Specific Cortisol Binding Protein in Porcine Follicular Fluid

A specific cortisol binding protein has been demonstrated in the follicular fluid of the porcine ovary. The binding of cortisol, progesterone, testosterone, Δ4-androstenedione and estradiol-17β was studied by equilibrium dialysis at 4°C in follicular fluid obtained from ovaries of mature pigs (6–12-months-old). The binding capacity of the follicular fluid protein with each steroid was variable. Cortisol demonstrated a specific binding to follicular fluid. The mean association constants of cortisol binding to follicular fluid from large, medium and small follicles were 1.99 ± 0.64 (SE) × 109 M−1 (N = 8), and 1.30 ± 0.62 (SE) × 109 M−1 (N = 6) and 2.05 × 109 M−1 (N = 2), respectively and were not significantly different from each other. The corresponding binding capacities were 0.60 ± 0 19 (SD) × 10−7, 1.06 ± 0.45 (SD) × 10−7 and 0.83 × 10−7 moles/l of undiluted follicular fluid. Tritiated cortisol bound to follicular fluid protein was displaced by corticosterone, deoxycorticosterone, progesterone and testosterone with relative affinities compared to cortisol (100%) of corticosterone 24%, deoxycorticosterone 20%, progesterone 11% and testosterone <1%. Dexamethasone did not bind to either follicular fluid or porcine serum at the concentrations of protein used for cortisol binding. Tritiated progesterone, testosterone and estradiol-17β did bind to follicular fluid, but were not displaced by 200 times the concentration of the respective unlabeled steroids with the experimental conditions used. The nonspecific binding properties of these steroids were similar to those of porcine serum except the serum had a lower binding capacity than that of follicular fluid. Calcium (0.05M Ca++), magnesium (0.05M Mg++), thioglycerol (0.01M), L-cysteine (0.01M) and dithiothreitol (0.01M) did not alter the cortisol binding properties. The follicular fluid was incubated with tritiated steroids and fractionated by polyacrylamide gel electrophoresis. The radioactive peak of cortisol was completely displaced by the addition of 700 times the concentration of unlabeled cortisol. The cortisol binding protein had a sedimentation of a 4.1 S fraction as determined by sucrose gradient centrifugation. These results suggest that porcine follicular fluid contains a specific cortisol binding α1-globulin fraction and, after stripping of the indigenous steroids, it appears to be similar to, but present in twice the concentration of that in serum. The cortisol concentration in follicular fluid (167 ± 29 (SE); 188 ± 43 (SE) and 181 ± 15 (SE) ng/ml in small, medium and large follicles, respectively) was also shown to be higher than the plasma (102 ± 14 (SE) ng/ml), (P<0.05), which may be partly bound to specific cortisol binding protein in the follicular fluid.