Proteolytic activity is involved in changes in intrafollicular insulin- like growth factor-binding protein levels during growth and atresia of ovine ovarian follicles

Abstract

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.

Intrafollicular Insulin-Like Growth Factor-Binding Protein Levels in Equine Ovarian Follicles during Preovulatory Maturation and Regression1

“…In other species, these changes in IGFBP-2 levels with follicular health status were shown to be related to both changes in mRNA expression (sheep [67], pigs [65,66]) and in proteolytic degradation (sheep [68], pigs [69]). …”

“…-~~~~* --·-and/or to change in proteolytic degradation (humans [70], sheep [68], pigs [69]). The precise role for the various IGFBPs in follicular function remains unclear.…”

Expression of Messenger Ribonucleic Acids of Insulin-Like Growth Factor Binding Proteins-2, -4, and -5 in the Ovine Ovary: Localization and Changes during Growth and Atresia of Antral Follicles1

“…Western ligand blots were quantified using the same image analysis system (see above) and as previously described [3,8]. The amount of radiolabeled IGF-II bound to the 35-kDa IGFBP-2, 30-kDa IGFBP-5, and 24-kDa IGFBP-4 was estimated from the ratio of the IOD (integrated optical density in arbitrary units) of these bands in the sample to that of the 35-30-kDa bands in a follicular fluid from bovine atretic follicle run simultaneously on each ligand blot and taken as internal standard.…”

“…Local paracrine processes are likely responsible for such changes in level of intrafollicular IGFBPs < 40 kDa observed during follicular growth and atresia. Particularly, in the sheep, we have recently shown that ovine follicular growth in vivo is characterized by a marked increase in intrafollicular proteolytic activity degrading IGFBP-4 and to a lesser extent IGFBP-2 and -5 [8]. Follicular atresia, in contrast, is characterized by a decrease in intrafollicular IGFBP-4 and -5 proteolytic activity [8].…”

“…Particularly, in the sheep, we have recently shown that ovine follicular growth in vivo is characterized by a marked increase in intrafollicular proteolytic activity degrading IGFBP-4 and to a lesser extent IGFBP-2 and -5 [8]. Follicular atresia, in contrast, is characterized by a decrease in intrafollicular IGFBP-4 and -5 proteolytic activity [8]. In the human as well, Chandrasekher et al [9] have reported the presence of proteolytic activity degrading IGFBP-4 in follicular fluid from dominant estrogenic but not from atretic follicles.…”