Step-specific Sorting of Mouse Spermatids by Flow Cytometry

Olivier Simard; Fr&#233;d&#233;ric Leduc; Genevi&#232;ve Acteau; M&#233;lina Arguin; Marie-Chantal Gr&#233;goire; Marc-Andr&#233; Brazeau; Isabelle Marois; Martin V. Richter; Guylain Boissonneault

doi:10.3791/53379

Passo-específico Seleção de mouse Espermátides por citometria de fluxo

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Developmental Biology

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JoVE Journal Developmental Biology

Step-specific Sorting of Mouse Spermatids by Flow Cytometry

Passo-específico Seleção de mouse Espermátides por citometria de fluxo

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06:31 min

December 31, 2015

DOI: 10.3791/53379-v

Olivier Simard*¹, Frédéric Leduc*¹, Geneviève Acteau¹, Mélina Arguin¹, Marie-Chantal Grégoire¹, Marc-André Brazeau¹, Isabelle Marois², Martin V. Richter², Guylain Boissonneault¹

¹Department of Biochemistry,Université de Sherbrooke, ²Department of Medicine,Université de Sherbrooke

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Overview

This article describes a flow cytometry sorting strategy for mouse spermatids, enabling the separation into four distinct populations. The method enhances the molecular study of spermiogenesis and minimizes cross-contamination.

Key Study Components

Area of Science

Neuroscience
Reproductive Biology
Cell Biology

Background

Mouse spermatids undergo various stages during spermiogenesis.
Understanding these stages is crucial for reproductive biology.
Chromatin remodeling impacts genomic integrity during spermatid development.
Current methods may not provide sufficient purity in cell populations.

Purpose of Study

To develop a method for sorting mouse spermatids into pure populations.
To facilitate molecular studies of spermiogenesis.
To investigate the effects of chromatin modeling on genomic integrity.

Methods Used

Flow cytometry for sorting spermatids.
DNA staining for selection parameters.
Sorting based on size and granulosity.
Preparation of tubes with heat-inactivated FBS for cell sorting.

Main Results

Successful separation of spermatids into four distinct populations.
High purity of sorted cells with minimal cross-contamination.
Enhanced understanding of spermiogenesis stages.
Potential for further research into chromatin remodeling effects.

Conclusions

The developed sorting strategy is effective for studying spermatids.
This method can advance research in reproductive biology.
Future studies can leverage this technique to explore genomic integrity.

Frequently Asked Questions

What are the main populations of spermatids sorted in this study?

The study sorts spermatids into round, early elongating, late elongating, and elongated populations.

How does this method improve upon previous techniques?

It provides higher purity of cell populations without cross-contamination.

What is the significance of chromatin remodeling in spermatids?

Chromatin remodeling affects genomic integrity, which is crucial for successful reproduction.

What parameters are used for sorting spermatids?

DNA staining, size, and granulosity are used as selection parameters.

Can this method be applied to other cell types?

While this study focuses on spermatids, the technique may be adaptable to other cell types in future research.

What are the next steps for research using this method?

Future studies can explore the molecular mechanisms of spermiogenesis and the effects of chromatin remodeling.

Descrevemos uma estratégia de classificação para espermátides de camundongos usando citometria de fluxo. As espermátides são classificadas em quatro populações altamente puras, incluindo espermogênese redonda (etapas 1-9 da espermiogênese), alongamento precoce (etapas 10-12 da espermiogênese), alongamento tardio (etapas 13-14 da espermiogênese) e espermátides alongadas (etapas 15-16 da espermiogênese). A coloração, o tamanho e a granulosidade do DNA são usados como parâmetros de seleção.

O objetivo geral deste procedimento é separar as espermátides de camundongos em quatro populações distintas, permitindo o estudo molecular da espermiogênese. Este método pode ajudar a responder a questões-chave no campo da reprodução de espelhos, como qual é o impacto da modelagem cromática na integridade genômica. A principal vantagem dessa técnica é que ela fornece separação de espermatozoides em população de células puras sem contaminação cruzada.

Tivemos a ideia para este método pela primeira vez quando observamos variações importantes na intensidade da coloração DN durante a remodelação da cromatina em parâmetros haplo no dia anterior à classificação celular. Adicione um a dois mililitros de FBS inativado por calor ao fundo redondo de polipropileno de cinco mililitros e tubos cônicos de polipropileno de 15 e 50 mililitros. Em seguida, revestir lentamente os tubos por inversão noturna a quatro graus Celsius em um rotador no dia seguinte.

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