The chambers were maintained in a water bath at 38°C throughout the complete period of perfusion. The medium was continuously exchanged at a flow rate of 15 ml/h. During incubation, follicles were perfused with Ringer’s solution at a flow rate of 1.8 ml/h. After 2 h of pre-perfusion, fractions of the perfusate were collected every 2 h up to 14 h. These conditions were selected because the follicular tissues were found to produce a constant release of steroids, PGE2, and ET-1. Collected samples were stored at -20°C until hormone determination.
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
Collection of Bovine Mature Follicles
Ovaries from Holstein cows containing a mature, presumably preovulatory follicle were collected within 20 min after slaughter from a local slaughterhouse and were transported to the laboratory in sterile saline solution (0.9% NaCl) containing 100 000 IU penicillin and 100 mg/L streptomycin at 38°C. Ovaries were visually inspected. The preovulatory stage was defined by the presence of a graafian follicle (between 1.5 and 2.0 cm in diameter) and a regressing corpus luteum in the ipsilateral or contralateral ovary. The uterine characteristics (size, color, tonus, consistency, and mucus) were also considered. Furthermore, at the end of the experiment, follicular fluid from each follicle was collected and the follicular wall was prepared for routine histological observation. buy asthma inhalers
The LH surge triggers drastic changes in the secretory function within the preovulatory follicles. It is well documented that LH regulates steroidogenesis and prostaglandin biosynthesis in granulosa, theca, and surrounding stromal cells. In several species, prostaglandin E2 (PGE2) has been considered an essential mediator for the LH action on the ovulatory processes. This concept is supported by the fact that inhibitors of prostaglandin synthesis such as in-domethacin can block ovulation in the rat, rabbit, and cow. Moreover, it has also been suggested that peptides such as endothelin and cytokines derived from the endothelium, fibroblasts, leukocytes, and follicular cells interact with LH and modify its action at the ovarian level.