Mouse Antral Oocyte Isolation: A Method to Isolate Antral Oocytes from Freshly Harvested Mouse Ovaries

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Animals

1. Maintain adult 3- to 6-week-old female mice in a room with controlled temperature and humidity (with phases of 12L:12D and fed ad libitum).
2. Inject mice intraperitoneally with 2.5 IU of Pregnant mare serum gonadotropin (PMSG) to stimulate follicular growth by mimicking the effects of follicle stimulating hormone (FSH). Isolate the ovaries for antral oocytes collection 48 h later. Euthanize the female mouse previously injected with PMSG, as per standard guidelines.
3. Quickly remove the ovaries from the body cavity and place them into the Petri dish containing M2 medium for subsequent oocytes isolation (see section 3).
   1. Make an incision on the skin covering the abdominal wall followed by an incision in the peritoneum by using sterile dissection scissors. Open the incision to expose the abdomen, move the guts on one side using sterile tweezers and localize the uterine tubes, forming a V-shape starting from the bladder.
   2. Observe the ovaries located below the kidneys. Hold each tube with sterile tweezer and make a small incision at the bottom of the ovaries to release them. Transfer both ovaries at the bottom of a Petri dish containing pre-equilibrated medium.

2. Hormone Preparation

1. Dilute PMSG (available in different stock concentrations) with sterile isotonic saline solution to obtain a working concentration of 2.5 I.U. hormone per 0.1 mL for an appropriate injection volume.      
   NOTE: Hormone solutions must be aliquoted in 0.5 mL tubes and stored at -20 °C until the day of use. Do not keep diluted aliquots longer than three months at -20 °C. Once thawed, if serial injections are performed, store the aliquots of hormone solutions at +4 °C and use them within 4 days.

3. Antral Oocytes Isolation

For proper oocytes isolation:

1. Equilibrate M2 medium (M2) at 5% CO₂ and 37 °C for at least 1 h before starting the oocytes isolation procedure. Prepare two Petri dishes (35 mm x 10 mm), one with 1 mL of M2 for ovary puncturing and the second one with small drops (20 µL) of M2 covered with mineral oil (~1.5 mL) for oocytes transfer after isolation from the ovary (see steps 3.4–3.6). Keep both Petri dishes in the incubator at 5% CO₂ and 37 °C until ready to be used.
2. Use a stereomicroscope during oocytes isolation procedures.
3. Prepare thin mouth glass pipettes to collect the oocytes by stretching glass Pasteur pipettes kept horizontally (holding the ends of the pipette with both hands), over a vertical flame coming from the Bunsen burner (Figure 1).
   1. Once the glass begins to melt, take the pipet out of the flame, and perform a quick pulling movement to obtain the desired pipette. Firmly cut the excess glass close to the narrow point of the pipette with fingernails to adjust the length and the diameter of the internal capillary. Ensure that the thin capillary has an internal diameter of ~100 µm, slightly greater than the oocyte's diameter (~80 µm).
   2. To mouth pipette, connect one end of the aspirator tube to the pulled pipette by using an appropriate tip and place the other end in the mouth, securing it with the teeth.
4. Collect the ovaries using sterile tweezers and deposit them at the bottom of a cell culture Petri dish (35 mm x 10 mm). Cover them with 1 mL of M2 previously equilibrated at 37 °C and 5% CO₂ (Figure 2).
5. Gently puncture the antral follicles of the ovaries with a sterile insulin syringe needle to release antral oocytes in M2 (Figure 3).
6. Gently mouth pipet the oocytes through a narrow Pasteur pipette to remove follicle cells and any other possible tissue debris (Figure 4A) and then settle them at the bottom of small drops (20 µL) of M2 covered with mineral oil suitable for embryo culture (previously prepared; Figure 4B).      
   NOTE: It is important to remove all the cumulus cells attached to the zona pellucida of the oocytes by continuous pipetting through several and different drops of M2. Perform this step as quick as possible to avoid alterations in the pH of the medium.