1. The purpose of the PMSG "priming" is to stimulate the growth of the ovarian tissue and hence increase the quantity of stage VI oocytes. Although the main purpose of this PMSG priming is to increase the population of stage VI oocytes, it also significantly shortens the time-course of GVBD. In oocytes from such stimulated females, GVBD is apparent within 2 h (for manually defolliculated oocytes) after steroid exposure, whereas GVBD in similar stage VI oocytes from unstimulated females may occur more than 6-8 h after exposure to progesterone (13).

2. In the original protocol, oocytes were incubated in half-strength oocyte incubation medium (equivalent of OR2 medium) for 5 min before an opening was made on the animal pole, and it usually took 10 to 20 min for the nucleus to be extruded. In our protocol, most nuclei will be extruded immediately, and the time during which an opened oocyte stays in half-strength OR2 is 1 to 2 min, which is much shorter. Another advantage of our protocol is that after 30-min incubation in half-strength OR2, the oo-cytes will be uniformly round and will automatically rotate (sometimes oocytes will slightly stick to the bottom of the plastic Petri dish, but this attachment can be easily disrupted by gently tapping the Petri dish) until the animal poles are uppermost, which is convenient for the later enucleation step. In contrast, in the original protocol, oocytes need to be adjusted individually to ensure that they stick to the bottom of the glass dish with animal poles uppermost.

3. Making the opening on the animal pole is the most critical step in the whole procedure. The first factor is the diameter: a minimum opening by the tip of a sharp watchmaker's forceps is sufficient for the nucleus to be extruded considering the osmotic pressure built up inside the oocyte and the plasticity of the nucleus, and an opening slightly larger will lead to substantial increase in the loss of cytoplasm. The second factor is the position of the opening: if the opening is made right at the animal pole, the nucleus will immediately emerge and prevent cytoplasm leakage by functioning as a temporary "seal." However, if the position is slightly away from the center, substantial loss of cytoplasm will occur before the nucleus appears underneath the opening. The correct way to make the opening is as follows: let the tip of the forceps touch the center of the animal pole, press down gently, and move the forceps backward immediately after breaking the plasma membrane.

The backward movement is necessary because otherwise it is very easy to break the nucleus, which pops out rapidly.

4. The precise role of the healing solution is not clear, but one apparent function is to prevent/minimize the exudation of cytoplasm from the wound (11). Oocytes will shrink in the healing medium, and the edge of the wound will contract and fold inward. Most enucleated oocytes will have some cytoplasm attached to the opening when placed in the healing solution. Because the removal of this piece of cytoplasm in the enucleation dish may enlarge the wound and lead to the exudation of more cytoplasm, the oocytes are transferred to the healing medium with the attached cytoplasm and left undisturbed until the end of incubation in healing solution. The whole piece can be easily detached from the oocyte by forceps before the oocytes are transferred into regular OR2 medium; at this time, the wounds underneath are healed.

5. When enucleated oocytes are incubated in the healing solution, they will not stick to the bottom of the Petri dish because there is always a layer of cytoplasm "coating" the bottom to prevent oocyte attachment. However, when healed enucleated oocytes are transferred to a new Petri dish containing OR2 medium, they will slightly stick to the bottom. This is problematic because attempts to dislodge them with a transfer pipet for further distribution into final assay wells may damage healed oocytes. To prevent this stickiness, PVA (1 mg/mL) is added to OR2 before the addition of the healed oocytes.

6. Oocyte cytoplasm is very rigid in Gall's medium and will remain in the oocyte even after the opening is made. Because the oocyte will shrink in the medium and there is no osmotic pressure to facilitate the extrusion of the nucleus, the opening should be bigger than the one made in half-strength OR2 medium, and it takes some time (usually 1-2 min per oocyte) to slowly squeeze the nucleus out.

7. The nucleus medium will quickly precipitate the nuclear content (P. J. DiMario, personal communication). The nuclei will appear opaque once transferred to the nucleus medium and can be easily observed and counted.

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