Oocyte Maturation Ovulation and Collection

Unfertilized eggs from Xenopus females are the source of interphase extracts used in the study of DNA replication in vitro. The injection of Xenopus females with hormone and the collection of unfertilized eggs is described in Subheadings 3.2.1 to 3.2.3. This includes: (1) the priming of Xenopus females by injection of PMSG to induce ovulation, (2) the induction of egg laying by injection of hCG, and (3) the collection and dejellying of the eggs for subsequent extract preparation.

3.2.1. Priming Xenopus Females With PMSG to Induce Ovulation

Females are primed with 75 U of PMSG at least 2 but not more than 8 d before the secondary injection with hCG, described in Subheading 3.2.2. The number of frogs to be injected (6-15) depends on the extract to be prepared (see Subheading 3.3.).

1. Prepare a 2500-U/mL stock of PMSG by slowly injecting 2 mL sterile water into a vial of 5000 U of PMSG using a 3-mL syringe and a 21-gage needle. Also, insert a 27-gage needle to relieve pressure. Mix by gentle inversion. This preparation can be stored at 4°C for up to 2 wk.

2. Transfer an appropriate amount of PMSG from the vial to a sterile Falcon 2059 tube using a 1-mL syringe. Dilute 10-fold with sterile water to a final concentration of 250 U/mL.

3. Fill the necessary number of 3-mL syringes with the diluted PMSG and inject each female subcutaneously along the leg with 0.3 mL using a 27-gage needle. You can use the same needle for about five frogs.

3.2.2. Induction of Egg Laying With hCG

1. Prepare a 2000-U/mL stock of hCG by slowly injecting 4.8 mL sterile water into a vial of 10000 U of hCG using a 5-mL syringe and a 21-gage needle. Also, insert a 27-gage needle to relieve pressure. Mix by gentle inversion. This preparation can be stored at 4°C for up to 2 wk.

2. Fill the necessary number of 3-mL syringes with the hCG and inject each female as above with 0.3 mL using a 27-gage needle. Injection is performed 18 to 22 h before the eggs are needed.

3. Each injected frog is placed into a separate bucket containing 100 mMNaCl (it is critical that the water not contain chlorine).

3.2.3. Harvesting and Dejellying the Eggs

Eggs are harvested 18 to 22 h after injection with HCG. Eggs from each frog are not combined until after they are dejellied and have been inspected under a dissection microscope.

1. Remove the frogs from the buckets and slowly decant all but about 100 mL of water from each bucket. Decant the remainder of the water and the eggs from each bucket into 250-mL beakers.

2. Decant as much of the water as possible from each beaker. Add 3 vol of 2.2% cysteine and mix every 30 s using a glass rod until the eggs are dejellied. Up to eight batches of eggs are dejellied at a time. Dejellying should be complete within 6 to 8 min. Dejellied eggs form a much more highly compact layer at the bottom of the beaker than eggs with jelly coats. The jelly coats can be seen as clear halos floating above the eggs before they dissolve completely in the cysteine.

3. Decant the cysteine and quickly wash the eggs three times with about 50 mL of 0.5X MMR at room temperature. Decant as much of the MMR as possible between washes.

4. Quickly wash the eggs twice with ELB, decanting as much of the ELB as possible between washes. Before decanting the last wash, remove any debris with a Pasteur pipet. Inspect each batch of eggs under a dissection microscope. Discard batches of eggs if the majority of eggs do not contain a visible germinal vesicle, if the majority of eggs have lysed, or if the pigment appears extensively mottled in appearance (see Note 3).

5. Combine the eggs and wash them once more with ELB. Eggs are now ready to be crushed for the production of LSE, described in Subheading 3.3.

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