Aneuploidy is the leading genetic cause of early miscarriage in humans. Most errors in chromosome segregation occur during meiosis in oocytes. Therefore, evaluating the spindle assembly checkpoint in oocytes is critical to understanding why oocytes are error-prone.
Here we describe three techniques to comprehensively evaluate SAC integrity in mouse oocytes by examining different critical steps at checkpoint using a combination of live imaging and immunofluorescence. Demonstrating the procedure will be Dr.Cecilia Blengini, a research associate from my laboratory. To begin prepare one milliliter of culture media containing nocodazole and reversine with DMSO as a control as described in the manuscript.
For oocyte maturation and live imaging use a pre-warmed 96-well plate in an incubator. And load the first, second and third well with 150 microliters of the control DMSO, nocodazole and nocodazole plus reversine treatment respectively. While keeping the plate in the incubator under the conditions as described in the manuscript.
To start myotic maturation, remove milrinone by sequentially transferring the oocytes through 6 drops of 100 microliters 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 while viewing the oocytes under a stereo microscope.
Image the oocytes using a bright-field microscope equipped with an incubator chamber with a controlled environment at 37 degrees Celsius, 5%carbon dioxide and 80%humidity. Capture images at the middle plane of the oocytes at intervals of 20 minutes for 24 hours. After quantifying the number of oocytes that extrude a polar body by identifying the cells that go through asymmetrical cytokines with a small cell next to the egg and within the shared zona pellucida.
View the images using imaging software. Microinject pro-phase one arrested oocytes with 100 nanograms per microliters of previously prepared securin-gfp cRNA. After microinjection, allow the oocytes to recover and translate the RNA in the carbon dioxide incubator for at least three hours.
Then load 150 microliters of culture media with or without five micromolar of nocodazole. And 150 microliters of culture media with 0.5 micromolar of reversine in three different wells of a 96-well plate. Wash the microinjected oocytes through six drops of milrinone free culture media And transfer one-third of the oocytes into each treatment.
Keep the plate in the incubator at 37 degrees Celsius with 5%carbon dioxide and 80%humidity until the oocytes resume meiosis, around three hours post milrinone washing. Record images of oocyte maturation using a fluorescence microscope equipped with an incubator chamber. Use image analysis software to quantify securin-gfp destruction and open the images of the GFP channel, starting at time 0.1.
In the preferred analysis software, use the selection or shape tool to mark each oocyte and generate a region of interest for each cell. Use the same region of interest to select a background area to be subtracted later and measure GFP pixel intensity in each region of interest at all the time points. Lastly to each GFP value, subtract the measurement of the region of interest in the background.
After splitting meiosis into three groups, transfer each group of meiosis into 100 microliter drops of milrinone free culture media. Cover the drops with mineral oil and place them in the incubator for three, five, and seven hours to reach the early prometaphase one, late prometaphase one and metaphase one stage respectively. At the assigned time points, fix the oocytes by transferring them into 500 microliters drops of 2%paraformaldehyde in PME buffer for 20 minutes at room temperature using a nine-well glass dish.
Then transfer the cells to a clean well containing a 500 microliter drop of blocking solution. To continue MAD2 detection, transfer the oocytes to a clean well containing a 500 microliter drop of permeabilization solution. After incubating for 20 minutes at room temperature, transfer the cells to a new well of blocking solution and incubate for 10 minutes.
Transfer the oocytes into a 30 microliter drop of blocking solution with anti-MAD2 antibody and anti-centromere antibody and incubate for two hours at room temperature. To wash excess primary antibodies after transferring the cells to 30 microliters drop of PME buffer supplemented with 0.5%Triton, incubate for 10 minutes in the humidified chamber. Transfer the cells to a 30 microliter drop of blocking solution containing secondary antibodies such as anti-human 633 and anti-rabbit 568.
And incubate for one hour at room temperature. To mount the cells on the microscope slide, transfer the cells to a 10 microliter drop of mounting media containing DAPI. Add small dots of petroleum jelly to each corner of the cover slip.
Carefully place them on top of the mounting media drop and press slowly to distribute. Seal the cover slip to the slide using clear nail polish. Image kinetochores using a confocal microscope equipped with a 40 or 63 X objective.
And using the anti-centromere antibody signal determine the Z range that allows imaging of the entire chromosome area. The DMSO treated control oocytes extruded a polar body around 14 hours after release from milrinone. After SAC activation, nocodazole treated oocytes were arrested in metaphase one and did not extrude a polar body.
However, in the absence of SAC activation, oocytes extruded a polar body despite having no kinetochore microtubule attachments. The rate of securin-gfp degradation during myotic maturation was evaluated. Wherein the control DMSO treated oocytes securin-gfp intensity starts to decrease at approximately 10 hours after milrinone washout.
When oocytes were matured in the presence of the SAC activating agent, nocodazole, securin-gfp levels were stable during the entire period of myotic maturation. In contrast, when preventing SAC activation with reversine treatment the securin-gfp pattern accelerated and degradation began at approximately four hours after milrinone washout consistent with SAC inhibition. Confocal imaging was employed to detect centromeres and MAD2 of oocytes matured to early prometaphase one, late prometaphase one and metaphase one.
To evaluate the strength of the SAC signal, the kinetochore localized, MAD2 pixel intensity was quantified. Significant levels of MAD2 recruitment to kinetochores occur in early prometaphase one when most kinetochores were not attached to microtubules. The levels of MAD2 reduced in later prometaphase and were nearly absent at metaphase one when all kinetochores were stably attached to microtubules.
It's important to highlight that each of these techniques have their limitations and their interpretation. And we suggest performing a combination of these methods to evaluate the SAC integrity. These three essays allows researchers to evaluate the stringency of the sac in oocytes giving insight about a potential cause of aneuploidy in human eggs.
