Live-Cell Imaging of Chromosome Segregation During Mouse Oocyte Meiosis

Summary

Here, we summarize live-cell imaging techniques for studying chromosome segregation in mouse oocytes, using H2B-RFP for chromatin and SiR-Tubulin for spindle visualization. We detail sample preparation, microinjection, culture, imaging setup, and analysis, offering a practical guide to achieve high-resolution and minimally phototoxic imaging of meiotic chromosome dynamics.

Abstract

Accurate chromosome segregation during oocyte meiosis is essential for ensuring proper embryonic development and preventing aneuploidy-related disorders. Live-cell imaging combined with fluorescence labelling techniques have become a powerful approach for studying meiotic chromosome dynamics with high spatiotemporal resolution. In this protocol, we summarize key methodologies for visualizing chromosome segregation in live mouse oocytes, focusing on the use of histone H2B-RFP for chromatin labelling and SiR-Tubulin for spindle tracking. We describe the procedures for sample preparation, mRNA microinjection, oocyte culture, live-cell imaging chamber setup, and confocal microscopy settings, all optimized to achieve high-resolution imaging while minimizing phototoxicity. Furthermore, we highlight critical experimental considerations such as phototoxicity, image processing, and quantitative analysis of meiotic events. By providing a comprehensive evaluation of current methodologies, this protocol serves as a practical guide for researchers seeking to investigate chromosome dynamics in live oocytes and improve the accuracy and reproducibility of meiotic studies.

Introduction

Accurate visualization of chromosomal and spindle dynamics during oocyte meiosis is critical for understanding the mechanisms that govern female gamete maturation and aneuploidy risk^1,2,3,4,5. Mammalian oocytes undergo a highly coordinated series of meiotic events, including germinal vesicle breakdown (GVBD), chromosome condensation, spindle assembly, and asymmetric division⁶. While fixed time-point imaging has provided valuable snapshots of these processes, it lacks temporal resolut....

Protocol

All experiments must be conducted in accordance with protocols approved by the local Animal Ethics Committee. Experimental procedures should adhere strictly to the 3Rs principles-Replacement, Reduction, and Refinement. Mice must be housed in an accredited animal facility under standardized environmental conditions (22 °C, 60% humidity) with free access to food and water.

1. Oocyte collection and preparation

NOTE: All equipment and reagents must be sterile. Perform all oocyte handling using a mouth pipette under a stereomicroscope (see Table of Materials). Cover all cultu....

Results

As shown in Figure 4A,B, this protocol enables high-quality live-cell imaging of chromosomes (H2B-RFP) and spindle microtubules (SiR-tubulin) in oocytes, thereby providing comprehensive spatiotemporal information throughout meiotic maturation.

The H2B-RFP channel in Figure 4A reveals all aspects of chromosome movement from initial alignment following GVBD (Figure 4A, 03:00 - 08:00) throug.......

Discussion

The protocol presented here outlines a robust method for the microinjection and live-cell imaging of mouse oocytes, enabling the visualization of chromosome dynamics during meiosis with high temporal and spatial resolution. Critical to the success of this approach is the meticulous handling of oocytes throughout each stage of the procedure, from collection and microinjection to imaging. Oocytes are highly sensitive to environmental fluctuations, and deviations in temperature, pH, or osmolarity can compromise their physio.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1-ml syringes	Terumo	SS+01T Tuberculin	Syringes for precise PMSG injections.
Agarose gel	Invitrogen	16500500	Used for DNA and RNA gel electrophoresis to separate nucleic acids by size, to conform H2B-RFP sequence
Alcohol burner	LabTek	LW15557-01	Used for sterilization and mouth pipitte preparation.
Bacterial culture media (LB broth)	Sigma-Aldrich	L3022	Standard medium used for growing E. coli and other bacterial cultures.
Cell culture dishes (35 × 10 mm and 60 × 15 mm)	Sigma-Aldrich	CLS430165, CLS430166	Used for culturing oocytes; available in various sizes.
Centrifuge (benchtop)	Eppendorf	5424R	A compact centrifuge for small sample volumes commonly used in research labs.
Centrifuge tubes (15-ml and 50-ml)	Corning	352096, 352070	Larger tubes for centrifugation, commonly used for cell and protein processing.
Chloroform	Sigma-Aldrich	C2432	A solvent commonly used in molecular biology protocols, especially in DNA/RNA extraction.
CO2 incubator	Sanyo	(model: MCO-18AIC)	Incubator designed to maintain a controlled CO2 atmosphere for oocyte culture.
Disposable ultra-fine needles (27 G × 1/2”)	Hanke Sass Wolf	4710004012	Fine needles for oocyte collection
DNase I (RNase-free)	Thermo Fisher Scientific	EN0521	Enzyme used to remove contaminating DNA from RNA preparations.
Dry heating block	Grant Instruments	(model: QBD4)	A block used for heating samples or reactions at precise temperatures without the need for water.
Ethanol (molecular biology grade)	Sigma-Aldrich	E7023	Used for DNA and RNA precipitation, as well as in sterilization procedures.
Fine dissecting scissors	Met-App Scientific and Surgical Instruments	2235	Used for precise dissection mouse oocytes
Fine forceps	Met-App Scientific and Surgical Instruments	2127	Used for precise dissection mouse oocytes
Gel electrophoresis system	Bio-Rad	1645050	A system for performing electrophoresis of nucleic acids or proteins in gels.
Gentamicin sulfate	Sigma-Aldrich	G1264	Antibiotic used to prevent bacterial contamination in cell cultures.
Glass Pasteur pipettes (230 mm)	VWR	6121702	Glass pipettes used for oocyte mouth pipette
IBMX	Sigma-Aldrich	I5879	A phosphodiesterase inhibitor used to prevent GVBD
Inverted Microscope	Nikon		A microscope designed for observing live cells or organisms from underneath the culture dish.
Light mineral oil suitable for mouse embryo culture	Sigma-Aldrich	M5310	Used for covering oocytes in culture to prevent evaporation and maintain temperature.
Lithium chloride	Sigma-Aldrich	L9650	Used in RNA precipitation methods.
Live-Cell Imaging System	Leica	LAS X	Imaging system designed for real-time observation of living cells using advanced microscopy techniques.
M16 medium (for in vitro culture)	Sigma-Aldrich	M7292	A medium designed for the culture of mouse oocytes and embryos and other in vitro applications.
Microcentrifuge tubes (0.6-ml and 1.6-ml)	Neptune	3735.X, 3745.X	Small-volume tubes for centrifugation, often used in molecular biology and biochemistry protocols.
Microcentrifuge tubes (RNase-free)	Eppendorf / Neptune	022431081 (Eppendorf)	RNase-free tubes for RNA work to prevent degradation of RNA.
Microinjection Needles	Sutter Instruments	P-30 Pipette Puller	Needles for precise microinjection into oocytes
Microinjection System	Harvard Apparatus	FemtoJet, PicoPump	Systems used for injecting cRNA into mouse oocytes with high precision.
Miniprep kit	Qiagen	27106 (QIAprep Spin Miniprep Kit)	A kit for isolating plasmid DNA from bacterial cultures for molecular biology applications.
Mouth aspirator tube assembly	Sigma-Aldrich	A5177	for aspiration oocytes
Pellet Paint	Merck Millipore	69049-3	A product for visualizing pellet formation during centrifugation.
Phenol:Chloroform:Isoamyl Alcohol	Thermo Fisher Scientific	15593031	A solution used in H2B-RFP extraction for phase separation.
Plasmid encoding H2B-RFP	Addgene	Plasmid #20972	Plasmid used for labeling chromosomes with red fluorescence protein (RFP)
PMSG	Prospec	HOR-272	For mouse superovalation
Proteinase K	Thermo Fisher Scientific	EO0491	An enzyme used to digest proteins, commonly used in DNA/RNA extraction procedures.
Restriction enzyme NotI	New England Biolabs (NEB)	R3189S	A restriction enzyme used in DNA cloning and molecular biology applications, for H2B-RFP plasmid linearize.
RNase inhibitor (optional)	New England Biolabs (NEB)	M0314S	Enzyme used to prevent RNA degradation during RNA-related experiments.
RNase-free gloves	VWR / Fisher Scientific	Varies by size	Gloves treated to prevent RNase contamination, used in RNA manipulation protocols.
RNase-free pipette tips	Axygen / Thermo Fisher	T-300-R-S	Pipette tips designed to be free of RNase contamination for RNA-related applications.
RNase-free water	Thermo Fisher Scientific	AM9932	Water purified to remove RNase enzymes, essential for RNA work to avoid degradation.
SDS (10%)	Sigma-Aldrich	L3771	Sodium dodecyl sulfate used for denaturing proteins in electrophoresis and other experiments.
SiR-Tubulin	Spirochrome	SC002	A fluorescent dye that binds specifically to tubulin for live cell imaging of the spindle
Sodium acetate (3 M)	Sigma-Aldrich	S7899	Used as a buffer and for precipitation of DNA in molecular biology procedures.
Spectrophotometer	Thermo Fisher (NanoDrop)	ND-ONE-W	Instrument used for quantifying nucleic acids, proteins, and other substances by measuring absorbance.
Stereomicroscope	Leica Microsystems	M165C	A microscope designed for observing specimens in three dimensions at low magnifications, useful for dissections.
Syringe filter unit (0.22-µm)	Merck	SLGP033RS	Used for sterile filtration of liquids, especially in microbiology and molecular biology applications.
Syringes and Needle Assembly	Terumo	SS+01T Tuberculin Syringes	used for precise PMSG delivery.
T7 mMESSAGE mMACHINE Kit	Thermo Fisher Scientific	AM1344	A kit for in vitro transcription of RNA from a T7 promoter for H2B-RFP cRNA preparation.
Water bath / heating block	Grant Instruments	QBD4	Used for maintaining precise temperatures in experiments requiring heat.