Name: cytological analysis of spermatogenesis: live and fixed preparations of drosophila testes
Uploaded: 2014-01-20T15:00:00.000+00:00
Duration: 10 min 30 s
Description: Watch this Scientific Journal Video about Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes at JoVE.com

Os autores gostariam de agradecer a Michael Anderson para a criação em laboratório Lee estes métodos aceitos para estudar espermatogênese com aconselhamento especializado de Karen Hales. H. Oda e Y. Akiyama-Oda generosamente forneceu a γ-tubulina-GFP voar estoque. Este trabalho foi apoiado por uma bolsa NIH R01 para LAL (GM074044).

1. Testes Dissection <ol><li> Anestesiar moscas em uma garrafa ou frasco, usando um fluxo de CO 2 e transferir para uma almofada de mosca. </li><li> Ordenar opera sob um microscópio de dissecação com um pequeno pincel, e recolher um número apropriado (dependendo do ensaio) de Drosophila, os machos dos genótipos desejáveis. Jovens do sexo masculino (0-2 dias de idade) são ideais para examinar as células ao longo das etapas anteriores da espermatogênese (por exemplo, espermatogônias, espermatócitos e espermátides iniciais pós-meióticas), enquanto que os homens um pouco mais velhos (2-5 dias de idade) são ideais para examinar células nos estágios finais da espermatogénese (em particular, o esperma maduro). </li><li> Use uma pinça para remover as asas de cada voar (para evitar que as moscas de flutuando no líquido durante a dissecção). </li><li> Adicionar ~ 500 ml de solução salina tamponada com fosfato (130 mM de NaCl, 7 mM de Na 2 HPO 4, 3 mM de NaH 2 PO 4; PBS) numa gota de um prato de dissecação com revestimento de silicone emum fundo preto. Outras soluções aquosas têm sido usados ​​com sucesso para os testículos dissecção 3. </li><li> Ponto de fórceps para o anterior da mosca, segure-o pelo tórax e mergulhá-lo na queda PBS. Durante a visualização através do microscópio de dissecação, use outro par de fórceps para agarrar e puxar a genitália externa (estrutura marrom escuro localizado na extremidade posterior do abdome ventral) posteriormente até que ele se separa do abdômen. Na maioria dos casos, os testículos, vesículas seminais e glândula acessória vai ser removido do abdómen, juntamente com os órgãos genitais externos e, se não, inserir um único par de fórceps para o abdómen e provocar os testículos. </li><li> Testículos separadas de glândula acessória e genitais externos utilizando dois pares de pinças na gota de PBS (Figura 2). De tipo selvagem testículos são facilmente distinguidos dos tecidos brancos vizinhos por sua cor amarela. Vá imediatamente para a etapa 2. </li><li> Para isolar os testículos dos machos pharate (i.. E fechada dentro da caixa de pupa), um passo adicional tem de ser realizada primeiramente que envolve a remoção da mosca do caso de pupa, este passo tem sido previamente descrita 31. Prossiga com dissecção como para os testículos adultos começando no passo 1.2. </li><li> Para isolar os testículos dos machos larvais, realizar uma modificação de um protocolo para isolar ovários de larvas de Drosophila 32. Resumidamente, as larvas do sexo masculino pode ser distinguido a partir de larvas femininas pela presença de um par de estruturas grandes, claros, ovais (testículos larva) incorporados no terço posterior do corpo gordo. Para isolar os testículos larvais, parcialmente esfolar abrir a larva do sexo masculino para isolar os testículos e o corpo gordo circundante do abdómen, tal como descrito para o isolamento de ovários de larvas. Prosseguir imediatamente para a etapa 2 do protocolo aqui descrito, os testículos pode depois ser removido do corpo gordo pouco antes da montagem (passos 2.3 ou 3.14) como descrito para os ovários 32. </li></ol><ol><li> Use uma pinça para colocar suavemente 2-3 pares de testículos em uma gota de 4-5 ml de PBS em uma praça lamínula de vidro. Note-se que a razão entre o número testículos ao volume PBS pode precisar de ser ajustado: muito líquido vai evitar que as células se espalhe corretamente quando esmagado, enquanto muito pouco líquido fará com que as células de estourar quando esmagado. Opcional: Use capa siliconizada desliza para minimizar a aderência de tecido para cobrir deslizamento no passo 3.2. </li><li> Utilizar um par de pinças para rasgar cada testículo numa posição apropriada de modo a maximizar a presença de tipos de células da linha germinal desejados na preparação (notar que o conteúdo do testículo principalmente vai egresso da região rasgada na lâmina durante o esmagamento etapa.) Para enriquecer para espermatogônias e espermatócitos, rasgar abrir o testículo adjacente à sua ponta apical (nível 1, Figura 2B). Para enriquecer para espermatócitos e espermátides, rasgar abrir o testículo em um position ligeiramente basal ao nível 1 (nível 2, Figura 2B). Para enriquecimento em células da linha germinal mais maduros, rasgar a testículo mais perto de onde a curvatura começa (nível 3, Figura 2B). </li><li> Delicadamente, coloque uma lâmina de vidro sobre a lamínula para esmagar os testículos; não aplique pressão manualmente, como o peso de só a lamínula é suficiente para obter uma amostra adequada esmagado. Tente evitar bolhas de interceptação. Opcional: Utilização de poli-L-lisina revestido lâminas de microscópio para promover a aderência do tecido a deslizar no passo 3.2. </li><li> Use preparação imediatamente (de preferência dentro de 15 minutos de preparação) para observar células vivas por microscopia de contraste de fase, pois moscas transgênicas com expressão de proteínas fluorescentes marcados nos testículos, células vivas podem ser examinados por microscopia de fluorescência nesta etapa. Alternativamente, proceder com a fixação e coloração de anticorpos (Protocolo 3). </li><li> Gentilmente pavio qualquer excesso de líquido sob a lamela usando uma limpeza wipe para permitir o achatamento de preparação até que as células germinais são claramente em foco. </li></ol> 3. Formaldeído Fixação e Coloração Anticorpo <ol><li> Encaixar congelar cada slide contendo testículos esmagados (das Protocolo 2) usando um par de pinças de metal para mergulhá-lo brevemente em nitrogênio líquido (até nitrogênio líquido pára borbulhando). </li><li> Retire a tampa de deslizamento imediatamente usando uma lâmina de barbear. </li><li> Use pinças metálicas para transferir lâminas para uma grade deslizante de vidro previamente arrefecido com gelo-etanol frio a 95% (grau de espectrofotometria, livre de metanol). Armazenar a -20 ° C durante 10 min. </li><li> Use pinças metálicas para transferir lâminas para uma grade deslizante de vidro cheio com 4% de formaldeído em PBS mais 0,1% de Triton X-100 (PBS-T). Armazenar à temperatura ambiente durante 7 min. </li><li> Use pinças de metal para transferir slides para um porta-lâminas de vidro cheio de PBS. Lavar as lâminas com PBS durante 5 minutos à temperatura ambiente. Repetir 1x. Executar todas as lavagens de solução descartando no rack lâmina de vidro ( <em&gt; Ie. derramando-o para fora) e substituindo com solução fresca. </li><li> Descartar o PBS e mergulhar as lâminas em PBS-T, durante 30 minutos à temperatura ambiente, para permeabilizar as membranas das células. </li><li> Lavar as lâminas com PBS durante 5 minutos à temperatura ambiente. Repetir 2x. </li><li> Bloqueio passo (opcional): mergulhar as lâminas em PBS mais 1% de BSA durante 45 min à temperatura ambiente. </li><li> Use uma caneta barreira hidrofóbica para desenhar um círculo no slide em torno de tecido amassado (facilmente visível a olho nu), a fim de limitar as soluções de anticorpos (adicionados em passos 3,9 e 3,11). O tecido deve ser mantido úmido em todos os momentos durante a execução de imunomarcação. </li><li> Adicionar 30-40 mL de anticorpo primário (diluído em PBS-T, 1:400 a 1:50, dependendo do anticorpo) para o tecido no interior do círculo. Se o bloqueio foi realizado, diluir o anticorpo primário em PBS-T + 1% de BSA. Anticorpo anti-gama-tubulina (por coloração de centrossomas na Figura 4) foi diluída de 1:100. Incubar em uma câmara escura úmida (caixa de plástico, por exemplo. Fechadocom toalhas de papel húmidas) durante 2 horas à temperatura ambiente ou durante a noite a 4 ° C. </li><li> Lavar as lâminas em PBS por 5 min a sala de 3x de temperatura. Se o bloqueio foi realizado, lavar duas vezes em PBS-T, e uma vez em PBS (5 min a temperatura ambiente, cada um). </li><li> Adicionar 30-40 mL de anticorpo secundário conjugado com fluoróforo (diluído 1:400 em PBS) para o tecido e incubar no escuro durante 1-2 horas à temperatura ambiente. </li><li> Lavar as lâminas com PBS durante 5 minutos à temperatura ambiente. Repetir 2x. </li><li> Adicionar 30-40 mL de solução DAPI (0,2 mg / ml em PBS) para o tecido no interior do círculo. </li><li> Delicadamente, coloque uma lamínula de vidro sobre o tecido, tomando cuidado para evitar bolhas de interceptação. Se bolhas de ar devem aparecer, cuidadosamente mover ao redor da lamínula sem destruir a abóbora até que as bolhas escapar dos lados da lamínula. </li><li> Use uma limpeza limpar para apagar o excesso de DAPI a partir das bordas do slide. </li><li> Selar a lamela para o slide usando claro unha polonês. </li><li> Use esta preparaçãono próximo 3-4 hr para ver células imunomarcadas por microscopia fluorescente. Para o armazenamento de longo prazo de lâminas (até, pelo menos, várias semanas), use uma mídia difícil montar à base de glicerol com DAPI, e armazenar a -20 ˚ lâminas C. </li><li> Em alternativa, fixar as amostras em metanol em vez de formaldeído (dependendo do antigénio). Depois de completar todo o protocolo até a etapa 3.2, mergulhe as lâminas de testículos esmagados em metanol por 10 min a -20 ° C e prossiga com o passo 3.5 em diante. </li></ol>

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H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118, 401-415 (1993).</li><li>White-Cooper, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+cell+type+and+stage-specific+ectopic+gene+expression+and+RNAi+induction+in+the+Drosophila+testis.">Tissue cell type and stage-specific ectopic gene expression and RNAi induction in the Drosophila testis.</a> Spermatogenesis. 2, 11-22 (2012).</li><li>Rebollo, E., Llamazares, S., Reina, J., Gonzalez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+noncentrosomal+microtubules+to+spindle+assembly+in+Drosophila+spermatocytes.">Contribution of noncentrosomal microtubules to spindle assembly in Drosophila spermatocytes.</a> PLoS Biol. 2, (2004).</li><li>Cheng, J., Hunt, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+live+imaging+of+stem+cells+in+Drosophila+testis.">Time-lapse live imaging of stem cells in Drosophila testis.</a> Curr. Protoc. Stem Cell. Biol.. 2, 10-1002 (2009).</li><li>Sheng, X. R., Matunis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+the+Drosophila+spermatogonial+stem+cell+niche+reveals+novel+mechanisms+regulating+germline+stem+cell+output.">Live imaging of the Drosophila spermatogonial stem cell niche reveals novel mechanisms regulating germline stem cell output.</a> Development. 138, 3367-3376 (2011).</li><li>Belloni, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+Cog7+affect+Golgi+structure,+meiotic+cytokinesis+and+sperm+development+during+Drosophila+spermatogenesis.">Mutations in Cog7 affect Golgi structure, meiotic cytokinesis and sperm development during Drosophila spermatogenesis.</a> J. Cell Sci. 125, 5441-5452 (2012).</li><li>Moon, S., Cho, B., Min, S. H., Lee, D., Chung, Y. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+THO+complex+is+required+for+nucleolar+integrity+in+Drosophila+spermatocytes.">The THO complex is required for nucleolar integrity in Drosophila spermatocytes.</a> Development. 138, 3835-3845 (2011).</li><li>Wang, Z., Mann, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+for+two+nearly+identical+TGIF-related+homeobox+genes+in+Drosophila+spermatogenesis.">Requirement for two nearly identical TGIF-related homeobox genes in Drosophila spermatogenesis.</a> Development. 130, 2853-2865 (2003).</li></ol>

Os autores declaram que não têm interesses financeiros concorrentes.

Embora os testículos de moscas do tipo selvagem pode ser facilmente identificado, devido à sua cor amarela (em contraste, os tecidos brancos vizinhos), os testículos de moscas mutantes brancos são brancos e, assim, eventualmente, pode ser confundido com o intestino. Mais estirpes transgénicas, que são tipicamente num fundo branco, também têm testículos branco porque o mini-gene de branco encontrada em P-elementos não promove a acumulação de pigmentos nos testículos. Quand...

Testículos de Drosophila são um sistema modelo ideal para o estudo de muitos processos biológicos incluindo a regulação das células estaminais, a meiose, e desenvolvimento do esperma 13-18. Os espermatócitos e os seus fusos meióticos são grandes e, portanto, conveniente para análise citológica e relaxado checkpoints do ciclo celular durante a espermatogênese facilitar o estudo de mutações em genes do ciclo celular. Diferentes tipos de células pode ser observada na progressão ordenada ao longo do comprimento dos testículos, e qualquer interrupção da espermatogénese pode conduzir a alterações neste arranjo global. Estas características combinadas com ferramentas genéticas Drosophila têm facilitado a análise mutacional da espermatogênese 21-23. As fases de Drosophila espermatogénese foram bem definidos. As células da linha germinativa que se desenvolvem de forma síncrona nos cistos progredir sequencialmente através das fases de espermatogénese ao longo do comprimento do testículo. Durante both da mitose e meiose divisões das células germinais masculinas, citocinese ocorre de forma incompleta de modo a que as células filhas permanecem ligados por pontes citoplasmáticos conhecidos como canais de anel (Figura 1). A extremidade apical do testículo contém uma população de células estaminais da linha germinativa, que dá origem a células spermatogonial, que se submetem a quatro divisões mitóticas com citocinese incompletos para gerar cistos de 16 células de espermatócitos primários. Após a fase S pré-meiótica, espermatócitos primários entrar G2, um período prolongado de crescimento ~ 90 horas, durante o qual o volume celular aumenta ~ 25 vezes. Progressão através da meiose I e meiose II resulta na formação de quistos de 32 células de espermatócitos secundários e de cistos de 64 células de espermatídeos haplóides, respectivamente. As espermátides arredondadas, imaturos passam por extensos remodelamento celular para formar espermatozóides maduros. Células pós-meióticas, em especial os molhos de alongamento e espermatídeos maduros, ocupam a maior parte do volume do testículo. Tele o transporte de sucesso de esperma funcional para moscas fêmeas requer a coordenação entre as diferentes partes do sistema reprodutor masculino, a qual é composta de várias estruturas emparelhados (os testículos, vesículas seminais e glândulas acessórias) e de uma única conduta ejaculatório (Figura 2). Os espermatozóides são produzidos dentro dos testículos e armazenado dentro das vesículas seminais até a cópula 24. As glândulas acessórias conter células secretoras de fluido seminal. O esperma migração das vesículas seminais são misturados com o fluido seminal dentro do ducto ejaculatório, a qual está ligada a ambas as vesículas seminais e glândulas acessórias. Esta mistura de esperma e fluido seminal é finalmente bombeado para fora do macho no interior da vagina da fêmea voar através do bulbo ejaculatório localizado na extremidade posterior do abdómen macho 25. As proteínas no fluido seminal, são essenciais para a armazenagem prolongada de esperma dentro de órgãos especializados conhecidos como Espermatecas na reptrato roductive de Drosophila fêmeas 26. Excelentes métodos para o isolamento de testículos de Drosophila e a visualização de células em vários estádios da espermatogénese estão disponíveis na literatura científica 3-12. Nós aqui acrescentar a este corpo de conhecimento, apresentando exemplos desses protocolos com uma demonstração em vídeo que o acompanha. O protocolo para a preparação de amostras de testículos vivos para microscopia de fase de contraste baseia-se num método anteriormente descrito 27. O protocolo para a fixação de formaldeído e imunocoloração de testículos também se baseia em um método previamente descrito 28. As abordagens aqui descritos têm sido utilizados em vários estudos de Drosophila espermatogénese (por exemplo, para avaliar as funções da dineína, um motor de microtúbulos menos dirigida-fim, durante a espermatogénese de Drosophila). Além dos protocolos básicos, são apresentadas sugestões para varying a dissecação, de modo a enriquecer a espermatogônias, espermatócitos, ou espermatozóides maduros. Diferentes métodos para o processamento dos testículos tais que cistos ou permanecem intactos ou são interrompidos quando necessário são descritos. Uma vantagem no uso de testículos de Drosophila como sistema modelo é que, em comparação com oócitos e embriões de Drosophila, os anticorpos e corantes podem facilmente penetrar nas células após a sua dispersão a partir dos testículos, e menos passos de lavagem são necessários, assim, os protocolos podem ser realizadas em um relativamente curto espaço de tempo.

<table><tbody><tr><td>Sylgard</td><td>World Precision Instruments</td><td>SYLG184</td><td>Two-part silicon elastomer for making silicone-coated dissection dish from Kimax Petri dish</td></tr><tr><td>PAP pen</td><td>Fisher Scientific</td><td>NC9888126</td><td>Ted Pella #22309</td></tr><tr><td>Clear nail protector</td><td>Wet n Wild</td><td>7780235001</td><td></td></tr><tr><td>ProLong Gold Antifade Reagent with DAPI</td><td>Life Technologies</td><td>P36931</td><td></td></tr><tr><td>Mouse anti-gamma-tubulin antibody (clone GTU-88)</td><td>Sigma-Aldrich</td><td>T6557</td><td></td></tr><tr><td>Cy3-AffiniPure Goat Anti-Mouse IgG&amp;nbsp;</td><td>Jackson ImmunoResearch</td><td>115-165-003</td><td></td></tr><tr><td>Triton X-100</td><td>Fisher Scientific</td><td>BP151-100</td><td></td></tr><tr><td>Ethanol</td><td>Fisher Scientific</td><td>AC61511-0040</td><td></td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A412-4</td><td></td></tr><tr><td>16% Formaldehyde</td><td>Thermo Fisher Scientific</td><td>28908</td><td></td></tr><tr><td>Sigmacote</td><td>Sigma-Aldrich</td><td>SL2</td><td>Use according to manufacturer&#039;s directions to siliconize cover slips</td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D-9542</td><td>0.5 mg/ml in 75% ethanol; store at -20 &amp;deg;C</td></tr><tr><td>NaCl</td><td>Research Products International Corp.</td><td>S23020</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S9763</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S0751</td><td></td></tr><tr><td>Kimwipes delicate task wipers</td><td>Fisher Scientific</td><td>S47299</td><td></td></tr><tr><td>BSA</td><td>Research Products International Corp.</td><td>A30075</td><td>Molecular biology grade</td></tr><tr><td>Glass Coplin staining jar, screw cap</td><td>Electron Microscopy Sciences</td><td>70315</td><td></td></tr><tr><td>Single frosted microscope slides</td><td>Corning</td><td>2948-75X25</td><td></td></tr><tr><td>Poly-L-lysine coated microscope slides</td><td>Polysciences, Inc.</td><td>22247-1</td><td>Optional (to replace untreated microscope slides )</td></tr><tr><td>Square cover glass</td><td>Corning</td><td>2865-22</td><td></td></tr><tr><td>Razor blades</td><td>Fisher Scientific</td><td>12-640</td><td></td></tr><tr><td>Kimax Petri dish</td><td>Fisher Scientific</td><td>S31473</td><td>Kimble #23060 10015 EMD</td></tr><tr><td>Forceps</td><td>Dumont</td><td>52100-51S</td><td>Pattern 5 INOX</td></tr><tr><td>Stemi 2000-CS stereoscope</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Eclipse 80i</td><td>Nikon</td><td></td><td></td></tr><tr><td>Plan-Fluor 40X objective</td><td>Nikon</td><td></td><td></td></tr><tr><td>Axiophot</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Plan-Neofluar Ph2 40X objective</td><td>Carl Zeiss</td><td></td><td></td></tr></tbody></table>

cytological analysis of spermatogenesis: live and fixed preparations of drosophila testes

Drosophila melanogaster é um poderoso sistema modelo que tem sido amplamente utilizado para elucidar de uma variedade de processos biológicos. Por exemplo, estudos, tanto do sexo feminino e as linhas germinativas masculinas de Drosophila têm contribuído grandemente para a compreensão atual da meiose, bem como a biologia de células-tronco. Excelente protocolos estão disponíveis na literatura para o isolamento e imagiologia de ovários e testículos 3-12 Drosophila. Aqui, os métodos para a dissecação e preparação de testes de Drosophila para análise microscópica são descritos com uma demonstração em vídeo que o acompanha. Um protocolo para o isolamento de testículos do abdómen de adultos do sexo masculino e preparar as lâminas de tecido vivo, para análise por microscopia de contraste de fase, bem como o protocolo para a fixação e imunocoloração testículos para análise por microscopia de fluorescência são apresentados. Estas técnicas podem ser aplicadas na caracterização de mutantes de Drosophila que exibem defects na espermatogénese, assim como na visualização de localizações subcelulares de proteínas.

Um exemplo de um par adequadamente dissecados dos órgãos reprodutores masculinos de Drosophila é mostrado na Figura 2A. Testículos do abdómen da mosca adulta masculina está normalmente ligado ao ducto ejaculatório (castanho, Figura 2A &#39;) e um par de glândulas acessórias (verde, Figura 2A&#39;), através de um par de vesículas seminais (azul, Figura 2A &#39;) . Para separar os testículos da maioria do tecido somático que o acompa...

Watch this Scientific Journal Video about Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes at JoVE.com

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Os métodos para isolar e preparar amostras de testículos de Drosophila (ao vivo e fixo) para geração de imagens por contraste de fase e microscopia de fluorescência são descritos aqui.

Citológica Análise da espermatogênese: Preparados ao vivo e fixo<em&gt; Drosophila</em&gt; Testes

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both ...

cytological-analysis-spermatogenesis-live-fixed-preparations

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

26.1K visualizações.  Vanderbilt University Medical Center.  Os métodos para isolar e preparar amostras de testículos de Drosophila (ao vivo e fixo) para geração de imagens por contraste de fase e microscopia de fluorescência são descritos aqui. 

Vídeo: Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Thin Sectioning of Slice Preparations for Immunohistochemistry

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been preserved, freshly removed, or excised but kept viable, as in slice preparations of brain tissue.  Subsequent microscopic studies of this material can be challenging, as the tissue samples may be difficult to handle.  Demonstrated here is a method for obtaining thin cryostat sections of tissue with a thickness that may range from 0.2-5.0 mm.  We routinely cut 400 micron thick Vibratome brain slices serially into 5-10 micron coronal cryostat sections.  The slices are typically first used for electrophysiology experiments and then require microscopic analysis of the cytoarchitecture of the region from which the recordings were observed.  We have constructed a simple device that allows controlled and reproducible preparation and positioning of the tissue slice.  This device consists of a cylinder 5 cm in length with a diameter of 1.2 cm, which serves as a freezing stage for the slice.  A ring snugly slides over the cylinder providing walls around the slice allowing the tissue to be immersed in freezing compound (e.g., OCT).  This is then quickly frozen with crushed dry ice and the resulting  wafer  can be position easily for cryostat sectioning.  Thin sections can be thaw-mounted onto coated slides to allow further studies to be performed, such as various staining methods, in situ hybridization, or immunohistochemistry, as demonstrated here.

The present method allows reproducible cryostat sectioning of small, difficult-to-manage, tissue pieces, such as biopsies and brain slices. We utilize a simple aluminum freezing stage to facilitate handling of tissue and a standard cryostat to routinely produce 5-10 micron serial sections from 400 micron thick brain slices.

Seccionamento fina de Preparações Slice para Imunohistoquímica

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been ...

Biologia

A Seminiferous Tubule Squash Technique for the Cytological Analysis of Spermatogenesis Using the Mouse Model

Meiotic progression in males is a process that requires the concerted action of a number of highly regulated cellular events. Errors occurring during meiosis can lead to infertility, pregnancy loss or genetic defects. Commencing at the onset of puberty and continuing throughout adulthood, continuous semi-synchronous waves of spermatocytes undergo spermatogenesis and ultimately form haploid sperm. The first wave of mouse spermatocytes undergoing meiotic initiation appear at day 10 post-partum (10 dpp) and are released into the lumen of seminiferous tubules as mature sperm at 35 dpp. Therefore, it is advantageous to utilize mice within this developmental time-window in order to obtain highly enriched populations of interest. Analysis of rare cell stages is more difficult in older mice due to the contribution of successive spermatogenic waves, which increase the diversity of the cellular populations within the tubules. The method described here is an easily implemented technique for the cytological evaluation of the cells found within the seminiferous tubules of mice, including spermatogonia, spermatocytes, and spermatids. The tubule squash technique maintains the integrity of isolated male germ cells and allows examination of cellular structures that are not easily visualized with other techniques. To demonstrate the possible applications of this tubule squash technique, spindle assembly was monitored in spermatocytes progressing through the prophase to metaphase I transition (G2/MI transition). In addition, centrosome duplication, meiotic sex chromosome inactivation (MSCI), and chromosome bouquet formation were assessed as examples of the cytological structures that can be observed using this tubule squash method. This technique can be used to pinpoint specific defects during spermatogenesis that are caused by mutation or exogenous perturbation, and thus, contributes to our molecular understanding of spermatogenesis.

The goal of this tubule squash technique is to rapidly assess cytological features of developing mouse spermatocytes while preserving cellular integrity. This method allows for the study of all stages of spermatogenesis, and can be easily implemented alongside other biochemical and molecular biological approaches for the study of mouse meiosis.

Uma técnica de Squash do tubo seminífero para a análise citológica da espermatogênese usando o modelo de Mouse

Meiotic progression in males is a process that requires the concerted action of a number of highly regulated cellular events. Errors occurring during ...

Biologia do Desenvolvimento

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

Imagens ao vivo de Drosophila melanogaster Embrionárias Migrações hemócitos

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with development, tissue homeostasis, and disease processes. Drosophila spermatocytes undergoing meiosis are ideal for this analysis because (1) whole Drosophila testes containing spermatocytes are relatively easy to prepare for microscopy, (2) the spermatocytes&#8217; large size makes them well suited for high resolution imaging, and (3) powerful Drosophila genetic tools can be integrated with in vivo analysis. Here, we present a readily accessible protocol for the preparation of whole testes from Drosophila third instar larvae and early pupae. We describe how to identify meiotic spermatocytes in prepared whole testes and how to image them live by time-lapse microscopy. Protocols for fixation and immunostaining whole testes are also provided. The use of larval testes has several advantages over available protocols that use adult testes for spermatocyte analysis. Most importantly, larval testes are smaller and less crowded with cells than adult testes, and this greatly facilitates high resolution imaging of spermatocytes. To demonstrate these advantages and the applications of the protocols, we present results showing the redistribution of the endoplasmic reticulum with respect to spindle microtubules during cell division in a single spermatocyte imaged by time-lapse confocal microscopy. The protocols can be combined with expression of any number of fluorescently tagged proteins or organelle markers, as well as gene mutations and other genetic tools, making this approach especially powerful for analysis of cell division mechanisms in the physiological context of whole tissues and organs.

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

The goal of this protocol is to analyze cell division in intact tissue by live and fixed cell microscopy using Drosophila meiotic spermatocytes. The protocol demonstrates how to isolate whole, intact testes from Drosophila larvae and early pupae, and how to process and mount them for microscopy.

Preparação de Drosophila Larval e Pupal Testes para Análise de Divisão Celular em Tecido Vivo e Intacto

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with ...

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

Preparação de Desenvolvimento e Adultos Drosophila Brains e retina de imagens ao vivo

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established cell isolation protocols using Hoechst 33342 staining in combination with fluorescence-activated cell sorting, it requires cell sorters equipped with an ultraviolet laser. Here we describe a cell isolation protocol using the DyeCycle Violet (DCV) stain, a low cytotoxicity DNA binding dye structurally similar to Hoechst 33342. DCV can be excited by both ultraviolet and violet lasers, which improves the flexibility of equipment choice, including a cell sorter not equipped with an ultraviolet laser. Using this protocol, one can isolate three live-cell subpopulations in meiotic prophase I, including leptotene/zygotene, pachytene, and diplotene spermatocytes, as well as post-meiotic round spermatids. We also describe a protocol to prepare single-cell suspension from mouse testes. Overall, the procedure requires a short time to complete (4-5 hours depending on the number of needed cells), which facilitates many downstream applications.

Here we present a simple and efficient method to isolate live meiotic and post-meiotic germ cells from adult mouse testes. Using a low-cytotoxicity, violet-excited DNA binding dye and fluorescence-activated cell sorting, one can isolate highly enriched spermatogenic cell populations for many downstream applications.

Isolamento de células spermatogênicas murinas usando um corante de ligação de DNA permeável celular-permeável violeta

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established ...

Imaging Centrosomes in Fly Testes

Centrosomes are conserved microtubule-based organelles whose structure and function change dramatically throughout the cell cycle and cell differentiation. Centrosomes are essential to determine the cell division axis during mitosis and to nucleate cilia during interphase. The identity of the proteins that mediate these dynamic changes remains only partially known, and the function of many of the proteins that have been implicated in these processes is still rudimentary. Recent work has shown that Drosophila spermatogenesis provides a powerful system to identify new proteins critical for centrosome function and formation as well as to gain insight into the particular function of known players in centrosome-related processes. Drosophila is an established genetic model organism where mutants in centrosomal genes can be readily obtained and easily analyzed. Furthermore, recent advances in the sensitivity and resolution of light microscopy and the development of robust genetically tagged centrosomal markers have transformed the ability to use Drosophila testes as a simple and accessible model system to study centrosomes. This paper describes the use of genetically-tagged centrosomal markers to perform genetic screens for new centrosomal mutants and to gain insight into the specific function of newly identified genes.

Imaging of centrosomal proteins during Drosophila spermatogenesis is a powerful method to identify new proteins critical for centrosome biology as well as to elucidate the particular function of known players in this process.

Centrossomas de imagem na mosca Testículos

Centrosomes are conserved microtubule-based organelles whose structure and function change dramatically throughout the cell cycle and cell ...

Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ

Studies performed in Drosophila melanogaster embryos and larvae provide crucial insight into developmental processes such as cell fate specification and organogenesis. Immunostaining allows for the visualization of developing tissues and organs. However, a protective cuticle that forms at the end of embryogenesis prevents permeation of antibodies into late-stage embryos and larvae. While dissection prior to immunostaining is regularly used to analyze Drosophila larval tissues, it proves inefficient for some analyses because small tissues may be difficult to locate and isolate. Sonication provides an alternative to dissection in larval Drosophila immunostaining protocols. It allows for quick, simultaneous processing of large numbers of late-stage embryos and larvae and maintains in situ morphology. After fixation in formaldehyde, a sample is sonicated. Sample is then subjected to immunostaining with antigen-specific primary antibodies and fluorescently labeled secondary antibodies to visualize target cell types and specific proteins via fluorescence microscopy. During the process of sonication, proper placement of a sonicating probe above the sample, as well as the duration and intensity of sonication, is critical. Additonal minor modifications to standard immunostaining protocols may be required for high quality stains. For antibodies with low signal to noise ratio, longer incubation times are typically necessary. As a proof of concept for this sonication-facilitated protocol, we show immunostains of three tissue types (testes, ovaries, and neural tissues) at a range of developmental stages.

Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ

Immunostaining is an effective technique for visualizing specific cell types and proteins within tissues. By utilizing sonication, the protocol described here alleviates the need to dissect Drosophila melanogaster tissues from late-stage embryos and larvae before immunostaining. We provide an efficient methodology for the immunostaining of formaldehyde-fixed whole mount larvae.

Facilitou-Sonication Imunofluorescência Coloração de Late-estágio embrionário e larval<em&gt; Drosophila</em&gt; Tecidos<em&gt; In Situ</em

Studies performed in Drosophila melanogaster embryos and larvae provide crucial insight into developmental processes such as cell fate specification and ...

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes differentiation from a stem cell population, mitotic amplification, and meiosis. In addition, post-meiotic germ cells undergo a dramatic morphological reshaping process as well as a global epigenetic reconfiguration of the germ line chromatin&#8212;the histone-to-protamine switch.
Studying the role of a protein in post-meiotic spermatogenesis using mutagenesis or other genetic tools is often impeded by essential embryonic, pre-meiotic, or meiotic functions of the protein under investigation. The post-meiotic phenotype of a mutant of such a protein could be obscured through an earlier developmental block, or the interpretation of the phenotype could be complicated. The model organism Drosophila melanogaster offers a bypass to this problem: intact testes and even cysts of germ cells dissected from early pupae are able to develop ex vivo in culture medium. Making use of such cultures allows microscopic imaging of living germ cells in testes and of germ-line cysts. Importantly, the cultivated testes and germ cells also become accessible to pharmacological inhibitors, thereby permitting manipulation of enzymatic functions during spermatogenesis, including post-meiotic stages.
The protocol presented describes how to dissect and cultivate pupal testes and germ-line cysts. Information on the development of pupal testes and culture conditions are provided alongside microscope imaging data of live testes and germ-line cysts in culture. We also describe a pharmacological assay to study post-meiotic spermatogenesis, exemplified by an assay targeting the histone-to-protamine switch using the histone acetyltransferase inhibitor anacardic acid. In principle, this cultivation method could be adapted to address many other research questions in pre- and post-meiotic spermatogenesis.

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

This protocol describes the dissection and cultivation of intact testes and germ-line cysts from Drosophila melanogaster pupae. This method allows microscopic observation of spermatogenesis ex vivo. Furthermore, we describe a pharmacological assay of the effect of inhibitors on specific stages of germ-cell development in pupal testes.

Ex vivo Cultura de Drosophila Pupal Testículo e único macho da linha germinal Cistos: Dissection, Imaging, eo tratamento farmacológico

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes ...

Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes

Drosophila testes are a powerful model system for studying biological processes including stem cell biology, nuclear architecture, meiosis and sperm development. However, immunolabeling of the whole Drosophila testis is often associated with significant non-uniformity of staining due to antibody penetration. Squashed preparations only partially overcome the problem since it decreases the 3D quality of the analyses. Herein, we describe a whole-mount protocol using NP40 and heptane during fixation together with immunolabeling in liquid media. It preserves the volume suitable for confocal microscopy together with reproducible and reliable labeling. We show different examples of 3D reconstitution of spermatocyte nuclei from confocal sections. The intra- and inter-testes reproducibility allows 3D quantification and comparison of fluorescence between single cells from different genotypes. We used different components of the intranuclear MINT structure (Mad1-containing Intra Nuclear Territory) as well as two components associated with the nuclear pore complex to illustrate this protocol and its applications on the largest cells of the testis, the S4-S5 spermatocytes.

Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes

We present here an improved protocol for the whole-mount preparation and immunostaining of Drosophila testes, suitable for confocal microscopy and also allowing reproducible and reliable labeling. We illustrate this protocol by 3D representation and quantification of colocalization experiments on the large S4-S5 spermatocytes.

Imunostaining de Testes Drosophila de Monte Inteiro para Análise Confocal 3D de Grandes Espermatocitos

Drosophila testes are a powerful model system for studying biological processes including stem cell biology, nuclear architecture, meiosis and sperm ...

Citológica Análise da espermatogênese: Preparados ao vivo e fixo

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