Proteolytic activity degrading insulin-like growth factor-binding protein-2, -3, -4, and -5 in healthy growing and atretic follicles in the pig ovary.
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.
Besnard N
，Pisselet C
，Monniaux D
，Monget P
... -
《BIOLOGY OF REPRODUCTION》
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 and a decrease in the level of intrafollicular insulin-like growth factor-binding protein-3 (IGFBP-3) and IGFBPs less than 40 kDa (IGFBP-2, -4, and -5), respectively. In contrast, follicular atresia is associated with a decrease and a large increase in levels of IGFBP-3 and IGFBPs less than 40 kDa, respectively. To assess whether intrafollicular proteases are involved in such changes, follicular fluid from follicles of different sizes and degrees of atresia was incubated alone or with pure 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 blotting and by immunoblotting using specific antisera. Ovine follicular fluid from different classes of follicles contained proteolytic activity degrading IGFBP-2, -3, -4, and -5. Degradation of IGFBPs was accompanied by the generation of small proteolytic fragments visualized by immunoblotting or after autoradiography using radiolabeled IGFBP-4. Moreover, follicular growth and atresia were characterized by changes in IGFBP proteolytic activity. Indeed, follicular growth (between 2 and 6 mm in diameter) was characterized by 1) a decrease in IGFBP-3 proteolytic activity and 2) a dramatic increase in proteolytic activity degrading IGFBP-4 and, to a lesser extent, IGFBP-2 and -5. Atresia, in contrast, was associated with a strong increase in IGFBP-3 proteolytic activity in small ( < 3-mm diameter) follicles and a decrease in IGFBP-4 and -5 proteolytic activity in large ( > 5-mm diameter) follicles. Regardless of the follicle class, IGFBP proteolytic activity was strongly inhibited by EDTA and 1,10-phenanthroline, but very slightly or not at all inhibited by tissue inhibitor of matrix metalloprotease-1 and-2 and BB-2116 (natural and synthetic inhibitors of matrix metalloproteases, respectively) as well as cysteine and serine proteases inhibitors, with the exception of phenylmethylsulfonylfluoride (1 mM) in atretic follicles. In addition, IGFBP proteolytic activity was dependent on the presence of zinc and calcium chloride. Zymography experiments showed the presence of 72- and 92- to 96-kDa gelatinases in follicular fluid; their levels were dramatically increased during follicular atresia. These results suggest that 1) changes in intrafollicular IGFBP proteolytic activity could be at least partly responsible for the changes in intrafollicular IGFBP levels that occur during follicular growth and atresia in the sheep; and 2) metalloprotease(s) in healthy and atretic follicles as well as serine protease(s) in atretic follicles are involved in IGFBP degradation.
Besnard N
，Pisselet C
，Zapf J
，Hornebeck W
，Monniaux D
，Monget P
... -
《ENDOCRINOLOGY》
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