MDS In Vitro
The MDS for bovine mature follicles was based on the method developed for porcine follicles in vivo, with some modifications for an in vitro organ culture system. Basically each follicle was dissected from surrounding stromal tissue, and four capillary dialysis membranes (Fre-senius SPS 900 Hollow Fibers, cutoff molecular size 1000 kDa, 0.2-mm diameter, 5 mm long; Fresenius AG, St. Wen-del, Germany) were implanted into the theca layer with at least a 5-mm distance between capillaries. buy flovent inhaler
The four capillaries (for control, LH, cytokines or ET-1, and LH+cytokine or LH+ET-1) were affixed to the surface of the follicular tissue by Histoacryl blue (B. Braun-Dexon GmbH, Spangenberg, Germany). Both ends of the capillary were glued to silicone elastomer tubing (i.d. 0.3 mm). For perfusion, one end of the tube was connected to a peristaltic pump and the other was routed to a fraction collector. The prepared follicles were then placed in organ culture chambers (modified 2070 tube; Falcon, Franklin Lakes, NJ) filled with 50 ml Medium 199 (Sigma Chemical Co., St. Louis, MO) containing Earle’s Salts, 10 mM NaHCO3, 365 mg/L L-glutamine, 25 mM Hepes, 5 g/L BSA, 60 mg/L penicillin, 100 mg/L streptomycin, 56 mg/L ascorbic acid, and 2 mg/ L amphotericin B at pH 7.4.