Three methods of extraction were compared for their efficiency in removing insulin-like growth factor binding proteins (IGFBPs) from bovine follicular fluid. Pools of follicular fluid collected from small (1-5 mm) and large (> 8 mm) bovine follicles were subjected to three extraction methods: 1) formic acid-acetone extraction at 25° C with no incubation, 2) acid-ethanol extraction at 25° C with 30 min incubation, and 3) acid-ethanol extraction at -20° C for 2 h. Methods 1 and 3 were the most efficient procedures for removal of IGFBP-3 whereas Methods 2 and 3 were the most efficient procedures for removal of IGFBP-2. Method 2 was nearly two-fold less efficient than the other methods at removing IGFBP-3, whereas Method 1 was five-fold less efficient than the other methods at removing IGFBP-2.

Ovaries of beef and dairy cattle were obtained at slaughter and follicular fluid collected from follicles 1 to 5 mm in diameter and from follicles 8 mm and greater in diameter. Aliquots of follicular fluid were stored at -20° C until used. Follicular fluid samples were thawed and extracted using three methods. Method 1: formic acid-acetone extraction procedures in which samples (50 m l) were added to tubes containing 25 m l of 8 M formic acid (0.5% Tween-20) and 175 m l of acetone, the contents vortexed for 10 sec and the samples were immediately centrifuged at 2500 x g for 15 min. Method 2: acid-ethanol extraction procedure in which samples (100 m l) were added to tubes containing 400 m l of acid-ethanol (12.5% 2 N HCl and 87.5% ethanol), the contents vortexed for 30 sec, incubated at 25° C for 30 min, and the samples centrifuged at 2000 x g for 30 min. Method 3: acid-ethanol extraction procedure as in Method 2 except that the incubation was for 2 h at -20° C.

To evaluate the presence of IGFBP-3 and -2 in the various samples, one-dimensional SDS-PAGE, under non-reducing conditions, was performed as described previously (Stewart et al., 1996). Briefly, proteins were separated on a 12% polyacrylamide separating gel and a 4% stacking gel, with 2 m l of unextracted follicular fluid and 22 m l of buffer loaded per lane or 10 m l of the various extraction samples, and 14 m l of buffer loaded per lane. After electrophoresis of samples, proteins were electrophoretically transferred to nitrocellulose, and ligand blotted for IGFBP activity with use of [¹²⁵I]IGF-II. Band intensity on autoradiographs was characterized by scanning densitometry.

All three extraction procedures resulted in removal of over 93% of IGFBP-3, with Methods 1 and 3 being the most efficient procedures for removal of IGFBP-3 (Figure 1). Method 2 was nearly two-fold less efficient than the other methods at removing IGFBP-3 (Figure 1). Methods 2 and 3 (acid-ethanol) were the most efficient procedures for removal of IGFBP-2, removing over 97% of the IGFBP-2. Method 1 (acid acetone) was five-fold less efficient than the other methods at removing IGFBP-2, with 84% removal of the IGFBP-2 (Figure 1). Previous studies have reported ³ 95% of the total IGFBPs are removed after a 16 h incubation at 4° C with acid-ethanol (Echternkamp et al., 1990). Also in agreement with previous work (Holly and Cwyfan-Hughes, 1994), IGFBP-3 appears to be more easily removed than IGFBP-2 with the formic acid-acetone extraction. Thus, the best of the three extraction methods reported here for removal of both IGFBP-2 and -3 is the 2 h incubation at 4° C with acid-ethanol.

Figure 1. Effect of various extraction methods on removal of IGFBP-2 and IGFBP-3 from bovine follicular fluid. Values are means of four to six follicular fluid samples and expressed as percentage of the total IGFBP-2 or IGFBP-3 in the original samples as assessed by ligand blotting. Method 1 = formic acid-acetone extraction; Method 2 = 30 min acid-ethanol extraction at 25° C; and Method 3 = 2 h acid-ethanol extraction at -20° C.