Genetic Reconstruction of Mouse Spermatogonial Stem Cell Self-Renewal In Vitro by Ras-Cyclin D2 Activation

Lee, Ji-Young; Kanatsu-Shinohara, Mito; Morimoto, Hiroko; Kazuki, Yasuhiro; Takashima, Seiji; Oshimura, Mitsuo; Toyokuni, Shinya; Shinohara, Takashi

doi:10.1016/j.stem.2009.04.020

Cited by 125 publications

(135 citation statements)

References 36 publications

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“…However, the regulation of SSC pool size has not been fully elucidated. It has been observed that the overexpression of GDNF in transgenic mice leads to the development of malignant testicular cancer, similar to the situation with classic human seminoma, and the Ras-cyclin D2 activation induces the tumorigenesis of SSCs [35,36]. Moreover, it is believed that human spermatocytic seminoma may originate from SSCs [37].…”

Section: Discussionmentioning

confidence: 98%

GABA exists as a negative regulator of cell proliferation in spermaogonial stem cells

Zheng

et al. 2013

Cellular and Molecular Biology Letters

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γ-amino butyric acid (GABA) is the main inhibitory neurotransmitter in the mammalian central nervous system. GABA is also found in many peripheral tissues, where it has important functions during development. Here, we identified the existence of the GABA system in spermatogonial stem cells (SSCs) and found that GABA negatively regulates SSC proliferation. First, we demonstrated that GABA and its synthesizing enzymes were abundant in the testes 6 days postpartum (dpp), suggesting that GABA signaling regulates SSCs function in vivo. In order to directly examine the effect of GABA on SSC proliferation, we then established an in vitro culture system for long-term expansion of SSCs. We showed that GABA A receptor subunits, including α1, α5, β1, β2, β3 and γ3, the synthesizing enzyme GAD67, and the transporter GAT-1, are expressed in SSCs. Using phosphorylated histone H3 (pH3) staining, we demonstrated that GABA or the GABA A R-specific agonist muscimol reduced the proliferation of SSCs. This GABA regulation of SSC proliferation was shown to be independent of apoptosis using the TUNEL assay. These results suggest that GABA acts as a negative regulator of SSC proliferation to maintain the homeostasis of spermatogenesis in the testes.

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Section: Discussionmentioning

confidence: 98%

GABA exists as a negative regulator of cell proliferation in spermaogonial stem cells

Zheng

et al. 2013

Cellular and Molecular Biology Letters

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“…The addition of GDNF and EGF and/or basic fibroblast growth factor (bFGF) induces SSC proliferation in vitro. Approximately 1-2% of the GS cells have SSC activity (4). GS cells from WT mice were infected with lentivirus vectors expressing candidate genes and Venus fluorescent marker gene under the control of an elongation factor2 promoter.…”

Section: Resultsmentioning

confidence: 99%

Transmission distortion by loss of p21 or p27 cyclin-dependent kinase inhibitors following competitive spermatogonial transplantation

Kanatsu-Shinohara

Takashima

Shinohara

2010

Proc. Natl. Acad. Sci. U.S.A.

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Spermatogonial stem cells (SSCs) undergo self-renewal division to support spermatogenesis. Although several positive regulators of SSC self-renewal have been identified, little is known about the mechanisms that negatively regulate SSCs. Here we developed a novel transplantation assay for SSCs and demonstrate that p21 and p27 cyclin-dependent kinase inhibitors play critical roles in SSC self-renewal and differentiation. Overexpression of p21 or p27 abrogated proliferation of cultured SSCs in vitro, and their expression levels were downregulated by exogenous self-renewal signals. In contrast, no apparent defects were found in p21 or p27-deficient SSCs by spermatogonial transplantation. However, competitive spermatogonial transplantation with WT SSCs revealed that the loss of either gene causes distortion of germline transmission: p21-deficiency facilitated mutant offspring production, whereas germline transmission was limited by p27-deficiency. Serial transplantation also showed that the loss of p27, but not p21, decreases secondary colony formation, suggesting that appropriate amounts of p27 are necessary for sustaining SSC selfrenewal. Thus, p21 and p27 cyclin-dependent kinase inhibitors play critical roles in germline transmission by regulating the balance between SSC self-renewal and differentiation, and competitive spermatogonial transplantation technique will be useful for analyzing subtle defects in spermatogenesis that are not evident by traditional spermatogonial transplantation.germline transmission | spermatogenesis | stem cell | cyclin-dependent kinase inhibitor | self-renewal

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“…Although HRAS mutations are predicted to be enriched by selfish selection and have been implicated in SSC growth regulation (22), no study has attempted to document their occurrence directly in the sperm of healthy males. To explore further the link between selfish selection and human disease, we quantified the levels in blood and sperm of spontaneous mutations around p.G12 of HRAS, the codon most frequently affected both by germline CS mutations and by somatically acquired oncogenic mutations.…”

Section: Significancementioning

confidence: 99%

Contributions of intrinsic mutation rate and selfish selection to levels of de novo HRAS mutations in the paternal germline

Giannoulatou

McVean

Taylor

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

117

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The RAS proto-oncogene Harvey rat sarcoma viral oncogene homolog (HRAS) encodes a small GTPase that transduces signals from cell surface receptors to intracellular effectors to control cellular behavior. Although somatic HRAS mutations have been described in many cancers, germline mutations cause Costello syndrome (CS), a congenital disorder associated with predisposition to malignancy. Based on the epidemiology of CS and the occurrence of HRAS mutations in spermatocytic seminoma, we proposed that activating HRAS mutations become enriched in sperm through a process akin to tumorigenesis, termed selfish spermatogonial selection. To test this hypothesis, we quantified the levels, in blood and sperm samples, of HRAS mutations at the p.G12 codon and compared the results to changes at the p.A11 codon, at which activating mutations do not occur. The data strongly support the role of selection in determining HRAS mutation levels in sperm, and hence the occurrence of CS, but we also found differences from the mutation pattern in tumorigenesis. First, the relative prevalence of mutations in sperm correlates weakly with their in vitro activating properties and occurrence in cancers. Second, specific tandem base substitutions (predominantly GC>TT/AA) occur in sperm but not in cancers; genomewide analysis showed that this same mutation is also overrepresented in constitutional pathogenic and polymorphic variants, suggesting a heightened vulnerability to these mutations in the germline. We developed a statistical model to show how both intrinsic mutation rate and selfish selection contribute to the mutational burden borne by the paternal germline.paternal age effect | male mutation bias | RASopathy | testis

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Genetic Reconstruction of Mouse Spermatogonial Stem Cell Self-Renewal In Vitro by Ras-Cyclin D2 Activation

Cited by 125 publications

References 36 publications

GABA exists as a negative regulator of cell proliferation in spermaogonial stem cells

GABA exists as a negative regulator of cell proliferation in spermaogonial stem cells

Transmission distortion by loss of p21 or p27 cyclin-dependent kinase inhibitors following competitive spermatogonial transplantation

Contributions of intrinsic mutation rate and selfish selection to levels of de novo HRAS mutations in the paternal germline

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