Pleiotropic activity of hepatocyte growth factor during embryonic mouse testis development

The hepatocyte growth factor (HGF) is a pleiotropic cytokine whose action is mediated by c-met, a glycoproteic receptor with tyrosine kinase activity which transduces its multiple biological activities including cell proliferation, motility and differentiation. During embryonic development HGF acts as a morphogenetic factor as previously demonstrated for metanephric and lung development. Recently, culturing male genital ridges, we demonstrated that HGF is able to support in vitro testicular cord formation. In the present paper we report the expression pattern of the HGF gene during embryonic testis development and the multiple roles exerted by this factor during the morphogenesis of this organ. Northern blot analysis reveals a positive signal in urogenital ridges isolated from 11.5 days post coitum (dpc) embryos and in testes isolated from 13.5 and 15.5 dpc male embryos. On the contrary HGF mRNA is undetectable in ovaries isolated from 13.5 and 15.5 dpc embryos. Moreover, we demonstrate that HGF is synthesized and secreted by the male gonad and is biologically active. These data indicate a male specific biological function of HGF during embryonic gonadal development. This hypothesis is supported by the in vitro demonstration that HGF acts as a migratory factor for male mesonephric cells which is a male specific event. In addition we demonstrate that during testicular development, HGF acts as a morphogenetic factor able to reorganize dissociated testicular cells which, under HGF stimulation, form a tridimensional network of cord-like structures. Finally, we demonstrate that HGF induces testicular cell proliferation in this way being responsible for the size increase of the testis. All together the data presented in this paper demonstrate that HGF is expressed during the embryonic development of the testis and clarify the multiple roles exerted by this factor during the morphogenesis of the male gonad.     

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.     

Inhibition of platelet-derived growth factor actions in the embryonic testis influences normal cord development and morphology

Platelet-derived growth factors (PDGFs) are paracrine factors with roles in mesenchymal-epithelial interactions during normal and pathologic processes. Previously, PDGF and its receptor (PDGFR) have been shown to be present in perinatal, peripubertal, and adult rat testes. The role of PDGF in embryonic testicular cord formation is not known. The hypothesis tested is that PDGFs and PDGFRs are expressed during cord formation and that inhibition of their action influences normal cord formation during embryonic testis development. Embryonic Day (E) 13 gonadal organ cultures were used. Organs were cultured for 3 days and treated daily with vehicle or a PDGFR-specific tyrosine phosphorylation inhibitor (i.e., the tyrphostin AG1295 or AG1296). Vehicle-treated testes formed normal cords, whereas tyrphostin-treated testes formed "swollen cords," a phenomenon characterized by a significant decrease in the number of cords per testis area and increased cord diameter due to fusion of cords. Expression of PDGF and PDGFR in E13, E14, E16, Postnatal Day (P) 0, and P20 testes was examined. Messenger RNAs for PDGF-A and -B and PDGF alpha- and beta-receptors were expressed in isolated testes during all developmental periods examined. Immunoreactivity for PDGF was present throughout the testicular compartment at E14, restricted primarily to testicular cords at E16, and present in cells of the testicular cords with a stronger immunoreactivity in certain interstitial cell types of P0 testis. PDGFR beta-receptor immunoreactivity was primarily localized to the mesonephros of E14 organs and the testicular interstitium of E16 and P0 testes. Tyrphostins did not affect apoptotic cell number in the testis. PDGF had no effect on cell growth in P0 testis cultures. The results show that PDGFs and PDGFRs are expressed in embryonic testis during cord formation in a tissue-specific manner. Inhibition of PDGF actions does not inhibit cord formation but does alter normal cord development and morphology. The observations provide insight into the factors involved in male sex differentiation and embryonic testis development.     

New testicular mechanisms involved in the prevention of fetal meiotic initiation in mice

In mammals, early fetal germ cells are unique in their ability to initiate the spermatogenesis or oogenesis programs dependent of their somatic environment. In mice, female germ cells enter into meiosis at 13.5 dpc whereas in the male, germ cells undergo mitotic arrest. Recent findings indicate that Cyp26b1, a RA-degrading enzyme, is a key factor preventing initiation of meiosis in the fetal testis. Here, we report evidence for additional testicular pathways involved in the prevention of fetal meiosis. Using a co-culture model in which an undifferentiated XX gonad is cultured with a fetal or neonatal testis, we demonstrated that the testis prevented the initiation of meiosis and induced male germ cell differentiation in the XX gonad. This testicular effect disappeared when male meiosis starts in the neonatal testis and was not directly due to Cyp26b1 expression. Moreover, neither RA nor ketoconazole, an inhibitor of Cyp26b1, completely prevented testicular inhibition of meiosis in co-cultured ovary. We found that secreted factor(s), with molecular weight greater than 10 kDa contained in conditioned media from cultured fetal testes, inhibited meiosis in the XX gonad. Lastly, although both Sertoli and interstitial cells inhibited meiosis in XX germ cells, only interstitial cells induced mitotic arrest in germ cell. In conclusion, our results demonstrate that male germ cell determination is supported by additional non-retinoid secreted factors inhibiting both meiosis and mitosis and produced by the testicular somatic cells during fetal and neonatal life.     

Effects of FGF9 on embryonic Sertoli cell proliferation and testicular cord formation in the mouse

Proliferation and cord formation by embryonic Sertoli cells are pivotal events involved in testis morphogenesis. A number of growth factors have been implicated in mediating these events. However, the exact level of involvement and importance of each as yet remains elusive. We have adopted an in vitro approach to assess developing mouse Sertoli cells, whereby they are cultured in the presence or absence of fibroblast growth factor (FGF9) and/or extracellular matrix (ECM) gel, since previous studies have shown that ECM gel aids Sertoli cell differentiation. The present findings corroborate this effect, but in addition demonstrate that in the presence of FGF9 (10 ng/ml), cells undergo greater proliferation than those cultured on gel alone. They also display a differentiated epithelial phenotype, with appositional contact of cell membranes in cord-like aggregations. In addition we have shown that cultured Sertoli cells generally express a smaller truncated, nuclear form of the FGFr3, although in the presence of FGF9 and absence of gel, the larger, cytoplasmic form of the receptor is also expressed. Immunolocalisation of FGFr3 in Sertoli cells of whole testes revealed a temporal expression pattern profile, with high levels being abundant in the embryonic testicular cords and at puberty, but an absence in adult Sertoli cells. Our findings suggest that FGF9 plays an important role in proliferation and organisation of embryonic Sertoli cells during testis morphogenesis.     

Genital ridges exert long-range effects on mouse primordial germ cell numbers and direction of migration in culture

The functional gametes of all vertebrates first arise in the early embryo as a migratory population of cells, the primordial germ cells (PGCs). These migrate to, and colonise, the genital ridges (GR) during the early organogenesis period, giving rise to the complete differentiating gonad. PGCs first become visible by alkaline phosphatase staining in the root of the developing allantois at 8.5 days post coitum (dpc). At 9.5 dpc they are found in the wall of the hind-gut and, during the following three days, they migrate along the hind-gut mesentery to the dorsal body wall, and then to the genital ridges. By 12.5 dpc, the great majority of PGCs have colonised the genital ridges. During this period the number of PGCs increases from less than 100 to approximately 4000. In a previous paper (Donovan et al. 1986), we showed that 10.5 dpc PGCs can be explanted from the hind-gut mesentery, and will spread and migrate on feeder cell layers. We showed also that the intrinsic ability of PGCs to spread and migrate changes as they colonise the genital ridges. In this paper, we examine extrinsic factors that control PGC behaviour in vitro. Using PGCs taken from 8.5 dpc embryos, at the beginning of their migratory phase, we show that culture medium conditioned by 10.5 dpc genital ridges causes an increase in the number of PGCs in these cultures. We also show that PGCs migrate towards 10.5 dpc genital ridges in preference to other explanted organs. These experiments show that genital ridges exert long-range effects on the migrating population of PGCs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Retinoic acid prevents germ cell mitotic arrest in mouse fetal testes

During mouse fetal development, meiosis is initiated in female germ cells only, with male germ cells undergoing mitotic arrest. Retinoic acid (RA) is degraded by Cyp26b1 in the embryonic testis but not in the ovary where it initiates the mitosis/meiosis transition. However the role of RA status in fetal germ cell proliferation has not been elucidated. As expected, using organ cultures, we observed that addition of RA in 11.5 days post-conception (dpc) testes induced Stra8 expression and meiosis. Surprisingly, in 13.5 dpc testes although RA induced Stra8 expression it did not promote meiosis. On 11.5 and 13.5 dpc, RA prevented male germ cell mitotic arrest through PI3K signaling. Therefore 13.5 dpc testes appeared as an interesting model to investigate RA effects on germ cell proliferation/differentiation independently of RA effect on the meiosis induction. At this stage, RA delayed SSEA-1 extinction, p63γ expression and DNA hypermethylation which normally occur in male mitotic arrested germ cells. In vivo, in the fetal male gonad, germ cells cease their proliferation and loose SSEA-1 earlier than in female gonad and RA administration maintained male germ cell proliferation. Lastly, inhibition of endogenous Cyp26 activity in 13.5 dpc cultured testes also prevented male germ cell mitotic arrest. Our data demonstrate that the reduction of RA levels, which occurs specifically in the male fetal gonad and was known to block meiosis initiation, is also necessary to allow the establishment of the germ cell mitotic arrest and the correct further differentiation of the fetal germ cells along the male pathway.     

The expression profiles of fibroblast growth factor 9 and its receptors in developing mice testes

An expressional lack of fibroblast growth factor 9 (FGF9) would cause male-to-female sex reversal in the mouse, implying the essential role of FGF9 in testicular organogenesis and maturation. However, the temporal expression of FGF9 and its receptors during testicular development remains elusive. In this study, immunohistochemistry was used to identify the localization of FGF9 and its receptors at different embryonic and postnatal stages in mice testes. Results showed that FGF9 continuously expressed in the testis during development. FGF9 had highest expression in the interstitial region at 17–18 d post coitum (dpc) and in the spermatocytes, spermatids and Leydig cell on postnatal days (pnd) 35–65. Regarding receptor expression, FGFR1 and FGFR4 were evenly expressed in the whole testis during the embryonic and postnatal stages. However, FGFR2 and FGFR3 were widely expressed during the embryonic testis development with higher FGFR2 expression in seminiferous tubules at 16–18 dpc and higher FGFR3 expression in interstitial region at 17–18 dpc. In postnatal stage, FGFR2 extensively expressed with higher expression at spermatids and Leydig cells on 35–65 pnd and FGFR3 widely expressed in the whole testis. Taken together, these results strongly suggest that FGF9 is correlated with the temporal expression profiles of FGFR2 and FGFR3 and possibly associated with testis development.     

Members of the ErbB receptor tyrosine kinases are involved in germ cell development in fetal mouse gonads.

To isolate the genes involved in mouse primordial germ cell (PGC) development, we carried out subtraction cDNA cloning between PGC-derived embryonic germ (EG) cells and inner cell mass-derived embryonic stem cells. Among the genes preferentially expressed in EG cells, we found a gene encoding a receptor tyrosine kinase ErbB3. By in situ hybridization and immunohistochemical staining, the expression of ErbB3 as well as that of ErbB2, a coreceptor for ErbB3, was detected in PGCs in genital ridges at 12.5 dpc (days postcoitum). The expression was, however, downregulated at 14.5 dpc when the PGCs underwent growth cessation. Neuregulin-beta, a ligand for ErbB2 and ErbB3, was also expressed in genital ridges. In addition, a recombinant Neuregulin-beta enhanced the number of PGCs in 12.5-dpc embryos in culture. Taken together, these observations suggest that ErbB signaling controls the growth or survival of PGCs in genital ridges.     

Retinoic acid receptors and retinoid X receptors in the rat testis during fetal and postnatal development: immunolocalization and implication in the control of the number of gonocytes

Retinoids have pleiotropic effects on embryonic development and are essential for spermatogenesis in the adult, where they act via nuclear retinoid receptors: retinoic acid receptors (RARs) and retinoid X receptors (RXRs). We used immunohistochemistry to examine the cellular localization of RARs and RXRs in the rat testis from Day 13.5 postconception (13.5 dpc) until Day 8 postpartum (8 dpp), and these findings were compared with those for immature and adult testes. RARalpha and RARbeta were detected in the interstitial tissue from 14.5 dpc, with intense staining in the gonocytes from 20. 5 dpc to 8 dpp. The nuclei of all cell types stained faintly for RARgamma from 8 dpp. Immunoreactivity for RXRalpha was intense in the gonocytes from 13.5 dpc and in the Leydig cells from 16.5 dpc, and persisted throughout the period studied. RXRbeta was always detected in the Leydig cells and during a short neonatal period in the gonocytes. RXRgamma gave a faint reaction in the nuclei of all cell types from 20.5 dpc. Unexpectedly, immunostaining for all the receptors tested, except RARgamma and RXRgamma, was detected in the cytoplasmic compartment of the cells of fetal and neonatal testes, while it was found in the nuclei in immature and adult testes. In cultures of dispersed testicular cells from 3 dpp pups, retinoic acid had a dose-dependent deleterious effect on the survival of the gonocytes and, to a lesser extent, of the somatic cells. These results suggest that retinoids act on the testicular development, especially on germ cells, via RARs and/or RXRs.     

Expression of the tudor-related gene Tdrd5 during development of the male germline in mice

Homologues of Drosophila germ cell determinant genes such as vasa, nanos and tudor have recently been implicated in development of the male germline in mice. In the present study, the mouse gene encoding Tudor domain containing protein 5 (TDRD5) was isolated from a 12.5-13.5 days post coitum (dpc) male-enriched subtracted cDNA library. Whole-mount in situ hybridization analysis of Tdrd5 expression in the mouse embryonic gonad indicated that this gene is upregulated in the developing testis from 12.5 dpc, with expression levels remaining higher in testis than ovary throughout embryogenesis. Expression of Tdrd5 was absent in testes isolated from We/We embryos, which lack germ cells. In situ hybridization (ISH) on cryosectioned 13.5 dpc testes suggests that expression of Tdrd5, like that of Oct4, is restricted to germ cells. Northern hybridization analysis of expression in adult tissues indicated that Tdrd5 is expressed in the testis only, implying that expression of this gene is restricted to the male germline throughout development to adulthood.     

Autonomous transition into meiosis of mouse fetal germ cells in vitro and its inhibition by gp130-mediated signaling

Mouse primordial germ cells (PGCs) arrive at the urogenital ridge (UGR) at around 10.5 days postcoitum (dpc). They proliferate until around 13.5 dpc, then enter into meiosis in the female or become mitotically arrested in the male gonads. In this study, meiotic transition of mouse PGCs was examined in vitro. Female PGCs obtained from UGRs or genital ridges at 10.5-11.5 dpc began to express meiosis-specific genes, Scp3 and Dmc1, after dissociation and cultivation on feeder cells for several days. Meiotic transition into the leptotene stage was confirmed by the formation of axial cores. Male PGCs at 10.5-11.5 dpc and migratory PGCs obtained from mesenteries at 10.5 dpc also expressed Scp3 and formed axial cores after several days of culture, supporting the hypothesis that PGCs are capable of entering meiosis before arriving at the UGR. gp130-mediated signaling, known to promote survival/growth of PGCs and also to inhibit the differentiation of embryonic stem cells, suppressed the expression of Scp3 in PGCs and inhibited the following formation of axial cores in vitro. This novel activity of gp130-mediated signaling may provide some clues for the understanding of pluripotency of mammalian germ-line cells and/or the sex differentiation of fetal germ cells.     

Sox15 is up regulated in the embryonic mouse testis

The mammalian sex determining region on the Y chromosome, SRY, is the founding member of the SOX gene family. SOX genes share a common DNA-binding motif termed the HMG box and have diverse roles in vertebrate embryonic development and tissue differentiation. Sox15 expression was analysed during mouse embryogenesis by whole-mount in situ hybridisation and Real Time RT-PCR. Sox15 was found to be expressed in developing mouse gonads from 11.5 dpc to 13.5 dpc with a peak of expression at 12.5 dpc. Expression was approximately twice as high in the male gonad as in the female gonad.     

The expression of neurotrophins and their receptors in the prenatal and adult human testis: evidence for functions in Leydig cells

Previous studies have demonstrated local functions for neurotrophins in the developing and mature testis of rodents. To examine whether these signaling molecules are present and also potentially active in the human testis, we characterized immunohistochemically the expression and cellular localization of the known neurotrophins and their receptors during prenatal testicular development as well as in the adult human testis. Results obtained revealed the presence of nerve growth factor (NGF), brain-derived neurotrophic factor, neurotrophin-3 and 4, as well as neurotrophin receptors p75^NTR, TrkA, TrkB, and TrkC during testis morphogenesis. These proteins were also detectable in the adult human testis, and their local expression could be confirmed largely by immunoblot and RT-PCR analyses. Remarkably, the Leydig cells were found to represent the predominant neurotrophin/receptor expression sites within both fetal and adult human testes. Functional assays performed with a mouse tumor Leydig cell line revealed that NGF exposure increases cellular steroid production, indicating a role in differentiation processes. These findings support previously-recognized neuronal characteristics of Leydig cells, provide additional evidence for potential roles of neurotrophins during testis morphogenesis and in the mature testis, and demonstrate for the first time a neurotrophin-induced functional activity in Leydig cells.     

R-Spondin 1/Dickkopf-1/Beta-Catenin Machinery Is Involved in Testicular Embryonic Angiogenesis

Testicular vasculogenesis is one of the key processes regulating male gonad morphogenesis. The knowledge of the molecular cues underlining this phenomenon is one of today’s most challenging issues and could represent a major contribution toward a better understanding of the onset of testicular morphogenetic disorders. R-spondin 1 has been clearly established as a candidate for mammalian ovary determination. Conversely, very little information is available on the expression and role of R-spondin 1 during testicular morphogenesis. This study aims to clarify the distribution pattern of R-spondin 1 and other partners of its machinery during the entire period of testicular morphogenesis and to indicate the role of this system in testicular development. Our whole mount immunofluorescence results clearly demonstrate that R-spondin 1 is always detectable in the testicular coelomic partition, where testicular vasculature is organized, while Dickkopf-1 is never detectable in this area. Moreover, organ culture experiments of embryonic male UGRs demonstrated that Dickkopf-1 acted as an inhibitor of testis vasculature formation. Consistent with this observation, real-time PCR analyses demonstrated that DKK1 is able to slightly but significantly decrease the expression level of the endothelial marker Pecam1. The latter experiments allowed us to observe that DKK1 administration also perturbs the expression level of the Pdgf-b chain, which is consistent with some authors’ observations relating this factor with prenatal testicular patterning and angiogenesis. Interestingly, the DKK1 induced inhibition of testicular angiogenesis was rescued by the co-administration of R-spondin 1. In addition, R-spondin 1 alone was sufficient to enhance, in culture, testicular angiogenesis.     

Identification of a fibroblast-derived epithelial morphogen as hepatocyte growth factor.

We have previously shown that Madin-Darby canine kidney (MDCK) epithelial cells grown in collagen gels in the presence of fibroblasts or fibroblast-conditioned medium (CM) form branching tubules, instead of the spherical cysts that develop under control conditions. We now report that the fibroblast-derived molecule responsible for epithelial tubulogenesis is hepatocyte growth factor (HGF). First, addition of exogenous HGF to cultures of MDCK cells induces formation of epithelial tubules. Second, the tubulogenic activity of fibroblast CM is completely abrogated by antibodies to HGF. These results demonstrate that HGF, a polypeptide that was identified as a mitogen for cultured hepatocytes, has the properties of a paracrine mediator of epithelial morphogenesis, and suggest that it may play important roles in the formation of parenchymal organs during embryonic development.