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Role and Gonadotrophic Regulation of X-Linked: MATERIALS AND METHODS(3)

METHODS(3)Follicular Isolation and Culture

All the animal work was carried out in compliance with the Guides of the Canadian Council on Animal Care. Ovaries from 22- to 24-day-old, immature female Sprague-Dawley rats were cut into small pieces and incubated at 37°C for 30 min in a-minimal essential medium (a-MEM) containing collagenase (type 1A, 4 mg/ml) and DNase 1 (0.3 mg/ml). The incubation was terminated with the transfer of the ovarian tissues into Leibowitz L-15 medium with 0.1% (w/v) BSA, and follicles (diameter, 160-210 |xm) were dissected out using 28.5-gauge needles. To minimize the experimental variation caused by damage incurred during the isolation procedures, only round follicles with an intact thecal layer on the day of isolation (Day 0) and on Day 1 of culture were selected for experimentation. generic amaryl

Confocal microscopic examination (Bio-Rad M500; Bio-Rad Laboratories Ltd., Hertfordshire, U.K.) of the selected follicles (following fixation with paraformaldehyde [4%, v/v; 30 min; room temperature [RT]) and staining with ethidium bromide (5 mg/ml, 15 min, RT) revealed that they were at the preantral (75%) and early antral (25%; as evidenced by the presence of an antral space as large as an area occupied by approximately three granulosa cells) stages of development. Follicles were cultured individually, and follicular diameter was measured daily before medium change for 4-6 days in a 96-well plate in 100 |xl of follicular culture medium (FCM; a-MEM supplemented with Hepes [10 mM], BSA [0.1%, w/v], rat serum [1%, v/v], bovine insulin [5 |xg/ml], transferrin [10 |xg/ ml], ascorbic acid [25 |xg/ml], sodium selenite anhydrous [2 ng/ml], and nonessential amino acids [1%, v/v], streptomycin-penicillin [0.5%, v/ v], and fungizone [0.25%, v/v]) with or without oFSH.