Oocyte enucleation

5 Transfer 20-30 oocytes into (5-10 ml) microdrops of cytochalasin B-supplemented M2 or FHM under oil, in the microinjection chamberb.

Protocol 8 continued

6 Place the microinjection chamber containing oocytes onto to the stage of the inverted microscope. Select an oocyte by micromanipulation and, using Nomarski optics, locate the small area of protuberance on the surface of the oocyte that contains the metaphase plate (Mil-phase chromosomes). Orient the egg with the holding pipette, and slit the zona pellucida overlying this protuberance using a finely drawn glass microinjection needle (see Protocol 5) (as shown in Figure 3A-C). After cutting the zona pellucida continue incubating the oocyte in cytochalasin B-supplemented M2 or FHM until all oocytes have been processed in this way.c

7 Commence enucleation of an oocyte, secured so that the holding pipette is oriented opposite to the slit in the zona pellucida, by inserting the enucleation pipette through the slit and into the perivitelline space (Figure 3D).

8 Remove the Mil-phase chromosomes from the oocyte as follows. Place the enucleation pipette in close contact with the ooplasm: the area of translucent cytoplasm containing the Mii-phase chromosomes can be observed to move and deform when the enucleation pipette is pushed against it. Aspirate this area into the enucleation pipette, and observe the chromosomes entering the enucleation pipette (Figure 3E).d

9 Repeat the procedure for the whole batch of oocytes. Pool and wash successfully enucleated oocytes through drops of fresh M2 or FHM.

10 To determine successful enucleation, stain manipulated oocytes by incubation in Hoechst 33342-supplemented medium for 5 min at 37 °C in the dark, wash through drops of fresh medium to remove residual dye, and confirm the absence of the metaphase plate or contaminating chromatin by fluorescence microscopy6.

a Nomarski optics are required to visualize clearly the oolemma and metaphase chromatin. bA short period of incubation (~5min) in cytochalasin B-supplemented medium is required prior to enucleation, to disrupt elements of the cytoskeleton (specifically the microfilaments) making the oocytes more plastic and less susceptible to damage by micromanipulation.

cThe areas of protuberance may become difficult to observe following cytochalasin B-treatment of oocytes.

d If the chromatin cannot be clearly visualized within the enucleation pipette, the ooplasm should not be used as a recipient cytoplasm.

e Staining of the ooplasm of enucleated oocytes is not recommended as a routine practice, because of the toxicity of Hoechst 33342 and UV irradiation.

(Subsequently a small, round fragment of the zona pellucida remains in the enucleation pipette.) Note that after enucleation and before direct injection of karyoplasts by Piezo-pulses, oocytes must be incubated in the absence of cytochalasin B, so that the oolemma can reform.

Until proficiency is acquired in the technique of oocyte enucleation, it is advisable to monitor the progress of enucleation by staining the enucleated oocyte

Figure 3 Cutting the zona pellucida of an oocyte in preparation for enucleation. Stages (A) to (C) of the procedure are explained in Protocol 8.

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