Methods

Ovarian oocytes are arrested at prophase of meiosis I and contain a prominent nucleus or GV. Oocytes resume meiosis (or mature) in response to hormonal stimulation (1,2). In X. laevis, the resumption of meiosis is followed 2 to 5 h later by the dissolution of the nuclear envelope (GVBD) and the outward appearance of a large

Fig. 1. The external morphology of Xenopus tropicalis oocytes. Treatment of stage VI oocytes with progesterone induces germinal vesicle breakdown and meiotic spindle formation. Where the first meiotic spindle contacts the cortex, (A) a small white spot appears, which subsequently alters appearance to (B) a dark ring as meiosis progresses beyond metaphase I. (C) Following collagenase defolliculation, stage VI oocytes (solid arrow) may be separated from immature oocytes (stage V, dashed arrow; stage IV, double arrowhead; stage III, dashed double arrowhead) using a Pasteur pipet.

Fig. 1. The external morphology of Xenopus tropicalis oocytes. Treatment of stage VI oocytes with progesterone induces germinal vesicle breakdown and meiotic spindle formation. Where the first meiotic spindle contacts the cortex, (A) a small white spot appears, which subsequently alters appearance to (B) a dark ring as meiosis progresses beyond metaphase I. (C) Following collagenase defolliculation, stage VI oocytes (solid arrow) may be separated from immature oocytes (stage V, dashed arrow; stage IV, double arrowhead; stage III, dashed double arrowhead) using a Pasteur pipet.

(100-|m) white spot. After completion of the first meiotic division and release of the first polar body, oocytes arrest at metaphase of the second meiotic division.

Similarly, maturation of X. tropicalis oocytes may be induced by continuous exposure of full-grown oocytes to MOM containing 10 |g/mL of progesterone, androstenedione (4-androstene-3,17-dione), corticosterone (4-pregnene-1ip,21-diol-3,20-dione), or Reichsten's substance (4-pregnene-17a,21-diol-3,20-dione). However, X. tropicalis oocytes mature much faster than X. laevis, with the majority of oocytes completing GVBD within 1 to 2 h of hormone treatment. In addition, rather than forming a large white spot, maturing X. tropicalis oocytes first form a small (10-|im) spot (Fig. 1A), which is subsequently replaced by a larger dark ring (Fig. 1B). Significantly, the white spot and dark ring act as external markers of progression through the first meiotic division because their appearance correlates with first meiotic spindle formation and exit from metaphase I, respectively (17).

3.1. Oocyte Isolation

Ovaries may be surgically removed from anesthetized or euthanized frogs. Unlike mammals, amphibian ovaries are capable of regenerating. Therefore, we can mini mize the number of animals used in any study by performing multiple survival surgeries on a single animal, allowing at least 1-3 mo between operations on the same frog.

To comply with the National Research Council Institute of Laboratory Animal Resources' guidelines for the breeding, care, and management of amphibians, we recommend the following procedures be employed for the isolation of ovarian oocytes from X. tropicalis. All surgical instruments should be heat sterilized prior to use. We make up ahead of time sterile packs containing one pair of surgical scissors, two pairs of fine forceps (one having tissue grips), two cotton swabs, ten 6-in. lengths of precut surgical thread, two surgeon's needles, and one needle holder.

To minimize the risk of infection during surgery, clean a space on a workbench with ethanol, perform the operations (preferably) on disposable padding, and wear disposable medical gloves during surgery. Although frogs may be immobilized by a 20- to 30-min immersion in an ice water slurry, current guidelines require that this procedure now be performed with anesthetic agents. Frogs should be immersed in MOM containing tricaine methanesulfonate (MS-222) (2 g/L) for 20 to 30 min until they become nonresponsive to handling, that is, when they show no signs of voluntary movement and fail to return to a proper orientation after turned over (if this is to be a nonsurvival study; see Note 2).

Place the frog in a dorsal recumbant position. To keep the frog's skin moist during the surgery, cover it with a tissue that has been soaked in MOM containing MS-222. Make a cut through the tissue to expose the frog's abdomen. Then, make a 1-cm incision in the epidermis and peritoneal wall, parallel to the dorsal-ventral midline and in the lower abdominal quadrant. Xenopus ovaries are multilobed, resembling gloves with oocytes lining the inner surfaces of the fingers or lobes.

Depending on experimental requirements, tease two to three ovarian lobes from the body cavity with a forceps and a sterile cotton swab. Securely ligate the ovary using 5-0 degradable sutures, excise the externalized portion, and place it in MOM. Next, gently push the still-exposed portions of the ovary back into the body cavity using a sterile cotton swab that has been moistened with MOM. Separately seal the peritoneal wall and epidermis with two sutures (5-0 degradable) each. The slimy nature of the outer surface of the epidermis of X. tropicalis can make it difficult to push a needle through, so we usually pierce the epidermis from the inner surface of the skin.

Typically, this surgery may be expected to take 10 min to complete. Postoperative care should include incubating frogs in shallow water containing 10 |g/mL gentami-cin until they have sufficiently recovered from anesthesia and housing them in a separate aquarium with daily monitoring for any signs of infection until the wound has healed.

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