oocyte collection, preparation, and microinjection in mouse for live imaging studies

Einfluss des Zytoskeletts auf die Kernform und Kondensatdynamik in Maus-Eizellen

Nuclear positioning is essential for multiple cellular and developmental functions1,2,3,4,5. Mammalian female germ cells named oocytes remodel their cytoplasm to position the nucleus at the cell center despite undergoing an asymmetric division in size, which relies on subsequent chromosome off-centering6 (Figure 1). This centering of the nucleus predicts successful oocyte development in mice and humans7, 8, and thus, their embryogenic potential (Figure 1).
Cytoplasmic remodeling in mouse oocytes is driven primarily by the actomyosin cytoskeleton9 (Figure 2). Its activity generates mechanical forces that agitate, reposition, and penetrate the nucleus10 (Figure 2). Consequently, this cytoplasmic-to-nucleoplasmic force transmission tunes the dynamics of nuclear messenger RNA-processing organelles named nuclear speckles11, one of several membrane-less organelles in the nucleus known as biomolecular condensates12,13,14,15,16 (Figure 2).
Live imaging has been decisive in deciphering the functional implications of nuclear agitation. Movies of nuclear migration over hours, as well as high-temporal resolution movies of the actin mesh and the bulk cytoplasm, largely contributed to the elaboration of a theoretical model for nuclear positioning, linking different timescales9. Also, high temporal resolution movies of the cytoplasm, nuclear outline and nuclear components such as chromatin and nuclear condensates, highlighted the role of cytoskeleton-based agitation of the nucleus on RNA-processing and gene expression in mouse oocytes, bridging different spatiotemporal scales within the cell10,11. Altogether, such a scale-crossing approach based on live imaging provided the first rationale linking cytoskeletal agitation of the nucleus to the developmental success of oocytes.
The protocol provides the imaging and image analysis pipeline used to study the transmission of cytoplasmic forces (generated primarily by F-actin and partly by microtubules) to the nucleus and its internal components in mouse oocytes. The outcome of these experiments is to capture the continuum of forces across spatial scales, from the cytoskeleton in the cytoplasm to the nuclear interior via high temporal resolution movies as shown in two recent studies10,11, that established the link between cytoplasmic active movements, fluctuations of the nuclear outline, as well as movement and surface fluctuations of a single type of nuclear biomolecular condensates: nuclear speckles. The same approach may be applied to other model systems where cytoplasmic forces are expected to change, such as in the context of malignant cancer cells17.

Autor im Rampenlicht: Aufklärung der Dynamik von Mechano-Transduktion und nuklearer Agitation in Maus-Eizellen

Erfassung der zytoskelettbasierten Agitation des Maus-Eizellkerns über räumliche Skalen hinweg

Eizellentnahme, -vorbereitung und -mikroinjektion in der Maus für Live-Imaging-Studien

Beginnen Sie damit, alle für das Experiment erforderlichen Materialien auf der Arbeitsplattform anzuordnen. Nach der Entnahme der Eierstöcke aus der Maus legen Sie sie in vorgewärmtes M2 plus Rinderserumalbuminmedium, das mit einem mikromolaren Milrinon ergänzt wird. Punktion der Eierstockfollikel mit chirurgischen Nadeln, um wachsende Eizellen aus Antralfollikeln freizusetzen.Sammeln Sie mit einer Mikropipette Eizellen der für Experimente benötigten Größe. Dann die Eizellen in eine Schale mit frischem Medium geben. Übertragen Sie Eizellen mit der Mikropipette von Tropfen zu Tropfen.Dies ermöglicht die mechanische Dissoziation von Eizellen von antralen Follikelzellen. Stabilisieren Sie die Eizelle eine Stunde lang bei 37 Grad Celsius. Zentrifugieren Sie die cRNA-Aliquote, Beschichtung für YFP-Rango und SRSF2 GFP.Injizieren Sie mit einem Mikroinjektor cRNA in das Zytoplasma von Eizellen in einem warmen M2 plus Rinderserumalbuminmedium, ergänzt mit Milrinon. Inkubieren Sie die Eizellen zwei Stunden lang in einem Kulturmedium bei 37 Grad Celsius für die cRNA-Translation. Als nächstes legen Sie die Eizellen in Tröpfchen des Kulturmediums auf einer 35-Millimeter-Gewebekulturschale mit einer mit Mineralöl bedeckten Deckglasbodenkammer ab.

oocyte-collection-preparation-microinjection-mouse-for-live-imaging

Research

JoVE Journal

Biology

Oocyte Collection, Preparation, and Microinjection in Mouse for Live Imaging Studies

117 Aufrufe. Beginnen Sie damit, alle für das Experiment erforderlichen Materialien auf der Arbeitsplattform anzuordnen. Nach der Entnahme der Eierstöcke aus der Maus legen Sie sie in vorgewärmtes M2 plus Rinderserumalbuminmedium, das mit einem mikromolaren Milrinon ergänzt wird. Punktion der Eierstockfollikel mit chirurgischen Nadeln, um wachsende Eizellen aus Antralfollikeln freizusetzen.Sammeln Sie mit einer Mikropipette Eizellen der für Experimente benötigten Größe. Dann die Eizellen in...

Serie: Eizellentnahme, -vorbereitung und -mikroinjektion in der Maus für Live-Imaging-Studien

Capturing Cytoskeleton-Based Agitation of the Mouse Oocyte Nucleus Across Spatial Scales

A major challenge in understanding the causes of female infertility is to elucidate mechanisms governing the development of female germ cells, named oocytes. Their development is marked by cell growth and subsequent divisions, two critical phases that prepare the oocyte for fusion with sperm to initiate embryogenesis. During growth, oocytes reorganize their cytoplasm to position the nucleus at the cell center, an event predictive of successful oocyte development in mice and humans and, thus, their embryogenic potential. In mouse oocytes, this cytoplasmic reorganization was shown to be driven by the cytoskeleton, the activity of which generates mechanical forces that agitate, reposition, and penetrate the nucleus. Consequently, this cytoplasmic-to-nucleoplasmic force transmission tunes the dynamics of nuclear RNA-processing organelles known as biomolecular condensates. This protocol provides an experimental framework to document, with high temporal resolution, the impact of the cytoskeleton on the nucleus across spatial scales in mouse oocytes. It details the imaging and image analysis steps and tools necessary to evaluate i) cytoskeletal activity in the oocyte cytoplasm, ii) cytoskeleton-based agitation of the oocyte nucleus, and iii) its effects on biomolecular condensate dynamics in the oocyte nucleoplasm. Beyond oocyte biology, the methods elaborated here can be adapted for use in somatic cells to similarly address cytoskeleton-based tuning of nuclear dynamics across scales.

This protocol provides an experimental framework to document the physical impact of the cytoskeleton on nuclear shape and the internal membrane-less organelles in the mouse oocyte system. The framework can be adapted for use in other cell types and contexts.

A major challenge in understanding the causes of female infertility is to elucidate mechanisms governing the development of female germ cells, named ...