Department of Biology, University of Pennsylvania
Stein, P., Schindler, K. Mouse Oocyte Microinjection, Maturation and Ploidy Assessment. J. Vis. Exp. (53), e2851, doi:10.3791/2851 (2011).
Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2.
In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy.
Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error.
1. Mouse oocyte collection
2. Oocyte microinjection
3. Oocyte maturation
4. Ploidy analysis
5. Representative Results:
Figure 2 is a Z-projection from a euploid egg. At metaphase of MII, euploid mouse eggs contain 20 pairs of chromosomes and therefore have 40 centromeres. Occasionally chromosomes fail to spread despite monastrol treatment. This situation makes it difficult to reliably count centromeres and we therefore do not include these eggs in our analyses. Occasionally, it may be challenging to determine whether a CREST-immunoreactive "spot" is 1 or 2 centromeres. Using programs such as Image J is useful because one can analyze each Z-section while carefully noting the orientation of the chromosomes, the number of sections in which a "spot" is detected and the pixel intensity the "spots" have. Depending where the meiotic spindle is positioned relative to the polar body, the regions of DNA can overlap and these samples should not be included in the analysis.
Unmanipulated, in vivo ovulated eggs from reproductively young mice have low rates of aneuploidy (˜1-2%). However, for reasons not understood, microinjection and in vitro maturation procedures can increase this rate upwards of 10%. Therefore, it is critical that control injected oocytes are included in any microinjection study.
Figure 1. Microinjection dish set up. A chamber slide with 5 μl drops of MEM/PVP + M just above 0.5μl drops of injection solution. Cover with mineral oil. In this example there are 3 drops for 3 different injection solutions and the slide is sitting on the stage of a microscope. To the left is the microinjection needle and to the right is the holding pipette. Note that there is a reflection of the needle holders.
Figure 2. Ploidy results. A Z-projection of a euploid metaphase II egg. DNA is colored in green and kinetochores are colored in red. The arrows point to 2 distinct chromatid arms indicating that kinetochores (#17 and #18) overlap. Euploid mouse eggs contain 20 kinetochore pairs (40 total "spots"). An aneuploid egg would contain any variation on this number. If this procedure were conducted on metaphase MI oocytes, there would be 40 kinetochore pairs (80 total "spots").
Table 1. Recipe for collection and microinjection medium (MEM/PVP + M). All materials are embryo-culture grade and from Sigma-Aldrich. Filter sterilize through 0.22μm PVDF filters (we use Milipore Stericups) and store at 4°C.
Table 2. Recipe for CZB medium. All materials are embryo-culture grade and from Sigma-Aldrich. Filter sterilize through 0.22μm PVDF filters (we use Milipore Stericups) and store at 4°C.
Microinjection of oocytes is a powerful method to study mechanisms that regulate meiotic maturation 10,11, 12, 13. This method provides an economical way to test hypotheses prior to making a large investment in developing transgenic and targeted mouse models. Oocyte collection and microinjection techniques require more time to master than typical cell biology procedures. Specific obstacles with collection often include controlling the mouth pipette, pulling the proper size glass pipette for collection and stripping of the somatic cells and increasing collection speed to minimize the time in which oocytes are outside of the incubator. We recommend practicing many times prior to doing experiments. Transferring oocytes between microdrops while maintaining the same number of cells is a great way to become comfortable with this method.
Cell death commonly occurs while learning microinjection. This could occur for a number of reasons, including injection of too large of a volume of material (i.e. the injection needle opening is too big), hitting the nucleus with the injection needle, piercing the opposite side of the oocyte or that the material injected is toxic to the oocyte. Practicing with injecting buffer into oocytes until your survival rate is at least 50% is key to mastering this technique. If oocytes fail to mature it is likely that the milrinone was not diluted enough. We recommend rinsing the oocytes through many large drops of milrinone-free CZB before maturation.
The ploidy analysis following microinjection is one of many assays to assess meiotic maturation. Other routine analyses we use in the laboratory include monitoring the kinetics by which the oocytes progress through meiosis, immunofluorescence to analyze spindle formation and chromosome alignment and egg activation or in vitro fertilization to assess the developmental consequences of the oocyte manipulation 14,15, 16, 17.
No conflicts of interest declared.
This work was conducted in Richard M. Schultz's laboratory. The authors would also like to acknowledge Michael Lampson for conceptualizing the centromere-counting assay and access to his confocal microscope. Teresa Chiang and Francesca Duncan assisted in optimizing the centromere-counting assay. Paula Stein is supported by HD022681 (to RMS) and Karen Schindler is supported by HD055822.
|Table of specific reagents:|
|Milrinone||Sigma-Aldrich||M4659||Resuspend in DMSO at 2.5mM|
|Mineral oil||Sigma-Aldrich||M5310||Only use embryo-tested, sterile-filtered|
|Anti-human Alexa 594||Invitrogen||A-11014|
|Albumin from bovine serum||Sigma-Aldrich||A3294|
|Monastrol||Sigma-Aldrich||M8515||Resuspend in DMSO at 100mM|
|Table of specific equipment:|
|Watchglass||Electron Microscopy Sciences||70543-30|
|Syringe||BD Biosciences||309623||1ml, 27G1/2|
|60 mm Petri Dish||Falcon BD||351007|
|Glass Pasteur pipets||Fisher Scientific||13-678-200|
|Side warmer||Fisher Scientific||Any standard model|
|Dissection microscope||Any standard model|
|Chamber Slide||Nalge Nunc international||177372|
|Capillary Tubing||Drummond Scientific||1-000-0500||microcaps|
|Pipette Puller||Flaming-Brown micropipette puller||Model P-97|
|Inverted microscope||Nikon Instruments||Any standard model|
|Micromanipulators||Eppendorf||Any standard model|
|Picoinjector||Harvard Apparatus||Model PLI-100||Any standard model|
|CO2 tanks||For incubator|
|N2 tank||For table and injector|
|Anti-vibration table||Technical Manufacturing Corp.||Any standard model|
|Incubator||Any standard model|
|Confocal microscope||Leica Microsystems||Any standard model|
|Dissection tools||Fine Science Tools||Any standard model|
|Humidified chamber||We use tupperware|
|Lid of 96 well plate||Nalge Nunc international||263339|
|Microscope slides||Fisher Scientific||12-544-3|
|Coverslips||Thomas Scientific||6663-F10||Thickness will vary for particular microscopes|
|Center well organ culture dish||Fisher Scientific||353037||60 X 15mm|