Cyclical expression of GDNF is required for spermatogonial stem cell homeostasis.
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ABSTRACT:
SUBMITTER: Sharma M
PROVIDER: S-EPMC5868990 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Spermatogonial stem cells (SSCs) are required for spermatogenesis. Earlier studies showed that glial cell line-derived neurotrophic factor (GDNF) was indispensable for SSC self-renewal by binding to the GFRA1/RET receptor. Mice with mutations in these molecules showed impaired spermatogenesis, which was attributed to SSC depletion. Here we show that SSCs undergo GDNF-independent self-renewal. A small number of spermatogonia formed colonies when testis fragments from a Ret mutant mouse strain were transplanted into heterologous recipients. Moreover, fibroblast growth factor 2 (FGF2) supplementation enabled in vitro SSC expansion without GDNF. Although GDNF-mediated self-renewal signaling required both AKT and MAP2K1/2, the latter was dispensable in FGF2-mediated self-renewal. FGF2-depleted testes exhibited increased levels of GDNF and were enriched for SSCs, suggesting that the balance between FGF2 and GDNF levels influences SSC self-renewal in vivo. Our results show that SSCs exhibit at least two modes of self-renewal and suggest complexity of SSC regulation in vivo.
| S-EPMC4375941 | biostudies-literature
Project description:Prepubertal boys treated with high-dose chemotherapy do not have an established means of fertility preservation because no established in vitro technique exists to expand and mature purified spermatogonial stem cells (SSCs) to functional sperm in humans. In this study, we define and characterize the unique testicular cellular niche required for SSC expansion using testicular tissues from men with normal spermatogenesis. Highly purified SSCs and testicular somatic cells were isolated by fluorescence-activated cell sorting using SSEA-4 and THY1 as markers of SSCs and somatic cells. Cells were cultured on various established niches to assess their role in SSC expansion in a defined somatic cellular niche. Of all the niches examined, cells in the SSEA-4 population exclusively bound to adult testicular stromal cells, established colonies, and expanded. Further characterization of these testicular stromal cells revealed distinct mesenchymal markers and the ability to undergo differentiation along the mesenchymal lineage, supporting a testicular multipotent stromal cell origin. In vitro human SSC expansion requires a unique niche provided exclusively by testicular multipotent stromal cells with mesenchymal properties. These findings provide an important foundation for developing methods of inducing SSC growth and maturation in prepubertal testicular tissue, essential to enabling fertility preservation for these boys.
| S-EPMC4149303 | biostudies-literature
Project description:Long-term mammalian spermatogenesis requires proper development of spermatogonial stem cells (SSCs) that replenish the testis with germ cell progenitors during adult life. TAF4b is a gonadal-enriched component of the general transcription factor complex, TFIID, which is required for the maintenance of spermatogenesis in the mouse. Successful germ cell transplantation assays into adult TAF4b-deficient host testes suggested that TAF4b performs an essential germ cell autonomous function in SSC establishment and/or maintenance. To elucidate the SSC function of TAF4b, we characterized the initial gonocyte pool and rounds of spermatogenic differentiation in the context of the Taf4b-deficient mouse testis. Here, we demonstrate a significant reduction in the late embryonic gonocyte pool and a deficient expansion of this pool soon after birth. Resulting from this reduction of germ cell progenitors is a developmental delay in meiosis initiation, as compared to age-matched controls. While GFRα1+ spermatogonia are appropriately present as Asingle and Apaired in wild-type testes, TAF4b-deficient testes display an increased proportion of long and clustered chains of GFRα1+ cells. In the absence of TAF4b, seminiferous tubules in the adult testis either lack germ cells altogether or are found to have missing generations of spermatogenic progenitor cells. Together these data indicate that TAF4b-deficient spermatogenic progenitor cells display a tendency for differentiation at the expense of self-renewal and a renewing pool of SSCs fail to establish during the critical window of SSC development.
| S-EPMC4376611 | biostudies-literature
Project description:Silver nanoparticles (Ag-NPs) are being utilized in an increasing number of fields and are components of antibacterial coatings, antistatic materials, superconductors, and biosensors. A number of reports have now described the toxic effects of silver nanoparticles on somatic cells; however, no study has examined their effects on the germ line at the molecular level. Spermatogenesis is a complex biological process that is particularly sensitive to environmental insults. Many chemicals, including ultrafine particles, have a negative effect on the germ line, either by directly affecting the germ cells or by indirectly acting on the somatic cells of the testis. In the present study, we have assessed the impact of different doses of Ag-NPs, as well as their size and biocompatible coating, on the proliferation of mouse spermatogonial stem cells (SSCs), which are at the origin of the germ line in the adult testis. At concentrations >OR= 10 microg/ml, Ag-NPs induced a significant decline in SSCs proliferation, which was also dependent on their size and coating. At the concentration of 10 microg/ml, reactive oxygen species production and/or apoptosis did not seem to play a major role; therefore, we explored other mechanisms to explain the decrease in cell proliferation. Because glial cell line-derived neurotrophic factor (GDNF) is vital for SSC self-renewal in vitro and in vivo, we evaluated the effects of Ag-NPs on GDNF-mediated signaling in these cells. Although the nanoparticles did not reduce GDNF binding or Ret receptor activity, our data revealed that already at a concentration of 10 microg/ml, silver nanoparticles specifically interact with Fyn kinase downstream of Ret and impair SSC proliferation in vitro. In addition, we demonstrated that the particle coating was degraded upon interaction with the intracellular microenvironment, reducing biocompatibility.
| S-EPMC2905406 | biostudies-literature
Project description:The objective of the present study was to investigate vascular endothelial growth factor A (VEGFA) isoform regulation of cell fate decisions of spermatogonial stem cells (SSC) in vivo. The expression pattern and cell-specific distribution of VEGF isoforms, receptors, and coreceptors during testis development postnatal d 1-180 suggest a nonvascular function for VEGF regulation of early germ cell homeostasis. Populations of undifferentiated spermatogonia present shortly after birth were positive for VEGF receptor activation as demonstrated by immunohistochemical analysis. Thus, we hypothesized that proangiogenic isoforms of VEGF (VEGFA(164)) stimulate SSC self-renewal, whereas antiangiogenic isoforms of VEGF (VEGFA(165)b) induce differentiation of SSC. To test this hypothesis, we used transplantation to assay the stem cell activity of SSC obtained from neonatal mice treated daily from postnatal d 3-5 with 1) vehicle, 2) VEGFA(164), 3) VEGFA(165)b, 4) IgG control, 5) anti-VEGFA(164), and 6) anti-VEGFA(165)b. SSC transplantation analysis demonstrated that VEGFA(164) supports self-renewal, whereas VEGFA(165)b stimulates differentiation of mouse SSC in vivo. Gene expression analysis of SSC-associated factors and morphometric analysis of germ cell populations confirmed the effects of treatment on modulating the biological activity of SSC. These findings indicate a nonvascular role for VEGF in testis development and suggest that a delicate balance between VEGFA(164) and VEGFA(165)b isoforms orchestrates the cell fate decisions of SSC. Future in vivo and in vitro experimentation will focus on elucidating the mechanisms by which VEGFA isoforms regulate SSC homeostasis.
| S-EPMC3275389 | biostudies-literature
Project description:Cytokine-dependent renewal of stem cells is a fundamental requisite for tissue homeostasis and regeneration. Spermatogonial progenitor cells (SPCs) including stem cells support life-long spermatogenesis and male fertility, but pivotal phosphorylation events that regulate fate decisions in SPCs remain unresolved. Here, we described a quantitative mass-spectrometry-based proteomic and phosphoproteomic analyses of SPCs following sustained stimulation with glial cell-derived neurotrophic factor (GDNF), an extrinsic factor supporting SPC proliferation. Stimulated SPCs contained 3382 identified phosphorylated proteins and 12141 phosphorylation sites. Of them, 325 differentially phosphorylated proteins and 570 phosphorylation sites triggered by GDNF were highly enriched for ERK1/2, GSK3, CDK1, and CDK5 phosphorylating motifs. We validated that inhibition of GDNF/ERK1/2-signaling impaired SPC proliferation and increased G2/M cell cycle arrest. Significantly, we found that proliferation of SPCs requires phosphorylation of the mTORC1 component Raptor at Ser863 Tissue-specific deletion of Raptor in mouse germline cells results in impaired spermatogenesis and progressive loss of spermatogonia, but in vitro increased phosphorylation of Raptor by raptor over-expression in SPCs induced a more rapidly growth of SPCs in culture. These findings implicate previously undescribed signaling networks in governing fate decision of SPCs, which is essential for the understanding of spermatogenesis and of potential consequences of pathogenic insult for male infertility.
| S-EPMC5461548 | biostudies-literature
Project description:Male mammals produce sperm for most of postnatal life and therefore require a robust germ line stem cell system, with precise balance between self-renewal and differentiation. Prior work established doublesex- and mab-3-related transcription factor 1 (Dmrt1) as a conserved transcriptional regulator of male sexual differentiation. Here we investigate the role of Dmrt1 in mouse spermatogonial stem cell (SSC) homeostasis. We find that Dmrt1 maintains SSCs during steady state spermatogenesis, where it regulates expression of Plzf, another transcription factor required for SSC maintenance. We also find that Dmrt1 is required for recovery of spermatogenesis after germ cell depletion. Committed progenitor cells expressing Ngn3 normally do not contribute to SSCs marked by the Id4-Gfp transgene, but do so when spermatogonia are chemically depleted using busulfan. Removal of Dmrt1 from Ngn3-positive germ cells blocks the replenishment of Id4-GFP-positive SSCs and recovery of spermatogenesis after busulfan treatment. Our data therefore reveal that Dmrt1 supports SSC maintenance in two ways: allowing SSCs to remain in the stem cell pool under normal conditions; and enabling progenitor cells to help restore the stem cell pool after germ cell depletion.
| S-EPMC5008761 | biostudies-literature
Project description:BACKGROUND: Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis, and reside within a specific microenvironment in the testes called "niche" which regulates stem cell properties, such as, self-renewal, pluripotency, quiescence and their ability to differentiate. METHODOLOGY/PRINCIPAL FINDINGS: Here, we introduce zebrafish as a new model for the study of SSCs in vertebrates. Using 5'-bromo-2'-deoxyuridine (BrdU), we identified long term BrdU-retaining germ cells, type A undifferentiated spermatogonia as putative stem cells in zebrafish testes. Similar to rodents, these cells were preferentially located near the interstitium, suggesting that the SSC niche is related to interstitial elements and might be conserved across vertebrates. This localization was also confirmed by analyzing the topographical distribution of type A undifferentiated spermatogonia in normal, vasa::egfp and fli::egfp zebrafish testes. In the latter one, the topographical arrangement suggested that the vasculature is important for the SSC niche, perhaps as a supplier of nutrients, oxygen and/or signaling molecules. We also developed an SSC transplantation technique for both male and female recipients as an assay to evaluate the presence, biological activity, and plasticity of the SSC candidates in zebrafish. CONCLUSIONS/SIGNIFICANCE: We demonstrated donor-derived spermato- and oogenesis in male and female recipients, respectively, indicating the stemness of type A undifferentiated spermatogonia and their plasticity when placed into an environment different from their original niche. Similar to other vertebrates, the transplantation efficiency was low. This might be attributed to the testicular microenvironment created after busulfan depletion in the recipients, which may have caused an imbalance between factors regulating self-renewal or differentiation of the transplanted SSCs.
| S-EPMC2942835 | biostudies-literature
Project description:GDNF-regulated gene expression was studied in cultures of actively self-renewing spermatogonial stem cells established from 6 day old male mice. GDNF is the essential growth factor regulating mouse spermatogonial stem cell self-renewal. Using a serum-free chemically defined culture system that supports mouse spermatogonial stem cell self-renewal for extended periods of time, GDNF-regulated genes were identified using microarray profiling. Keywords: GDNF withdrawal and time-course replacement
2006-05-11 | GSE4799 | GEO
Project description:Glial cell line-derived neurotrophic factor (GDNF) plays a crucial role in regulating the proliferation of spermatogonial stem cells (SSC). The signaling pathways mediating the function of GDNF in SSC remain unclear. This study was designed to determine whether GDNF signals via the Ras/ERK1/2 pathway in the C18-4 cells, a mouse SSC line. The identity of this cell line was confirmed by the expression of various markers for germ cells, proliferating spermatogonia, and SSC, including GCNA1, Vasa, Dazl, PCNA, Oct-4, GFRalpha1, Ret, and Plzf. Western blot analysis revealed that GDNF activated Ret tyrosine phosphorylation. All 3 isoforms of Shc were phosphorylated upon GDNF stimulation, and GDNF induced the binding of the phosphorylated Ret to Shc and Grb2 as indicated by immunoprecipitation and Western blotting. The active Ras was induced by GDNF, which further activated ERK1/2 phosphorylation. GDNF stimulated the phosphorylation of CREB-1, ATF-1, and CREM-1, and c-fos transcription. Notably, the increase in ERK1/2 phosphorylation, c-fos transcription, bromodeoxyuridine incorporation, and metaphase counts induced by GDNF, was completely blocked by pretreatment with PD98059, a specific inhibitor for MEK1, the upstream regulator of ERK1/2. GDNF stimulation eventually upregulated cyclin A and CDK2 expression. Together, these data suggest that GDNF induces CREB/ATF-1 family member phosphorylation and c-fos transcription via the Ras/ERK1/2 pathway to promote the proliferation of SSC. Unveiling GDNF signaling cascades in SSC has important implications in providing attractive targets for male contraception as well as for the regulation of stem cell renewal vs. differentiation.
| S-EPMC2905627 | biostudies-literature