The overall goal of this procedure is to detail all of the steps necessary for characterizing mouse antral oocytes based on their chromatin organization, according to their ability to fully sustain a prospective embryonic development. This method can help answer key questions in the field of developmental biology, such as how to distinguish and isolate two different kinds of antral ooyctes, SN, surrounded nucleolus, and NSN, non-surrounded nucleolus, residing in the antral compartment of the mammalian ovary. To harvest the ovaries, begin by injecting three to six week old female mice IP with 2.5 international units of pregnant mare serum gonadotropin.
Two days later, about an hour before the harvest, fill one sterile Petri dish with M2 medium and add several 20 micriliter drops of M2 medium to a second Petri dish, covering each drop with 1.5 milliliters of mineral oil. Then transfer all of the Petri dishes to a 37 degree Celsius, 5%carbon dioxide incubator to allow the medium to equilibrate. Next, use sterile dissection scissors to make an incision in the skin covering the abdominal wall, followed by an incision in the peritoneum of the first hormone-injected mouse.
Use sterile tweezers to move the intestines aside to localize the uterine tubes, which form a V-shape starting from the bladder. After locating the ovaries below the kidneys, grasp one uterine tube and release the corresponding ovary with a small incision in the bottom of the tissue. Repeat the process to harvest the other ovary, and then place both ovaries in the Petri dish filled with pre-equilibrated M2 medium.
Before isolating the antral oocyte, make glass mouth pipettes by grasping both ends of a glass Pasteur pipette and holding the pipette horizontally over a Bunsen burner flame. When the glass begins to melt, remove the pipette from the flame and quickly pull the instrument apart. Using a fingernail, firmly snip the excess glass close to the narrow point of the pipette to shorten the tip, and to achieve an internal capillary diameter of about 100 microns.
It's important to have the right pipette diameter. If it's too narrow, the ooyctes will be stuck, fragmented, and die. Then connect the unmodified end of the pipette to an aspirator tube.
Next, transfer the ovaries into the Petri dish containing one milliliter of pre-equilibrated M2 medium, and use a sterile insulin syringe needle to gently puncture the antral follicles of the ovaries. As the oocytes are released, gently collect them by mouth pipette, taking care not to aspirate any follicle cells or other tissue debris. Then quickly pipette each oocyte through multiple pre-equilibrated M2 medium drops to remove all of the cumulus cells attached to the zona pellucida of the oocytes.
Once the oocytes have been denuded, carefully settle the oocytes at the bottom of individual drops of fresh equilibrated M2 medium. To stain the oocytes, use a mouth pipette to transfer the cells into 20 microliter drops of Hoechst 33342, diluted in M2 medium. Then transfer the oocytes into a new Petri dish containing five microliter drops of M2 medium covered with mineral oil, taking care to settle the oocytes individually at the bottom of each drop.
When all of the oocytes have been transferred, place the dish under the fluorescent microscope and excite the cells just long enough to visualize the presence or absence of a ring around the nucleolus. Take care not to overexpose the oocytes, as the chromatin can be damaged and the oocytes can die if they are exposed too long. After the SN and NSN oocytes have been identified, sort the cells into the appropriate corresponding Petri dish in the relevant reagent for the desired downstream analysis, according to their chromatin organization.
Hoechst 33342 staining for chromatin organization is probably the most critical step of the procedure. Five to 10 seconds of excitation should be long enough to sort the oocytes based on the presence or absence of chromatin surrounding the nucleolus of each oocyte. Once mastered, this technique can be completed in less than three hours if performed properly.
While attempting this procedure, it is important to work at room temperature and to remember to treat both ovaries and oocytes gently. Following this procedure, the oocytes can then be used for in vitro maturation and in vitro fertilization. After its development, this technique paved the way for researchers in the field of developmental biology to answer to a still-open question, how to recognize and to distinguish between two kinds of antral oocytes residing in the mammalian ovaries.
After watching this video, you will have a good understanding on how to make and use your own mouth pipette, how to isolate and sort antral oocytes by means of Hoechst 33342 staining. Don't forget that working with animal and cells requires special precautions, like wearing your personal protective equipment and using sterilized instruments.
