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.
Bovine LH (USDA-bLH-B-6), human ET-1 (Peptide Institute Inc., Osaka, Japan), recombinant human TNFa, and recombinant human IL-1p (both donated by Dainippon Pharm. Co., Osaka, Japan) were diluted in Ringer’s solution to obtain the required final concentrations of 5 ^g/ml, 250 ng/ml, 100 ng/ml, and 10 ng/ml, respectively. The doses were determined based on the result of a preliminary experiment and on the transfer capacity of the membrane, which was previously estimated to be 1% for steroids and prostaglandins and 0.1% for peptides and LH. The solutions were then infused into the MDS for 2 h (LH between 4 and 6 h; ET-1, TNFa, or IL-p between 8 and 10 h). buy antibiotics online
Steroids and PGE2 Extraction
The perfusates (3 ml) and follicular fluids (300 ^l) were adjusted to pH 3.5 using 1 N HCl and 5 N HCl, respectively, and allowed to stand at room temperature for 1 h. Then 3 volumes of diethyl ether was added and the mixture was shaken for 30 min at a frequency of 250 cycles/min (using Shaker SA 31; Yamato Co., Tokyo, Japan). The samples were then allowed to stand for 15 min and placed in a – 80°C freezer for 1 h. The upper diethyl ether fraction was decanted and evaporated. The residue was dissolved in 300 ^l assay buffer for steroid and prostaglandin enzyme immunoassays (EIAs) (40 mM PBS, 0.1% BSA, pH 7.2). To estimate the recovery rate, A, P4, E2, and PGE2 were added to the Ringer’s solution (30, 100, 20, and 100 pg/ml, respectively), and the obtained values were 85%, 88%, 75%, and 72%, respectively.