Fetal calf serum (FCS; 5%) was added prior to gassing. A gaseous atmosphere of 45% O2:50% N2:5% CO2 was used according to the method of Moor et al.. Selected follicles were transferred into 6-well culture dishes with or without netwell inserts of 500-^m pore diameter and containing the culture medium described above. The culture medium was gassed using an anaerobic jar and warmed to 39°C in an incubator prior to culture. After the follicles were transferred into the culture dishes, they were gassed again by the same system for 5 min. Culture was continued for up to 2 days without changing the medium; however, during longer periods of culture the medium was changed every 2 days. buy antibiotics online
Culture of Oocytes in Hemisections of Follicular Wall
Follicles were dissected from ovaries as described above, and selected follicles (3- to 8-mm diameter) were completely trimmed from the remaining stromal tissues. The position of the oocyte was located within each follicle using a ste-reomicroscope. The follicle wall was then pierced at the side opposite to the oocyte. After the follicular fluid was gently flushed out, the collapsed follicle was cut in half using fine scissors; the oocyte remained in one half of the follicle. Oocytes that detached during the process and others isolated by aspiration were placed inside the follicle hemisections. Two oocytes were placed into each hemisec-tion. Three follicle hemisections were transferred to 500-^l drops of Waymouth medium supplemented with 5% FCS under mineral oil (Sigma) in a 35-mm tissue culture dish and were maintained in an atmosphere of 5% CO2 in air at 39°C for 24 h. In preliminary experiments, various volumes of culture medium (200 ^l to 1 ml) were used to find the most effective volume.