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

Summary

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.

Abstract

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.

Introduction

Nuclear positioning is essential for multiple cellular and developmental functions^1,2,3,4,5. 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 humans^{7, 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 condensates^{12,13,14,15,16} (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 cell^10,11. 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 studies^10,11, 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.

Protocol

All animal experiments were performed in accordance with the guidelines of the European Community and were approved by the French Ministry of Agriculture (authorization No. 75-1170) and by the Direction Générale de la Recherche et de l'Innovation (DGRI; GMO agreement number DUO-5291). Mice were housed in the animal facility on a 12 h light/dark cycle, with an ambient temperature of 22-24 °C and humidity of 40%-50%. Mice used here include female OF1 (Oncins France 1, 8 to 12 weeks old) and female C57BL/…

Representative Results

Image panels in Figure 3 show examples of a typical fully grown oocyte (Figure 3A), the nucleoplasm in a fully grown oocyte expressing YFP-Rango (Figure 3B), the nucleoplasm in a fully grown oocyte expressing a correct (left panel; Figure 3C) or an excessive (right panel; Figure 3C) dose of SRSF2-GFP cRNA, and an immunostaining of nuclear speckles in a fully grown oocyte us…

Discussion

Key steps in this protocol include proper microinjection of oocytes without affecting their survival or normal function^9,10,11, as well as microinjecting predefined amounts of cRNA that would allow correct visualization of relevant structures, like nuclear speckles.

Establishing the link between cytoplasmic and (intra)-nuclear dynamics is essential when studying how the cytoskeleton agitates the nucle…

Materials

Bovine Serum Albumin (BSA)	Sigma	A3311
CSU-X1-M1 spinning disk	Yokogawa
DMI6000B microscope	Leica
Femtojet microinjector	Eppendorf
Fiji
Filter wheel	Sutter Instruments Roper Scientific
Fluorodish	World Precision Instruments	FD35-100
Metamorph software	Universal Imaging,		version 7.7.9.0
Mineral oil	Sigma Aldrich	M8410-1L
NanoDrop 2000	Thermo Scientific
OF1 and C57BL/6 mice	Charles River Laboratories
Poly(A) Tailing kit	Thermo Fisher	AM1350
Retiga 3 CCD camera	QImaging
RNAeasy kit	Qiagen	74104
SC35 antibody	Abcam	ab11826	Nuclear speckle antibody; mouse IgG1 anti-SRSF2/SC35 (1:400)
SRSF2-GFP plasmid	OriGene Technologies	MG202528	NM_011358
Stripper Micropipette	XLAB Solutions		specialized for oocyte collection
T3 mMessage mMachine	Thermo Fisher	AM1384
T7 mMessage mMachine	Thermo Fisher	AM13344
Thermostatic chamber	Life Imaging Service
Windows Excel	Windows