PROTEOLYTIC ACTIVITY DEGRADING INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN-2, GROWTH FACTOR-BINDING PROTEIN-3, GROWTH FACTOR-BINDING PROTEIN-4, AND GROWTH FACTOR-BINDING PROTEIN-5 IN HEALTHY GROWING AND ATRETICFOLLICLES IN THE PIG OVARY

In the pig, ovarian follicular growth is characterized by an increase in intrafollicular levels of insulin-like growth factor-binding protei n (IGFBP)-3 and a decrease in the levels of IGFBPs < 40 kDa (IGFBP-2, -4 and, to a lesser extent, a 30-kDa IGFBP likely corresponding to IGF BP-5). In contrast, atresia is primarily associated with a strong incr ease in intrafollicular levels of IGFBP-2 and -4, with intrafollicular levels of IGFBP-3 and -5 varying slightly or not at all. The purpose of the present study was to determine whether intrafollicular protease s are involved in such changes, Porcine follicular development was syn chronized with a progestin, and individual follicles were isolated 12 h and 96 h after progestin withdrawal. Follicular fluid from follicles of various sizes and qualities was collected and incubated alone or w ith a source of exogenous bovine IGFBP-2 or human IGFBP-3, -4, or -5 f or 20 h at 37 degrees C. Samples were then analyzed by Western ligand blotting and by immunoblotting using specific antisera. Porcine follic ular fluid from various classes of follicles contained proteolytic act ivity degrading IGFBP-2, -4, and -5. In contrast, intrafollicular IGFB P-3 proteolytic activity was very low or nondetectable. in preovulator y follicles, degradation of IGFBPs < 40 kDa was 1) accompanied by the generation of small proteolytic fragments visualized by immunoblotting , 2) strongly inhibited by EDTA and 1,10-phenanthroline, and 3) depend ent on the presence of zinc and calcium chloride. PMSF (1 mM, serine p rotease inhibitor) inhibited degradation of IGFBP-2 and to a lesser ex tent IGFBP-4, but not IGFBP-5. Other serine and cysteine protease inhi bitors as well as TIMP-2 and BB-2116 (natural tissue inhibitor-2 and s ynthetic inhibitor of matrix metalloproteinases [MMPs], respectively) were ineffective. Gelatin-substrate zymography revealed the presence o f two major intrafollicular gelatinase MMPs at 60 kDa and 76-85 kDa (l ikely MMPs 2 and 9, respectively), the levels of which decreased (76-8 5 kDa) or strongly increased (60 kDa) during follicular atresia. Folli cular growth at diameters between 2 and 6-7 mm was characterized by a dramatic increase in proteolytic activity degrading IGFBP-2, -5 and, t o a lesser extent, IGFBP-4. Atresia, in contrast, was associated with a marked decrease in proteolytic activity degrading IGFBP-2, -4, and - 5. These results suggest that 1) changes in proteolytic activity of in trafollicular IGFBPs < 40 kDa are at least partly responsible for the changes in intrafollicular IGFBP levels during follicular growth and a tresia in the pig and 2) calcium- and zinc-dependent metalloprotease(s ) as well as serine protease(s) are involved in degradation of intrafo llicular IGFBPs < 40 kDa.