Method Article

Evaluation of the Spindle Assembly Checkpoint Integrity in Mouse Oocytes

DOI:

10.3791/64459

⸱

September 13th, 2022

 ,  , 
1Department of Genetics, Rutgers, The State University of New Jersey, 2Human Genetics Institute of New Jersey, 3Department of Theriogenology, Faculty of Veterinary Medicine, Mansoura University

In This Article

Summary

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Error in chromosome segregation is a common feature in oocytes. Therefore, studying the spindle assembly checkpoint gives important clues about the mechanisms needed to produce healthy eggs. The present protocol describes three complementary assays to evaluate spindle assembly checkpoint integrity in mouse oocytes.

Abstract

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Aneuploidy is the leading genetic abnormality causing early miscarriage and pregnancy failure in humans. Most errors in chromosome segregation that give rise to aneuploidy occur during meiosis in oocytes, but why oocyte meiosis is error-prone is still not fully understood. During cell division, cells prevent errors in chromosome segregation by activating the spindle assembly checkpoint (SAC). This control mechanism relies on detecting kinetochore (KT)-microtubule (MT) attachments and sensing tension generated by spindle fibers. When KTs are unattached, the SAC is activated and prevents cell-cycle progression. The SAC is activated first by MPS1 kinase, which triggers the recruitment and formation of the mitotic checkpoint complex (MCC), composed of MAD1, MAD2, BUB3, and BUBR1. Then, the MCC diffuses into the cytoplasm and sequesters CDC20, an anaphase-promoting complex/cyclosome (APC/C) activator. Once KTs become attached to microtubules and chromosomes are aligned at the metaphase plate, the SAC is silenced, CDC20 is released, and the APC/C is activated, triggering the degradation of Cyclin B and Securin, thereby allowing anaphase onset. Compared to somatic cells, the SAC in oocytes is not as effective because cells can undergo anaphase despite having unattached KTs. Understanding why the SAC is more permissive and if this permissiveness is one of the causes of chromosome segregation errors in oocytes still needs further investigation. The present protocol describes the three techniques to comprehensively evaluate SAC integrity in mouse oocytes. These techniques include using nocodazole to depolymerize MTs to evaluate the SAC response, tracking SAC silencing by following the kinetics of Securin destruction, and evaluating the recruitment of MAD2 to KTs by immunofluorescence. Together these techniques probe mechanisms needed to produce healthy eggs by providing a complete evaluation of SAC integrity.

Introduction

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Aneuploidy, which arises from errors in chromosome segregation, is the leading cause of early miscarriages and is highly linked to mistakes in meiosis1. Meiosis is distinct from mitosis because it consists of two rounds of cell division without an intervening DNA replication step. In meiosis I, homologous chromosomes separate while sister chromatids remain together. In oocytes, this step is error-prone, leading to aneuploid egg production2.

To prevent chromosome segregations errors, most cell types activate a surveillance mechanism that pauses the cell cycle, called the spindle assembly checkpoint (SAC). This mechanism senses kinetochore (KT)-microtubule (MT) attachments and the tension is generated when chromosomes are oriented in a bipolar manner3. Unattached kinetochores trigger a SAC response which starts with the recruitment of MPS1, the master regulator of the SAC, to kinetochores3,4. MPS1 initiates the recruitment of other SAC components, acting as a platform to form the mitotic checkpoint complex (MCC). The MCC, composed of MAD1, MAD2, BUB3, and BUBR1, diffuses into the cytoplasm and inhibits APC/C activation by sequestrating its activator CDC20. Once all kinetochores are stably attached to MTs and chromosomes are aligned at the metaphase plate, the SAC is silenced, and the MCC disassembles and releases CDC20, thereby allowing APC/C activation. Active APC/C degrades Securin and Cyclin B, two key steps in triggering anaphase onset5,6. In somatic cells, the SAC is stringent because it is activated by a single unattached kinetochore and is sufficient to induce cell-cycle arrest6. However, during oocyte meiosis, the SAC is more permissive, and oocytes can enter anaphase I with one or more unattached kinetochores6,7,8,9,10. Understanding why the SAC is more permissive in oocytes is an ongoing area of focus in the field. Mechanisms that cause defects in SAC activation or SAC silencing could lead to errors in chromosome segregation or prolonged cell cycle arrest and cell death. Therefore, evaluating the mechanisms that maintain SAC integrity in oocytes is important to understanding the process of forming healthy, euploid eggs.

This protocol describes techniques to comprehensively evaluate the SAC integrity in mouse oocyte meiosis by examining different critical steps of the checkpoint. First, the evaluation of the SAC response after inducing SAC activation is described. This activation is achieved by generating unattached kinetochores using nocodazole, a drug that depolymerizes MTs11. Second, a method to monitor SAC silencing is described by tracking the dynamics of Securin degradation during oocyte maturation. Finally, an immunofluorescence-based assay is employed to measure the recruitment of MAD2, one of the MCC components, to kinetochores. Together, these assays comprehensively assess SAC integrity during oocyte meiotic maturation.

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Protocol

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All mice used in these protocols were housed and raised according to the Rutgers University Institutional Animal Use and Care Committee (Protocol 201702497) and National Institutes of Health guidelines. These regulatory bodies approved all experimental procedures involving animal studies. All mice used in the present study were 6-8-week-old CF-1 females.

1. Experimental preparation

  1. Before starting the SAC evaluations, collect mouse oocytes following the previously published report12. Divide collected oocytes into three evenly sized groups and keep them in Chatot, Ziomek, and Bavister (CZB) culture media containing 2.5 µM milrinone (see Table of Materials) to avoid meiotic resumption13.
    NOTE: CZB composition: 81.6 mM NaCL; 4.8 mM KCl; 1.2 mM KH2PO4; 1.2 mM MgSO4-7H2O; 0.27 mM Pyruvate; 1.7 mM CaCl2; 30.8 mM DL-Lactic Acid; 7 mM Taurine; 0.1 mM EDTA; 25 mM NaHCO3; Gentamicin; and 0.3% BSA.
  2. Prepare the cRNA for microinjection as described previously12,14. Amplify the mouse Securin gene from the cDNA cloned from mouse germinal vesicle oocytes, followed by subcloning into the pMDL2 vector containing Gfp sequence15,16.
    1. To prepare Securin-Gfp cRNA, linearize the plasmid with Nde I digestion. Perform in vitro transcription by using T3 RNA polymerase and purifying the cRNA16.
      ​NOTE: Store cRNA in aliquots of 2-3 µL at -80 °C until use.

2. Nocodazole treatment and live imaging

  1. Prepare 1 mL of culture media (CZB) containing 5 µM nocodazole (NOC), 1 mL of culture media with 5 µM of NOC plus 0.5 µM reversine (NOC + REV), and 1 mL of culture media with Dimethyl sulfoxide (DMSO) (1:2000) as a control (see Table of Materials).
  2. For oocyte maturation and live imaging, use a 96-well plate pre-warmed in the incubator at 37 °C, 5% CO2, and 80% humidity. In the first well, load 150 µL of the control DMSO treatment; in the second well, load 150 µL of NOC treatment; and in the third well, load 150 µL of NOC + REV treatment. Keep the plate in the incubator under the same conditions as above until needed.
    NOTE: In the 96-well plate, avoid using the first row and the first column because the shadow of the border interferes with the quality of the images.
  3. To start meiotic maturation, remove milrinone. While viewing the oocytes under a stereomicroscope using magnification between 30x-64x, wash milrinone out of the media by sequentially transferring the oocytes through six drops of 100 µL of milrinone-free culture media containing DMSO and place them into the corresponding well of the 96-well plate using a hand- or mouth-operated pipette.
    1. Count oocytes by picking them up using a hand- or mouth-operated pipette and delivering them to the next drop to avoid losing any. The oocytes are single, large (~80 µm in diameter), and round cells. The nucleus will appear like a button in the center of the cell, and the membrane and zona pellucida surround the oocyte.
      NOTE: Transfer the oocytes among drops while moving the least amount of liquid possible. This allows for optimal removal of the milrinone from the culture media. If oocytes fail to resume meiosis, it is likely that the milrinone was not effectively removed.
  4. Repeat the same process (step 2.3) for NOC and NOC + REV treatment.
  5. Image the oocytes using a brightfield microscope equipped with an incubator chamber with a controlled environment under these conditions: 37 °C, 5% CO2, and 80% humidity. Capture images at the middle plane of the oocytes at intervals of 20 min for 24 h.
  6. Quantify the number of oocytes that extrude a polar body (PB) by identifying the cells that go through asymmetrical cytokinesis. The result will be a small cell (PB) next to the egg and within the shared zona pellucida. View the images using imaging software (ImageJ, see Table of Materials).
  7. Import image sequence into the image analysis software and advance the frames until one or more cells go through asymmetrical cytokinesis. Calculate the percentage of oocytes that extrude a PB of the total of oocytes17.

3. Monitoring the pattern of Securin-gfp degradation during meiotic maturation

  1. Centrifuge 3 µL of Securin-Gfp cRNA(step 1.2) at 19,283 x g for 30 min at 4 °C18.
    NOTE: Use the supernatant to avoid loading impurities that could cause needle blockage during microinjection.
  2. Follow step-by-step oocyte microinjection, extensively described in reference12. Microinject prophase I-arrested oocytes with 100 ng/µL of Securin-gfp cRNA prepared in step 1.2.
  3. After microinjection, allow the oocytes to recover and translate the RNA in the CO2 incubator for at least 3 h. Check the expression by observing the oocytes in an automated multichannel fluorescence imaging system (see Table of Materials) to view the GFP signal.
  4. As described in step 2.2, load 150 µL of culture media with or without 5 µM of nocodazole and 150 µL of culture media with 0.5 µM of reversine in three different wells of a 96-well plate.
  5. Wash the microinjected oocytes through six drops of milrinone-free culture media and transfer 1/3 of the oocytes into each treatment.
  6. Keep the plate in the incubator at 37 °C, with 5% CO2 and 80% humidity until the oocytes resume meiosis, around 3 h post milrinone washing.
    NOTE: Use a stereomicroscope with magnification between 30x-64x to evaluate the nuclear envelope breakdown as a hallmark of meiotic resumption.
  7. Record images of oocyte maturation using a fluorescence microscope equipped with an incubator chamber as described in step 2.6. Use brightfield settings to find the oocytes in the well. Use a 488 filter to detect the Securin-gfp signal and adjust the fluorescence intensity to avoid over-exposing the cells.
    1. If the imaging system is automated, save the positions of the oocytes in each well. Because Securin-gfp is evenly distributed into the cytoplasm, capture images in the middle plane of the oocytes at intervals of 20 min for 24 h. To capture groups of oocytes in one picture, use a lower magnification objective lens such as 10x.
  8. Use image analysis software to quantify Securin-gfp destruction. Open the images of the GFP channel. Start at time point 1. In the preferred analysis software, use a selection or shape tool to mark each oocyte and generate a region of interest (ROI) for each cell.
    1. Use the same ROI to select a background area to be subtracted later. Measure GFP pixel intensity in each ROI.
  9. Using the ROIs for each oocyte generated in step 3.8, measure the GFP intensity in all the time points.
    NOTE: In some software programs, an "Analyze" tab allows the selection of the Measure function.
    1. Then, advance to the next time frame and repeat this process to measure the intensity of GFP in each time frame. Lastly, to each GFP value, subtract the measurement of the ROI in the background selected in step 3.8. This analysis will give a value for Securin-gfp intensity for each oocyte at each time point.
  10. Extract different parameters from the pattern of Securin destruction: (a) the time that Securin-gfp degradation began. This tells when SAC silencing started; (b) the time of Securin-gfp minimum signal. This tells when SAC silencing has dropped below a threshold level to allow high APC/C activation and full Securin-GFP degradation; (c) the rate of Securin-gfp destruction19. This tells how rapidly SAC activity drops below a threshold level for APC/C activation and Securin-GFP degradation.

4. Recruitment of MAD2 at kinetochores by immunofluorescence during meiotic maturation

NOTE: For oocyte collection and maturation, refer to the previously published report12.

  1. Split oocytes into three groups. Transfer each group of oocytes into 100 µL drops of milrinone-free culture media. Cover the drops with mineral oil and place them in the incubator (37 °C, 5% CO2, and 80% humidity) for 3 h, 5 h, and 7 h to reach early prometaphase I, late prometaphase I, and metaphase I stage, respectively.
    NOTE: Place each time point in a different dish to avoid taking out the same dish from the incubator several times, which affects the regular timing of meiotic maturation.
  2. At the assigned time points, fix the oocytes by transferring them into 500 µL drops of 2% PFA in 1x PHEM buffer for 20 min at room temperature, using a 9-well glass dish as previously described20. Then, transfer the cells to a clean well containing a 500 µL drop of blocking solution (PBS + 0.3% BSA + 0.01% Tween-20 + 0.02% NaN3).
    NOTE: PHEM composition: 60 mM PIPES; 25 mM HEPES; 10 mM EGTA; and 2 mM MgCl2.
    NOTE: One can stop at this point and store the oocytes in blocking solution in the 9-well plate at 4 °C until the convenient time to complete immunofluorescence.
  3. To continue MAD2 detection, transfer the oocytes to a clean well containing a 500 µL drop of permeabilization solution (PBS + 0.3% BSA + 0.1% TritonX-100 + 0.02% NaN3). Incubate for 20 min at room temperature, and then transfer the cells to a new well of blocking solution and incubate for 10 min.
  4. For the rest of the immunofluorescence steps, use a 96-well dish lid with indentations as previously described20. Use a humidified dark chamber to avoid light exposure and evaporation. Transfer the oocytes into a 30 µL drop of blocking solution with anti-MAD2 antibody (1:1000, rabbit) and anti-centromeric antibody (ACA) (1:30, human) (see Table of Materials) and incubate for 2 h at room temperature.
    NOTE: The 96-well dish lid allows room for the processing of several proteins and groups at the same time.
  5. To wash excess primary antibodies, transfer the cells to a 30 µL drop of 1x PHEM buffer supplemented with 0.5% Triton and incubate for 10 min in the humidified chamber. Repeat this step two more times.
  6. Perform the fourth wash, transferring the cells to a 30 µL drop of 1x PHEM buffer without 0.5% 1x Triton, and incubate for 10 min.
  7. Transfer the cells to a 30 µL drop of blocking solution containing secondary antibodies such as anti-human-633 (1:200) and anti-rabbit-568 (1:200) (see Table of Materials) and incubate for 1 h at room temperature.
    NOTE: Choose the combination of fluorophores based on your microscope lasers or filters.
  8. To wash excess secondary antibodies, repeat steps 4.5-4.6.
  9. To mount the cells on the microscope slide, transfer the cells to a 10 µL drop of mounting media containing DAPI (0.1 mg/mL) (see Table of Materials). Add small dots of petroleum jelly to each corner of the coverslip, carefully place them on top of the mounting media drop, and press slowly to distribute. Use clear nail polish to seal the coverslip to the slide. For a more detailed description of the mounting process, see reference20.
  10. Image kinetochores using a confocal microscope (see Table of Materials) equipped with a 40x or 63x objective. Using the ACA signal, determine the z-range that allows imaging of the entire chromosome area.
    NOTE: An optical zoom of 4.0 and z-step size of 0.5 µm can be used on some imaging systems. These parameters may vary from system to system, and optimization will be necessary.
  11. Analyze MAD2 intensity at kinetochores using image analysis software. Make a z-stack maximum projection and then split the channels.
  12. First, create a mask using the ACA channel by selecting the ACA channel to establish a threshold that identifies all the kinetochore signals. Go to the edit tab and create a selection. Then, create an ROI with this selection.
  13. Select the MAD2 channel and bring in the selection created in step 4.12. Select a threshold method that better adapts to the MAD2 signal. Measure the intensity.
    NOTE: Select the threshold method in the control treatment and keep it constant when analyzing different treatments.
  14. To make relative pixel intensity calculations, divide the intensity of each cell by the average intensity of the WT oocytes in the experiment.

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Results

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Evaluation of SAC responsiveness by nocodazole treatment
The purpose of this experiment is to evaluate SAC activation and strength. By using nocodazole to depolymerize spindle microtubules, all kinetochores will be unattached, which will cause a SAC-mediated cell-cycle arrest. In the present imaging system, DMSO-treated control oocytes extruded a polar body around 14 h after release from milrinone (Figure 1, top panels). Consistent with SAC activation, nocodazole-treated...

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Discussion

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The spindle assembly checkpoint is a critical control mechanism during cell division designed to prevent chromosome segregation errors. It allows the cell to have enough time to correct improper KT-MT attachments. Meiosis in oocytes is an error-prone process, where most of the chromosome mis-segregation occurs during meiosis I, leading to the generation of aneuploid eggs that are the main cause of early miscarriages and infertility in humans1,24. Several hypothes...

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Disclosures

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The authors have no conflicts to disclose.

Acknowledgements

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Funding for this project was provided by the National Institutes of Health (R35GM136340 to KS).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Bovine serum albumin (BSA)SigmaA4503
DAPILife TechnologiesD1306
Dimethyl-sulfoxide (DMSO)SigmaD5879
Donkey-anti-rabbit-Alexa-568Life TechnologiesA10042
EVOS FL Auto Imaging SystemLife TechnologiesFluorescence microscope
EVOS Onstage IncubatorLife TechnologiesIncubator chamber
Glass Bottom 96- well plates N 1.5 uncoatedMatTek CorporationP96G-1.5-5-F
goat-anti-human-Alexa-633Life TechnologiesA21091
HEPESSigmaH3537
Human anti-ACAAntibodies Incorporated15-234Dilution 1/30
ImageJNIH
KClSigmaP5405
KH2PO4SigmaP5655
Leica SP8 equipped with a 63×, 1.40 NA oil immersion objectiveLeica
MgSO4·7H20SigmaM7774
MilrinoneSigmaM4659
Na2HPO4SigmaS2429
NaClSigmaS5886
NaN3SigmaS2002
NocodazoleSigmaM1404
Paraformaldhyde (PFA)SigmaP6148
PIPESSigmaP6757
Rabbit anti- MAD2Biolegend924601Dilution 1/1000; previously Covance #PRB-452C
ReversineCayman Chemical10004412
Triton-XSigma274348
Tween-20SigmaX100
VectashieldVector laboratoriesH-1000

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Tags

Spindle Assembly CheckpointMouse OocytesChromosome SegregationAneuploidy MechanismsSAC IntegrityLive ImagingImmunofluorescence AnalysisSecurin DegradationMAD2 RecruitmentConfocal Microscopy

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