Extraction and Preparation of Oocytes from Xenopus laevis

Detailed instructions on the care of Xenopus laevis frogs are available elsewhere (10). To maintain consistent oocyte quality and minimize seasonal variations, we have found it advisable to maintain the same animals over 2-3 yr in a constant temperature, 12-h day/light cycle. It is also advisable to keep new frogs in separate tanks to avoid synchronization of seasonal cycles. Oocytes should not be removed from each frog more often than once every 3 mo to ensure animal health and avoid depletion of the oocytes.

1. Submerge the frog in a chilled solution of 0.15% MS222. The anesthetic is absorbed through the skin. Place a wet paper towel on top of a bed of ice. When the frog is nonresponsive to touch, place it face up on the wet paper towel and ice.

2. Perform an abdominal laparotomy with a 2-cm horizontal incision to the left or right of the midline and remove three to five lobes of oocytes. Place the oocytes in a 35-mm Petri dish containing OR-2 solution (see Note 6).

3. Using suture, stitch the endodermal layer first. Use two or three stitches, connecting the first two stitches and doubling the suture on the second stitch. Stitch the skin layer. Return the frog to a cold, shallow aquarium tank to recover as the water slowly returns to room temperature.

4. Separate the oocytes into clumps (5-10 oocytes/clump is best). Use forceps and remove as much sac as possible. Place the clumped oocytes in a 15-mL conical tube containing 15 mg of collagenase dissolved in 5 mL of OR-2 solution. After the oocytes are added bring the volume to 10 mL with OR-2 solution and start timing while gently rocking the tube on a shaker. Collagenasing times will vary between

25 and 45 min depending on the collagenase and the oocytes. New collagenase should be tested on a few oocytes prior to use, as efficiency and toxicity vary between lots.

5. When the oocyte clumps look like they might be starting to separate, rinse them with OR-2 solution—first in the tube 4 to 5x, then in a 10-cm Petri dish another 4 to 5x using a shortened, polished pasteur pipet. Rinse once with L15 medium and store in sterile culture dishes containing L15 in a 17° C incubator.

6. Oocytes usually remain healthy for up to a week if the medium is changed daily. Removing the follicular membrane prolongs the health of the oocytes, as does removal of early- and late-stage oocytes from the preparation.

7. Using fine forceps remove the outer layer of follicle cells surrounding each oocyte. In most cases this layer is an opaque off-white color and is clearly visible. Often fine, branching capillaries can be seen in the follicular layer. The follicular layer is degraded during the collagenase treatment and may therefore be partially or totally absent from some oocytes. To preserve the health of the oocyte, it should be noted that while it is customary to remove the follicular layer manually after a short collagenase treatment, longer collagenase treatments can be used to completely defolliculate the oocytes. However, these prolonged treatments can also compromise the long-term health of the oocytes.

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