Culture of Antral Follicles
Ovaries were obtained from local abattoirs and maintained at 28-35°C during transport to the laboratory (1-2 h). Ovaries were dissected from the rest of the reproductive tract and transferred into a beaker containing 200-300 ml of Dulbecco’s PBS (pH = 7.4) at 39°C (Unipath Ltd., Basingstoke, England) containing 0.5 ml of gentamycin solution (Sigma Chemical Co., St. Louis, MO) in a clean glass beaker. They were then washed once in industrial methylated spirits and transferred to PBS at 39°C. Using a pair of scissors and forceps, follicles of 3- to 8-mm diameter (Fig. 1a) were dissected from the surrounding connective tissues. Buy Asthma Inhalers Online
Individual follicles were transferred to PBS at 39°C and completely trimmed from remaining connective tissues. Nonatretic follicles were selected on the basis of morphological criteria including translucency, lack of free particles, and the presence of blood vessels (Fig. 1b). Selected follicles were cultured in Waymouth medium MB752/1 (Gibco BRL, Oakville, ON, Canada) supplemented with 2240 mg/L sodium bicarbonate (Sigma), 0.23 mM pyruvic acid, 50 mg/L streptomycin sulfate, 75 mg/L penicillin G, 3 mg/ml BSA (A-6003; Sigma), 5 ^g/ml insulin, 5 ^g/ml transferrin, and 5 ng/ml selenium (Sigma; pH 7.4, osmolarity 280 mOsmol).