Mito Kanatsu-Shinohara

Akt mediates self-renewal division of mouse spermatogonial stem cells

Spermatogonial stem cells have unique properties to self-renew and support spermatogenesis throughout their lifespan. Although glial cell line-derived neurotrophic factor (GDNF) has recently been identified as a self-renewal factor for spermatogonial stem cells, the molecular mechanism of spermatogonial stem cell self-renewal remains unclear. In the present study, we assessed the role of the phosphoinositide-3 kinase (PI3K)-Akt pathway using a germline stem (GS) cell culture system that allows in vitro expansion of spermatogonial stem cells. Akt was rapidly phosphorylated when GDNF was added to the GS cell culture, and the addition of a chemical inhibitor of PI3K prevented GS cell self-renewal. Furthermore, conditional activation of the myristoylated form of Akt-Mer (myr-Akt-Mer) by 4-hydroxy-tamoxifen induced logarithmic proliferation of GS cells in the absence of GDNF for at least 5 months. The myr-Akt-Mer GS cells expressed spermatogonial markers and retained androgenetic imprinting patterns. In addition, they supported spermatogenesis and generated offspring following spermatogonial transplantation into the testes of infertile recipient mice, indicating that they are functionally normal. These results demonstrate that activation of the PI3K-Akt pathway plays a central role in the self-renewal division of spermatogonial stem cells.

Pluripotency of a Single Spermatogonial Stem Cell in Mice

Abstract Although pluripotent stem cells were recently discovered in postnatal testis, attempts to analyze their developmental potential have led to conflicting claims that spermatogonial stem cells are pluripotent or that they lose spermatogenic potential after conversion into pluripotent stem cells. To examine this issue, we analyzed the developmental fate of a single spermatogonial stem cell that appeared during transfection experiments. After transfection of a neomycin-resistance gene into germline stem cells, we obtained an embryonic stem-like, multipotent germline stem cell line. Southern blot analysis revealed that the germline stem and multipotent germline stem clones have the same transgene integration pattern, demonstrating their identical origin. The two lines, however, have different DNA methylation patterns. The multipotent germline stem cells formed chimeras after blastocyst injection but did not produce sperm after germ cell transplantation, whereas the germline stem cells could produce only spermatozoa and did not differentiate into somatic cells. Interestingly, the germline stem cells expressed several transcription factors (Pou5f1, Sox2, Myc, and Klf4) required for reprogramming fibroblasts into a pluripotent state, suggesting that they are potentially pluripotent. Thus, our study provides evidence that a single spermatogonial stem cell can acquire pluripotentiality but that conversion into a pluripotent cell type is accompanied by loss of spermatogenic potential.

Long-Term Culture of Male Germline Stem Cells From Hamster Testes

Abstract Spermatogonial stem cells provide the foundation for spermatogenesis in male animals. We recently succeeded in culturing and genetically engineering mouse spermatogonial stem cells, but little is known regarding the culture and growth requirements of spermatogonial stem cells in other animal species. In this study, we report the successful long-term culture of spermatogonial stem cells from hamster testes. Spermatogonial stem cells were purified using an anti-ITGA6 antibody and cultured in the presence of glial cell line-derived neurotrophic factor. The cells continued to proliferate for at least 1 year. During this period, they were genetically modified using a lentivirus and underwent spermatogenesis after transplantation into the testes of immunodeficient nude mice. However, germ cells generated in the surrogate xenogeneic recipients did not differentiate beyond the spermatid stage, and these round spermatids could not produce offspring through in vitro microinsemination. These results suggest that the germ cells may not have acquired characteristics necessary for fertility in the xenogeneic microenvironment. Nevertheless, the successful establishment of culture conditions conducive for hamster spermatogonial stem cell growth and maintenance indicates that this technique can be extended to other animal species in which current genetic modification techniques are impossible or inefficient.

Homing of Mouse Spermatogonial Stem Cells to Germline Niche Depends on β1-Integrin

Spermatogonial stem cells (SSCs) provide the foundation for spermatogenesis. In a manner comparable to hematopoietic stem cell transplantation, SSCs colonize the niche of recipient testes and reinitiate spermatogenesis following microinjection into the seminiferous tubules. However, little is known about the homing mechanism of SSCs. Here we examined the role of adhesion molecules in SSC homing. SSCs isolated from mice carrying loxP-tagged beta1-integrin alleles were ablated for beta1-integrin expression by in vitro adenoviral cre transduction. The beta1-integrin mutant SSCs showed significantly reduced ability to recolonize recipient testes in vivo and to attach to laminin molecules in vitro. In contrast, genetic ablation of E-cadherin did not impair homing, and E-cadherin mutant SSCs completed normal spermatogenesis. In addition, the deletion of beta1-integrin on Sertoli cells reduced SSC homing. These results identify beta1-integrin as an essential adhesion receptor for SSC homing and its association with laminin is critical in multiple steps of SSC homing.

Transcriptional repression and DNA hypermethylation of a small set of ES cell marker genes in male germline stem cells

BackgroundWe previously identified a set of genes called ECATs (ES cell-associated transcripts) that are expressed at high levels in mouse ES cells. Here, we examine the expression and DNA methylation of ECATs in somatic cells and germ cells.ResultsIn all ECATs examined, the promoter region had low methylation levels in ES cells, but higher levels in somatic cells. In contrast, in spite of their lack of pluripotency, male germline stem (GS) cells expressed most ECATs and exhibited hypomethylation of ECAT promoter regions. We observed a similar hypomethylation of ECAT loci in adult testis and isolated sperm. Some ECATs were even less methylated in male germ cells than in ES cells. However, a few ECATs were not expressed in GS cells, and most of them targets of Oct3/4 and Sox2. The Octamer/Sox regulatory elements were hypermethylated in these genes. In addition, we found that GS cells express little Sox2 protein and low Oct3/4 protein despite abundant expression of their transcripts.ConclusionOur results suggest that DNA hypermethylation and transcriptional repression of a small set of ECATs, together with post-transcriptional repression of Oct3/4 and Sox2, contribute to the loss of pluripotency in male germ cells.

Spermatogonial Stem Cell Self-Renewal and Development

Spermatogenesis originates from spermatogonial stem cells (SSCs). Development of the spermatogonial transplantation technique in 1994 provided the first functional assay to characterize SSCs. In 2000, glial cell line-derived neurotrophic factor was identified as a SSC self-renewal factor. This discovery not only provided a clue to understand SSC self-renewing mechanisms but also made it possible to derive germline stem (GS) cell cultures in 2003. In vitro culture of GS cells demonstrated their potential pluripotency and their utility in germline modification. However, in vivo SSC analyses have challenged the traditional concept of SSC self-renewal and have revealed their relationship with the microenvironment. An improved understanding of SSC self-renewal through functional assays promises to uncover fundamental principles of stem cell biology and will enable us to use these cells for applications in animal transgenesis and medicine.

Allogeneic Offspring Produced by Male Germ Line Stem Cell Transplantation into Infertile Mouse Testis

Abstract The testis is one of several immune-privileged organs and is known for its unique ability to support allogeneic or xenogeneic tissue transplants. We investigated the possibility of deriving offspring from mice that underwent transplantation with allogeneic male germ line stem cells in the testis. Although mature adult mice rejected allogeneic germ cells and were infertile, offspring were obtained by intracytoplasmic germ cell injection using partially differentiated donor cells. In contrast, complete spermatogenesis occurred when allogeneic germ cells were transplanted into immature pup testes. Tolerance induction by monoclonal antibody administration allowed the pup transplant recipients to produce allogeneic offspring by natural mating, whereas no spermatozoa were found in the epididymis of untreated recipients. Thus, these results indicate that a histoincompatible recipient can serve as a “surrogate father” to propagate the genetic information of heterologous male donors.

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

Spermatogonial stem cells (SSCs) undergo self-renewal division and support spermatogenesis. Although several cytokines coordinate to drive SSC self-renewal, little is known about the mechanisms underlying this process. We investigated the molecular mechanism by reconstructing SSC self-renewal in vitro without exogenous cytokines. Activation of Ras or overexpression of cyclins D2 and E1, both of which were induced by Ras, enabled long-term self-renewal of cultured spermatogonia. SSCs with activated Ras responded properly to differentiation signals and underwent spermatogenesis, whereas differentiation was abrogated in cyclin transfectants after spermatogonial transplantation. Both Ras- and cyclin-transfected cells produced seminomatous tumors, suggesting that excessive self-renewing stimulus induces oncogenic transformation. In contrast, cells that overexpressed cyclin D1 or D3 failed to make germ cell colonies after transplantation, which indicated that cyclin expression pattern is an important determinant to long-term SSC recolonization. Thus, the Ras-cyclin D2 pathway regulates the balance between tissue maintenance and tumorigenesis in the SSC population.

FGF2 mediates mouse spermatogonial stem cell self-renewal via upregulation of Etv5 and Bcl6b through MAP2K1 activation

Fibroblast growth factor 2 (FGF2) and glial cell line-derived neurotrophic factor (GDNF) are required to recapitulate spermatogonial stem cell (SSC) self-renewal in vitro. Although studies have revealed the role of the GDNF signaling pathway in SSCs, little is known about how FGF2 is involved. In the present study, we assessed the role of the FGF2 signaling pathway using a mouse germline stem (GS) cell culture system that allows in vitro expansion of SSCs. Adding GDNF or FGF2 induced phosphorylation of MAPK1/3, and adding the MAP2K1 inhibitor PD0325091 reduced GS cell proliferation and MAPK1/3 phosphorylation. Moreover, GS cells transfected with an activated form of Map2k1 not only upregulated Etv5 and Bcl6b gene expression, but also proliferated in an FGF2-independent manner, suggesting that they act downstream of MAP2K1 signaling to drive SSC self-renewal. Although GS cells transfected with Map2k1, Etv5 or Bcl6b showed normal spermatogonial markers, transplanting GS cells expressing Bcl6b into infertile mouse testes resulted in the formation of a germ cell tumor, suggesting that excessive self-renewal signals causes tumorigenic conversion. These results show that FGF2 depends on MAP2K1 signaling to drive SSC self-renewal via upregulation of the Etv5 and Bcl6b genes.

Clonal Origin of Germ Cell Colonies after Spermatogonial Transplantation in Mice

Abstract Spermatogenesis originates from a small number of spermatogonial stem cells that can reinitiate spermatogenesis and produce germ cell colonies following transplantation into infertile recipient testes. Although several previous studies have suggested a single-cell origin of germ cell colonies, only indirect evidence has been presented. In this investigation, we tested the clonal origin hypothesis using a retrovirus, which could specifically mark an individual spermatogonial stem cell. Spermatogonial stem cells were infected in vitro with an enhanced green fluorescence protein-expressing retrovirus and subsequently transplanted into infertile recipient mice. Live haploid germ cells were recovered from individual colonies and were microinjected into eggs to create offspring. In total, 45 offspring were produced from five colonies, and 23 (51%) of the offspring were transgenic. Southern blot analysis indicated that the transgenic offspring from the single colony carried a common integration site, and the integration site was different among the transgenic offspring from different colonies. These results provide evidence that germ cell colonies develop from single spermatogonial stem cells.