Culture of Oocytes Attached to Part of the Follicle Wall
Follicles were dissected from the ovaries as explained above. Selected follicles were cut according to De Loos et al.. Briefly, the COC was located using a stereomicro-scope. Directly opposite the point of attachment of the COC to the follicle wall, the follicle was pierced with a needle, and the follicular fluid was gently flushed out, leaving a collapsed follicle. Three areas of the collapsed follicle were cut away using fine scissors to leave a sandwich of follicular wall with its COC positioned centrally. To obtain a preparation of follicular wall (2-3 mm2) with a COC attached, the upper part of this sandwich was pulled back and the follicular wall around the COC was trimmed (Fig. 2). buy prednisone
An alternative method employed for the isolation of oocytes attached to part of the follicle wall compartment was a vacuum aspiration system. An 18-gauge needle (1.2-mm internal diameter) connected to a vacuum line was inserted into individual follicles; the needle was used to gently scratch the follicular wall during aspiration. Using this technique, the majority of the oocytes isolated were attached to part of the follicular wall compartments. Five to ten isolated COC follicular wall complexes were cultured in 500 ^l Waymouth maturation medium supplemented with 5% FCS in an atmosphere of 5% CO2 in air at 39°C for 24 h.