Our research aims to understand how the cell cycle is regulated in mammalian oocytes and in pre-implantation embryos. A key aspect of our research involves examination of repair capacity and DNA damage response in different maturation states in oocytes and also in pre-implantation embryos. We are particularly interested in understanding how the checkpoints respond, and which repair mechanisms established after potential DNA damage.
Our lab was the first to mechanistically examine the DNA damage response in mammalian oocytes. We have shown the absence of the G2 prophase DNA damage checkpoint in mammalian oocytes, and we observed the importance of the spindle assembly checkpoint in establishing metaphase arrest after damage. By using this protocol, it is possible to detect DNA double strand breaks with accuracy and to determine whether oocytes have DNA damage and whether DNA damage is being repaired.
This protocol involves the use of immunofluorescence, which allows us to identify the exact sites of damage to DNA. To begin, prepare drops of M2 IBMX medium in a plastic tissue culture dish, and place the dish on a hot block at 37 degrees Celsius for at least 30 minutes before oocyte isolation. Dissect the ovaries of the euthanized mice and place them in a five milliliter round bottom tube with M2 IBMX.
Transfer the ovaries to a plastic lid containing 1.5 milliliters of M2 IBMX. Remove any periovarian adipose tissue or fallopian tube segments and release the cumulus oocyte complexes by mechanical perforation of the ovaries with a 27 gauge needle. Transfer the cumulus oocyte complexes to a culture dish with drops of M2 IBMX, and remove the cumulus cells by repeated pipetting bsing a narrow bore glass pasture pipette.
Select the surrounded nucleolus germinal vesicle or GV stage oocytes and transfer them in a drop of M2 IBMX medium on hot block at 37 degrees Celsius, protected from light. After inducing double strand breaks using etoposide, place the GV stage oocytes in drops of the genotoxic agent for one hour on the hot block in the dark. Add drops of M16 culture medium supplemented with 400 micromolar IBMX to the oocytes to keep them arrested for a prolonged period.
Place the ides in an incubator at 37 degrees Celsius and 5%carbon dioxide. Place the control and etoposide-treated germinal vesicles or GV oocytes in different plastic tissue culture dishes with PFA-Tx-100 buffer for 40 minutes at room temperature. Rinse the oocytes in three distinct 50 microliter washing buffers at room temperature, and place them in 25 microliters of blocking buffer on a hot block for one hour.
Next, prepare the primary antibody that recognizes gamma H2AX. Dilute the primary antibody with the blocking buffer at a one to 200 ratio, and immerse the oocytes in 15 microliter drops at four degrees Celsius overnight. Wash the oocytes in three different 50 microliter washing buffers.
Prepare the Alexa Fluor 488 conjugated goat anti-rabbit secondary antibody. After diluting it with the blocking buffer, immerse the oocytes in 15 microliter drops of the antibody for one hour on a 37 degrees Celsius hot block protected from light. Take a far red fluorescent DNA dye DRAQ7 and transfer the oocytesto it for 10 minutes at room temperature in the dark before washing them with three different washing buffers.
Add small drops of washing buffer to the oocytes in a 35 millimeter glass bottom Petri dish for confocal microscopy. Switch on the laser controller and the lasers in the confocal system. After turning on the microscope controller and the lamps, open the confocal software, and choose the 40X oil lens.
Place the dish with the oocytes into the specimen holder, and try to focus on the oocytes by moving the stage on X, Y, and Z axes using the joystick. Set the laser power, the gain, and the pinhole size independently for each experiment to minimize any saturation. For each oocyte, set the area of interest specifically in the nucleus at the DNA area.
Define the borders of the DNA area, and adjust the Z step size to three micrometers. Then start the scanning. Save the images for each cell in the selected folder.
Open the ImageJ software and click on Image, followed by Color and Split Channels to split all the channels. Click on Lookup Table and choose the preferred colors for each channel. Click on Image, Color, and Merge Channels to merge the channels for gamma H2AX and DNA.
In the surrounded nucleus oocytes with high levels of DNA damage, click on Image, Stacks, Z Project, and with the Freehand Selections command, select the entire DNA area. Click on Analyze, followed by Measure to measure the gamma H2AX fluorescence, and copy the measurements into an Excel file. The gamma H2AX fluorescence in the surrounded nucleolus GV stage oocytes zero hours after the etoposide treatment is shown in this figure.
The gamma H2AX increases immediately after the exposure at all the etoposide concentrations and the increase is concentration-dependent. The gamma H2AX fluorescence in the surrounded nucleus GV stage oocytes 20 hours after the etoposide treatment is shown here. Gamma H2AX reduces 20 hours after the exposure at all the etoposide concentrations.
After protracted prophase arrest, the GV stage oocytes showed the capacity to reduce the gamma H2AX foci number and intensity, implying the presence of active repair processes in the GV stage arrested oocytes.
