This article details the process of isolating denuded mouse antral oocytes, which are crucial for in vitro embryogenesis. The methodology includes the preparation of female mice and the careful extraction of oocytes from antral follicles.
In a mammalian ovary, fully grown oocytes derived from the antral follicles only need a short period of maturation in culture, a crucial factor for in vitro embryogenesis. These oocytes are surrounded by a cluster of somatic cells, which must be removed through denudation to facilitate sperm injection.
To isolate denuded mouse antral oocytes, begin with an anesthetized female mouse pre-injected with pregnant mare serum gonadotropin hormone to stimulate follicular growth. Prep the mouse and incise the lower abdomen.
Exteriorize the intestine to reveal ovaries at the top of V-shaped uterine tubes. Dissect an ovary. Transfer it to a pre-equilibrated culture medium to maintain the viability of oocytes. Under a stereomicroscope, identify the antral follicles by their distinctive fluid-filled cavity.
Using a sterile needle, puncture the antral follicles of the ovary to release oocytes. Using a pipette of an optimum diameter, carefully aspirate the oocytes to protect them from mechanical damage. Avoid aspirating follicular cells and other tissue debris.
Subsequently, transfer the oocytes into multiple media drops covered with mineral oil. This step helps remove the surrounding somatic cells and generate denuded oocytes. Lastly, transfer the denuded oocytes into fresh media drops with mineral oil and preserve them for further experiments.
To harvest the ovaries, begin by injecting 3- to 6-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-microliter 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 degrees 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 oocytes 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 1 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.
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