The overall goal of this series of assays is to investigate human gene function during Meiosis I using mouse oocytes. These methods can help answer key questions in the human reproductive field, such as whether there's a genetic predisposition to maternal aneuploidy. The main advantage of this technique is that it uses mouse oocytes as a model for mammalian meiosis, which is useful given the inaccessibility of human eggs for gene function studies.
We first had the idea for this method when we sequenced the exomes of women who had undergone in vitro fertilization treatments and who's embryonic aneuploidy rates could not be explained by their maternal age. Visual demonstration of this method is critical, as the image analysis steps are difficult to learn and defining strict analysis parameters is key to rigor and reproducibility. Using a validated cloning strategy, insert the full-length coding sequence of the gene of interest into the multiple cloning region of the in vitro transcription vector.
Mutagenize the DNA if generating single nucleotide polymorphisms or insertions and deletions, as described in the text protocol. Next, transform the mutagenized DNA into a bacterial host. Use a kit to isolate and purify the plasmid, following the manufacturer's instructions.
Then, linearize the final products using single restriction enzyme digestion of the purified DNA. Purify the digested product using a validated DNA cleanup protocol or a kit. And elute in 30 microliters of RNase/DNase-free water.
In vitro, transcribe DNA using T7 polymerase following the manufacturer's protocol. Finally, purify and elute RNA in 30 microliters of RNase/DNase-free water using a silica matrix spin-column based nucleic acid purification kit following the manufacturer's protocol. Divide oocytes into equal groups for microinjection.
Microinject one group with experimental mRNA and the other group with the wild-type control NPBs or GFP mRNA. Using a hand or mouth-operated pipetting apparatus where the glass pipette opening is slightly larger than the diameter of an oocyte, transfer oocytes into a 100 microliter drop of milrinone-free culture medium in a Petri dish covered in embryo-quality mineral oil. Then, incubate the oocytes for seven hours at 37 degrees Celsius in 5%CO2 to allow sufficient maturation to metaphase of Meiosis I.After incubation, use the same pipette apparatus to transfer the oocytes into a center-well organ culture dish containing 700 microliters of pre-chilled MEM collection medium in the center well and 500 microliters of water in the outside ring.
Place the dish on ice for seven minutes. Fix the oocytes by transferring them into a well of a clear glass spot plate containing 400 microliters of 2%paraformaldehyde in 1x PBS. Incubate for 20 minutes at room temperature.
Following immunostaining, as described in the text protocol, image the oocytes using a 40 to 63x objective on a confocal microscope. Capture small steps in the Z-plane optimized for the working objective, and image the entire region of the metaphase spindle. To identify the kinetochore microtubule attachment status, first open the image by dragging the file into the ImageJ toolbar.
In the opened Z-series, navigate to the image drop down menu, and under the Color sub tab, click Merge Channels to make all channels visible. It is critical that the analyst be familiar with what type of attachments to kinetochores are possible in mouse oocytes. The quality of the image will greatly influence your ability to properly analyze these data.
Using the toggle at the bottom of the image, move through each Z slice and determine kinetochore microtubule attachment. Using the same pipette apparatus, transfer the oocytes into a 100 microliter drop of milrinone-free culture medium under oil. Incubate the oocytes for seven hours to allow sufficient maturation to metaphase of Meiosis I as before.
Then, transfer the oocytes to a center-well organ culture dish containing 700 microliters of culture medium containing monastrol in the center well and 400 microliters of water in the surrounding ring. Incubate at 37 degrees Celsius for two hours. Rinse out the monastrol by transferring oocytes through three drops of 100 microliters of CZB culture medium.
Then transfer the oocytes to a new center-well organ culture dish containing 700 microliters of culture medium plus proteasome inhibitor in the center ring. Add 400 microliters of water to the outside ring and incubate for three hours. Next, fix the oocytes by transferring them into a well of a clear glass spot plate containing 400 microliters of 2%paraformaldehyde in PBS for 20 minutes at room temperature.
Image the oocytes using a 40 to 63x objective on a confocal microscope, capturing less than one micron steps in the Z-plane and making sure to image the entire region of the metaphase spindle. To identify chromosome alignment status, open the image files using imaging software. Then drag the image into the ImageJ control panel.
In the open Z-series, use the point tool to mark points at the end of each spindle pole in their respective Z slice by placing the tool over the spindle pole in the image and clicking on the image. Add these points to the ROI Manager by simultaneously pressing the command button and the T button on the keyboard. Determine the specific coordinates for each of the set positions by highlighting the specific point in the ROI Manager and clicking the Measure button.
This will provide a Results table containing X and Y coordinates for each point, which are the X1, Y1 and X1, Y2 points, respectively. Next, determine the true Z coordinate. This is done by multiplying the slice number of the specific Z slice in the Z stack in which the spindle pole was identified by the stack thickness.
These will be the Z1 and Z2 points, respectively. Determine the spindle length using the Pythagorean theorem equation with the previously defined X, Y, and Z coordinates. Next, determine the spindle midzone.
This is calculated as 1/2 the length of the spindle. Click the line icon in the ImageJ toolbar and draw a line that is from one spindle pole to the spindle midzone. The length will be displayed on the ImageJ toolbar.
Defining strict chromosome alignment parameters allows for a robust quantitative analysis, important in data reproducibility. Determine the chromosome alignment status by assessing chromosome distance from the spindle midzone using the line measurement tool. Click the line icon in the ImageJ toolbar, and draw a line from the spindle midzone to the chromosome.
The length will be displayed on the ImageJ toolbar. Chromosomes greater than four microns from the spindle midzone are considered unaligned. After the oocytes have undergone immunostaining and imaging, correct microtubule kinetochore attachments can be assessed.
In a normal attachment, each sister kinetochore pair of the bivalent is attached to spindle microtubules from opposite poles. Up to three different types of abnormal attachments may be observed. One example occurs when there are no microtubules contacting the kinetochore.
Alternatively, microtubules can bind incorrectly, producing syntelic or merotelic attachments. Syntelic attachments form with both pairs of sister kinetochores attached to microtubules emanating from a single pole. Merotelic attachments occur when the sister kinetochores in a pair are attached to opposite poles.
After oocytes recover from the induced misalignment, the ability for chromosomes to realign is assessed by imaging. In this example, one chromosome bivalent is considered misaligned since it is located at more than four microns from the spindle midzone. When choosing which assay is appropriate for assessing gene variant function, it's important to remember that the human gene variants under study are being overexpressed and that the endogenous mouse homologs may need to be depleted at the same time.
Following this procedure, other methods like CRISPR-Cas9 to create humanized mouse's strains can be performed to answer additional questions, such as understanding how the gene variant influences egg and embryo quality and if it impacts reproductive health of the individual. After its development, this technique paved the way for researchers in the field of reproductive genetics to explore other causative factors to maternal aneuploidy in humans. Gene identification could provide powerful diagnostics and a basis for studies at correcting aneuploidies.
Thank you for watching. Good luck with your experiments.
