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1.
Nat Commun ; 13(1): 2500, 2022 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-35523793

RESUMO

Maintenance of male fertility requires spermatogonial stem cells (SSCs) that self-renew and generate differentiating germ cells for production of spermatozoa. Germline cells are sensitive to genotoxic drugs and patients receiving chemotherapy can become infertile. SSCs surviving treatment mediate germline recovery but pathways driving SSC regenerative responses remain poorly understood. Using models of chemotherapy-induced germline damage and recovery, here we identify unique molecular features of regenerative SSCs and characterise changes in composition of the undifferentiated spermatogonial pool during germline recovery by single-cell analysis. Increased mitotic activity of SSCs mediating regeneration is accompanied by alterations in growth factor signalling including PI3K/AKT and mTORC1 pathways. While sustained mTORC1 signalling is detrimental for SSC maintenance, transient mTORC1 activation is critical for the regenerative response. Concerted inhibition of growth factor signalling disrupts core features of the regenerative state and limits germline recovery. We also demonstrate that the FOXM1 transcription factor is a target of growth factor signalling in undifferentiated spermatogonia and provide evidence for a role in regeneration. Our data confirm dynamic changes in SSC functional properties following damage and support an essential role for microenvironmental growth factors in promoting a regenerative state.


Assuntos
Fosfatidilinositol 3-Quinases , Espermatogênese , Diferenciação Celular/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Espermatogênese/genética , Espermatogônias , Células-Tronco/metabolismo , Testículo/metabolismo
2.
J Vis Exp ; (164)2020 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-33104058

RESUMO

Spermatogenesis is a unique differentiation process that ultimately gives rise to one of the most distinct cell types of the body, the sperm. Differentiation of germ cells takes place in the cytoplasmic pockets of somatic Sertoli cells that host 4 to 5 generations of germ cells simultaneously and coordinate and synchronize their development. Therefore, the composition of germ cell types within a cross-section is constant, and these cell associations are also known as stages (I-XII) of the seminiferous epithelial cycle. Importantly, stages can also be identified from intact seminiferous tubules based on their differential light absorption/scatter characteristics revealed by transillumination, and the fact that the stages follow each other along the tubule in a numerical order. This article describes a transillumination-assisted microdissection method for the isolation of seminiferous tubule segments representing specific stages of mouse seminiferous epithelial cycle. The light absorption pattern of seminiferous tubules is first inspected under a dissection microscope, and then tubule segments representing specific stages are cut and used for downstream applications. Here we describe immunostaining protocols for stage-specific squash preparations and for intact tubule segments. This method allows a researcher to focus on biological events taking place at specific phases of spermatogenesis, thus providing a unique tool for developmental, toxicological, and cytological studies of spermatogenesis and underlying molecular mechanisms.


Assuntos
Células Epiteliais/citologia , Túbulos Seminíferos/citologia , Coloração e Rotulagem , Transiluminação , Acrossomo/metabolismo , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Núcleo Celular/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Microdissecção , Células de Sertoli/citologia , Espermatogênese , Espermatozoides/citologia
3.
Sci Rep ; 10(1): 6751, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32317665

RESUMO

SOX3 is a transcription factor expressed within the developing and adult nervous system where it mostly functions to help maintain neural precursors. Sox3 is also expressed in other locations, notably within the spermatogonial stem/progenitor cell population in postnatal testis. Independent studies have shown that Sox3 null mice exhibit a spermatogenic block as young adults, the mechanism of which remains poorly understood. Using a panel of spermatogonial cell marker genes, we demonstrate that Sox3 is expressed within the committed progenitor fraction of the undifferentiated spermatogonial pool. Additionally, we use a Sox3 null mouse model to define a potential role for this factor in progenitor cell function. We demonstrate that Sox3 expression is required for transition of undifferentiated cells from a GFRα1+ self-renewing state to the NGN3 + transit-amplifying compartment. Critically, using chromatin immunoprecipitation, we demonstrate that SOX3 binds to a highly conserved region in the Ngn3 promoter region in vivo, indicating that Ngn3 is a direct target of SOX3. Together these studies indicate that SOX3 functions as a pro-commitment factor in spermatogonial stem/progenitor cells.


Assuntos
Células-Tronco Germinativas Adultas/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Tecido Nervoso/genética , Regiões Promotoras Genéticas , Fatores de Transcrição SOXB1/genética , Espermatogônias/metabolismo , Testículo/metabolismo , Células-Tronco Germinativas Adultas/citologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Masculino , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Ligação Proteica , Fatores de Transcrição SOXB1/deficiência , Transdução de Sinais , Espermatogênese/genética , Espermatogônias/citologia , Espermatogônias/crescimento & desenvolvimento , Testículo/citologia , Testículo/crescimento & desenvolvimento , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
4.
Cell Mol Life Sci ; 76(20): 4071-4102, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31254043

RESUMO

Mammalian spermatogenesis is a highly complex multi-step process sustained by a population of mitotic germ cells with self-renewal potential known as spermatogonial stem cells (SSCs). The maintenance and regulation of SSC function are strictly dependent on a supportive niche that is composed of multiple cell types. A detailed appreciation of the molecular mechanisms underpinning SSC activity and fate is of fundamental importance for spermatogenesis and male fertility. However, different models of SSC identity and spermatogonial hierarchy have been proposed and recent studies indicate that cell populations supporting steady-state germline maintenance and regeneration following damage are distinct. Importantly, dynamic changes in niche properties may underlie the fate plasticity of spermatogonia evident during testis regeneration. While formation of spermatogenic colonies in germ-cell-depleted testis upon transplantation is a standard assay for SSCs, differentiation-primed spermatogonial fractions have transplantation potential and this assay provides readout of regenerative rather than steady-state stem cell capacity. The characterisation of spermatogonial populations with regenerative capacity is essential for the development of clinical applications aimed at restoring fertility in individuals following germline depletion by genotoxic treatments. This review will discuss regulatory mechanisms of SSCs in homeostatic and regenerative testis and the conservation of these mechanisms between rodent models and man.


Assuntos
Fertilidade/genética , Infertilidade Masculina/genética , Espermatogênese/genética , Espermatogônias/citologia , Células-Tronco/citologia , Testículo/citologia , Animais , Diferenciação Celular , Regulação da Expressão Gênica , Homeostase/genética , Humanos , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Infertilidade Masculina/terapia , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Modelos Genéticos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Espermatogônias/metabolismo , Nicho de Células-Tronco/genética , Células-Tronco/metabolismo , Testículo/metabolismo
5.
Nat Commun ; 10(1): 2278, 2019 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-31123254

RESUMO

Mammalian spermatogenesis is sustained by mitotic germ cells with self-renewal potential known as undifferentiated spermatogonia. Maintenance of undifferentiated spermatogonia and spermatogenesis is dependent on tightly co-ordinated transcriptional and post-transcriptional mechanisms. The RNA helicase DDX5 is expressed by spermatogonia but roles in spermatogenesis are unexplored. Using an inducible knockout mouse model, we characterise an essential role for DDX5 in spermatogonial maintenance and show that Ddx5 is indispensable for male fertility. We demonstrate that DDX5 regulates appropriate splicing of key genes necessary for spermatogenesis. Moreover, DDX5 regulates expression of cell cycle genes in undifferentiated spermatogonia post-transcriptionally and is required for cell proliferation and survival. DDX5 can also act as a transcriptional co-activator and we demonstrate that DDX5 interacts with PLZF, a transcription factor required for germline maintenance, to co-regulate select target genes. Combined, our data reveal a critical multifunctional role for DDX5 in regulating gene expression programmes and activity of undifferentiated spermatogonia.


Assuntos
RNA Helicases DEAD-box/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Splicing de RNA/fisiologia , Espermatogênese/genética , Espermatogônias/metabolismo , Animais , Ciclo Celular/genética , Proliferação de Células/genética , Técnicas de Cocultura , RNA Helicases DEAD-box/genética , Embrião de Mamíferos , Fertilidade/genética , Fibroblastos , Regulação da Expressão Gênica/fisiologia , Masculino , Camundongos , Camundongos Knockout , Modelos Animais , Cultura Primária de Células , Testículo/citologia
6.
Development ; 145(18)2018 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-30126904

RESUMO

Male fertility is dependent on spermatogonial stem cells (SSCs) that self-renew and produce differentiating germ cells. Growth factors produced within the testis are essential for SSC maintenance but intrinsic factors that dictate the SSC response to these stimuli are poorly characterised. Here, we have studied the role of GILZ, a TSC22D family protein and spermatogenesis regulator, in spermatogonial function and signalling. Although broadly expressed in the germline, GILZ was prominent in undifferentiated spermatogonia and Gilz deletion in adults resulted in exhaustion of the GFRα1+ SSC-containing population and germline degeneration. GILZ loss was associated with mTORC1 activation, suggesting enhanced growth factor signalling. Expression of deubiquitylase USP9X, an mTORC1 modulator required for spermatogenesis, was disrupted in Gilz mutants. Treatment with an mTOR inhibitor rescued GFRα1+ spermatogonial failure, indicating that GILZ-dependent mTORC1 inhibition is crucial for SSC maintenance. Analysis of cultured undifferentiated spermatogonia lacking GILZ confirmed aberrant activation of ERK MAPK upstream mTORC1 plus USP9X downregulation and interaction of GILZ with TSC22D proteins. Our data indicate an essential role for GILZ-TSC22D complexes in ensuring the appropriate response of undifferentiated spermatogonia to growth factors via distinct inputs to mTORC1.


Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Espermatogênese/fisiologia , Espermatogônias/citologia , Fatores de Transcrição/metabolismo , Animais , Células Cultivadas , Proteínas de Ligação a DNA , Endopeptidases/biossíntese , Regulação da Expressão Gênica no Desenvolvimento/genética , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Infertilidade Masculina/genética , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Espermatogênese/genética , Células-Tronco/citologia , Ubiquitina Tiolesterase
7.
Nat Commun ; 9(1): 2819, 2018 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-30026551

RESUMO

The role of stem cells in tissue maintenance is appreciated and hierarchical models of stem cell self-renewal and differentiation often proposed. Stem cell activity in the male germline is restricted to undifferentiated A-type spermatogonia (Aundiff); however, only a fraction of this population act as stem cells in undisturbed testis and Aundiff hierarchy remains contentious. Through newly developed compound reporter mice, here we define molecular signatures of self-renewing and differentiation-primed adult Aundiff fractions and dissect Aundiff heterogeneity by single-cell analysis. We uncover an unappreciated population within the self-renewing Aundiff fraction marked by expression of embryonic patterning genes and homeodomain transcription factor PDX1. Importantly, we find that PDX1 marks a population with potent stem cell capacity unique to mature, homeostatic testis and demonstrate dynamic interconversion between PDX1+ and PDX1- Aundiff states upon transplant and culture. We conclude that Aundiff exist in a series of dynamic cell states with distinct function and provide evidence that stability of such states is dictated by niche-derived cues.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Espermatogônias/metabolismo , Células-Tronco/metabolismo , Testículo/metabolismo , Transativadores/genética , Animais , Diferenciação Celular , Linhagem da Célula/genética , Efeito Fundador , Perfilação da Expressão Gênica , Genes Reporter , Proteínas de Homeodomínio/metabolismo , Integrases/genética , Integrases/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Análise de Célula Única , Espermatogônias/citologia , Células-Tronco/citologia , Testículo/citologia , Testículo/crescimento & desenvolvimento , Transativadores/metabolismo , Proteína Vermelha Fluorescente
8.
Stem Cell Reports ; 9(3): 956-971, 2017 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-28867346

RESUMO

Sustained spermatogenesis in adult males and fertility recovery following germ cell depletion are dependent on undifferentiated spermatogonia. We previously demonstrated a key role for the transcription factor SALL4 in spermatogonial differentiation. However, whether SALL4 has broader roles within spermatogonia remains unclear despite its ability to co-regulate genes with PLZF, a transcription factor required for undifferentiated cell maintenance. Through development of inducible knockout models, we show that short-term integrity of differentiating but not undifferentiated populations requires SALL4. However, SALL4 loss was associated with long-term functional decline of undifferentiated spermatogonia and disrupted stem cell-driven regeneration. Mechanistically, SALL4 associated with the NuRD co-repressor and repressed expression of the tumor suppressor genes Foxl1 and Dusp4. Aberrant Foxl1 activation inhibited undifferentiated cell growth and survival, while DUSP4 suppressed self-renewal pathways. We therefore uncover an essential role for SALL4 in maintenance of undifferentiated spermatogonial activity and identify regulatory pathways critical for germline stem cell function.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição Forkhead/genética , Inativação Gênica , Genes Supressores de Tumor , Células Germinativas/citologia , Proteínas Tirosina Fosfatases/genética , Células-Tronco/citologia , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular , Fatores de Transcrição Forkhead/metabolismo , Deleção de Genes , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Fenótipo , Regiões Promotoras Genéticas/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Regeneração , Espermatogônias/citologia , Espermatogônias/metabolismo , Testículo/citologia
9.
EMBO Rep ; 16(4): 467-80, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25700280

RESUMO

Adult tissue maintenance is often dependent on resident stem cells; however, the phenotypic and functional heterogeneity existing within this self-renewing population is poorly understood. Here, we define distinct subsets of undifferentiated spermatogonia (spermatogonial progenitor cells; SPCs) by differential response to hyperactivation of mTORC1, a key growth-promoting pathway. We find that conditional deletion of the mTORC1 inhibitor Tsc2 throughout the SPC pool using Vasa-Cre promotes differentiation at the expense of self-renewal and leads to germline degeneration. Surprisingly, Tsc2 ablation within a subset of SPCs using Stra8-Cre did not compromise SPC function. SPC activity also appeared unaffected by Amh-Cre-mediated Tsc2 deletion within somatic cells of the niche. Importantly, we find that differentiation-prone SPCs have elevated mTORC1 activity when compared to SPCs with high self-renewal potential. Moreover, SPCs insensitive to Tsc2 deletion are preferentially associated with mTORC1-active committed progenitor fractions. We therefore delineate SPC subsets based on differential mTORC1 activity and correlated sensitivity to Tsc2 deletion. We propose that mTORC1 is a key regulator of SPC fate and defines phenotypically distinct SPC subpopulations with varying propensities for self-renewal and differentiation.


Assuntos
Células-Tronco Adultas/metabolismo , Linhagem da Célula/genética , Complexos Multiproteicos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células-Tronco Adultas/citologia , Animais , Diferenciação Celular , Proliferação de Células , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Feminino , Deleção de Genes , Regulação da Expressão Gênica , Engenharia Genética , Integrases/genética , Integrases/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Transgênicos , Complexos Multiproteicos/genética , Fenótipo , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/genética
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