Role of neurotropins in rat embryonic testis morphogenesis (cord formation)

The process of seminiferous cord formation is the first morphological event that differentiates a testis from an ovary and indicates male sex determination. Cord formation occurs by embryonic Day 14 (Day 0 = plug date; E14) in the rat. A series of experiments were conducted to determine if neurotropins and their receptors are important for the process of rat embryonic cord formation. The expression of low affinity neurotropin receptor (p75/LNGFR) was determined by immunohistochemistry on sections of both testis and ovary from E13 through birth (Day 0, P0) with an antibody to p75/LNGFR. The staining for p75/LNGFR was present in the mesonephros of E13 gonads and in a sex-specific manner appeared around developing cords at E14 in the embryonic testis. At birth, staining for p75/LNGFR was localized to a single layer of cells (i.e., peritubular cells) that surrounded the seminiferous cords. The genes for both neurotropin 3 (NT3) and for corresponding high affinity neurotropin trkC receptor were found to be expressed in the E14 rat testis, as well as other neurotropins and receptors. Immunocytochemical analysis of E14 rat testis demonstrated that NT3 was localized to the Sertoli cells and trkC was present in individual cells of the interstitium at E16 and in selected preperitubular cells at E18. Previously, the peritubular cells adjacent to the cords were demonstrated to be derived from migrating mesonephros cells around the time of cord formation. To determine if neurotropins were involved in cord formation, the actions of neurotropins were inhibited. A high affinity neurotropin receptor (trk)-specific kinase inhibitor, K252a, was used to treat organ cultures of testes from E13 rats prior to cord formation. Treatment of E13 testis organ cultures with K252a completely inhibited cord formation. K252a-treated organ cultures of E14 testis that contained cords did not alter cord morphology. A second experiment to inhibit neurotropin actions utilized a specific antagonist trk-IgG chimeric fusion protein and E13 testis organ cultures. The trk-IgG molecules dimerize with endogenous trk receptors and inhibit receptor signaling and activation of ligand function. Forty percent of E13 testis organ cultures treated with trkC-IgG had significantly reduced cord formation. TrkA-IgG had no effect on initiation of cords; however, in fifty percent of the treated organs, a "swollen" appearance of the cord structures was observed. Experiments using trkB-IgG chimeric protein on E13 organ cultures had no effect on cord formation or cord morphology. The testes from trkC and NT3 knockout mice were examined to determine if there were any morphological differences in the testis. NT3 knockouts appeared to have normal cord morphology in E15 and E17 testis. TrkC knockout mice also had normal cord morphology in E14 and P0 testis. Both NT3 and trkC knockout-mice testis had less interstitial area than wild-type controls. In addition, the trkC knockout mice have an increased number of cells expressing p75LNGFR within the cords when compared to controls or NT3 knockout mice. Combined observations suggest compensation between the different neurotropin ligands, receptors, and/or possibly different growth factors for this critical biological process. In summary, results suggest a novel nonneuronal role for neurotropins in the process of cord formation during embryonic rat testis development. The hypothesis developed is that neurotropins are involved in the progression of male sex differentiation and are critical for the induction of embryonic testis cord formation.     

PDGF-PDGFR network differentially regulates the fate,migration, proliferation,and cell cycle progression of myogenic cells

Platelet-derived growth factors (PDGFs) regulate embryonic development, tissue regeneration, and wound healing through their binding to PDGF receptors, PDGFRα and PDGFRβ. However, the role of PDGF signaling in regulating muscle development and regeneration remains elusive, and the cellular and molecular responses of myogenic cells are understudied. Here, we explore the PDGF-PDGFR gene expression changes and their involvement in skeletal muscle myogenesis and myogenic fate. By surveying bulk RNA sequencing and single-cell profiling data of skeletal muscle stem cells, we show that myogenic progenitors and muscle stem cells differentially express PDGF ligands and PDGF receptors during myogenesis. Quiescent adult muscle stem cells and myoblasts preferentially express PDGFRβ over PDGFRα. Remarkably, cell culture- and injury-induced muscle stem cell activation altered PDGF family gene expression. In myoblasts, PDGF-AB and PDGF-BB treatments activate two pro-chemotactic and pro-mitogenic downstream transducers, RAS-ERK1/2 and PI3K-AKT. PDGFRs inhibitor AG1296 inhibited ERK1/2 and AKT activation, myoblast migration, proliferation, and cell cycle progression induced by PDGF-AB and PDGF-BB. We also found that AG1296 causes myoblast G0/G1 cell cycle arrest. Remarkably, PDGF-AA did not promote a noticeable ERK1/2 or AKT activation, myoblast migration, or expansion. Also, myogenic differentiation reduced the expression of both PDGFRα and PDGFRβ, whereas forced PDGFRα expression impaired myogenesis. Thus, our data highlight PDGF signaling pathway to stimulate satellite cell proliferation aiming to enhance skeletal muscle regeneration and provide a deeper understanding of the role of PDGF signaling in non-fibroblastic cells.     

Developmental roles of platelet-derived growth factors

Platelet-derived growth factor (PDGF) was originally identified in platelets and in serum as a mitogen for fibroblasts, smooth muscle cells (SMC) and glia cells in culture. PDGF has since expanded to a family of dimers of at least four gene products, whose biological actions are mediated through two receptor tyrosine kinases, PDGFRs. The present review summarizes and discusses the biological functions of PDGFs and PDGFRs in developmental processes, mainly as revealed through genetic analysis in mice. Such studies have demonstrated multiple critical roles of PDGFs and PDGFRs in embryonic and postnatal development. PDGFs seem to act upon specific populations of progenitor cells that give rise to several different cell types with distinct functions in a variety of developmental processes. Analogies are seen between the cell functions and the developmental processes controlled by PDGFs. This suggests that ancestral PDGF and PDGFR expression patterns and functions may have been iterated in related sets of morphogenetic processes in the course of evolution.     

Embryonic mouse testis development: role of platelet derived growth factor (PDGF-BB)

Platelet-derived growth factors (PDGFs) are paracrine growth factors mediating epithelial-mesenchymal interactions and exerting multiple biological activities which include cell proliferation, motility, and differentiation. As previously demonstrated, PDGFs act during embryonic development and recently, by culturing male genital ridges, we have demonstrated that PDGF-BB is able to support in vitro testicular cord formation. In the present paper, we report that PDGF-BB is present during embryonic testis development and, in organ culture, induces cord formation although with reduced diameters compared with the cords formed in the genital ridges cultured in the presence of HGF. Moreover we have analyzed the roles exerted by this growth factor during the morphogenesis of the testis. We demonstrate by immunohistochemical experiments that PDGF-BB and its receptors are synthesized by the male UGRs isolated from 11.5 and 13.5 dpc embryos and by Western blot that the factor is secreted in a biologically active form by testicular cells isolated from 13.5 dpc embryos. The biological roles of the factor have also been studied and we demonstrate that PDGF-BB acts as a migratory factor for male mesonephric cells whose migration is a male specific event necessary for a normal testicular morphogenesis. In addition we demonstrate that during testicular development, PDGF-BB induces testicular cell proliferation being in this way responsible for the increase in size of the testis. Finally we demonstrate that PDGF-BB is able to reorganize dissociated testicular cells inducing the formation of large cellular aggregates. However the structures formed in vitro under PDGF-BB stimulation never had a cord-like morphology similar to the cord-like structures formed in the presence of HGF (Ricci et al., 2002, Mech Dev 118:19-28), suggesting that this factor does not act as a morphogenetic factor during testicular development. All together the data presented in this paper demonstrate that PDGF-BB and its receptors (alpha- and beta-subunits) are present during the crucial ages of embryonic mouse testis morphogenesis and indicate the multiple roles exerted by this factor during the development of the male gonad.     

血小板源生长因子受体与肿瘤

血小板源生长因子(platelet-derived growth factor,PDGF)经由其受体(platelet-derived growth fac tor receptor,PDGFR)表现细胞效应。PDGF和PDGFR涉及多种肿瘤的发病机制并在血管生成中起重要作用。PDGF在肿瘤中的自分泌刺激、PDGFR的过表达或过度活化或者刺激肿瘤内血管生成都会促进肿瘤生长;PDGFR的阻断可以降低实体瘤中组织间质液压而增强药物传送。这些机制可能提示在肿瘤治疗中PDGFR抑制剂单用、与化疗药物或者和其他靶点药物联合用药的可能性和可行性。随着PDGFR拮抗剂,如imatinib的上市,PDGFR作为抗肿瘤药物的靶点备受瞩目。     

Testis developmental phenotypes in neurotropin receptor trkA and trkC null mutations: role in formation of seminiferous cords and germ cell survival

The objective of the present study was to determine if the neurotropin receptors trkC and trkA are involved in embryonic testis development. These receptors bind neurotropin 3 and nerve growth factor, respectively. The hypothesis tested was that the absence of trkC or trkA receptors will have detrimental effects on testis development and morphology. The trkA and trkC homozygote knockout (KO) mice generally die either at or shortly after birth. Therefore, heterozygote mice were mated to obtain homozygote gene KO mice at Embryonic Day (E) 13, E14, E17, and E19 of gestation, with E0 being the plug date. Gonads from approximately 80 embryos were collected and fixed, and each embryo was genotyped. To determine gonadal characteristics for each genotype, the number of germ cells, number of seminiferous cords, seminiferous cord area, and interstitial area were calculated at each developmental age. Germ cell numbers varied in trkA gene KO mice from those of wild-type mice at each age evaluated. In trkC gene KO mice, differences were detected in germ cell numbers when compared to wild-type mice at E17 and E19. At E19, germ cell numbers were reduced in both trkA and trkC gene KO mice when compared to wild-type animals. Apoptosis was evaluated in testes of wild-type, trkC gene KO, and trkA gene KO mice to determine if the alteration in germ cell numbers at each developmental age was influenced by different patterns of germ cell survival or apoptosis. No differences were found in germ cell apoptosis during embryonic testis development. Interestingly, trkA gene KO mice that survived to Postnatal Day 19 had a 10-fold increase in germ cell apoptosis when compared to germ cells in wild-type mice. Evaluation of other morphological testis parameters demonstrated that trkC KO testes had reduced interstitial area at E13, reduced number of seminiferous cords at E14, and reduced seminiferous cord area at E19. The trkA gene KO testes had a reduction in the number of seminiferous cords at E14. Histology of both trkA and trkC gene KO testes demonstrated that these gonads appear to be developmentally delayed when compared to their wild-type testis counterparts at E13 during testis development. The current study demonstrates that both trkA and trkC neurotropin receptors influence germ cell numbers during testis development and events such as seminiferous cord formation.     

Platelet-derived growth factors and their receptors: Structural and functional perspectives

The four types of platelet-derived growth factors (PDGFs) and the two types of PDGF receptors (PDGFRs, which belong to class III receptor tyrosine kinases) have important functions in the development of connective tissue cells. Recent structural studies have revealed novel mechanisms of PDGFs in propeptide loading and receptor recognition/activation. The detailed structural understanding of PDGF–PDGFR signaling has provided a template that can aid therapeutic intervention to counteract the aberrant signaling of this normally silent pathway, especially in proliferative diseases such as cancer. This review summarizes the advances in the PDGF system with a focus on relating the structural and functional understandings, and discusses the basic aspects of PDGFs and PDGFRs, the mechanisms of activation, and the insights into the therapeutic antagonism of PDGFRs. This article is part of a Special Issue entitled: Emerging recognition and activation mechanisms of receptor tyrosine kinases.     

Chemotactic role of neurotropin 3 in the embryonic testis that facilitates male sex determination

The first morphological event after initiation of male sex determination is seminiferous cord formation in the embryonic testis. Cord formation requires migration of pre-peritubular myoid cells from the adjacent mesonephros. The embryonic Sertoli cells are the first testicular cells to differentiate and have been shown to express neurotropin-3 (NT3), which can act on high-affinity trkC receptors expressed on migrating mesonephros cells. NT3 expression is elevated in the embryonic testis during the time of seminiferous cord formation. A trkC receptor tyrophostin inhibitor, AG879, was found to inhibit seminiferous cord formation and mesonephros cell migration. Beads containing NT3 were found to directly promote mesonephros cell migration into the gonad. Beads containing other growth factors such as epidermal growth factor (EGF) did not influence cell migration. At male sex determination the SRY gene promotes testis development and the expression of downstream sex differentiation genes such as SOX-9. Inhibition of NT3 actions caused a reduction in the expression of SOX-9. Combined observations suggest that when male sex determination is initiated, the developing Sertoli cells express NT3 as a chemotactic agent for migrating mesonephros cells, which are essential to promote embryonic testis cord formation and influence downstream male sex differentiation.     

Seminiferous cord formation is regulated by hedgehog signaling in the marsupial

The signaling molecule DHH, secreted by Sertoli cells, has essential regulatory functions in testicular differentiation. DHH is required for the differentiation of peritubular myoid cells that line the seminiferous cords and steroidogenic Leydig cells. The testicular cords in Dhh-null male mice lack a basal lamina and develop abnormally. To date, the DHH-signaling pathway has never been examined outside of any eutherian mammals. This study examined the effects of inhibition of DHH signaling in a marsupial mammal, the tammar wallaby, by culturing gonads in vitro in the presence of the hedgehog-signaling inhibitors cyclopamine and forskolin. Disruption of hedgehog signaling in the tammar testes caused highly disorganized cord formation. SOX9 protein remained strongly expressed in Sertoli cells, laminin distribution was highly fragmented, and germ cells were distributed around the cortical regions of treated testes in an ovarianlike morphology. This suggests that hedgehog signaling regulates cord formation in the tammar wallaby testis as it does in eutherian mammals. These data demonstrate that the hedgehog pathway has been highly conserved in mammals for at least 160 million years.     

Vascular endothelial growth factor and kinase domain region receptor are involved in both seminiferous cord formation and vascular development during testis morphogenesis in the rat

Morphological male sex determination is dependent on migration of endothelial and preperitubular cells from the adjacent mesonephros into the developing testis. Our hypothesis is that VEGFA and its receptor KDR are necessary for both testicular cord formation and neovascularization. The Vegfa gene has 8 exons with many splice variants. Vegfa120, Vegfa164, and Vegfa188 mRNA isoforms were detected on Embryonic Day (E) 13.5 (plug date=E0) in the rat. Vegfa120, Vegfa144, Vegfa164, Vegfa188, and Vegfa205 mRNA were detected at E18 and Postnatal Day 3 (P3). Kdr mRNA was present on E13.5, whereas Fms-like tyrosine kinase 1 receptor (Flt1) mRNA was not detected until E18. VEGFA protein was localized to Sertoli cells at cord formation and KDR to germ and interstitial cells. The VEGFA signaling inhibitors SU1498 (40 microM) and VEGFR-TKI (8 microM) inhibited cord formation in E13 testis cultures with 90% reduced vascular density (P<0.01) in VEGFR-TKI-treated organs. Furthermore, Je-11 (10 microM), an antagonist to VEGFA, also perturbed cord formation and inhibited vascular density by more than 50% (P<0.01). To determine signal transduction pathways involved in VEGFA's regulation of testis morphogenesis, E13 testis were treated with LY 294002 (15 microM), a phosphoinositide 3-kinase (PI3K) pathway inhibitor, resulting in inhibition of both vascular density (46%) and cord formation. Thus, we support our hypothesis and conclude that VEGFA, secreted by the Sertoli cell, is involved in both neovascularization and cord formation and potentially acts through the PI3K pathway during testis morphogenesis to elicit its effects.     

Mouse brains deficient in neuronal PDGF receptor-beta develop normally but are vulnerable to injury

Platelet-derived growth factors (PDGFs) and PDGF receptors (PDGFRs) are widely expressed in the mammalian CNS, though their functional significance remains unclear. The corresponding null-knockout mutations are lethal. Here, we developed novel mutant mice in which the gene encoding the beta subunit of PDGFR (PDGFR-beta) was genetically deleted in CNS neurons to elucidate the role of PDGFR-beta, particularly in the post-natal stage. Our mutant mice reached adulthood without apparent anatomical defects. In the mutant brain, immunohistochemical analyses showed that PDGFR-beta detected in neurons and in the cells in the subventricular zone of the lateral ventricle in wild-type mice was depleted, but PDGFR-beta detected in blood vessels remained unaffected. The cerebral damage after cryogenic injury was severely exacerbated in the mutants compared with controls. Furthermore, TdT-mediated dUTP-biotin nick end labeling (TUNEL)-positive neuronal cell death and lesion formation in the cerebral hemisphere were extensively exacerbated in our mutant mice after direct injection of NMDA without altered NMDA receptor expression. Our results clearly demonstrate that PDGFR-beta expressed in neurons protects them from cryogenic injury and NMDA-induced excitotoxicity.     

Embryonic testis cord formation and mesonephric cell migration requires the phosphotidylinositol 3-kinase signaling pathway

Mesonephric cell migration and seminiferous cord formation are critical processes in embryonic testis development at the time of male sex determination. Extracellular growth factors shown to influence seminiferous cord formation such as neurotropin-3 utilize in part the phosphotidylinositol 3-kinase (PI3K) signal transduction pathway. The current study investigates the hypothesis that the PI3K pathway is critical in seminiferous cord formation and testis development. The role of the PI3K signaling pathway in testicular cord formation was examined using an Embryonic Day 13 organ culture system and a PI3K-specific inhibitor LY294002. The actions of a mitogen-activated protein (MAP) kinase-specific inhibitor PD98059 was also examined. The PI3K inhibitor blocked cord formation or reduced the number of cords in a concentration-dependent manner. The actions of LY294002 were found to have a developmental stage specificity in that cord inhibition was observed in organs from embryos with 16-17 tail somites, while organs from embryos with 19 or more tail somites had no block in cord formation and only a small reduction in cord number. In contrast, the MAP kinase inhibitor PD98059 did not block cord formation and only caused a slight reduction in cord number. Neither PI3K or MAP kinase inhibitor altered apoptotic cell number, suggesting apoptosis was not the reason for the inhibition of cord formation. Embryonic testis cell migration assays showed that the PI3K inhibitor LY294002 blocked mesonephros cell migration into the testis, while the MAP kinase inhibitor had no effect. Observations suggest the interference of cell migration is the cause for the inhibition of cord formation. Western blot analysis confirmed that LY294002 and PD98509 inhibited phosphorylation of Akt and ERK1/ERK2, respectively. Combined observations demonstrate that the PI3K signaling pathway is involved in embryonic testis cord formation and mesonephros cell migration.     

Reaggregates of cells from rat testis resemble developing gonads

Reaggregates prepared from newborn rat testis cells in Moscona-type rotation cultures were analyzed and compared with normal fetal (12-21 days) and newborn testes at the light and electron microscope level. After 25 h of culture, the aggregates resembled normal testicular tissue. The cells of the surface layer were spindle-shaped and connected by adherent junctions. The epithelial cords were composed exclusively of Sertoli cells and were surrounded by elongated cells resembling the developing myoid cells in newborn testes. The basal aspect of the cords was covered by a layer of flocculent material which, in places, was organized like an ordinary basement membrane. Individual spermatogonia with pseudopodes were observed in the interstitial tissue. Some Leydig cells were organized into small clusters like those typical in newborn testes. The present observations indicate that, histologically, the reaggregation of separated testicular cells resembles the differentiation of embryonic male gonads.     

Hepatocyte growth factor (HGF) receptor expression and role of HGF during embryonic mouse testis development

The hepatocyte growth factor (HGF) receptor, c-met, transduces the HGF multiple biological activities. During embryonic development the system HGF/c-met regulates the morphogenesis of different organs and tissues. In this study we examined c-met gene expression during mouse testis development and, by means of Northern blot and in situ hybridization, we report the receptor expression pattern. C-met expression is not detectable in male genital ridges isolated from embryos at 11.5 days postcoitum (dpc). In testes isolated from 12.5 and 13.5 dpc, c-met expression is detectable and essentially localized in the developing cords. Male genital ducts do not express c-met at the reported ages, whereas female ducts appear c-met positive. Moreover, we report that HGF is able to induce testicular morphogenesis in vitro. Male genital ridges isolated from embryos at 11.5 dpc are morphologically nonorganized. Culturing 11.5 dpc urogenital ridges in the presence of HGF we obtained testis organization and testicular cord formation. Our data demonstrate that c-met is expressed during the beginning period of testis differentiation and that HGF is able to support testicular differentiation in vitro. All these data indicate that this growth factor, besides its role as mitogenic factor, plays a fundamental role during testicular cord formation probably inducing cell migration and/or cell differentiation.     

Sex reversal and aromatase in chicken.

Aromatase inhibitors administered before sexual differentiation of the gonads can induce sex reversal in female chickens. To analyze the process of sex reversal, we have followed for several months the changes induced by Fadrozole, a nonsteroidal aromatase inhibitor, in gonadal aromatase activity and in morphology and structure of the female genital system. Fadrozole was injected into eggs on day four of incubation, and its effects were examined during the embryonic development and for eight months after hatching. In control females, aromatase activity in the right and the left gonad was high in the middle third of embryonic development, and then decreased up to hatching. After hatching, aromatase activity increased in the left ovary, in particular during folliculogenesis, whereas in the right regressing gonad, it continued to decrease to reach testicular levels at one month. In treated females, masculinization of the genital system was characterized by the maintenance of the right gonad and its differentiation into a testis, and by the differentiation of the left gonad into an ovotestis or a testis; however, in all individuals, the left Müllerian duct and the posterior part of the right Müllerian duct were maintained. In testes and ovotestes, aromatase activity was lower than in gonads of control females (except in the right gonad as of one month after hatching) but remained higher than in testes of control and treated males. Moreover, in ovotestes, aromatase activity was higher in parts displaying follicles than in parts devoid of follicles. The main structural changes in the gonads during sex reversal were partial (in ovotestes) or complete (in testes) degeneration of the cortex in the left gonad, and formation of an albuginea and differentiation of testicular cords/tubes in the two gonads. Testicular cords/tubes transdifferentiated from ovarian medullary cords and lacunae whose epithelium thickened and became Sertolian. Transdifferentiation occurred all along embryonic and postnatal development; thus, new testicular cords/tubes were continuously formed while others degenerated. The sex reversed gonads were also characterized by an abundant fibrous interstitial tissue and abnormal medullary condensations of lymphoid-like cells; in the persisting testicular cords/tubes, spermatogenesis was delayed and impaired. Related to aromatase activity, persistence of too high levels of estrogens can explain the presence of oviducts, gonadal abnormalities and infertility in sex reversed females.