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 resolution and cannot capture transient or rapid events that occur between sampling intervals. In contrast, live-cell imaging of fluorescently labelled chromosomes and microtubules offers a powerful approach to monitor these dynamic processes in real time^2,3,7. This allows direct observation of the timing, duration, and sequence of meiotic events within individual oocytes, and enabling identification of transient events that might otherwise be missed in fixed samples. However, maintaining oocyte viability and physiological integrity during microinjection and imaging requires meticulous handling and optimized conditions⁸.

This protocol describes a comprehensive workflow for microinjecting mouse germinal vesicle (GV)-stage oocytes with H2B-RFP cRNA, enabling fluorescent labelling of chromatin, followed by time-lapse confocal imaging of chromosomes and spindle behavior throughout meiosis⁴. The method integrates key steps including hormonal priming, oocyte isolation, microinjection under controlled temperature conditions, and live imaging using confocal microscopy with environmental control^4,9. The overall goal of this paper is to present a robust and reproducible workflow for real-time visualization of chromosomal and spindle dynamics in live mouse oocytes, providing a platform for investigating the molecular and cellular mechanisms underlying meiotic regulation and aneuploidy risk.

While this protocol provides high temporal and spatial resolution, it does rely on exogenous reporter expression, which may not fully replicate endogenous protein dynamics and could potentially interfere with native meiotic processes if overexpressed. Researchers should consider these limitations and validate findings using complementary approaches where possible.

This protocol is designed to support studies investigating meiotic mechanisms, chromosome segregation errors, and the impact of genetic or environmental perturbations on oocyte quality. It provides a robust platform for both basic research and translational applications in reproductive biology.

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 culture medium droplets with mineral oil (Figure 1A-E).

1. Hormonal stimulation
   1. Inject 3-4-week-old female mice intraperitoneally with 5 international units (IU) of pregnant mare's serum gonadotropin (PMSG; see Table of Materials) 46-48 h prior to oocyte collection.
      ​NOTE: B6CBAF1 mice (F1 offspring of C57BL/6J × CBA/CaCrl) are preferred due to superior oocyte yield and favorable maturation characteristics.
2. Preparation of culture media
   1. Filter M2 and M16 media (see Table of Materials) using a 0.2 µm syringe filter. Supplement with IBMX to a final concentration of 100 µM by diluting from a 100 mM stock.
   2. Prepare 5-6 droplets of M2-IBMX (~30 µL each) and 3 droplets of M16-IBMX in 35 mm plastic tissue culture dishes. Overlay all droplets with mineral oil. Place M2-IBMX dishes on a 37 °C hot block (Figure 1A) and M16-IBMX dishes in a 37 °C, 5% CO₂ incubator for at least 30 min before oocyte isolation (Figure 1C).
   3. Prepare an additional dish containing 800-1,000 µL of M2-IBMX (no oil) for ovary dissection. Place on a 37 °C heating plate mounted over a stereomicroscope (Figure 1B).
3. Oocyte isolation
   1. Sacrifice mice by cervical dislocation. Wipe the lower abdomen with 70% ethanol, then make a small horizontal incision in the skin and peritoneum to enter the peritoneal cavity. Extend the incision by pulling apart the edges of the incision with fingers.
   2. Using forceps, sweep the bowel out of the way to expose the ovaries in the upper abdomen, located at the superior pole of the kidneys. Using fine forceps and micro-scissors, carefully excise the ovaries while avoiding damage to the tissue. Immediately transfer ovaries into a 1.5 mL tube containing 100-200 µL of pre-warmed M2-IBMX medium.
   3. Transfer ovaries to 1 mL of pre-warmed M2-IBMX medium and move to a heated dissection microscope stage (Figure 1B). Carefully remove adipose tissue and oviductal remnants.
      NOTE: Handle ovaries gently to prevent damage to follicles or oocytes. Maintain ovaries in a warm medium during transfer from the animal room to the laboratory.
   4. Release cumulus-oocyte complexes (COCs) by puncturing follicles using a 27 G needle (Figure 1A-B). Collect COCs and transfer into 25-30 µL droplets of M2-IBMX (Figure 1D).
   5. Dissociate cumulus cells by repeated pipetting with a narrow-bore glass Pasteur pipette (Figure 1E).
      NOTE: While mouth pipetting is commonly used for oocyte handling, mechanical pipettors or micromanipulators provide alternatives, especially in settings where mouth pipetting is restricted or not feasible. These alternatives have been successfully employed in similar protocols^10,11.
   6. Identify and select fully grown GV-stage oocytes based on a larger diameter and centrally located GV using a stereomicroscope at 20x-40x magnification. Transfer to fresh 30 µL of M2-IBMX droplets on a 37 °C hot block, protected from light.
      NOTE: Maintain oocytes at 37 °C throughout all procedures to preserve physiological integrity. Pre-warm all media and use temperature-controlled stages for all microscope-based handling and microinjection.
   7. Allow oocytes to recover for 20 min on a 37 °C heated stage before microinjection.

2. H2B-RFP cRNA preparation

NOTE: Perform all steps wearing gloves in a clean, RNase-free environment. Use only RNase-free water and reagents, and preferably work in the presence of RNase inhibitors.

1. Sequence-verify the H2B-RFP plasmid (see Table of Materials) by Sanger sequencing using primers flanking the insert. Amplify the plasmid by transforming competent E. coli (e.g., DH5α), selecting a single colony, and growing it in LB broth with appropriate antibiotics. Purify the plasmid using a standard midi or maxi prep kit. For detailed procedures, see Green and Sambrook¹².
2. Purify plasmid DNA using a commercial miniprep kit according to the manufacturer's instructions. Quantify DNA concentration using a spectrophotometer.
3. Linearize 2-4 µg of plasmid DNA downstream of the insert using a suitable restriction enzyme that cuts once in the vector backbone (NotI was used for H2B-RFP). Set up the digestion reaction using the appropriate buffer and enzyme concentration as described in Table 1 and incubate at 37 °C for 1-2 h. Confirm complete linearization by agarose gel electrophoresis. Multiple bands indicate incomplete digestion. If using a commercial kit, follow the manufacturer's instructions.
   NOTE: Choose a restriction enzyme that cuts precisely downstream of the insert and under optimal digestion conditions. Verify linearization by running a small aliquot on an agarose gel.
4. Remove RNase contamination by incubating the digestion reaction with 1 µL of Proteinase K (10 µg/mL), 5.3 µL of 10% SDS, and 50 µL of nuclease-free water at 50 °C for 1 h.
   NOTE: This step is essential for eliminating RNase and protein contaminants. Use only RNase-free reagents and maintain clean working conditions.
5. Extract the digested DNA with phenol:chloroform:isoamyl alcohol (25:24:1), followed by a chloroform extraction.
   NOTE: To minimize toxicity and streamline sample handling, commercial column or bead-based purification methods (e.g., silica membrane or magnetic bead systems) may also be used in place of phenol/chloroform extraction.
6. Precipitate DNA by adding 3 M sodium acetate and 100% ethanol. Incubate at −80 °C for at least 2 h.
7. Pellet the DNA by centrifugation at 12,000-14,000 x g for 10 min at 4 °C. Wash the pellet with 70% ethanol, centrifuge again at the same speed for 5 min at 4 °C, air-dry briefly (2-5 min), and resuspend in 6 µL of RNase-free water.
   NOTE: After ethanol precipitation and centrifugation, a visible translucent or white pellet should form at the bottom of the tube. If no pellet is seen, repeat centrifugation at a higher speed or reassess the DNA concentration.
8. Perform in vitro transcription using the T7 mMESSAGE mMACHINE kit (see Table 2), following the manufacturer's protocol.
9. Incubate the transcription reaction at 37 °C for at least 2 h. Extend incubation if transcript length exceeds 5 kb.
10. Remove template DNA by DNase treatment using 1 U of DNase I per 10 µL reaction volume, incubated at 37 °C for 15 min.
11. Purify the synthesized cRNA using lithium chloride precipitation¹³. After precipitation and centrifugation, a visible translucent or white pellet should form at the bottom of the tube. Wash the RNA pellet with 70% ethanol.
12. Resuspend the final cRNA pellet in 20 µL of RNase-free water. Measure the RNA concentration and confirm the presence and length of the poly(A) tail by performing RT-PCR followed by sequencing of the PCR product. Aliquot the cRNA into 1 µL volumes and store at −80 °C.

3. Preparation and microinjection of oocytes

NOTE: Throughout the procedure, maintain oocytes at 37 °C using prewarmed media and temperature-controlled microscope stages to preserve oocyte quality.

1. Preparation
   1. Thaw H2B-RFP cRNA aliquots on ice, briefly centrifuge, and dilute to the desired concentration (e.g., 250 ng/µL).
      ​NOTE: The optimal cRNA concentration may vary depending on the construct, expression system, and experimental goals. We recommend titrating the cRNA concentration (e.g., within the range of 50-300 ng/µL) to minimize overexpression artifacts while ensuring sufficient signal. Users should also monitor meiotic progression (e.g., GVBD, spindle formation, and polar body extrusion) as a functional readout of oocyte health and viability following construct expression. It has been reported that injection concentrations of cRNA typically range from 0.2 to 1 µg/µL, with optimal results often achieved at 0.5 µg/µL^{2,3,4,5,7,8,9}.
   2. Prepare microinjection needles from borosilicate glass capillaries (OD = 1.5 mm, ID = 0.86 mm, length = 100 mm) using a pipette puller (Figure 2A).
      NOTE: We use a vertical pipette puller (see Table of Materials; Figure 2A). Optimize pulling parameters empirically. The needle tip should be fine enough to minimize oocyte damage but wide enough to avoid clogging and allow smooth cRNA delivery.
   3. Load 0.5-1.5 µL of cRNA solution into the needle tip using RNase-free microloader tips. Remove any air bubbles prior to injection.
2. Microinjection dish setup
   1. Prepare a microinjection dish with 100 µL of M2-IBMX medium and overlay completely with mineral oil to prevent evaporation and osmolarity shifts.
3. Oocyte microinjection
   1. Mount the holding and injection pipettes onto the micromanipulator system (Figure 2B,C).
   2. Place the microinjection dish on the microscope stage. Using a mouth pipette, transfer oocytes into the M2-IBMX droplet.
   3. Bring the holding and injection pipettes into the same horizontal plane as the oocytes.
   4. Capture one oocyte using gentle suction with the holding pipette (Figure 2B and C).
   5. Insert the injection needle through the zona pellucida and oolemma, targeting the cytoplasm and avoiding the nucleus.
   6. Deliver H2B-RFP cRNA using a brief pulse of negative capacitance through an intracellular electrometer (Figure 2B-E). Keep the injection volume approximately 5% of the oocyte volume. A successful injection is typically visible as a small but distinct dispersal of cytoplasm. Adjust injection pressure and injection timing to avoid membrane damage.
   7. Withdraw the needle swiftly and smoothly at the same angle to minimize membrane tearing.
   8. Release the oocyte from the holding pipette and reposition it to a separate area of the dish to distinguish injected from uninjected oocytes (Figure 2F). For this setup, organize the workflow by keeping uninjected oocytes at the top of the dish and moving injected ones to the bottom.
   9. Repeat steps 3.3.4-3.3.8 for all remaining oocytes. Survival rates post-injection vary with technique and mRNA properties. Expect 60%-80% viability.
4. Post-injection handling
   1. Transfer injected oocytes to a fresh droplet of M16-IBMX medium in a clean culture dish.
   2. Incubate at 37 °C for 1-4 h to allow H2B-RFP expression. Adjust incubation duration based on cRNA concentration, transcript size, and expression efficiency.

4. Timelapse imaging of H2B-RFP-labeled chromosomes during meiosis

1.  Preparation of equipment and media
   1. Prewarm the confocal microscope stage incubator and environmental chamber to 37 °C for ≥ 30 min before use (Figure 3A). Turn on the gas controller and initiate gas flow to maintain the chamber at 5% CO₂ and ambient O₂. Ensure the chamber is humidified to support oocyte health during extended live-cell imaging.
   2. Prepare culture drops: Place 5-6 drops of 50 µL of M16 medium supplemented with SiR-Tubulin (100 nM final concentration) in a 35 mm Petri dish. Overlay with mineral oil. In a separate glass-bottom imaging dish, place three 5 µL drops of M16 + SiR-Tubulin and overlay with mineral oil. Prewarm both dishes at 37 °C for ≥ 30 min before use.
2. Oocyte transfer and wash
   1. Washing oocytes: Transfer injected oocytes sequentially through the 50 µL M16 + SiR-Tubulin drops to wash away residual IBMX (5 washes recommended). Perform washes gently to avoid oocyte damage.
      ​NOTE: Residual IBMX can inhibit meiosis resumption, so thorough washing is critical.
   2. Final transfer: After washes, transfer 15-20 oocytes into the 5 µL drop of M16 + SiR-Tubulin in the prewarmed glass-bottom imaging dish.
3. Microscope setup
   1. Mounting imaging dish: Place the glass-bottom dish on the confocal microscope stage (Figure 3A). Allow oocytes to settle for ~10 min.
   2. Stage positioning: Using brightfield imaging, center oocytes in the field of view. Save positions using microscope software.
      ​NOTE: In this study, we used a Leica TCS SP8 confocal microscope with an HC PL APO 20x/1.0 NA water immersion objective lens. However, other confocal systems and objective lenses with similar specifications can be used depending on availability.
   3. Water pump setup for water immersion objectives: If using a water immersion objective lens, operate the water pump system to supply water intermittently at intervals of 5-10 min. This periodic flow maintains the water column between the lens and the glass-bottom imaging dish, preventing drying and ensuring stable imaging conditions during long-term experiments. For oil immersion lenses, this water flow setup is not required.
4. Imaging settings
   1. Switch to laser-scanning mode. Set laser intensities as follows: 561 nm at 0.5% (~100 µW at the objective) for H2B-RFP (chromosomes); 633 nm at 3% (~300 µW at the objective) for SiR-Tubulin (spindle microtubules; Figure 3B,E).
      NOTE: Follow proper laser safety guidelines. Minimize laser exposure to protect sample viability.
5. Z-stack acquisition
   1. Acquire Z-stacks spanning 30-50 µm with a 2-4 µm step size (Figure 3B,C).
   2. Capture images at 5-10 min intervals using a scan speed of 600 Hz (Figure 3B,D). Total imaging durations can span 12-16 h, depending on the meiotic stage being monitored, and are limited by phototoxicity and oocyte tolerance to extended in vitro culture.
6. Sequential image acquisition
   1. On the microscope systems, perform sequential scanning with two HyD detectors: H2B-RFP: Excitation at 561 nm; emission collected at 580-630 nm; SiR-Tubulin Excitation at 633 nm; emission collected at 650-700 nm (Figure 3E).
7. Initiating time-lapse imaging
   1. Before starting long-term time-lapse acquisition, verify signal quality by taking a test image. Fluorescent signals should be evenly distributed: H2B-RFP should label chromatin distinctly, and SiR-tubulin should highlight microtubule structures without excessive background. Confirm autofocus stability and minimal photobleaching in short preliminary imaging runs.
   2. Start time-lapse imaging by clicking Start.
      NOTE: GVBD, marking M-phase entry, typically occurs 60-90 min post-IBMX washout (Figure 4A). In this setup, C57BL/6 oocytes extrude the first polar body around 8-9 h post-GVBD (Figure 4A). Timing may vary with mouse strain and culture conditions.

5. Post-imaging processing

1. Open the imaging file in an image analysis software by selecting File > Open and choosing the .lif File from the imaging session.
2. Generate Z projections for the H2B RFP and SiR-Tubulin channels by selecting Processing > Generate Projection. Set the projection type to Maximum Intensity Projection. Select both the RFP and SiR Tubulin channels, then click Run.
   NOTE Do not perform a Z projection for the DIC channel, as it can result in a blurred image. Instead, identify and select the single DIC plane that is most sharply focused. Use this focused DIC image in combination with the maximum intensity projections of the H2B-RFP and SiR-Tubulin channels to achieve optimal visualization.
3. Extract a single brightfield focal plane. Return to Process Tools and select Edit > Crop. Choose the Brightfield channel, use the Z slider to find the optimal focal plane, and click Apply.
4. Merge the projection and brightfield images by opening Process Tools, selecting Merge > Add Channels. Add the RFP projection, SiR-Tubulin projection, and the single brightfield image. Right-click the merged set and then click Apply.
5. Export the final merged image by selecting File > Export > Image. Choose TIFF format for high-quality, uncompressed output.
   NOTE: Exporting in TIFF format is recommended for downstream analysis and figure preparation.

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) through to segregation at anaphase I (Figure 4A, 09:00 yellow arrow) and ending with chromosome alignment at metaphase II (Figure 4A, 13:30). The high-resolution images allow researchers to visualize fine structures such as spindle microtubule rearrangement and subtle chromosome positioning, facilitating studies of meiotic progression, spindle positioning, and polar body extrusion.

Moreover, this protocol supports functional studies for interrogating the effects of small molecule inhibitors or gene editing (such as RNAi or CRISPR) on chromosome and spindle behavior under live-cell conditions that are designed to minimize disruption to the oocyte microenvironment. As shown in Figure 4B, treatment of the oocyte with the CDK1 small molecule inhibitor, flavopiridol^3,14, markedly accelerated the initiation of anaphase (Figure 4B, 0:20 yellow arrow) and subsequent formation of the first polar body (Figure 4B, 0:40-2:40). This acceleration contrasts with the timing observed in normal meiosis I, as shown in Figure 4A (5:00-9:00, yellow arrow). Typically, anaphase occurs approximately 3-5 h after spindle formation, depending on the experimental conditions and maturation environment¹⁵, whereas in the flavopiridol-treated group (Figure 4B), anaphase onset occurred in just ~20 min (Figure 4B, 0:20 yellow arrow). Significantly, following polar body extrusion, the oocyte failed to progress into metaphase II (Figure 4A, 13:30) and instead underwent chromosome decondensation reflecting entry into an interphase-like state (Figure 4B, 2:40). This observation demonstrates that acute CDK1 inhibition triggers premature exit from metaphase I and failure to enter metaphase II thereby underlining the critical importance of CDK1 control for mediating the transition between first and second meiotic M-phases.

These results highlight the capability of this technique to generate high-resolution temporal and spatial data that directly correlate meiotic events with experimental manipulations. For example, the flavopiridol experiment (Figure 4B) illustrates how the method captures altered chromosome behaviours following CDK1 inhibition. In treated oocytes, chromosomes separate prematurely and fail to achieve stable alignment at metaphase II, instead undergoing decondensation and dispersal into an interphase-like configuration. This contrasts with the orderly chromosome alignment, segregation, and metaphase II arrest seen in control oocytes (Figure 4A). Such differences in chromosome movement and structural organization are readily detected by the optimized live-cell imaging setup described here. By enabling continuous observation of individual oocytes, the protocol makes it possible to pinpoint the exact timing of key transitions, quantify the duration of specific phases, and detect subtle structural changes that fixed-sample methods cannot capture. Such outcomes can be analyzed by time-lapse quantification, spindle morphology assessment, or chromosomal alignment scoring, providing a powerful framework for both mechanistic studies and comparative analyses under different experimental conditions.

Figure 1: Preparation and handling of mouse oocytes. (A) Heat block for handling mouse oocytes under in vitro conditions, maintained at 37 °C. The dark cover protects isolated oocytes from direct light exposure to preserve viability. Shown as well is a culture dish containing M2 medium overlaid with mineral oil. (B) Temperature-controlled platform for isolating mouse oocytes from the ovary. A stereomicroscope equipped with a heat plate set at 37 °C is used to maintain thermal stability. The temperature controller maintains the platform at the desired temperature for extended periods. (C) Incubator for oocyte culture, maintained at 37 °C with 5% CO₂ and adequate humidity. (D) Cumulus-oocyte complexes (COCs) isolated from the ovary. (E) Denuded oocytes with prominent GVs obtained after removal of cumulus cells using a mouth pipette. Scale bar = 100 µm. Please click here to view a larger version of this figure.

Figure 2. Set up and components of the mouse oocyte microinjection system. (A) Injection needle puller. (B) Mouse oocyte microinjection station, including the inverted microscope (B1), microinjection stage (B2), micromanipulator (B3), and microinjector (B4).(C) Detailed view of the microinjection stage, showing the holding pipette (C1), microinjection needle (C2), and microinjection dish containing M2-IBMX medium covered with oil (C3). (D) Components of the microinjector, including the intracellular electrometer (D1) and PV820 Pneumatic PicoPump (D2). (E) Micromanipulator and temperature control setup, comprising the hydraulic X-Y-Z micromanipulator (E1), electric X-Y-Z micromanipulator (E2), temperature controller (E3), and microscope power supply control (E4). (F) Close-up view of the microinjection process. The holding pipette (F1) secures a mouse oocyte, while the injection needle (F2) is used for cytoplasmic injection. The upper portion of the image shows oocytes that have already been injected. Please click here to view a larger version of this figure.

Figure 3: Confocal live-cell imaging setup for mouse oocytes. (A) Temperature-controlled incubation chamber for live-cell imaging. (A1) Plastic enclosure surrounding most of the microscope. Note that oocytes are enclosed within a separate smaller chamber on the microscope stage; (A2) Control panel of the Leica confocal microscope; (A3) Joystick for controlling stage movement in two dimensions; (A4) Water pump providing continuous water flow to the objective to maintain optimal imaging conditions during long-time-course experiments. (B) The confocal microscope control station and monitor. (B1) Temperature controller maintaining 37 °C; (B2) CO₂ controller maintaining 5% CO₂. The temperature and CO₂ controllers work together to regulate the incubation chamber, creating an optimal environment for oocyte culture. (B3) Monitor displaying experimental settings and live-cell images; (B4) quick-access panel for controlling key microscope parameters. (C) Confocal imaging setup parameters. (C1) Experiment mode set to xyzt; (C2) Pixel resolution set to 512 × 512 with a scan speed of 600-1000; (C3) Scanning parameters set to 1. (D) Imaging acquisition parameters. (D1) Z-stack range set to 50 µm; (D2) Time-lapse interval set to 5 min over a total imaging period of 12 h. (E) Laser control panel. (E1) Laser wavelengths set to 561 nm for H2B-RFP and 633 nm for SiR-tubulin; (E2) Emission detection set using two HyD detectors to simultaneously capture H2B-RFP and SiR-tubulin signals. Please click here to view a larger version of this figure.

Figure 4: Live-cell imaging of chromosome and spindle dynamics in mouse oocytes. (A) Images depict a live mouse oocyte expressing H2B-RFP (following cRNA microinjection) and stained with SiR-tubulin. The top panel shows merged images from the RFP, SiR-tubulin, and DIC channels, while the bottom panel shows the H2B-RFP channel alone, highlighting chromosome dynamics during meiotic maturation. The yellow arrow indicates anaphase. Time is displayed in hours and minutes (hh: mm), with 00:00 marking the start of live imaging. Scale bar = 10 µm. (B) Mouse oocyte treated with the CDK1 inhibitor Flavopiridol (5 µM) at metaphase I. The top panel shows merged images; the lower panel shows the H2B-RFP channel, displaying chromosome dynamics. Time is displayed in hours and minutes (h:mm), with 0:00 marking the time of Flavopiridol treatment. (A) serves as the untreated control for comparison with (B). Scale bar=10 µm. Please click here to view a larger version of this figure.

Component	Volume
Plasmid DNA	x µL
Restriction enzyme	2 µL
10x Reaction buffer	10 µL
Nuclease-free water	y µL (to 100 µL total)

Table 1: Table of component combinations for linearization of the H2B-RFP plasmid.

Component	Volume
Linearized DNA	6 µL
2x NTP/CAP mix	10 µL
10x Reaction buffer	2 µL
Enzyme mix	2 µL
Nuclease-free water	y µL (to 20 µL total)

Table 2: Table of component combinations for H2B-RFP cRNA preparation.

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 physiological integrity and developmental potential^3,4,5,16,17. Therefore, maintaining optimal conditions-such as continuous incubation at 37 °C and the use of pre-warmed media and temperature-controlled microscope stages-is essential for preserving oocyte viability during extended manipulations and imaging sessions^3,4.

A key strength of this protocol is its integration of fluorescent markers (H2B-RFP for chromosomes and SiR-tubulin for spindles) with time-lapse confocal microscopy, allowing simultaneous visualization of chromatin and microtubule structures in living oocytes. This dual-labeling approach facilitates detailed tracking of key meiotic events, including germinal vesicle breakdown, spindle assembly, chromosome alignment, and polar body extrusion. Highresolution imaging studies in mouse oocytes by Schuh and Ellenberg⁷ and Kitajima et al.¹⁸ have demonstrated similar strategies for minimizing phototoxicity while capturing spindle selforganization and kinetochore dynamics. Our imaging parameters -- such as low laser power and minimal exposure times -- were optimized to balance signal detection with phototoxicity reduction, ensuring that oocytes could proceed through meiosis without artificial arrest or damage.

While the data strongly support that this protocol maintains oocyte viability and meiotic integrity, we also note that it is important for individual users to validate oocyte survival and developmental competence according to their specific imaging systems, fluorescent constructs, and experimental goals. Variations in equipment configuration, imaging duration, or cRNA constructs may influence outcomes and should be assessed empirically.

The procedure also highlights the technical challenges inherent in microinjection of large mammalian oocytes. Successful delivery of cRNA into the cytoplasm without compromising the plasma membrane or meiotic competence requires precise micromanipulation and injection pressure control¹⁷. In our hands, post-injection survival rates ranged from 60%-80%, consistent with prior reports using similar techniques^19,20. Variability in survival and expression efficiency underscores the need for operator experience and ongoing optimization, particularly when adapting the protocol to different mouse strains or molecular constructs^20,21.

While the protocol is robust, several aspects can be modified to accommodate specific experimental goals or to resolve technical challenges. These modifications, however, may affect experimental outcomes and should be implemented with caution. For instance, the concentration of cRNA can be adjusted within a typical range of 0.1-1 µg/µL to balance expression efficiency with minimal cytotoxicity. In our hands, and with the given imaging system, 0.1-0.25 µg/µL typically yields strong fluorescence with minimal toxicity, whereas higher concentrations (up to 1 µg/µL⁹) may enhance signal intensity but increase the risk of overexpression artifacts or meiotic arrest. Labeling strategies may also be adapted. Alternatives such as transgenic reporter mice or fluorophore-conjugated antibodies can provide more physiological expression levels and reduce concerns of ectopic protein load, though they may offer less flexibility in experimental design and temporal resolution. Additionally, the choice of culture medium can be tailored to optimize oocyte health. Depending on the mouse strain and laboratory environment, supplementing media with antioxidants or culturing under lowoxygen conditions -- as demonstrated by Yamagata et al. in preimplantation embryos -- may improve oocyte viability during prolonged imaging sessions²².

Troubleshooting strategies for common problems
Low oocyte survival post-injection: This may result from excessive injection pressure, suboptimal needle shape/size, or cold media. Ensuring that needles are properly sculpted and polished, using minimal injection pressure, and pre-warming all media and tools are recommended practices.

Weak or inconsistent fluorescent signal: This can occur due to low-quality or degraded cRNA. Preparation of capped, polyadenylated cRNA, followed by gel verification, and storage at −80 °C in aliquots, helps maintain consistency.

Injection failure or cytoplasmic leakage: A visible cytoplasmic dispersal should be observed upon successful injection. Absence of such dispersal may suggest incorrect needle position (external to the oolemma) or a blocked needle. These issues may be resolved by adjusting micromanipulator positioning, flushing the needle, or checking the air pressure system.

Pellet not visible during DNA precipitation: This may indicate low DNA yield or inefficient precipitation. Repeat plasmid preparation using a high-quality E. coli culture system. To enhance DNA recovery, include glycogen as a co-precipitant and extend centrifugation time at high speed.

Potential loss of inhibitor efficacy due to oil overlay: This is an important consideration when using drug inhibitors under an oil overlay. As reported by Rémillard-Labrosse et al.²³, drugs may leach into the oil, reducing their effective concentration within the culture media. We recommend users test the actual effective concentration of inhibitors under oil before conducting their formal experiments. Additionally, planning shorter-term experiments can help minimize potential loss of drug efficacy. After experiments performed under oil, it is also advisable to check the inhibitor's effect again to confirm that it remains active.

Overall, this protocol provides a valuable platform for studying the dynamic processes governing chromosome behavior and spindle assembly during mammalian oocyte meiosis. By enabling high-resolution, live-cell imaging in a physiologically supportive environment, it offers insights into the mechanisms of meiotic error and reproductive failure and lays the groundwork for future investigations into factors affecting oocyte quality and developmental competence.

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.