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.

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