ binds PDGF-AB, PDGF-BB, PDGF-CC and PDGF-DD (Fig. 1). PDGF receptors have partially overlapping and partially distinct signaling capabilities, which could have specific biological consequences. For example, chemotaxis of fibroblasts and smooth muscle cells is stimulated by PDGFR-β
PDGFs and PDGF receptors are produced in the testis during prenatal and postnatal life in a timeand space-dependent manner. In the rat, transcripts from the Pdgfa, Pdgfb, Pdgfra and Pdfgrb genes are found on embryonic day 18 (E18), reach high levels by postnatal day 5 (P5) and then decline to lower levels in older animals [27]. These age-related changes were confirmed for Pdgfra – the highest levels of transcript were observed in the testes of five-day-old animals, followed by a decline in relative abundance with increasing age [28]. Localization studies have shown that, in the prenatal period, Sertoli cells are the primary source of PDGF-A and PDGF-B molecules, whereas the cells expressing PDGFR-α and PDGFR-β
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are scattered in the tissue between the testicular cords [29]. During the first week of postnatal life, the rat testis shows an intense staining for PDGF-A and PDGF-B in the cytoplasm of the Sertoli cells, and PDGFR-α- and -β-subunit immunoreactivity in the peritubular myoid cells (PMCs) [27,29]. Li and colleagues also reported PDGFR production in some but not all gonocytes during the first five days after birth [29]. In pubertal animals, only minimal positive staining is seen for PDGF-A and PDGF-B in Sertoli cells and for PDGFR-α- and -β-subunits in PMCs. Finally, in the adult, Leydig cells are the only cellular component showing a positive reaction for PDGF-A, PDGF-B, PDGFR-α and PDGFR-β [27,30]. These results confirmed those from studies on isolated cells. In fact, a small percentage of gonocytes purified from three-day-old rat testis produces PDGF receptors [29]; rat PMCs from prepubertal animals show specific high-affinity PDGF receptors mainly of the β-type [31]; Pdgfa, Pdgfb, Pdgfra and Pdgfrb mRNA was extracted from purified adult rat Leydig cells [27,32]; and cultured adult Leydig cells produce PDGF-like molecules and bind PDGF [27]. In the mouse, high amounts of Pdgfra and Pdgfrb mRNA are found from E12.5 to E17.5 in the mesenchyme cells between the testicular tubules [25,33,34], whereas PDGF-A is localized to the tubular epithelium. These results were confirmed for PDGFR-β by both immunoblot analysis and immunohistochemistry. Indeed, E12.5 and E13.5 mouse testes produce increasing amounts of PDGFR-β protein along with a progressive increase of PDGFR-β-positive mesenchymal cells (A. Puglianiello et al. *) More recently, expression of Pdgfc was found on the ectodermic surface of the mouse genital regions at E12.5 [35]. Only PDGF-A and PDGFR-α localization data are available for the postnatal mouse testis. In situ hybridization and immunohistochemistry have shown PDGFR-α-positive cells in the interstitium of pubertal and adult animals. PDGF-A is seen in the Sertoli cells of all the testicular tubules before puberty and in a subset of tubules after puberty. A weak PDGF-A immunohistochemical signal is also localized in some interstitial cells at this age [25]. In summary, the spatial and temporal expression patterns PDGFR-α and PDGFR-β suggest that, with the noticeable exception of gonocytes, mesenchymal PMCs and Leydig cells are the probable targets for an autocrine or paracrine action of PDGF in the testis. The cellular origin of the PDGF ligands depends on the developmental period and the animal species. Sertoli cells are the main source of PDGF ligands before birth until puberty in both the rat and mouse. In the adult rat, there is a shift in the production of PDGF-A and PDGF-B from the Sertoli cells to the *Puglianiello, A. et al. (2000) The PDGF/PDGFR system is involved in testis morphogenesis in the mouse embryo. 11th European Workshop on Molecular and Cellular Endocrinology of the Testis. Saint-Malo, France A2 (abstract). http://tem.trends.com
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Leydig cells. However, in the adult mouse, Sertoli cells continue to produce PDGF-A even when Leydig cells start to synthesize this ligand. Information on the expression of PDGF genes in the human testis is still incomplete. However, a strong PDGF-B and PDGFR-β immunoreactivity was seen in sex cords of the human fetus after 12 weeks of gestation [36], and northern analysis of adult testicular samples revealed the presence of full-length PDGFA [9], PDGFB [11], PDGFC [9,10,37], PDGFD [11] and PDGFRA [38,39] mRNAs, suggesting that PDGF might play a role in the local control of testicular function in humans. Testicular actions of PDGF in vitro and in vivo In vitro studies
The testicular localization of the PDGF receptors suggests that PMCs, Leydig cells and possibly gonocytes might be targets for the local action of PDGF, a hypothesis that has been confirmed by in vitro and in vivo studies. PDGF is a potent chemoattractant for PMCs in culture [27] and stimulates contractility [40,41], protein synthesis [41] and the production and proliferation of extracellular matrix components in PMCs [31]. The chemotactic response of PMCs to PDGF is rapid, and PDGF dimers show different potencies, with PDGF-BB being the most active. This behavior confirms that, similar to the classic target cells for PDGF (fibroblasts and smooth muscle cells), PMCs express both α- and β-receptors, although there are generally higher levels of PDGFR-β. Moreover, PMCs show a migratory response to early postnatal Sertoli cellconditioned medium that is inhibited by anti-PDGF antibodies [27], demonstrating that this effect is PDGF mediated. The strong mitogenic and attractive properties of PDGF-B indicate that its secretion by Sertoli cells could be ideally suited to chemotactically attracting and mitogenically stimulating PMCs in close proximity of the tubule, and that the production of PDGF-B by Sertoli cells could be crucial in seminiferous tubule formation through paracrine interactions with PMC precursors. The response of PMCs to PDGF is analogous to that which has been well documented for vascular smooth muscle cells, a cell type in which PDGF is regarded as a powerful chemotactic and proliferative signal involved in vascular pathological changes [42]. Unexpectedly, a hypertrophic rather than hyperplastic response of PMC primary cultures to PDGF-BB has also been reported [41]. However, the high concentration of cells used in these experiments could have masked the PDGF-mediated proliferative effect by contact inhibition. Interestingly, the reciprocal expression of PDGF receptors by PMCs and the production of PDGFs by Sertoli cells correlates, in temporal sequence, with the timing of recruitment of PMCs from the undifferentiated intertubular stromalfibroblast population and the migration of PMCs from the interstitium to the peritubulum [43]. The
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Sertoli cells might direct the development of their neighboring PMC precursors via PDGF. The ability of rat Leydig cells to produce both PDGFs and to express PDGF receptors in adulthood suggests an autocrine effect of the PDGF system on this cell type. Accordingly, PDGF-BB inhibits the activity of 5-α-reductase and ∆5-3β-hydroxysteroid dehydrogenase in cultured Leydig cells from 25-day-old rats [48] and increases luteinizing hormone-stimulated testosterone production in vitro [28,49]. These observations are consistent with a current working model where, during the early phases of postnatal development, PDGFR-β-positive PMC precursors relocate to the peritubulum and proliferate in response to PDGF-BB produced by Sertoli cells. As testicular development progresses, the shift of production of PDGFs and PDGF receptors towards Leydig cells could modulate testosterone production in an autocrine manner (Fig. 2). PDGF-BB was also shown to induce the proliferation of isolated neonatal rat and mouse gonocytes [29,50]. These data indicate that PDGF might have functional importance for cells of non-mesenchymal origin, such as gonocytes, within the testis. However, further studies are required to confirm this hypothesis because only a small percentage of gonocytes were PDGF receptor positive and PDGF responsive. In vivo studies
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Fig. 2. Schematic illustration of the proposed model for the role of PDGF during seminiferous tubule formation in the rat. (a) During the perinatal period, PMC progenitors, scattered in the interstitium, express PDGF receptors, mainly PDGFR-β, and Sertoli cells secrete PDGF ligands. (b) Under the influence of PDGF secreted by the Sertoli cells, and in conjunction with the maturation of the tubule, PDGFR-β-positive PMC progenitors are chemotactically attracted and spread to acquire positions surrounding the tubule, proliferate and, in association with Sertoli cells, form the basal membrane. At puberty, the secretion of PDGF by Sertoli cells drops, whereas the PMCs, as part of their differentiation program, lose the ability to express PDGF receptors on their surface and acquire the ability to contract. (c) In the adult testis, the mature Leydig cells start to secrete PDGF and express PDGF receptors, modulating testosterone production autocrinally. Abbreviations: PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor; PMC, peritubular myoid cell.
expression of the PDGF receptor by PMCs also correlates with the period of highest proliferative activity of these cells [44] and the ability of PMCs to secrete extracellular matrix components to form, in cooperation with Sertoli cells, the basal membrane of the tubule [45]. PMCs cease to express PDGF receptors as soon as they shift from a synthetic to a contractile phenotype, which corresponds with the morphological and functional maturation of PMCs and the development of the ability of the tubule to contract [46,47]. This evidence supports the view that http://tem.trends.com
The description of the testicular phenotype of the Pdgfa−/− mouse has provided important insights into the in vivo function of PDGF in the male gonad, attributing a crucial role to PDGF-A in adult Leydig cell development [25]. In the fetus, Leydig cells appear in the interstitium shortly after the differentiation of the testicular cords. This population of fetal Leydig cells produces androgens that are required for masculinization during fetal and neonatal life. They regress thereafter and are replaced during puberty by adult Leydig cells, which supply the testosterone necessary for the completion of spermatogenesis and maintenance of male reproductive function. Adult Leydig cells are not derived from preexisting fetal cells but from undifferentiated mesenchymal stem cells [51]. Testicular development before birth was found to be normal in Pdgfa−/− mice, and the fetal population of Leydig cells is present and functional as demonstrated by normal androgenization of the animals [25]. Postnatally, Pdgfa-deficient mice develop a progressive reduction in testicular size, loss of Leydig cells, reduced circulating testosterone and arrested spermatogenesis, despite normal plasma levels of luteinizing hormone [25]. The cellular mechanisms responsible for these effects of null mutation can be ascribed to the lack of recruitment of the adult Leydig cell population from undifferentiated PDGFR-α-positive interstitial mesenchymal cells, putative precursors of the adult Leydig cells [25] (Fig. 3). Lack of PDGF-A could lead to adult Leydig cell deficiency through proliferative arrest and progressive
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(a)
Adult Leydig cell precursor PDGFR-α
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GF
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Fig. 3. Model of Leydig cell development. (a) Adult Leydig cell PDGFR-α-positive precursors originate from PDGFR-α-positive mesenchymal cells located in the interstitium together with the fetal population of Leydig cells. (b) In early puberty, fetal Leydig cells are progressively lost and a new generation of maturing Leydig cells, not derived from preexisting fetal Leydig cells, appears. (c) In the adult, no more fetal Leydig cells are present and the interstitium is filled with mature testosterone-producing adult Leydig cells. (d) In Pdgfa−/− testis, the substitution of the fetal Leydig cells with the adult generation of cells does not occur and consequently, (e) in the adult, there is a progressive reduction in testicular size, lack of Leydig cells and arrested spermatogenesis. Abbreviations: PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor; PMC, peritubular myoid cell.
depression of the Leydig cell precursors. Alternatively, PDGF-A could directly control Leydig cell differentiation. This evidence, coupled with the distribution of PDGF-A and PDGFR-α in the normal testis, suggests that this system is essential in epithelial–mesenchymal interactions that are crucial for testicular organogenesis. http://tem.trends.com
Conclusions and perspectives
The results described here provide clear evidence that PDGF has an important role during testicular development. PDGF-B–PDGFR-β interactions might be involved in the proliferation and migration of PMC precursors from the intertubular space to the peritubulum. PDGF-A–PDGFR-α interactions drive the commitment of the precursors of the adult Leydig cells to develop. In both cases, the expression pattern of the genes encoding ligands and receptors follows the overall consensus, with ligands found in the epithelium and receptors in the mesenchyme. Genetic analysis of Pdgfa-null mice has furnished the in vivo evidence for the crucial role of PDGF-A–PDGFR-α in Leydig cell development, although further studies are required for a better understanding of the intimate cellular mechanisms of Leydig cell loss in these mice. Pdgfb- or Pdgfrbknockout mice die during late gestation and no data are available on the reproductive system of these
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Acknowledgements This work was supported by grants from the Italian Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR) and progetti di ricerca di Ateneo (ex quota 60%). S.M. and S.B. are recipients of a postdoctoral fellowship of the University of Rome ‘La Sapienza’.
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animals [19,52]. The most conspicuous phenotype of PDGF-B and PDGFR-β mutants is the absence of vascular smooth muscle cells and/or pericytes and of mesangial cells of the kidney glomerulus caused by failure of these cell progenitors to locate to their appropriate final destination, expand and differentiate. The functional and structural analogy between vascular smooth muscle cells, kidney mesangial cells and testicular PMCs suggests analogous developmental roles of PDGF-B/PDGFR-β for these cell types. The significance of the PDGF–PDGF receptor system in testicular physiology is further suggested by studies identifying important transcription factors, implicated in the male sex determination pathway, testicular development and disease [3], as regulators of PDGF chains and PDGF receptor subunit synthesis. For instance, the pattern of synthesis of PDGF-A in early and differentiated testicular epithelial structures is identical to that reported for the Wilms’ tumor suppressor gene product (WT1) in developing and adult testis [53,54]. WT1, a transcriptional factor that regulates genes essential for gonadogenesis [2], can either repress or activate PDGFA [55–58], and has been suggested to be subject to post-transcriptional processing within the Sertoli cell [59]. Whether PDGFA is one of the transcriptional targets through which the profound effects of WT1 on testicular organogenesis and function could be exerted needs further investigation. However, it is worth mentioning that the lack of adult Leydig cell development, normal appearance of Sertoli cells and seminiferous tubules with spermatogenic arrest seen in Pdgfa-knockout mice [25] closely resembles forms of male pseudohermaphroditism, which is the most common genital abnormality in XY individuals with WT1 mutations [60,61]. GATA-4, a member of the GATA
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