Isolation of Glycosaminoglycans from Follicular Fluid
GAGs were extracted from follicular fluid by alkaline borohydride. To 1 ml of follicular fluid, 3 ml borohydride buffer (containing 15 mM NaBH4 dissolved in 1 M NaOH) was added. After 1 h at 73°C, the mixture was neutralized by addition of 6 M HCl. Proteins were precipitated by addition of trichloroacetic acid to a final concentration of 6% (w/ v). The mixture was incubated for 2 h at —20°C, and proteins were pelleted by centrifugation at 2000 X g for 15 min at 4°C. The supernatant was diluted in 100% ethanol in a ratio 1:5 to precipitate GAG and incubated for 18 h at —20°C.
GAGs were pelleted by centrifugation at 25 000 X g for 30 min at 4°C. The resulting pellet was dried and GAG were redissolved in demineralized water and stored at 4°C over night.
Quantification of Total Sulfated Glycosaminoglycans
Concentrations of total sulfated GAG in follicular fluid and GAG isolated from follicular fluid were determined using a DMMB assay. To 10 |xl of sample, 200 |xl of DMMB reagent (46 |xM DMMB, 40 mM glycine, and 42 mM NaCl, pH 3.0) was added and absorbance was measured at 525 nm. Chondroitin 4-sulfate was used as a standard.