記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND: Many animals switch between asexual and sexual reproduction in nature. We previously established a system for the sexual induction of planarian Dugesia ryukyuensis by feeding asexual planarians with minced sexual planarians. We identified DL-tryptophan (Trp) as one of the sex-inducing substances. DL-Trp can induce ovarian development, the first and essential step of sexual induction. D-Trp must act as a principal bioactive compound in terms of ovarian development, because the ovary-inducing activity of D-Trp was 500 times more potent than that of L-Trp. However, how Trp controls sexual induction is still unknown. RESULTS: In this study, qRT-PCR analyses suggested that the putative amino acid transporter gene Dr-SLC38A9 is highly expressed in sexual worms, especially in the yolk glands. In situ hybridization analyses showed that Dr-SLC38A9 is expressed in the ovarian primordia of asexual worms and in the mature ovaries, testes, and yolk glands of sexual worms. In addition, Dr-SLC38A9 RNA interference during sexual induction resulted in the suppression of the development of reproductive organs. These results suggest that Dr-SLC38A9 is involved in the development of these organs. Moreover, we demonstrated that the reproductive organ-specific expression of Dr-SLC38A9 is enhanced by the addition of D-Trp. CONCLUSION: We propose that D-Trp activates the expression of Dr-SLC38A9 to promote sexual induction in the planarian D. ryukyuensis.

DOI： 10.1186/s40851-021-00173-z

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Guaranteeing the sustainability of gametogenesis is a fundamental issue for perpetuating the species. In the mammalian ovary, sustainability is accomplished by keeping a number of oocytes "stocked" in the dormant state. Despite the evident importance of this state, the mechanisms underlying the oocyte dormancy are not fully understood, although it is presumed that both intrinsic and extrinsic factors are involved. Here, we review environmental factors involved in the regulation of oocyte dormancy. Consideration of the environmental factors illustrates the nature of the ovarian compartment, in which primordial follicles reside. This should greatly improve our understanding of the mechanisms and also assist in reconstitution of the dormant state in culture. Accumulating knowledge on the dormant state of oocytes will contribute to a wide range of research in fields such as developmental biology, reproductive biology and regenerative medicine.

DOI： 10.1111/dgd.12653

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

A set of sex chromosomes is required for gametogenesis in both males and females, as represented by sex chromosome disorders causing agametic phenotypes. Although studies using model animals have investigated the functional requirement of sex chromosomes, involvement of these chromosomes in gametogenesis remains elusive. Here, we elicit a germ cell-intrinsic effect of sex chromosomes on oogenesis, using a novel culture system in which oocytes were induced from embryonic stem cells (ESCs) harboring XX, XO or XY. In the culture system, oogenesis using XO and XY ESCs was severely disturbed, with XY ESCs being more strongly affected. The culture system revealed multiple defects in the oogenesis of XO and XY ESCs, such as delayed meiotic entry and progression, and mispairing of the homologous chromosomes. Interestingly, Eif2s3y, a Y-linked gene that promotes proliferation of spermatogonia, had an inhibitory effect on oogenesis. This led us to the concept that male and female gametogenesis appear to be in mutual conflict at an early stage. This study provides a deeper understanding of oogenesis under a sex-reversal condition.

DOI： 10.1371/journal.pgen.1008676

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Respiratory failure is a life-threatening problem for pre-term and term infants, yet many causes remain unknown. Here, we present evidence that whey acidic protein (WAP) four-disulfide core domain protease inhibitor 2 (Wfdc2), a protease inhibitor previously unrecognized in respiratory disease, may be a causal factor in infant respiratory failure. Wfdc2 transcripts are detected in the embryonic lung and analysis of a Wfdc2-GFP knock-in mouse line shows that both basal and club cells, and type II alveolar epithelial cells (AECIIs), express Wfdc2 neonatally. Wfdc2-null-mutant mice display progressive atelectasis after birth with a lethal phenotype. Mutant lungs have multiple defects, including impaired cilia and the absence of mature club cells from the tracheo-bronchial airways, and malformed lamellar bodies in AECIIs. RNA sequencing shows significant activation of a pro-inflammatory pathway, but with low-quantity infiltration of mononuclear cells in the lung. These data demonstrate that Wfdc2 function is vitally important for lung aeration at birth and that gene deficiency likely causes failure of the lung mucosal barrier.

DOI： 10.1242/dmm.040139

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Nanog is a core transcription factor specifically expressed not only in the pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs), embryonic germ cells (EGCs), and induced PSCs (iPSCs), but also in the unipotent primordial germ cells (PGCs). Although Nanog promoter/enhancer regions are well characterized by in vitro analyses, direct correlations between the regulatory elements for Nanog expression and in vivo expression patterns of Nanog have not been fully clarified. In this study, we generated Nanog-RFP transgenic (Tg) mice in which expression of red fluorescent protein (RFP) is driven by a 5.2 kb Nanog promoter/enhancer region. As expected, RFP was expressed in the inner cell mass of blastocysts, ESCs, and iPSCs. However, RFP fluorescence was not observed in PGCs, although Nanog was expressed in PGCs. Because RFP fluorescence was visible in the PGC-derived pluripotent EGCs in culture, it was suggested that the reporter gene expression was specifically activated in PSCs. In conclusion, we have generated a novel Nanog-RFP Tg mouse line that can selectively tag PSCs over unipotent PGCs.

DOI： 10.1002/dvg.23334

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

In mammals, most immature oocytes remain dormant in the primordial follicles to ensure the longevity of female reproductive life. A precise understanding of mechanisms underlying the dormancy is important for reproductive biology and medicine. In this study, by comparing mouse oogenesis in vivo and in vitro, the latter of which bypasses the primordial follicle stage, we defined the gene-expression profile representing the dormant state of oocytes. Overexpression of constitutively active FOXO3 partially reproduced the dormant state in vitro. Based on further gene-expression analysis, we found that a hypoxic condition efficiently induced the dormant state in vitro. The effect of hypoxia was severely diminished by disruption of the Foxo3 gene and inhibition of hypoxia-inducible factors. Our findings provide insights into the importance of environmental conditions and their effectors for establishing the dormant state.

DOI： 10.1073/pnas.1817223116

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The most immature oocytes remain dormant in primordial follicles in the ovary, ensuring the longevity of female reproductive life. Despite its biological and clinical importance, knowledge of mechanisms regulating the dormant state remains limited. Here, we show that mechanical stress plays a key role in maintaining the dormant state of the oocytes in primordial follicles in mice. Transcriptional and histological analyses revealed that oocytes were compressed by surrounding granulosa cells with extracellular matrix. This environmental state is functionally crucial, as oocytes became activated upon loosening the structure and the dormancy was restored by additional compression with exogenous pressure. The nuclei of oocytes in primordial follicles rotated in response to the mechanical stress. Pausing the rotation triggered activation of oocytes through nuclear export of forkhead box O3 (FOXO3). These results provide insights into the mechanisms by which oocytes are kept dormant to sustain female reproductive life.

DOI： 10.1126/sciadv.aav9960

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Reconstitution in culture of biological processes, such as differentiation and organization, is a key challenge in regenerative medicine, and one in which stem cell technology plays a central role. Pluripotent stem cells and spermatogonial stem cells are useful materials for reconstitution of germ cell development in vitro, as they are capable of differentiating into gametes. Reconstitution of germ cell development, termed in vitro gametogenesis, will provide an experimental platform for a better understanding of germ cell development, as well as an alternative source of gametes for reproduction, with the potential to cure infertility. Since germ cells are the cells for 'the next generation', both the culture system and its products must be carefully evaluated. In this issue, we summarize the progress in in vitro gametogenesis, most of which has been made using mouse models, as well as the future challenges in this field.

DOI： 10.1530/REP-17-0510

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Primordial germ cells (PGCs) are specified from epiblast cells in mice. Genes associated with naive pluripotency are repressed in the transition from inner cell mass to epiblast cells, followed by upregulation after PGC specification. However, the molecular mechanisms underlying the reactivation of pluripotency genes are poorly characterized. Here, we exploited the in vitro differentiation of epiblast-like cells (EpiLCs) from embryonic stem cells (ESCs) to elucidate the molecular and epigenetic functions of PR domain-containing 14 (PRDM14). We found that Prdm14 overexpression in EpiLCs induced their conversion to ESC-like cells even in the absence of leukemia inhibitory factor in adherent culture. This was impaired by the loss of Kruppel-like factor 2 and ten-eleven translocation (TET) proteins. Furthermore, PRDM14 recruited OCT3/4 to the enhancer regions of naive pluripotency genes via TET-base excision repair-mediated demethylation. Our results provide evidence that PRDM14 establishes a transcriptional network for naive pluripotency via active DNA demethylation.

DOI： 10.1016/j.stemcr.2016.10.007

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The female germ line undergoes a unique sequence of differentiation processes that confers totipotency to the egg. The reconstitution of these events in vitro using pluripotent stem cells is a key achievement in reproductive biology and regenerative medicine. Here we report successful reconstitution in vitro of the entire process of oogenesis from mouse pluripotent stem cells. Fully potent mature oocytes were generated in culture from embryonic stem cells and from induced pluripotent stem cells derived from both embryonic fibroblasts and adult tail tip fibroblasts. Moreover, pluripotent stem cell lines were re-derived from the eggs that were generated in vitro, thereby reconstituting the full female germline cycle in a dish. This culture system will provide a platform for elucidating the molecular mechanisms underlying totipotency and the production of oocytes of other mammalian species in culture.

DOI： 10.1038/nature20104

PubMed

記述言語：日本語   出版者・発行元：(公社)日本繁殖生物学会  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Hematopoietic stem cells (HSCs) maintain quiescence by activating specific metabolic pathways, including glycolysis. We do not yet have a clear understanding of how this metabolic activity changes during stress hematopoiesis, such as bone marrow transplantation. Here, we report a critical role for the p38MAPK family isoform p38α in initiating hematopoietic stem and progenitor cell (HSPC) proliferation during stress hematopoiesis in mice. We found that p38MAPK is immediately phosphorylated in HSPCs after a hematological stress, preceding increased HSPC cycling. Conditional deletion of p38α led to defective recovery from hematological stress and a delay in initiation of HSPC proliferation. Mechanistically, p38α signaling increases expression of inosine-5'-monophosphate dehydrogenase 2 in HSPCs, leading to altered levels of amino acids and purine-related metabolites and changes in cell-cycle progression in vitro and in vivo. Our studies have therefore uncovered a p38α-mediated pathway that alters HSPC metabolism to respond to stress and promote recovery.

DOI： 10.1016/j.stem.2016.05.013

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Primordial germ cells (PGCs) are lineage-restricted unipotent cells that can dedifferentiate into pluripotent embryonic germ cells (EGCs). Here we performed whole-transcriptome analysis during the conversion of PGCs into EGCs, a process by which cells acquire pluripotency. To examine the molecular mechanism underlying this conversion, we focused on Blimp-1 and Akt, which are involved in PGC specification and dedifferentiation, respectively. Blimp-1 overexpression in embryonic stem cells suppressed the expression of downstream targets of the pluripotency network. Conversely, Blimp-1 deletion in PGCs accelerated their dedifferentiation into pluripotent EGCs, illustrating that Blimp-1 is a pluripotency gatekeeper protein in PGCs. AKT signaling showed a synergistic effect with basic fibroblast growth factor plus 2i+A83 treatment on EGC formation. AKT played a major role in suppressing genes regulated by MBD3. From these results, we defined the distinct functions of Blimp-1 and Akt and provided mechanistic insights into the acquisition of pluripotency in PGCs.

DOI： 10.1016/j.stemcr.2015.05.007

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Reactivation of the endogenous telomerase reverse transcriptase (TERT) catalytic subunit and telomere elongation occur during the reprogramming of somatic cells to induced pluripotent stem (iPS) cells. However, the role of TERT in the reprogramming process is unclear. To clarify its function, the reprogramming process was examined in TERT-KO somatic cells. To exclude the effect of telomere elongation, tail-tip fibroblasts (TTFs) from first generation TERT-KO mice were used. Although iPS cells were successfully generated from TERT-KO TTFs, the efficiency of reprogramming these cells was markedly lower than that of WT TTFs. The gene expression profiles of iPS cells induced from TERT-KO TTFs were similar to those of WT iPS cells and ES cells, and TERT-KO iPS cells formed teratomas that differentiated into all three germ layers. These data indicate that TERT plays an extratelomeric role in the reprogramming process, but its function is dispensable. However, TERT-KO iPS cells showed transient defects in growth and teratoma formation during continuous growth. In addition, TERT-KO iPS cells developed chromosome fusions that accumulated with increasing passage numbers, consistent with the fact that TERT is essential for the maintenance of genome structure and stability in iPS cells. In a rescue experiment, an enzymatically inactive mutant of TERT (D702A) had a positive effect on somatic cell reprogramming of TERT-KO TTFs, which confirmed the extratelomeric role of TERT in this process.

DOI： 10.1074/jbc.M113.536037

PubMed

記述言語：英語   掲載種別：（MISC）研究発表要旨（国際会議）   出版者・発行元：ELSEVIER SCIENCE INC  

DOI： 10.1016/j.juro.2014.02.291

Web of Science

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Nucleostemin is a nucleolar protein known to play a variety of roles in cell-cycle progression, apoptosis inhibition, and DNA damage protection in embryonic stem cells and tissue stem cells. However, the role of nucleostemin in hematopoietic stem cells (HSCs) is yet to be determined. Here, we identified an indispensable role of nucleostemin in mouse HSCs. Depletion of nucleostemin using short hairpin RNA strikingly impaired the self-renewal activity of HSCs both in vitro and in vivo. Consistently, nucleostemin depletion triggered apoptosis rather than cell-cycle arrest in HSCs. Furthermore, DNA damage accumulated during cultivation upon depletion of nucleostemin. The impaired self-renewal activity of HSCs induced by nucleostemin depletion was partially rescued by p53 deficiency but not by p16(Ink4a) or p19(Arf) deficiency. Taken together, our study demonstrates that nucleostemin protects HSCs from DNA damage accumulation and is required for the maintenance of HSCs.

DOI： 10.1016/j.bbrc.2013.10.032

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Considering the similarities between the transcriptional programming involved in cancer progression and somatic cell reprogramming, we tried to identify drugs that would be effective against malignant cancers. We used the early transposon Oct4 and Sox2 enhancer (EOS) system to select human prostate cancer (PCA) cells expressing high levels of OCT4. Patients with metastatic castration-resistant PCA that does not respond to treatment with docetaxel have few therapeutic options. The OCT4-expressing PCA cells selected using the EOS system showed increased tumorigenicity and high resistance to docetaxel, both in vitro and in vivo. By using their gene expression data, expression signature-based prediction for compound candidates identified an antiviral drug, ribavirin, as a conversion modulator from drug resistance to sensitivity. Treatment of PCA cells with ribavirin decreased their resistance against treatment with docetaxel. This indicated that ribavirin reversed the gene expression, including that of humoral factors, in the OCT4-expressing PCA cells selected using the EOS system. Thereby, ribavirin increased the efficacy of docetaxel for cancer cells. We propose a novel cell reprogramming approach, named drug efficacy reprogramming, as a new model for identifying candidate antitumor drugs.

DOI： 10.1111/cas.12183

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Germ cells are similar to pluripotent stem cells in terms of gene expression patterns and the capacity to convert to pluripotent stem cells in culture. The factors involved in germ cell development are also able to reprogram somatic cells. This suggests that germ cells are useful tools for investigating the mechanisms responsible for somatic cell reprograming. In this study, the expression of reprograming factors in primordial germ cells (PGCs) was analyzed. PGCs expressed Oct3/4, Sox2, and c-Myc but not Klf4. However, Klf2, Klf5, Essrb, or Essrg, which were expressed in PGCs, could compensate for Klf4 during somatic cell reprograming. Furthermore, PGCs could be converted to a pluripotent state by infection with any of the known reprogramming factors (Oct3/4, Sox2, Klf4, and c-Myc). These cells were designated as multipotent PGCs (mPGCs). Contrary to differences in the origins of somatic cells in somatic cell reprogramming, we hypothesized that the gene expression levels of the reprogramming factors would vary in mPGCs. Candidate genes involved in the regulation of tumorigenicity and/or reprogramming efficiency were identified by comparing the gene expression profiles of mPGCs generated by the exogenous expression of c-Myc or L-Myc.

DOI： 10.1002/stem.1303

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The epigenetic status of germ cells changes dynamically during development. In this study, we analyzed the dynamics of histone H3 lysine 9 dimethylation (H3K9me2), a highly conserved mark of epigenetic silencing, and the expression of two lysine methyltransferases, G9a/Ehmt2/KMT1C and GLP/Ehmt1/KMT1D, in murine male embryonic germ cells after sex determination. Our previous studies established that G9a and GLP are the primary enzymes for H3K9me2 and predominantly exist as a G9a-GLP heteromeric complex that appears to be a functional H3K9 methyltransferase in vivo. During the period from Embryonic Day (E) 13.5 to E18.5 in mice, gonadal H3K9me2 levels were substantially lower in germ cells than in cells of nongerm lineage. Immunohistochemical analysis showed that during this phase in development, GLP level, but not G9a level, was also significantly lower in male germ cells. However, GLP mRNA was present in E13 and E16 male germ cells, with levels similar to those in cells of nongerm lineage. Interestingly, GLP is upregulated in embryonic male germ cells deficient for Nanos2, which encodes a germ cell-specific RNA-binding protein. Our data suggest that GLP protein expression is posttranscriptionally regulated in murine embryonic male germ cells after sex determination and that low H3K9me2 level results from the absence of GLP (severe reduction of the G9a-GLP heteromeric complex).

DOI： 10.1095/biolreprod.112.103572

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Defining the metabolic programs that underlie stem cell maintenance will be essential for developing strategies to manipulate stem cell capacity. Mammalian hematopoietic stem cells (HSCs) maintain cell cycle quiescence in a hypoxic microenvironment. It has been proposed that HSCs exhibit a distinct metabolic phenotype under these conditions. Here we directly investigated this idea using metabolomic analysis and found that HSCs generate adenosine-5'-triphosphate by anaerobic glycolysis through a pyruvate dehydrogenase kinase (Pdk)-dependent mechanism. Elevated Pdk expression leads to active suppression of the influx of glycolytic metabolites into mitochondria. Pdk overexpression in glycolysis-defective HSCs restored glycolysis, cell cycle quiescence, and stem cell capacity, while loss of both Pdk2 and Pdk4 attenuated HSC quiescence, glycolysis, and transplantation capacity. Moreover, treatment of HSCs with a Pdk mimetic promoted their survival and transplantation capacity. Thus, glycolytic metabolic status governed by Pdk acts as a cell cycle checkpoint that modulates HSC quiescence and function.

DOI： 10.1016/j.stem.2012.10.011

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Primordial germ cells (PGCs) are unipotent cells committed to germ lineage: PGCs can only differentiate into gametes in vivo. However, upon fertilization, germ cells acquire the capacity to differentiate into all cell types in the body, including germ cells. Therefore, germ cells are thought to have the potential for pluripotency. PGCs can convert to pluripotent stem cells in vitro when cultured under specific conditions that include bFGF, LIF, and the membrane-bound form of SCF (mSCF). Here, the culture conditions which efficiently convert PGCs to pluripotent embryonic germ (EG) cells are described, as well as methods used for identifying pluripotent candidate cells during culture.

DOI： 10.1007/7651_2013_24

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Somatic cell reprogramming is achieved by four reprogramming transcription factors (RTFs), Oct3/4, Sox2, Klf4, and c-Myc. However, in addition to the induction of pluripotent cells, these RTFs also generate pseudo-pluripotent cells, which do not show Nanog promoter activity. Therefore, it should be possible to fine-tune the RTFs to produce only fully pluripotent cells. For this study, a tagging system was developed to sort induced pluripotent stem (iPS) cells according to the expression levels of each of the four RTFs. Using this system, the most effective ratio (Oct3/4-high, Sox2-low, Klf4-high, c-Myc-high) of the RTFs was 88 times more efficient at producing iPS cells than the worst effective ratio (Oct3/4-low, Sox2-high, Klf4-low, c-Myc-low). Among the various RTF combinations, Oct3/4-high and Sox2-low produced the most efficient results. To investigate the molecular basis, microarray analysis was performed on iPS cells generated under high (Oct3/4-high and Sox2-low) and low (Oct3/4-low and Sox2-high) efficiency reprogramming conditions. Pathway analysis revealed that the G protein-coupled receptor (GPCR) pathway was up-regulated significantly under the high efficiency condition and treatment with the chemokine, C-C motif ligand 2, a member of the GPCR family, enhanced somatic cell reprogramming 12.3 times. Furthermore, data from the analysis of the signature gene expression profiles of mouse embryonic fibroblasts at 2 days after RTF infection revealed that the genetic modifier, Whsc1l1 (variant 1), also improved the efficiency of somatic cell reprogramming. Finally, comparison of the overall gene expression profiles between the high and low efficiency conditions will provide novel insights into mechanisms underlying somatic cell reprogramming.

DOI： 10.1074/jbc.M112.380683

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

To understand mechanisms underlying acquisition of pluripotency, it is critical to identify cells that can be converted to pluripotent stem cells. For this purpose, we focused on unipotent primordial germ cells (PGCs), which can be reprogrammed into pluripotent embryonic germ (EG) cells under defined conditions. Treatment of PGCs with combinations of signaling inhibitors, including inhibitors of MAP2K (MEK), GSK3B (GSK-3beta), and TGFB (TGFbeta) type 1 receptors, induced cells to enter a pluripotent state at a high frequency (12.1%) by Day 10 of culture. When we employed fluorescence-activated cell sorting to monitor conversion of candidate cells to a pluripotent state, we observed a cell cycle shift to S phase, indicating enrichment of pluripotent cells, during the early phase of EG formation. Transcriptome analysis revealed that PGCs retained expression of some pluripotent stem cell-associated genes, such as Pou5f1 and Sox2, during EG cell formation. On the other hand, PGCs lost their germ lineage characteristics and acquired expression of pluripotent stem cell markers, such as Klf4 and Eras. The overall gene expression profiles revealed by this system provide novel insight into how pluripotency is acquired in germ-committed cells.

DOI： 10.1095/biolreprod.111.096792

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

During cell division, one of the major features of somatic cell reprogramming by defined factors, cells are potentially exposed to DNA damage. Inactivation of the tumor suppressor gene p53 raised reprogramming efficiency but resulted in an increased number of abnormal chromosomes in established iPS cells. Ataxia-telangiectasia mutated (ATM), which is critical in the cellular response to DNA double-strand breaks, may also play an important role during reprogramming. To clarify the function of ATM in somatic cell reprogramming, we investigated reprogramming in ATM-deficient (ATM-KO) tail-tip fibroblasts (TTFs). Although reprogramming efficiency was greatly reduced in ATM-KO TTFs, ATM-KO iPS cells were successfully generated and showed the same proliferation activity as WT iPS cells. ATM-KO iPS cells had a gene expression profile similar to ES cells and WT iPS cells, and had the capacity to differentiate into all three germ layers. On the other hand, ATM-KO iPS cells accumulated abnormal genome structures upon continuous passages. Even with the abnormal karyotype, ATM-KO iPS cells retained pluripotent cell characteristics for at least 20 passages. These data indicate that ATM does participate in the reprogramming process, although its role is not essential.

DOI： 10.1016/j.bbrc.2011.03.013

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Germ cells possess the unique ability to acquire totipotency during development in vivo as well as give rise to pluripotent stem cells under the appropriate conditions in vitro. Recent studies in which somatic cells were experimentally converted into pluripotent stem cells revealed that genes expressed in primordial germ cells (PGCs), such as Oct3/4, Sox2, and Lin28, are involved in this reprogramming. These findings suggest that PGCs may be useful for identifying factors that successfully and efficiently reprogram somatic cells into toti- and/or pluripotent stem cells. Here, we show that Blimp-1, Prdm14, and Prmt5, each of which is crucial for PGC development, have the potential to reprogram somatic cells into pluripotent stem cells. Among them, Prmt5 exhibited remarkable reprogramming of mouse embryonic fibroblasts into which Prmt5, Klf4, and Oct3/4 were introduced. The resulting cells exhibited pluripotent gene expression, teratoma formation, and germline transmission in chimeric mice, all of which were indistinguishable from those induced with embryonic stem cells. These data indicate that some of the factors that play essential roles in germ cell development are also active in somatic cell reprogramming.

DOI： 10.1074/jbc.M110.216390

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The nucleostemin (NS) gene encodes a nucleolar protein found at high levels in several types of stem cells and tumor cell lines. The function of NS is unclear but it may play a critical role in S-phase entry by stem/progenitor cells. Here we characterize NS expression in murine male germ cells. Although NS protein was highly expressed in the nucleoli of all primordial germ cells, only a limited number of gonocytes showed NS expression in neonatal testes. In adult testes, NS protein was expressed at high levels in the nucleoli of spermatogonia and primary spermatocytes but at only low levels in round spermatids. To evaluate the properties of cells expressing high levels of NS, we generated transgenic reporter mice expressing green fluorescent protein (GFP) under the control of the NS promoter (NS-GFP Tg mice). In adult NS-GFP Tg testes, GFP and endogenous NS protein expression were correlated in spermatogonia and spermatocytes but GFP was also ectopically expressed in elongated spermatids and sperm. In testes of NS-GFP Tg embryos, neonates, and 10-day-old pups, however, GFP expression closely coincided with endogenous NS expression in developing germ cells. In contrast to a previous report, our results support the existence in neonatal testes of spermatogonial stem cells with long-term repopulating capacity. Furthermore, our data show that NS expression does not correlate with cell-cycle status during prepuberty, and that strong NS expression is essential for the maintenance of germline stem cell proliferation capacity. We conclude that NS is a marker of undifferentiated status in the germ cell lineage during prepubertal spermatogenesis.

DOI： 10.1634/stemcells.2008-0506

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Mammalian spermatogenesis is maintained by stem cell capacity within undifferentiated spermatogonial subpopulation. Here, using a combination of surface markers, we describe a purification method for undifferentiated spermatogonia. Flow cytometric analysis revealed that this population is composed of Plzf-positive cells and exhibits quiescence and the side population phenotype, fulfilling general stem cell criteria. We then applied this method to analyze undifferentiated spermatogonia and stem cell activity of Atm(-/-) mice. Atm(-/-) testis shows progressive depletion of undifferentiated spermatogonia accompanied by cell-cycle arrest. In Atm(-/-) undifferentiated spermatogonia, a self-renewal defect was observed in vitro and in vivo. Accumulation of DNA damage and activation of the p19(Arf)-p53-p21(Cip1/Waf1) pathway were observed in Atm(-/-) undifferentiated spermatogonia. Moreover, suppression of p21(Cip1/Waf1) in an Atm(-/-) background restored transplantation ability of undifferentiated spermatogonia, indicating that ATM plays an essential role in maintenance of undifferentiated spermatogonia and their stem cell capacity by suppressing DNA damage-induced cell-cycle arrest.

DOI： 10.1016/j.stem.2007.10.023

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Lifelong spermatogenesis is maintained by coordinated sequential processes including self-renewal of stem cells, proliferation of spermatogonial cells, meiotic division, and spermiogenesis. It has been shown that ataxia telangiectasia-mutated (ATM) is required for meiotic division of the seminiferous tubules. Here, we show that, in addition to its role in meiosis, ATM has a pivotal role in premeiotic germ cell maintenance. ATM is activated in premeiotic spermatogonial cells and the Atm-null testis shows progressive degeneration. In Atm-null testicular cells, differing from bone marrow cells of Atm-null mice, reactive oxygen species-mediated p16(Ink4a) activation does not occur in Atm-null premeiotic germ cells, which suggests the involvement of different signaling pathways from bone marrow defects. Although Atm-null bone marrow undergoes p16(Ink4a)-mediated cellular senescence program, Atm-null premeiotic germ cells exhibited cell cycle arrest and apoptotic elimination of premeiotic germ cells, which is different from p16(Ink4a)-mediated senescence.

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Stem cells are maintained in an undifferentiated state by interacting with a microenvironment known as the "niche," which is comprised of various secreted and membrane proteins. Our goal was to identify niche molecules participating in stem cell-stem cell and/or stem cell-supporting cell interactions. Here, we isolated genes encoding secreted and membrane proteins from purified male germ stem cells using a signal sequence trap approach. Among the genes identified, we focused on the junctional adhesion molecule 4 (JAM4), an immunoglobulin type cell adhesion molecule. JAM4 protein was actually localized to the plasma membrane in male germ cells. JAM4 expression was downregulated as cells differentiated in both germ cell and hematopoietic cell lineages. To analyze function in vivo, we generated JAM4-deficient mice. Histological analysis of testes from homozygous nulls did not show obvious abnormalities, nor did liver and kidney tissues, both of which strongly express JAM4. The numbers of hematopoietic stem cells in bone marrow were indistinguishable between wild-type and mutant mice, as was male germ cell development. These results suggest that JAM4 is expressed in stem cells and progenitor cells but that other cell adhesion molecules may substitute for JAM4 function in JAM4-deficient mice both in male germ cell and hematopoietic lineages.

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Mammalian spermatogenesis is maintained by a continuous supply of differentiating cells from self-renewing stem cells. The stem cell activity resides in a small subset of primitive germ cells, the undifferentiated spermatogonia. However, the relationship between the establishment of this population and the initiation of differentiation in the developing testes remains unclear. In this study, we have investigated this issue by using the unique expression of Ngn3, which is expressed specifically in the undifferentiated spermatogonia, but not in the differentiating spermatogonia or their progenitors, the gonocytes. Our lineage analyses demonstrate that the first round of mouse spermatogenesis initiates directly from gonocytes, without passing through the Ngn3-expressing stage (Ngn3- lineage). By contrast, the subsequent rounds of spermatogenesis are derived from Ngn3-positive undifferentiated spermatogonia, which are also immediate descendents of the gonocytes and represent the stem cell function (Ngn3+ lineage). Thus, in mouse spermatogenesis, the state of the undifferentiated spermatogonia is not an inevitable step but is a developmental option that ensures continuous sperm production. In addition, the segregation of gonocytes into undifferentiated spermatogonia (Ngn3+ lineage) or differentiating spermatogonia (Ngn3- lineage) is topographically related to the establishment of the seminiferous epithelial cycle, thus suggesting a role of somatic components in the establishment of stem cells.

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Questions persist regarding male germ stem cells and how they mature during the prespermatogenic period of testicular development. We successfully labeled the prespermatogonia with green fluorescence protein (GFP) by using Oct-4 enhancer/promoter. This study shows that GFP was specifically expressed in prespermatogonia, spermatogonia and spermatids that faithfully reproduce the endogenous expression of Oct-4. Histochemical analysis revealed that most of the TRA98-positive gonocytes are also positive for GFP. However, the frequency of GFP expressing cells out of TRA98 expressing cells decreased together with the maturation of gonocytes in the first week after birth. To compare the stem cell activity between GFP-positive and -negative populations, we performed a transplantation of sorted cells into testes from an individual population. Colonization efficiency of germ cells from a GFP-positive population resulted in a 30-fold increase in colonization compared with a GFP-negative population. Since the expression of Oct-4 in prespermtogonia correlates well with the stem activity, Oct-4 might be a crucial molecule in the stem cell property of spermatogonia but not in cell survival.

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Mouse interferon-induced transmembrane protein (IFITM) gene, Ifitm3 (previously known as mil-1 and fragilis), is expressed in primordial germ cells (PGCs), in their precursors, and in germ cells of the fetal gonads (Saitou et al. [2002] Nature 418:293-300; Tanaka and Matsui [2002] Mech Dev 119S:S261-S267). By examining the expression of green fluorescent protein transgene under the control of DNA sequences flanking exon 1, we have identified domains that direct Ifitm3 transcription in PGCs and their precursors in gastrula stage and 13.5 days post coitum embryos. Germ cell-specific expression is achieved by the activity of a consensus element unique to the Ifitm genes, which may act to suppress Ifitm3 expression in somatic tissues. The lack of any influence of the interferon-stimulable response elements on transgene expression in the germ-line suggests that interferon-mediated response is not critical for activating Ifitm3.

PubMed