Proteolytic Activity Degrading Insulin-like Growth Factor-Binding Protein−2, −3, −4, and −5 in Healthy Growing and Atretic Follicles in the Pig Ovary1

Abstract

In the pig, ovarian follicular growth is characterized by an increase in intrafollicular levels of insulin-like growth factor-binding protein (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 IGFBP-5). In contrast, atresia is primarily associated with a strong increase 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 proteases are involved in such changes. Porcine follicular development was synchronized 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 with a source of exogenous bovine IGFBP-2 or human IGFBP-3, -4, or -5 for 20 h at 37 degrees C. Samples were then analyzed by Western ligand blotting and by immunoblotting using specific antisera. Porcine follicular fluid from various classes of follicles contained proteolytic activity degrading IGFBP-2, -4, and -5. In contrast, intrafollicular IGFBP-3 proteolytic activity was very low or nondetectable. In preovulatory 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) dependent on the presence of zinc and calcium chloride. PMSF (1 mM, serine protease inhibitor) inhibited degradation of IGFBP-2 and to a lesser extent IGFBP-4, but not IGFBP-5. Other serine and cysteine protease inhibitors as well as TIMP-2 and BB-2116 (natural tissue inhibitor-2 and synthetic inhibitor of matrix metalloproteinases [MMPs], respectively) were ineffective. Gelatin-substrate zymography revealed the presence of two major intrafollicular gelatinase MMPs at 60 kDa and 76-85 kDa (likely MMPs 2 and 9, respectively), the levels of which decreased (76-85 kDa) or strongly increased (60 kDa) during follicular atresia. Follicular growth at diameters between 2 and 6-7 mm was characterized by a dramatic increase in proteolytic activity degrading IGFBP-2, -5 and, to 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 intrafollicular IGFBPs < 40 kDa are at least partly responsible for the changes in intrafollicular IGFBP levels during follicular growth and atresia in the pig and 2) calcium- and zinc-dependent metalloprotease(s) as well as serine protease(s) are involved in degradation of intrafollicular IGFBPs < 40 kDa.