Fertilization of In Vitro Matured Oocytes by Nuclear Transplantation

1. Prime female adult X. laevis with 50 U PMSG, and leave at 18 °C for about 48 h before isolating oocytes. Techniques for obtaining ovary tissue, isolating oocytes by manual defolliculation and culturing and injecting oocytes have been described elsewhere (16,22). Maintain oocytes in 1X modified barth's saline (MBS) + 1 mg/mL (w/v) BSA, and inject and manipulate as desired. Alternatively, oocytes can be maintained in oocyte culture medium (OCM). Prepare fresh for each experiment. This medium is preferable if oocytes will be cultured for an extended period (>24 h) before maturation. Transfer defolliculated oocytes to fresh media, and change these media several times to remove traces of yolk and debris.

2. Prepare sperm nuclei as previously described (Subheading 3.1.1.)

3. Add 1-5 |im progesterone to oocytes maintained in MBS + 1 mg/mL BSA to begin maturation.

4. Determine when sperm nuclei should be transplanted. A general rule to follow is that oocytes should be ready for fertilization in about 2X the amount of time taken to get from progesterone addition to germinal vesicle breakdown (GVBD; appearance of white spot in the animal hemisphere). We typically add 5 |im progesterone in the evening after defolliculating oocytes (5-7 pm), incubate oocytes at 18°C overnight and during the next day, and inject sperm 20-25 h after progesterone addition (see Note 2).

The most common mistake made is not allowing oocytes sufficient time after maturation before injecting the sperm nuclei. Oocytes must be able to respond to pricking by a needle with a vigorous cortical contraction before sperm are transplanted, or no development will occur. Even after oocytes first become responsive to pricking, they are probably not fully competent to support embryonic development immediately and should be incubated an additional 3-4 h at 18°C. Since there is probably quite a bit of variability between batches of oocytes from different frogs and between frogs from different colonies, the optimal timing should be determined by prick-activating a small number of test oocytes at several times during the incubation period to determine when they become responsive.

5. Dilute and transplant sperm nuclei as described in the transgenesis protocol. There is no need to swell the nuclei in interphase extract. We have used slightly lower dilutions of sperm than are used for transgenesis for this protocol (such that two to three sperm may be deposited into some eggs) and have done these injections in 0.4X MMR without Ficoll. Use a 40-60 |im wide needle tip to transplant the sperm as described for transgenesis. When successful, oocytes should pierce very easily for injection, and membrane texture should not seem at all rubbery. There should be a normal cortical contraction in the animal hemisphere after activation, and the injected, matured oocytes (eggs now) should look and later cleave like fertilized eggs. When testing this method, approx 25% of the in vitro matured oocytes developed into blastula-gastrula stages. Of these, the majority developed into tadpoles, and were apparently morphologically normal and raised for months.

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