PROTEOLYTIC ACTIVITY IS INVOLVED IN CHANGES IN INTRAFOLLICULAR INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN-LEVELS DURING GROWTH AND ATRESIA OF OVINE OVARIAN FOLLICLES

In the sheep, follicular growth is characterized by both an increase a nd a decrease in the level of intrafollicular insulin-like growth fact or-binding protein-3 (IGFBP-3) and IGFBPs less than 40 kDa (IGFBP-2, - 4, and -5), respectively. In contrast, follicular atresia is associate d with a decrease and a large increase in levels of IGFBP-3 and IGFBPs less than 40 kDa, respectively. To assess whether intrafollicular pro teases are involved in such changes, follicular fluid from follicles o f different sizes and degrees of atresia was incubated alone or with p ure human IGFBP-3, -4, or -5 or serum (as a source of exogenous IGFBP- 2) for 20 h at 37 C. Samples were then analyzed by Western ligand blot ting and by immunoblotting using specific antisera. Ovine follicular f luid from different classes of follicles contained proteolytic activit y degrading IGFBP-2, -3, -4, and -5. Degradation of IGFBPs was accompa nied by the generation of small proteolytic fragments visualized by im munoblotting or after autoradiography using radiolabeled IGFBP-4. More over, follicular growth and atresia were characterized by changes in I GFBP proteolytic activity. Indeed, follicular growth (between 2 and 6 mm in diameter) was characterized by 1) a decrease in IGFBP-3 proteoly tic activity and 2) a dramatic increase in proteolytic activity degrad ing IGFBP-4 and, to a lesser extent, IGFBP-2 and -5. Atresia, in contr ast, was associated with a strong increase in IGFBP-3 proteolytic acti vity in small (<3-mm diameter) follicles and a decrease in IGFBP-4 and -5 proteolytic activity in large (>5-mm diameter) follicles. Regardle ss of the follicle class, IGFBP proteolytic activity was strongly inhi bited by EDTA and 1,10-phenanthroline, but very slightly or not at all inhibited by tissue inhibitor of matrix metalloprotease-l and -2 and BB-2116 (natural and synthetic inhibitors of matrix metalloproteases, respectively) as well as cysteine and serine proteases inhibitors, wit h the exception of phenylmethylsulfonylfluoride (1 mM) in atretic foll icles. In addition, IGFBP proteolytic activity was dependent on the pr esence of zinc and calcium chloride. Zymography experiments showed the presence of 72- and 92- to 96-kDa gelatinases in follicular fluid; th eir levels were dramatically increased during follicular atresia. Thes e results suggest that 1) changes in intrafollicular IGFBP proteolytic activity could be at least partly responsible for the changes in intr afollicular IGFBP levels that occur during follicular growth and atres ia in the sheep; and 2) metalloprotease(s) in healthy and atretic foll icles as well as serine protease(s) in atretic follicles are involved in IGFBP degradation.