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1.
Nature ; 2024 Jul 17.
Article in English | MEDLINE | ID: mdl-39020172

ABSTRACT

Telomerase is intimately associated with stem cells and cancer, because it catalytically elongates telomeres-nucleoprotein caps that protect chromosome ends1. Overexpression of telomerase reverse transcriptase (TERT) enhances the proliferation of cells in a telomere-independent manner2-8, but so far, loss-of-function studies have provided no evidence that TERT has a direct role in stem cell function. In many tissues, homeostasis is shaped by stem cell competition, a process in which stem cells compete on the basis of inherent fitness. Here we show that conditional deletion of Tert in the spermatogonial stem cell (SSC)-containing population in mice markedly impairs competitive clone formation. Using lineage tracing from the Tert locus, we find that TERT-expressing SSCs yield long-lived clones, but that clonal inactivation of TERT promotes stem cell differentiation and a genome-wide reduction in open chromatin. This role for TERT in competitive clone formation occurs independently of both its reverse transcriptase activity and the canonical telomerase complex. Inactivation of TERT causes reduced activity of the MYC oncogene, and transgenic expression of MYC in the TERT-deleted pool of SSCs efficiently rescues clone formation. Together, these data reveal a catalytic-activity-independent requirement for TERT in enhancing stem cell competition, uncover a genetic connection between TERT and MYC and suggest that a selective advantage for stem cells with high levels of TERT contributes to telomere elongation in the male germline during homeostasis and ageing.

2.
Cell Death Dis ; 14(8): 501, 2023 08 04.
Article in English | MEDLINE | ID: mdl-37542070

ABSTRACT

Gonadal sex determination and differentiation are controlled by somatic support cells of testes (Sertoli cells) and ovaries (granulosa cells). In testes, the epigenetic mechanism that maintains chromatin states responsible for suppressing female sexual differentiation remains unclear. Here, we show that Polycomb repressive complex 1 (PRC1) suppresses a female gene regulatory network in postnatal Sertoli cells. We genetically disrupted PRC1 function in embryonic Sertoli cells after sex determination, and we found that PRC1-depleted postnatal Sertoli cells exhibited defective proliferation and cell death, leading to the degeneration of adult testes. In adult Sertoli cells, PRC1 suppressed specific genes required for granulosa cells, thereby inactivating the female gene regulatory network. Chromatin regions associated with female-specific genes were marked by Polycomb-mediated repressive modifications: PRC1-mediated H2AK119ub and PRC2-mediated H3K27me3. Taken together, this study identifies a critical Polycomb-based mechanism that suppresses ovarian differentiation and maintains Sertoli cell fate in adult testes.


Subject(s)
Histones , Polycomb Repressive Complex 1 , Female , Male , Humans , Polycomb Repressive Complex 1/genetics , Polycomb Repressive Complex 1/metabolism , Histones/genetics , Histones/metabolism , Testis/metabolism , Gene Regulatory Networks , Polycomb Repressive Complex 2/genetics , Polycomb Repressive Complex 2/metabolism , Chromatin , Polycomb-Group Proteins/genetics , Polycomb-Group Proteins/metabolism , Cell Differentiation/genetics
3.
Biol Open ; 11(4)2022 04 15.
Article in English | MEDLINE | ID: mdl-35394008

ABSTRACT

In the mouse testis, sperm originate from spermatogonial stem cells (SSCs). SSCs give rise to spermatogonial progenitors, which expand their population until entering the differentiation process that is precisely regulated by a fixed time-scaled program called the seminiferous cycle. Although this expansion process of progenitors is highly important, its regulatory mechanisms remain unclear. NANOS3 is an RNA-binding protein expressed in the progenitor population. We demonstrated that the conditional deletion of Nanos3 at a later embryonic stage results in the reduction of spermatogonial progenitors in the postnatal testis. This reduction was associated with the premature differentiation of progenitors. Furthermore, this premature differentiation caused seminiferous stage disagreement between adjacent spermatogenic cells, which influenced spermatogenic epithelial cycles, leading to disruption of the later differentiation pathway. Our study suggests that NANOS3 plays an important role in timing progenitor expansion to adjust to the proper differentiation timing by blocking the retinoic acid (RA) signaling pathway.


Subject(s)
Adult Germline Stem Cells , Spermatogonia , Adult Germline Stem Cells/metabolism , Animals , Cell Differentiation/genetics , Male , Mice , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Spermatogenesis/genetics , Spermatogonia/metabolism , Testis
4.
J Cell Sci ; 131(17)2018 09 03.
Article in English | MEDLINE | ID: mdl-30097555

ABSTRACT

Spermatogenesis involves the progressive reorganization of heterochromatin. However, the mechanisms that underlie the dynamic remodeling of heterochromatin remain unknown. Here, we identify SCML2, a germline-specific Polycomb protein, as a critical regulator of heterochromatin organization in spermatogenesis. We show that SCML2 accumulates on pericentromeric heterochromatin (PCH) in male germ cells, where it suppresses PRC1-mediated monoubiquitylation of histone H2A at Lysine 119 (H2AK119ub) and promotes deposition of PRC2-mediated H3K27me3 during meiosis. In postmeiotic spermatids, SCML2 is required for heterochromatin organization, and the loss of SCML2 leads to the formation of ectopic patches of facultative heterochromatin. Our data suggest that, in the absence of SCML2, the ectopic expression of somatic lamins drives this process. Furthermore, the centromere protein CENP-V is a specific marker of PCH in postmeiotic spermatids, and SCML2 is required for CENP-V localization on PCH. Given the essential functions of PRC1 and PRC2 for genome-wide gene expression in spermatogenesis, our data suggest that heterochromatin organization and spermatogenesis-specific gene expression are functionally linked. We propose that SCML2 coordinates the organization of heterochromatin and gene expression through the regulation of Polycomb complexes.


Subject(s)
Heterochromatin/metabolism , Polycomb-Group Proteins/metabolism , Spermatids/metabolism , Spermatogenesis , Animals , Gene Expression Regulation, Developmental , Heterochromatin/genetics , Histones/genetics , Histones/metabolism , Male , Meiosis , Mice, Inbred C57BL , Mice, Knockout , Polycomb Repressive Complex 1/genetics , Polycomb Repressive Complex 1/metabolism , Polycomb-Group Proteins/genetics , Spermatids/cytology
5.
Proc Natl Acad Sci U S A ; 115(19): 4957-4962, 2018 05 08.
Article in English | MEDLINE | ID: mdl-29686098

ABSTRACT

Repressive H3K27me3 and active H3K4me2/3 together form bivalent chromatin domains, molecular hallmarks of developmental potential. In the male germline, these domains are thought to persist into sperm to establish totipotency in the next generation. However, it remains unknown how H3K27me3 is established on specific targets in the male germline. Here, we demonstrate that a germline-specific Polycomb protein, SCML2, binds to H3K4me2/3-rich hypomethylated promoters in undifferentiated spermatogonia to facilitate H3K27me3. Thus, SCML2 establishes bivalent domains in the male germline of mice. SCML2 regulates two major classes of bivalent domains: Class I domains are established on developmental regulator genes that are silent throughout spermatogenesis, while class II domains are established on somatic genes silenced during late spermatogenesis. We propose that SCML2-dependent H3K27me3 in the male germline prepares the expression of developmental regulator and somatic genes in embryonic development.


Subject(s)
Histones/metabolism , Polycomb-Group Proteins/metabolism , Promoter Regions, Genetic , Spermatogenesis/physiology , Spermatogonia/metabolism , Animals , Embryonic Development , Gene Expression Regulation, Developmental , Histones/genetics , Male , Mice , Mice, Knockout , Polycomb-Group Proteins/genetics , Spermatogonia/cytology
6.
Stem Cell Reports ; 10(2): 553-567, 2018 02 13.
Article in English | MEDLINE | ID: mdl-29337115

ABSTRACT

Undifferentiated spermatogonia comprise a pool of stem cells and progenitor cells that show heterogeneous expression of markers, including the cell surface receptor GFRα1. Technical challenges in isolation of GFRα1+ versus GFRα1- undifferentiated spermatogonia have precluded the comparative molecular characterization of these subpopulations and their functional evaluation as stem cells. Here, we develop a method to purify these subpopulations by fluorescence-activated cell sorting and show that GFRα1+ and GFRα1- undifferentiated spermatogonia both demonstrate elevated transplantation activity, while differing principally in receptor tyrosine kinase signaling and cell cycle. We identify the cell surface molecule melanocyte cell adhesion molecule (MCAM) as differentially expressed in these populations and show that antibodies to MCAM allow isolation of highly enriched populations of GFRα1+ and GFRα1- spermatogonia from adult, wild-type mice. In germ cell culture, GFRα1- cells upregulate MCAM expression in response to glial cell line-derived neurotrophic factor (GDNF)/fibroblast growth factor (FGF) stimulation. In transplanted hosts, GFRα1- spermatogonia yield GFRα1+ spermatogonia and restore spermatogenesis, albeit at lower rates than their GFRα1+ counterparts. Together, these data provide support for a model of a stem cell pool in which the GFRα1+ and GFRα1- cells are closely related but show key cell-intrinsic differences and can interconvert between the two states based, in part, on access to niche factors.


Subject(s)
Cell Differentiation/genetics , Glial Cell Line-Derived Neurotrophic Factor Receptors/genetics , Spermatogenesis/genetics , Spermatogonia/cytology , Animals , CD146 Antigen/genetics , Cell Lineage/genetics , Fibroblast Growth Factors/genetics , Flow Cytometry , Glial Cell Line-Derived Neurotrophic Factor/genetics , Glial Cell Line-Derived Neurotrophic Factor/metabolism , Male , Mice , Signal Transduction/genetics , Spermatogonia/growth & development , Stem Cell Niche/genetics , Stem Cells/cytology , Testis/cytology
7.
Genes Dev ; 31(16): 1693-1703, 2017 08 15.
Article in English | MEDLINE | ID: mdl-28924034

ABSTRACT

During spermatogenesis, a large number of germline genes essential for male fertility are coordinately activated. However, it remains unknown how timely activation of this group of germline genes is accomplished. Here we show that Polycomb-repressive complex 1 (PRC1) directs timely activation of germline genes during spermatogenesis. Inactivation of PRC1 in male germ cells results in the gradual loss of a stem cell population and severe differentiation defects, leading to male infertility. In the stem cell population, RNF2, the dominant catalytic subunit of PRC1, activates transcription of Sall4, which codes for a transcription factor essential for subsequent spermatogenic differentiation. Furthermore, RNF2 and SALL4 together occupy transcription start sites of germline genes in the stem cell population. Once differentiation commences, these germline genes are activated to enable the progression of spermatogenesis. Our study identifies a novel mechanism by which Polycomb directs the developmental process by activating a group of lineage-specific genes.


Subject(s)
Polycomb Repressive Complex 1/physiology , Spermatogenesis/genetics , Transcriptional Activation , Animals , Cell Line , DNA-Binding Proteins/metabolism , Epigenesis, Genetic , Male , Mice , Mice, Transgenic , Polycomb Repressive Complex 1/genetics , Polycomb Repressive Complex 1/metabolism , Spermatogonia/cytology , Spermatogonia/metabolism , Transcription Factors/metabolism , Transcription Initiation Site , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism
8.
Genes Dev ; 29(23): 2420-34, 2015 Dec 01.
Article in English | MEDLINE | ID: mdl-26584619

ABSTRACT

Telomerase inactivation causes loss of the male germline in worms, fish, and mice, indicating a conserved dependence on telomere maintenance in this cell lineage. Here, using telomerase reverse transcriptase (Tert) reporter mice, we found that very high telomerase expression is a hallmark of undifferentiated spermatogonia, the mitotic population where germline stem cells reside. We exploited these high telomerase levels as a basis for purifying undifferentiated spermatogonia using fluorescence-activated cell sorting. Telomerase levels in undifferentiated spermatogonia and embryonic stem cells are comparable and much greater than in somatic progenitor compartments. Within the germline, we uncovered an unanticipated gradient of telomerase activity that also enables isolation of more mature populations. Transcriptomic comparisons of Tert(High) undifferentiated spermatogonia and Tert(Low) differentiated spermatogonia by RNA sequencing reveals marked differences in cell cycle and key molecular features of each compartment. Transplantation studies show that germline stem cell activity is confined to the Tert(High) cKit(-) population. Telomere shortening in telomerase knockout strains causes depletion of undifferentiated spermatogonia and eventual loss of all germ cells after undifferentiated spermatogonia drop below a critical threshold. These data reveal that high telomerase expression is a fundamental characteristic of germline stem cells, thus explaining the broad dependence on telomerase for germline immortality in metazoans.


Subject(s)
Adult Stem Cells/enzymology , Gene Expression Regulation, Enzymologic , Spermatogonia/enzymology , Telomerase/genetics , Telomerase/metabolism , Animals , Cell Differentiation/genetics , Embryonic Stem Cells/enzymology , Flow Cytometry , Gene Knock-In Techniques , Male , Mice , Mice, Inbred C57BL , Promoter Regions, Genetic/genetics
9.
BMC Biol ; 13: 53, 2015 Jul 22.
Article in English | MEDLINE | ID: mdl-26198001

ABSTRACT

BACKGROUND: The male germline transcriptome changes dramatically during the mitosis-to-meiosis transition to activate late spermatogenesis genes and to transiently suppress genes commonly expressed in somatic lineages and spermatogenesis progenitor cells, termed somatic/progenitor genes. RESULTS: These changes reflect epigenetic regulation. Induction of late spermatogenesis genes during spermatogenesis is facilitated by poised chromatin established in the stem cell phases of spermatogonia, whereas silencing of somatic/progenitor genes during meiosis and postmeiosis is associated with formation of bivalent domains which also allows the recovery of the somatic/progenitor program after fertilization. Importantly, during spermatogenesis mechanisms of epigenetic regulation on sex chromosomes are different from autosomes: X-linked somatic/progenitor genes are suppressed by meiotic sex chromosome inactivation without deposition of H3K27me3. CONCLUSIONS: Our results suggest that bivalent H3K27me3 and H3K4me2/3 domains are not limited to developmental promoters (which maintain bivalent domains that are silent throughout the reproductive cycle), but also underlie reversible silencing of somatic/progenitor genes during the mitosis-to-meiosis transition in late spermatogenesis.


Subject(s)
Chromatin/genetics , Epigenesis, Genetic , Germ Cells/cytology , Meiosis , Mitosis , Sex Chromosomes/genetics , Spermatogenesis , Animals , Cells, Cultured , Chromatin/chemistry , Gene Expression Regulation, Developmental , Genes, X-Linked , Germ Cells/metabolism , Histones/chemistry , Histones/genetics , Male , Mice, Inbred C57BL , Sex Chromosomes/chemistry , Transcriptional Activation , Transcriptome , X Chromosome Inactivation
10.
Dev Cell ; 34(1): 96-107, 2015 Jul 06.
Article in English | MEDLINE | ID: mdl-26120033

ABSTRACT

In many adult tissues, homeostasis relies on self-renewing stem cells that are primed for differentiation. The reconciliation mechanisms of these characteristics remain a fundamental question in stem cell biology. We propose that regulation at the post-transcriptional level is essential for homeostasis in murine spermatogonial stem cells (SSCs). Here, we show that Nanos2, an evolutionarily conserved RNA-binding protein, works with other cellular messenger ribonucleoprotein (mRNP) components to ensure the primitive status of SSCs through a dual mechanism that involves (1) direct recruitment and translational repression of genes that promote spermatogonial differentiation and (2) repression of the target of rapamycin complex 1 (mTORC1), a well-known negative pathway for SSC self-renewal, by sequestration of the core factor mTOR in mRNPs. This mechanism links mRNA turnover to mTORC1 signaling through Nanos2-containing mRNPs and establishes a post-transcriptional buffering system to facilitate SSC homeostasis in the fluctuating environment within the seminiferous tubule.


Subject(s)
Gene Expression Regulation/physiology , RNA-Binding Proteins/genetics , RNA/metabolism , Spermatogonia/cytology , Stem Cells/cytology , Animals , Cell Differentiation/physiology , Cell Proliferation/physiology , Male , Mice , Signal Transduction/physiology
11.
Dev Cell ; 32(5): 574-88, 2015 Mar 09.
Article in English | MEDLINE | ID: mdl-25703348

ABSTRACT

Gametogenesis is dependent on the expression of germline-specific genes. However, it remains unknown how the germline epigenome is distinctly established from that of somatic lineages. Here we show that genes commonly expressed in somatic lineages and spermatogenesis-progenitor cells undergo repression in a genome-wide manner in late stages of the male germline and identify underlying mechanisms. SCML2, a germline-specific subunit of a Polycomb repressive complex 1 (PRC1), establishes the unique epigenome of the male germline through two distinct antithetical mechanisms. SCML2 works with PRC1 and promotes RNF2-dependent ubiquitination of H2A, thereby marking somatic/progenitor genes on autosomes for repression. Paradoxically, SCML2 also prevents RNF2-dependent ubiquitination of H2A on sex chromosomes during meiosis, thereby enabling unique epigenetic programming of sex chromosomes for male reproduction. Our results reveal divergent mechanisms involving a shared regulator by which the male germline epigenome is distinguished from that of the soma and progenitor cells.


Subject(s)
Epigenomics , Gene Expression Regulation, Developmental , Histones/metabolism , Polycomb Repressive Complex 1/metabolism , Testis/metabolism , Ubiquitin/metabolism , Adaptor Proteins, Signal Transducing , Animals , Biomarkers/metabolism , Blotting, Western , Cell Cycle Proteins , Cells, Cultured , Chromatin Immunoprecipitation , Chromosomal Proteins, Non-Histone , Female , Gene Expression Profiling , Gene Silencing , Germ Cells , Immunoenzyme Techniques , Intracellular Signaling Peptides and Proteins/physiology , Male , Meiosis/genetics , Mice , Mice, Knockout , Oligonucleotide Array Sequence Analysis , Polycomb Repressive Complex 1/genetics , Polycomb Repressive Complex 1/physiology , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Repressor Proteins/physiology , Reverse Transcriptase Polymerase Chain Reaction , Sex Chromosomes/genetics , Spermatogenesis , Testis/cytology , Ubiquitin-Protein Ligases/physiology , Ubiquitination
12.
Biol Reprod ; 91(6): 145, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25359900

ABSTRACT

In mammalian testes, spermatogonial stem cells (SSCs) maintain spermatogenesis over a long period of time by undergoing self-renewal and differentiation. SSCs are among the most primitive of spermatogenic cells (undifferentiated spermatogonia), and their activities are strictly regulated by extrinsic niche factors. However, the factors that constitute a testicular niche remain poorly understood. In this study, we demonstrate that fibroblast growth factor (FGF) signaling maintains undifferentiated spermatogonia through activating ERK1/2 signaling in vivo. Undifferentiated spermatogonia comprise GFRA1(+) and NANOS3(+) subpopulations, which are likely to undergo self-renewal and enter the differentiation pathway, respectively. In the testis, Fgfr1 was expressed in the entire population of undifferentiated spermatogonia, and deleting FGFR1 in spermatogenic cells partially inactivated ERK1/2 and resulted in reduced numbers of both GFRA1(+) and NANOS3(+) cells. In addition, Fgf8 was expressed in spermatogenic cells, and loss- and gain-of-function models of FGF8 demonstrated that FGF8 positively regulated the numbers of undifferentiated spermatogonia through FGFR1, particularly among NANOS3(+) cells. Finally we show a possible involvement of FGF signaling in the reversion from NANOS3(+) into GFRA1(+) undifferentiated spermatogonia. Taken together, our data suggest that FGF signaling is an important component of the testicular niche and has a unique function for maintaining undifferentiated spermatogonia.


Subject(s)
Fibroblast Growth Factor 8/physiology , Receptor, Fibroblast Growth Factor, Type 1/physiology , Spermatogonia/cytology , Stem Cell Niche/genetics , Testis/cytology , Animals , Cell Differentiation/genetics , Female , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Pregnancy , Signal Transduction/genetics , Spermatogonia/metabolism , Testis/metabolism
13.
J Cell Biol ; 205(5): 663-75, 2014 Jun 09.
Article in English | MEDLINE | ID: mdl-24914237

ABSTRACT

During meiosis, DNA damage response (DDR) proteins induce transcriptional silencing of unsynapsed chromatin, including the constitutively unsynapsed XY chromosomes in males. DDR proteins are also implicated in double strand break repair during meiotic recombination. Here, we address the function of the breast cancer susceptibility gene Brca1 in meiotic silencing and recombination in mice. Unlike in somatic cells, in which homologous recombination defects of Brca1 mutants are rescued by 53bp1 deletion, the absence of 53BP1 did not rescue the meiotic failure seen in Brca1 mutant males. Further, BRCA1 promotes amplification and spreading of DDR components, including ATR and TOPBP1, along XY chromosome axes and promotes establishment of pericentric heterochromatin on the X chromosome. We propose that BRCA1-dependent establishment of X-pericentric heterochromatin is critical for XY body morphogenesis and subsequent meiotic progression. In contrast, BRCA1 plays a relatively minor role in meiotic recombination, and female Brca1 mutants are fertile. We infer that the major meiotic role of BRCA1 is to promote the dramatic chromatin changes required for formation and function of the XY body.


Subject(s)
BRCA1 Protein/physiology , DNA Damage , Heterochromatin/genetics , Meiosis , Recombination, Genetic , X Chromosome/genetics , Alleles , Animals , Chromosome Pairing , Chromosomes/metabolism , Exons , Female , Gene Deletion , Gene Silencing , Male , Mice , Mutation , Phenotype , Spermatogenesis
14.
Stem Cells ; 31(11): 2517-27, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23897718

ABSTRACT

Coordination of stem cell fate is regulated by extrinsic niche signals and stem cell intrinsic factors. In mammalian testes, spermatogonial stem cells maintain constant production of abundant spermatozoa by alternating between self-renewal and differentiation at regular intervals according to a periodical program known as the seminiferous epithelial cycle. Although retinoic acid (RA) signaling has been suggested to direct the cyclical differentiation of spermatogonial stem cells, it remains largely unclear how their cycle-dependent self-renewal/proliferation is regulated. Here, we show that MEK/ERK signaling contributes to the cyclical activity of spermatogonial stem cells. We found that ERK1/2 is periodically activated in Sertoli cells during the stem cell self-renewal/proliferation phase, and that MEK/ERK signaling is required for the stage-related expression of the critical niche factor GDNF. In addition, ERK1/2 is activated in GFRα1-positive spermatogonial stem cells under the control of GDNF and prevent them from being differentiated. These results suggest that MEK/ERK signaling directly and indirectly maintains spermatogonial stem cells by mediating a signal that promotes their periodical self-renewal/proliferation. Conversely, RA signaling directly and indirectly induces differentiation of spermatogonial stem cells. We propose that temporally regulated activations of RA signaling and a signal regulating MEK/ERK antagonistically coordinates the cycle-related activity of spermatogonial stem cells.


Subject(s)
MAP Kinase Signaling System/physiology , Spermatogonia/cytology , Spermatogonia/enzymology , Stem Cells/cytology , Stem Cells/enzymology , Animals , Cell Differentiation/physiology , Glial Cell Line-Derived Neurotrophic Factor/biosynthesis , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Sertoli Cells/cytology , Sertoli Cells/enzymology
15.
Development ; 139(23): 4347-55, 2012 Dec 01.
Article in English | MEDLINE | ID: mdl-23095883

ABSTRACT

Mammalian spermatogenesis contributes a constant production of large numbers of spermatozoa, which is achieved by a cyclically regulated program known as the seminiferous epithelial cycle. Sertoli cells, functionally unique somatic cells, create a microenvironment to support the continuous differentiation of germ cells especially through the formation of a blood-testis barrier (BTB). The BTB is essential for maintaining homeostasis in seminiferous tubules and opens transiently at stages VII-VIII to ensure constant differentiation of spermatogenic cells. However, it is poorly understood how the dynamic organization of BTB is regulated. In our current study, we find that the overexpression of a dominant-negative form of RARα (dnRARα) in Sertoli cells disrupts the BTB at stages VII-XII and causes the large-scale apoptosis of differentiating germ cells. These abnormal events are found to be associated with cyclical gene expression changes in Sertoli cells, which can be represented by abnormal activation and repression of genes showing peaks of expression during stages I-VI and VII-XII, respectively. We find that one such gene, Ocln, encoding a tight junction component, partly contributes to the BTB disruption caused by dnRARα. Taken together, our data suggest that the cyclical activation of RA signaling in Sertoli cells during stages VII-XII contributes to a periodic organization of the BTB through changes in stage-dependent gene expression.


Subject(s)
Blood-Testis Barrier/physiology , Gene Expression Regulation, Developmental , Occludin/genetics , Receptors, Retinoic Acid/metabolism , Sertoli Cells/metabolism , Tretinoin/metabolism , Animals , Apoptosis , Cell Communication , Cell Differentiation , Gene Knockout Techniques , Male , Mice , Mice, Inbred C57BL , Retinoic Acid Receptor alpha , Seminiferous Epithelium/metabolism , Seminiferous Tubules/metabolism , Signal Transduction , Spermatogenesis/physiology , Tight Junctions/metabolism
16.
Mol Cell Biol ; 32(1): 206-15, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22037762

ABSTRACT

Mammalian spermatogenesis is a highly regulated system dedicated to the continuous production of spermatozoa from spermatogonial stem cells, and the process largely depends on microenvironments created by Sertoli cells, unique somatic cells that reside within a seminiferous tubule. Spermatogenesis progresses with a cyclical program known as the "seminiferous epithelial cycle," which is accompanied with cyclical gene expression changes in Sertoli cells. However, it is unclear how the cyclicity in Sertoli cells is regulated. Here, we report that Notch signaling, which is known to play an important role for germ cell development in Drosophila and Caenorhabditis elegans, is cyclically activated in Sertoli cells and regulates stage-dependent gene expression of Hes1. To elucidate the regulatory mechanism of stage-dependent Hes1 expression and the role of Notch signaling in mouse spermatogenesis, we inactivated Notch signaling in Sertoli cells by deleting protein O-fucosyltransferase 1 (Pofut1), using the cre-loxP system, and found that stage-dependent Hes1 expression was dependent on the activation of Notch signaling. Unexpectedly, however, spermatogenesis proceeded normally. Our results thus indicate that Notch signaling regulates cyclical gene expression in Sertoli cells but is dispensable for mouse spermatogenesis. This highlights the evolutionary divergences in regulation of germ cell development.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , Gene Expression Regulation, Developmental , Homeodomain Proteins/genetics , Receptors, Notch/metabolism , Sertoli Cells/metabolism , Spermatogenesis , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Fucosyltransferases/genetics , Germ Cells/cytology , Germ Cells/metabolism , Homeodomain Proteins/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Sertoli Cells/cytology , Signal Transduction , Transcription Factor HES-1
17.
Stem Cells ; 30(2): 280-91, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22102605

ABSTRACT

Stem cells are maintained by both stem cell-extrinsic niche signals and stem cell-intrinsic factors. During murine spermatogenesis, glial cell line-derived neurotrophic factor (GDNF) signal emanated from Sertoli cells and germ cell-intrinsic factor NANOS2 represent key regulators for the maintenance of spermatogonial stem cells. However, it remains unclear how these factors intersect in stem cells to control their cellular state. Here, we show that GDNF signaling is essential to maintain NANOS2 expression, and overexpression of Nanos2 can alleviate the stem cell loss phenotype caused by the depletion of Gfra1, a receptor for GDNF. By using an inducible Cre-loxP system, we show that NANOS2 expression is downregulated upon the conditional knockout (cKO) of Gfra1, while ectopic expression of Nanos2 in GFRA1-negative spermatogonia does not induce de novo GFRA1 expression. Furthermore, overexpression of Nanos2 in the Gfra1-cKO testes prevents precocious differentiation of the Gfra1-knockout stem cells and partially rescues the stem cell loss phenotypes of Gfra1-deficient mice, indicating that the stem cell differentiation can be suppressed by NANOS2 even in the absence of GDNF signaling. Taken together, we suggest that NANOS2 acts downstream of GDNF signaling to maintain undifferentiated state of spermatogonial stem cells.


Subject(s)
Carrier Proteins/physiology , Cell Differentiation , Nerve Growth Factors/metabolism , Neuroglia/metabolism , Signal Transduction , Spermatogonia/physiology , Animals , Apoptosis , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Proliferation , Cells, Cultured , Female , Gene Expression Regulation , Genes, Reporter , Glial Cell Line-Derived Neurotrophic Factor/metabolism , Glial Cell Line-Derived Neurotrophic Factor Receptors/genetics , Glial Cell Line-Derived Neurotrophic Factor Receptors/metabolism , Luminescent Proteins/biosynthesis , Luminescent Proteins/genetics , Male , Mice , Mice, Transgenic , RNA-Binding Proteins , Spermatogonia/metabolism , Testis/cytology , Testis/metabolism
18.
Blood ; 118(5): 1374-85, 2011 Aug 04.
Article in English | MEDLINE | ID: mdl-21628412

ABSTRACT

c-Maf is one of the large Maf (musculoaponeurotic fibrosarcoma) transcription factors that belong to the activated protein-1 super family of basic leucine zipper proteins. Despite its overexpression in hematologic malignancies, the physiologic roles c-Maf plays in normal hematopoiesis have been largely unexplored. On a C57BL/6J background, c-Maf(-/-) embryos succumbed from severe erythropenia between embryonic day (E) 15 and E18. Flow cytometric analysis of fetal liver cells showed that the mature erythroid compartments were significantly reduced in c-Maf(-/-) embryos compared with c-Maf(+/+) littermates. Interestingly, the CFU assay indicated there was no significant difference between c-Maf(+/+) and c-Maf(-/-) fetal liver cells in erythroid colony counts. This result indicated that impaired definitive erythropoiesis in c-Maf(-/-) embryos is because of a non-cell-autonomous effect, suggesting a defective erythropoietic microenvironment in the fetal liver. As expected, the number of erythroblasts surrounding the macrophages in erythroblastic islands was significantly reduced in c-Maf(-/-) embryos. Moreover, decreased expression of VCAM-1 was observed in c-Maf(-/-) fetal liver macrophages. In conclusion, these results strongly suggest that c-Maf is crucial for definitive erythropoiesis in fetal liver, playing an important role in macrophages that constitute erythroblastic islands.


Subject(s)
Erythroblasts/cytology , Erythroblasts/physiology , Erythropoiesis/genetics , Fetus/cytology , Liver/cytology , Proto-Oncogene Proteins c-maf/physiology , Animals , Cell Communication/genetics , Cell Movement/genetics , Cell Proliferation , Embryo, Mammalian , Erythroblasts/metabolism , Fetus/metabolism , Gene Expression Profiling , Liver/embryology , Liver/metabolism , Macrophages/metabolism , Macrophages/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Microarray Analysis , Proto-Oncogene Proteins c-maf/genetics
19.
Gene ; 445(1-2): 66-72, 2009 Sep 15.
Article in English | MEDLINE | ID: mdl-19539733

ABSTRACT

c-Maf, which is one of the large Maf transcription factors, can bind to Maf recognition element (MARE) and activates transcription of target genes. Although c-Maf is expressed in macrophages and directly regulates the expression of interleukin-10, detailed information regarding its function in the null mutant phenotype of tissue macrophages remain unknown. In this study, we demonstrated that c-Maf is specifically expressed in the F4/80 positive fetal liver and adult macrophages. The expression of F4/80, which is a tissue macrophage-specific seven trans-membrane receptor, was dramatically suppressed in the c-Maf-deficient macrophage, whereas the expression of Mac-1 was not affected, suggesting that c-Maf is not necessary for the lineage commitment of macrophages. Luciferase reporter and EMSA showed that c-Maf directly regulates the expression of F4/80 by interacting with the half-MARE site of the F4/80 promoter. These results suggest that c-Maf is required for the F4/80 expression in macrophages in vivo.


Subject(s)
Antigens, Differentiation/genetics , Macrophages/metabolism , Proto-Oncogene Proteins c-maf/physiology , Animals , Antigens, Differentiation/metabolism , Base Sequence , Cells, Cultured , Embryo, Mammalian , Gene Expression Regulation , Liver/embryology , Liver/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Molecular Sequence Data , Proto-Oncogene Proteins c-maf/genetics , Proto-Oncogene Proteins c-maf/metabolism , Sequence Homology, Nucleic Acid
20.
Blood ; 111(8): 4075-80, 2008 Apr 15.
Article in English | MEDLINE | ID: mdl-18250229

ABSTRACT

Targeted disruption of the Runx1/ AML1 gene in mice has demonstrated that it is required for the emergence of definitive hematopoietic cells but that it is not essential for the formation of primitive erythrocytes. These findings led to the conclusion that Runx1 is a stage-specific transcription factor acting only during definitive hematopoiesis. However, the zebrafish and Xenopus homologs of Runx1 have been shown to play roles in primitive hematopoiesis, suggesting that mouse Runx1 might also be involved in the development of primitive lineages. In this study, we show that primitive erythrocytes in Runx1(-/-) mice display abnormal morphology and reduced expression of Ter119, Erythroid Kruppel-like factor (EKLF, KLF1), and GATA-1. These results suggest that mouse Runx1 plays a role in the development of both primitive and definitive hematopoietic cells.


Subject(s)
Core Binding Factor Alpha 2 Subunit/metabolism , Erythropoiesis , Animals , Cell Differentiation , Core Binding Factor Alpha 2 Subunit/deficiency , Down-Regulation , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Erythrocytes/metabolism , Erythrocytes/pathology , Erythrocytes/ultrastructure , Erythroid Precursor Cells/cytology , Erythroid Precursor Cells/metabolism , GATA1 Transcription Factor/metabolism , Kruppel-Like Transcription Factors/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Oligonucleotide Array Sequence Analysis , RNA/metabolism , Yolk Sac/cytology , Yolk Sac/metabolism
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