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
Steroid (progesterone, P4; androstenedione, A; and estradiol-17p, E2), PGE2, and ET-1 concentrations in the follicular fluid were determined after extraction as described below. The concentrations are shown in Table 1. Only dominant follicles that had not yet been exposed to the endogenous LH surge were used in this experiment. Such follicles were identified by the presence of a PGE2 concentration in the follicular fluid of less than 2000 pg/ml. The position of implantation of the MDS capillary membrane in the theca layer was also confirmed with a light microscope. Furthermore, to confirm that the MDS was functional in the theca layer, pregnenolone (10-7 M) was infused for 4 h into the MDS, and A and E2 releases were determined. A significant increase in A release (179 ± 19%, mean ± SEM, of baseline; p < 0.01) but not E2 release (108 ± 14% of baseline) was observed, which is a typical response of theca cells.