Fucoidan was biotinylated with biotin-hydrazide after partial oxidation of the carbohydrate moieties according to the manufacturer’s instructions. Briefly, 10 mg fucoidan was dissolved in 200 |xl demineralized water and partially oxidized by the addition of 50 |xl of 100 mM NaIO4 (containing 100 mM CH3COONa, pH 5.5). After 15 min, the excess periodate was removed with a Sephadex G50 desalting column that was equilibrated with 100 mM sodium acetate buffer (pH 5.5). To the eluted fucoidan, biotin-hydrazide was coupled (final concentration 5 mM) by agitation for 2 h at room temperature.
Nonconjugated biotin was removed from both fucoi-dan-biotin and mannan-BSA-biotin by means of a Sephadex G 50 desalting column.
Follicular Fluid Collection
Bovine ovaries were obtained in a local slaughterhouse (Gosschalk and Zn BV, Epe, The Netherlands) and transported to the laboratory in PBS at 22°C within 2 h postmortem. Fluids from dominant follicles (>15 mm) were aspirated into a sterile 15-ml plastic tube. Follicular fluid of 10 dominant follicles was pooled and centrifuged at 1300 X g for 15 min, and the supernatant was subsequently stored at —20°C until use.