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1.
EMBO Rep ; 24(6): e56316, 2023 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-37099396

RESUMO

Spermatozoa have a unique genome organization. Their chromatin is almost completely devoid of histones and is formed instead of protamines, which confer a high level of compaction and preserve paternal genome integrity until fertilization. Histone-to-protamine transition takes place in spermatids and is indispensable for the production of functional sperm. Here, we show that the H3K79-methyltransferase DOT1L controls spermatid chromatin remodeling and subsequent reorganization and compaction of the spermatozoon genome. Using a mouse model in which Dot1l is knocked-out (KO) in postnatal male germ cells, we found that Dot1l-KO sperm chromatin is less compact and has an abnormal content, characterized by the presence of transition proteins, immature protamine 2 forms and a higher level of histones. Proteomic and transcriptomic analyses performed on spermatids reveal that Dot1l-KO modifies the chromatin prior to histone removal and leads to the deregulation of genes involved in flagellum formation and apoptosis during spermatid differentiation. As a consequence of these chromatin and gene expression defects, Dot1l-KO spermatozoa have less compact heads and are less motile, which results in impaired fertility.


Assuntos
Cromatina , Histonas , Animais , Masculino , Diferenciação Celular/genética , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Expressão Gênica , Histonas/metabolismo , Proteômica , Sêmen/metabolismo , Espermatogênese/genética , Espermatozoides/metabolismo , Camundongos
2.
Hum Mol Genet ; 31(1): 97-110, 2021 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-34368842

RESUMO

Fanconi anemia (FA) is a rare human genetic disorder characterized by bone marrow failure, predisposition to cancer and developmental defects including hypogonadism. Reproductive defects leading to germ cell aplasia are the most consistent phenotypes seen in FA mouse models. We examined the role of the nuclear FA core complex gene Fancg in the development of primordial germ cells (PGCs), the embryonic precursors of adult gametes, during fetal development. PGC maintenance was severely impaired in Fancg-/- embryos. We observed a defect in the number of PGCs starting at E9.5 and a strong attrition at E11.5 and E13.5. Remarkably, we observed a mosaic pattern reflecting a portion of testicular cords devoid of PGCs in E13.5 fetal gonads. Our in vitro and in vivo data highlight a potential role of Fancg in the proliferation and in the intrinsic cell motility abilities of PGCs. The random migratory process is abnormally activated in Fancg-/- PGCs, altering the migration of cells. Increased cell death and PGC attrition observed in E11.5 Fancg-/- embryos are features consistent with delayed migration of PGCs along the migratory pathway to the genital ridges. Moreover, we show that an inhibitor of RAC1 mitigates the abnormal migratory pattern observed in Fancg-/- PGCs.


Assuntos
Anemia de Fanconi , Animais , Movimento Celular/genética , Anemia de Fanconi/genética , Proteína do Grupo de Complementação G da Anemia de Fanconi/metabolismo , Células Germinativas/metabolismo , Gônadas/metabolismo , Camundongos , Transdução de Sinais
3.
Development ; 146(6)2019 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-30824552

RESUMO

Neonatal germ cell development provides the foundation of spermatogenesis. However, a systematic understanding of this process is still limited. To resolve cellular and molecular heterogeneity in this process, we profiled single cell transcriptomes of undifferentiated germ cells from neonatal mouse testes and employed unbiased clustering and pseudotime ordering analysis to assign cells to distinct cell states in the developmental continuum. We defined the unique transcriptional programs underlying migratory capacity, resting cellular states and apoptosis regulation in transitional gonocytes. We also identified a subpopulation of primitive spermatogonia marked by CD87 (plasminogen activator, urokinase receptor), which exhibited a higher level of self-renewal gene expression and migration potential. We further revealed a differentiation-primed state within the undifferentiated compartment, in which elevated Oct4 expression correlates with lower expression of self-renewal pathway factors, higher Rarg expression, and enhanced retinoic acid responsiveness. Lastly, a knockdown experiment revealed the role of Oct4 in the regulation of gene expression related to the MAPK pathway and cell adhesion, which may contribute to stem cell differentiation. Our study thus provides novel insights into cellular and molecular regulation during early germ cell development.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Análise de Sequência de RNA , Espermatogônias/citologia , Animais , Animais Recém-Nascidos , Apoptose , Adesão Celular , Diferenciação Celular , Perfilação da Expressão Gênica , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Microscopia de Fluorescência , Fator 3 de Transcrição de Octâmero/fisiologia , Receptores do Ácido Retinoico/fisiologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/fisiologia , Espermatogênese/genética , Transcriptoma , Tretinoína/fisiologia , Receptor gama de Ácido Retinoico
4.
Int J Mol Sci ; 20(22)2019 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-31744138

RESUMO

Ongoing progress in genomic technologies offers exciting tools that can help to resolve transcriptome and genome-wide DNA modifications at single-cell resolution. These methods can be used to characterize individual cells within complex tissue organizations and to highlight various molecular interactions. Here, we will discuss recent advances in the definition of spermatogonial stem cells (SSC) and their progenitors in humans using the single-cell transcriptome sequencing (scRNAseq) approach. Exploration of gene expression patterns allows one to investigate stem cell heterogeneity. It leads to tracing the spermatogenic developmental process and its underlying biology, which is highly influenced by the microenvironment. scRNAseq already represents a new diagnostic tool for the personalized investigation of male infertility. One may hope that a better understanding of SSC biology could facilitate the use of these cells in the context of fertility preservation of prepubertal children, as a key component of regenerative medicine.


Assuntos
Medicina Regenerativa , Células-Tronco/metabolismo , Transcriptoma , Humanos , Infertilidade Masculina/diagnóstico , Infertilidade Masculina/terapia , Masculino , Análise de Célula Única , Espermatogênese , Espermatogônias/citologia , Transplante de Células-Tronco , Células-Tronco/citologia
6.
Blood ; 124(24): 3613-23, 2014 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-25261197

RESUMO

Fanconi anemia (FA) is an inherited chromosomal instability syndrome that is characterized by progressive bone marrow failure. One of the main causes of morbidity and mortality in FA is a bleeding tendency, resulting from low platelet counts. Platelets are the final products of megakaryocyte (MK) maturation. Here, we describe a previously unappreciated role of Fanconi anemia group A protein (Fanca) during the endomitotic process of MK differentiation. Fanca deficiency leads to the accumulation of MKs with low nuclear ploidy and to decreased platelet production. We show, for the first time, that Fanca(-/-) mice are characterized by limited number and proliferative capacity of MK progenitors. Defective megakaryopoiesis of Fanca(-/-) cells is associated with the formation of nucleoplasmic bridges and increased chromosomal instability, indicating that inaccurate endoreplication and karyokinesis occur during MK polyploidization. Sustained DNA damage forces Fanca(-/-) MKs to enter a senescence-like state. Furthermore, inhibition of the Rho-associated kinase, a regulator of cytokinesis, improves the polyploidization of Fanca(-/-) MKs but greatly increases their genomic instability and diminishes their differentiation potential, supporting the notion that accumulation of DNA damage through endomitotic cycles affects MK maturation. Our study indicates that Fanca expression during endomitosis is crucial for normal megakaryopoiesis and platelet production.


Assuntos
Proteína do Grupo de Complementação A da Anemia de Fanconi , Regulação da Expressão Gênica/genética , Megacariócitos/metabolismo , Trombocitopenia , Trombopoese/genética , Animais , Senescência Celular/genética , Instabilidade Cromossômica/genética , Proteína do Grupo de Complementação A da Anemia de Fanconi/biossíntese , Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Megacariócitos/patologia , Camundongos , Camundongos Knockout , Mitose , Trombocitopenia/genética , Trombocitopenia/metabolismo , Trombocitopenia/patologia
7.
Hum Mol Genet ; 21(1): 121-35, 2012 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-21968513

RESUMO

Fanconi anemia (FA) is a human rare genetic disorder characterized by congenital defects, bone marrow (BM) failure and predisposition to leukemia. The progressive aplastic anemia suggests a defect in the ability of hematopoietic stem cells (HSC) to sustain hematopoieis. We have examined the role of the nuclear FA core complex gene Fancg in the functionality of HSC. In Fancg-/- mice, we observed a decay of long-term HSC and multipotent progenitors that account for the reduction in the LSK compartment containing primitive hematopoietic cells. Fancg-/- lymphoid and myeloid progenitor cells were also affected, and myeloid progenitors show compromised in vitro functionality. HSC from Fancg-/- mice failed to engraft and to reconstitute at short and long term the hematopoiesis in a competitive transplantation assay. Fancg-/- LSK cells showed a loss of quiescence, an impaired migration in vitro in response to the chemokine CXCL12 and a defective homing to the BM after transplantation. Finally, the expression of several key genes involved in self-renewal, quiescence and migration of HSC was dysregulated in Fancg-deficient LSK subset. Collectively, our data reveal that Fancg should play a role in the regulation of physiological functions of HSC.


Assuntos
Proteína do Grupo de Complementação G da Anemia de Fanconi/deficiência , Anemia de Fanconi/fisiopatologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Animais , Medula Óssea/metabolismo , Movimento Celular , Quimiocina CXCL12/metabolismo , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação G da Anemia de Fanconi/genética , Feminino , Hematopoese , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos
8.
EMBO J ; 27(5): 770-81, 2008 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-18239686

RESUMO

Although brain development abnormalities and brain cancer predisposition have been reported in some Fanconi patients, the possible role of Fanconi DNA repair pathway during neurogenesis is unclear. We thus addressed the role of fanca and fancg, which are involved in the activation of Fanconi pathway, in neural stem and progenitor cells during brain development and adult neurogenesis. Fanca(-/-) and fancg(-/-) mice presented with microcephalies and a decreased neuronal production in developing cortex and adult brain. Apoptosis of embryonic neural progenitors, but not that of postmitotic neurons, was increased in the neocortex of fanca(-/-) and fancg(-/-) mice and was correlated with chromosomal instability. In adult Fanconi mice, we showed a reduced proliferation of neural progenitor cells related to apoptosis and accentuated neural stem cells exhaustion with ageing. In addition, embryonic and adult Fanconi neural stem cells showed a reduced capacity to self-renew in vitro. Our study demonstrates a critical role for Fanconi pathway in neural stem and progenitor cells during developmental and adult neurogenesis.


Assuntos
Encéfalo/citologia , Proteína do Grupo de Complementação A da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação G da Anemia de Fanconi/deficiência , Neurônios/citologia , Células-Tronco/citologia , Animais , Apoptose , Encéfalo/embriologia , Proliferação de Células , Aberrações Cromossômicas , Reparo do DNA , Desenvolvimento Embrionário , Anemia de Fanconi , Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Proteína do Grupo de Complementação G da Anemia de Fanconi/genética , Feminino , Camundongos , Camundongos Knockout , Gravidez
9.
Blood ; 115(3): 443-52, 2010 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-19797522

RESUMO

Few techniques are available to characterize in vivo the early cellular dynamics of long-term reconstitution of hematopoiesis after transplantation of hematopoietic stem cells (HSCs) after lethal irradiation. Using a fiber-optic imaging system, we track the early steps of in vivo recruitment and proliferation of Lin(-)Sca-1(+)c-Kit(+)CD34(-) (LSKCD34(-)) HSCs highly enriched in HSCs and transplanted into lethally irradiated mice. Recruitment of the transplanted LSKCD34(-) hematopoietic cells first occurs in the femoral head and is continuous during 24 hours. Quantification of the fluorescence emitted by the transplanted hematopoietic cells shows that proliferation of LSKCD34(-) hematopoietic cells in the femoral head was potent 3 days after transplantation. Using a development of this fiber-optic imaging system, we show that the transplanted LSKCD34(-) hematopoietic cells are associated with vascularized structures as early as 5 hours after transplantation. This early association is dependent on reactive oxygen species (ROS) partly through the regulation of vascular cell adhesion molecule-1 expression on endothelial cells and is followed by a ROS-dependent proliferation of LSKCD34(-) hematopoietic cells. This new in vivo imaging technique permits the observation of the early steps of hematopoietic reconstitution by HSCs in long bones and shows a new role of ROS in the recruitment of HSCs by bone marrow endothelial cells.


Assuntos
Hematopoese/efeitos dos fármacos , Imagem Molecular/métodos , Espécies Reativas de Oxigênio/farmacologia , Animais , Antígenos CD34/metabolismo , Antígenos Ly/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Fêmur/citologia , Fêmur/metabolismo , Fêmur/fisiologia , Tecnologia de Fibra Óptica/métodos , Hematopoese/fisiologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Proteínas Proto-Oncogênicas c-kit/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Estudos de Validação como Assunto , Irradiação Corporal Total
10.
Stem Cell Res ; 60: 102723, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35247845

RESUMO

In adult testis, the cell mobility is essential for spermatogonia differentiation and is suspected to regulate spermatogonial stem cell fate. Netrin-1 controls cell migration and/or survival according to the cellular context. Its involvement in some self-renewing lineages raises the possibility that Netrin-1 could have a role in spermatogenesis. We show that in addition to Sertoli cells, a fraction of murine undifferentiated spermatogonia express the Netrin-1 receptor UNC5c and that UNC5c contributes to spermatogonia differentiation. Receptor loss in Unc5crcm males leads to the concomitant accumulation of transit-amplifying progenitors and short syncytia of spermatogonia. Without altering cell death rates, the consequences of Unc5c loss worsen with age: the increase in quiescent undifferentiated progenitors associated with a higher spermatogonial stem cell enriched subset leads to the spermatocyte I decline. We demonstrate in vitro that Netrin-1 promotes a guidance effect as it repulses both undifferentiated and differentiating spermatogonia. Finally, we propose that UNC5c triggers undifferentiated spermatogonia adhesion/ migration and that the repulsive activity of Netrin-1 receptors could regulate spermatogonia differentiation, and maintain germ cell homeostasis.


Assuntos
Espermatogênese , Espermatogônias , Animais , Diferenciação Celular/fisiologia , Homeostase , Masculino , Camundongos , Receptores de Netrina/metabolismo , Netrina-1/metabolismo , Espermatogênese/fisiologia , Testículo
11.
Nat Genet ; 54(4): 469-480, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35410378

RESUMO

DNA methylation plays a critical role in spermatogenesis, as evidenced by the male sterility of DNA methyltransferase (DNMT) mutant mice. Here, we report a division of labor in the establishment of the methylation landscape of male germ cells and its functions in spermatogenesis. Although DNMT3C is essential for preventing retrotransposons from interfering with meiosis, DNMT3A broadly methylates the genome (with the exception of DNMT3C-dependent retrotransposons) and controls spermatogonial stem cell (SSC) plasticity. By reconstructing developmental trajectories through single-cell RNA sequencing and profiling chromatin states, we found that Dnmt3A mutant SSCs can only self-renew and no longer differentiate in association with spurious enhancer activation that enforces an irreversible stem cell gene program. Our findings therefore highlight a key function of DNA methylation in male fertility: the epigenetic programming of SSC commitment to differentiation and lifelong spermatogenesis supply.


Assuntos
Metilação de DNA , Espermatogênese , Espermatogônias , Animais , Metilação de DNA/genética , Metilases de Modificação do DNA/genética , Masculino , Camundongos , Retroelementos , Espermatogênese/genética , Espermatogônias/metabolismo , Células-Tronco/metabolismo
12.
Stem Cell Reports ; 17(4): 936-952, 2022 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-35334216

RESUMO

Male infertility is responsible for approximately half of all cases of reproductive issues. Spermatogenesis originates in a small pool of spermatogonial stem cells (SSCs), which are of interest for therapy of infertility but remain not well defined in humans. Using multiparametric analysis of the side population (SP) phenotype and the α-6 integrin, THY1, and ß-2 microglobulin cell markers, we identified a population of human primitive undifferentiated spermatogonia with the phenotype ß-2 microglobulin (ß-2M)-SPα-6+THY1+, which is highly enriched in stem cells. By analyzing the expression signatures of this SSC-enriched population along with other germinal progenitors, we established an exhaustive transcriptome of human spermatogenesis. Transcriptome profiling of the human ß-2M-SPα-6+THY1+ population and comparison with the profile of mouse undifferentiated spermatogonia provide insights into the molecular networks and key transcriptional regulators regulating human SSCs, including the basic-helix-loop-helix (bHLH) transcriptional repressor HES1, which we show to be implicated in maintenance of SSCs in vitro.


Assuntos
Células-Tronco Germinativas Adultas , Espermatogênese , Animais , Perfilação da Expressão Gênica , Humanos , Masculino , Camundongos , Espermatogênese/genética , Espermatogônias/metabolismo , Células-Tronco/metabolismo , Testículo/metabolismo , Fatores de Transcrição/metabolismo
13.
Dev Biol ; 342(1): 74-84, 2010 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-20346356

RESUMO

During testis development, proliferation and death of gonocytes are highly regulated to establish a standard population of adult stem spermatogonia that maintain normal spermatogenesis. As Transforming Growth Factor beta (TGFbeta) can regulate proliferation and apoptosis, we investigated its expression and functions during testis development. We show that TGFbeta2 is only expressed in quiescent gonocytes and decreases gonocyte proliferation in vitro. To study the functions of TGFbeta2, we developed conditional mice that invalidate the TGFbeta receptor type II in germ cells. Most of the knock-out animals die during fetal life, but the surviving adults show a reduced pool of spermatogonial stem/progenitor cells and become sterile with time. Using an organ culture system mimicking in vivo development, we show higher proportions of proliferating and apoptotic gonocytes from 13.5 dpc until 1 dpp, suggesting a reduction of germinal quiescence in these animals. Conversely, a 24-hour TGFbeta2-treatment of explanted wild-type testes, isolated every day from 13.5 dpc until 1 dpp, increased the duration of quiescence. These data show that the TGFbeta signaling pathway plays a physiological role during testis development by acting directly as a negative regulator of the fetal and neonatal germ cell proliferation, and indicate that the TGFbeta signaling pathway might regulate the duration of germ cell quiescence and is necessary to maintain adult spermatogenesis.


Assuntos
Células Germinativas/metabolismo , Transdução de Sinais/genética , Espermatogênese/genética , Espermatogônias/metabolismo , Fator de Crescimento Transformador beta/genética , Animais , Apoptose/genética , Proliferação de Células , Fertilidade/genética , Masculino , Camundongos , Camundongos Knockout , Técnicas de Cultura de Órgãos , Proteínas Serina-Treonina Quinases/fisiologia , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/fisiologia , Espermatogônias/citologia , Células-Tronco/metabolismo , Testículo/citologia , Testículo/crescimento & desenvolvimento , Testículo/metabolismo , Fator de Crescimento Transformador beta/metabolismo
15.
Mol Cell Neurosci ; 38(4): 569-77, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18583149

RESUMO

Neurogenesis persists in the adult brain subventricular zone where neural stem/progenitor cells (NSPCs) lie close to brain endothelial cells (BECs). We show in mouse that BECs produce bone morphogenetic proteins (BMPs). Coculture of embryonic and adult NSPCs with BECs activated the canonical BMP/Smad pathway and reduced their proliferation. We demonstrate that coculture with BECs in the presence of EGF and FGF2 induced a reversible cell cycle exit of NSPCs (LeX+) and an increase in the amount of GFAP/LeX-expressing progenitors thought to be stem cells. Levels of the phosphatidylinositol phosphatase PTEN were upregulated in NSPCs after coculture with BECs, or treatment with recombinant BMP4, with a concomitant reduction in Akt phosphorylation. Silencing Smad5 with siRNA or treatment with Noggin, a BMP antagonist, demonstrated that upregulation of PTEN in NSPCs required BMP/Smad signaling and that this pathway regulated cell cycle exit of NSPCs. Therefore, BECs may provide a feedback mechanism to control the proliferation of NSPCs.


Assuntos
Proteínas Morfogenéticas Ósseas/fisiologia , Encéfalo/metabolismo , Proliferação de Células , Células Endoteliais/metabolismo , Neurônios/fisiologia , Células-Tronco/metabolismo , Animais , Encéfalo/citologia , Diferenciação Celular/fisiologia , Linhagem Celular , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/citologia , Células-Tronco/citologia
16.
Cell Stem Cell ; 24(1): 1-2, 2019 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-30609395

RESUMO

Mechanisms regulating maintenance of the stem cell pool in facultative niches of the mammalian testis are poorly understood. In this issue of Cell Stem Cell, Kitadate et al. (2019) propose a minimal model in which stem cells compete for limited resources of fibroblast growth factors, which predicts their density during homeostasis and regenerative conditions.


Assuntos
Mitógenos , Células-Tronco , Animais , Diferenciação Celular , Células Germinativas , Homeostase , Masculino
17.
Nat Commun ; 10(1): 3858, 2019 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-31451685

RESUMO

The Polycomb group of proteins is required for the proper orchestration of gene expression due to its role in maintaining transcriptional silencing. It is composed of several chromatin modifying complexes, including Polycomb Repressive Complex 2 (PRC2), which deposits H3K27me2/3. Here, we report the identification of a cofactor of PRC2, EZHIP (EZH1/2 Inhibitory Protein), expressed predominantly in the gonads. EZHIP limits the enzymatic activity of PRC2 and lessens the interaction between the core complex and its accessory subunits, but does not interfere with PRC2 recruitment to chromatin. Deletion of Ezhip in mice leads to a global increase in H3K27me2/3 deposition both during spermatogenesis and at late stages of oocyte maturation. This does not affect the initial number of follicles but is associated with a reduction of follicles in aging. Our results suggest that mature oocytes Ezhip-/- might not be fully functional and indicate that fertility is strongly impaired in Ezhip-/- females. Altogether, our study uncovers EZHIP as a regulator of chromatin landscape in gametes.


Assuntos
Proteínas Oncogênicas/metabolismo , Óvulo/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Espermatozoides/metabolismo , Adulto , Animais , Linhagem Celular Tumoral , Cromatina/metabolismo , Feminino , Células HEK293 , Histonas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Knockout , Mutação , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/isolamento & purificação , Oogênese , Ovário/citologia , Ovário/patologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Células Sf9 , Espermatogênese , Testículo/citologia , Testículo/patologia
18.
Toxicology ; 247(2-3): 80-7, 2008 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-18407394

RESUMO

We investigated whether uranium intoxication affects female fertility by assessing its effects on ovarian function and on the oocyte. We treated two groups of female mice for 15 weeks with 5, 50 or 400 mg/L of uranyl nitrate in drinking water. In the first group, mice were euthanized immediately after intoxication. Mice of the second group were paired after intoxication with untreated males. Dams and their female pups were euthanized 3 months after the end of intoxication. We assayed the kidneys, femurs and one ovary per female for U content and collected the other ovary for histology. The number and size of all the ovarian follicles were analyzed. Mice from the first group and female pups had significantly fewer large antral follicles (Ø > 200 microm) than the untreated mice. By contrast, dams in the second group had more secondary and early preantral follicles (Ø 70-110 microm) than untreated mice. However, U had no effect on follicle atresia. We then analyzed the in vitro effects of U on oocyte maturation and fragmentation. GV-oocytes were cultured in the presence of 1mM uranyl acetate and observed for 72 h. Oocyte maturation was slowed down by U during resumption of meiosis and at metaphase II. However, the rhythm and rate of oocyte fragmentation were similar to those of control mice. Our findings demonstrate that U induces changes in folliculogenesis and oocyte maturation in mice and could consequently represent a risk for women who are chronically exposed.


Assuntos
Meiose/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Urânio/toxicidade , Animais , Apoptose/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Oócitos/patologia , Oócitos/fisiologia , Folículo Ovariano/patologia , Folículo Ovariano/fisiologia , Urânio/farmacocinética
19.
Mutat Res ; 641(1-2): 58-60, 2008 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-18423770

RESUMO

Mouse expanded simple tandem repeat (ESTR) loci are the most unstable loci in the mouse genome. Despite the fact that over the last decade these loci have been extensively used for studying germline mutation induction in mice, to date little is known about the mechanisms underlying spontaneous and induced ESTR mutation. Here we used flow cytometry and single-molecule PCR to compare the frequency of ESTR mutation in four flow-sorted fractions of the mouse male germ cells - spermatogonia, spermatocytes I, round and elongated spermatids. The frequency and the spectrum of ESTR mutation did not significantly differ between different stages of mouse spermatogenesis. Considering these data and the results of other publications, we propose that spontaneous ESTR mutation is mostly attributed to replication slippage in spermatogonia and these loci may be regarded as a class of expanded microsatellites.


Assuntos
Mutação em Linhagem Germinativa/genética , Espermátides/fisiologia , Espermatogênese/genética , Sequências de Repetição em Tandem/genética , Animais , Citometria de Fluxo , Masculino , Camundongos , Camundongos Endogâmicos CBA , Reação em Cadeia da Polimerase , Espermatócitos/citologia , Espermatócitos/metabolismo , Espermatogônias/citologia , Espermatogônias/fisiologia
20.
Oncotarget ; 8(6): 10050-10063, 2017 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-28052023

RESUMO

The male germinal lineage, which is defined as unipotent, produces sperm through spermatogenesis. However, embryonic primordial germ cells and postnatal spermatogonial stem cells (SSCs) can change their fate and convert to pluripotency in culture when they are not controlled by the testicular microenvironment. The mechanisms underlying these reprogramming processes are poorly understood. Testicular germ cell tumors, including teratoma, share some molecular characteristics with pluripotent cells, suggesting that cancer could result from an abnormal differentiation of primordial germ cells or from an abnormal conversion of SCCs to pluripotency in the testis. Here, we investigated whether the somatic reprogramming factors Oct3/4, Sox2, Klf4 and c-Myc (OSKM) could play a role in SSCs reprogramming and induce pluripotency using a doxycycline-inducible transgenic Col1a1-4F2A-OSKM mouse model. We showed that, in contrast to somatic cells, SSCs from adult mice are resistant to this reprogramming strategy, even in combination with small molecules, hypoxia, or p53 deficiency, which were previously described to favour the conversion of somatic cells to pluripotency. This finding suggests that adult SSCs have developed specific mechanisms to repress reprogramming by OSKM factors, contributing to circumvent testicular cancer initiation events.


Assuntos
Células-Tronco Germinativas Adultas/metabolismo , Técnicas de Reprogramação Celular , Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Células-Tronco Germinativas Adultas/efeitos dos fármacos , Animais , Hipóxia Celular , Linhagem da Célula , Células Cultivadas , Reprogramação Celular/efeitos dos fármacos , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Proteína-1 Reguladora de Fusão/genética , Proteína-1 Reguladora de Fusão/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Células-Tronco Pluripotentes Induzidas/patologia , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Fator 3 de Transcrição de Octâmero/genética , Fenótipo , Proteínas Proto-Oncogênicas c-myc/genética , Fatores de Transcrição SOXB1/genética , Transfecção , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
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