Proteolytic degradation of IGF-binding protein (IGFBP)-2 in equine ovarian follicles: involvement of pregnancy-associated plasma protein-A (PAPP-A) and association with dominant but not subordinated follicles

Abstract

In the ovary of mammalian species, terminal follicular growth is accompanied by a decrease in intrafollicular levels of IGF-binding protein-2 (IGFBP-2) and IGFBP-4. The decrease in IGFBP-4 levels is essentially due to an increase in proteolytic cleavage by intrafollicular pregnancy-associated plasma protein-A (PAPP-A) in growing healthy follicles. The decrease in IGFBP-2 levels is partly due to a decrease in mRNA expression by follicular cells. In addition, we have recently shown that IGFBP-2 is also proteolytically cleaved by PAPP-A in bovine and porcine growing follicles. In the present work, we showed that follicular fluid from late dominant equine follicles (35 mm diameter) contains a proteolytic activity against IGFBP-2. First follicular fluid from dominant follicles contained lower levels of native IGFBP-2 than the corresponding serum, as assessed by Western ligand blotting. In contrast, immunoblotting experiments showed much higher levels of a 12 kDa proteolytic fragment in dominant follicular fluid than in the serum. Moreover, equine dominant follicular fluid was able to induce proteolysis of exogenous recombinant bovine (rb)IGFBP-2, this degradation being dose-dependently enhanced by IGFs. The proteolytic activity against IGFBP-2 in equine follicles was partially immunoneutralized by a polyclonal antibody raised against human PAPP-A. Moreover, cleavage of rbIGFBP-2 by equine follicular fluid was dosedependently inhibited by a peptide derived from the heparin-binding domain of IGFBP-5, as well as by peptides derived from other heparin-binding domaincontaining proteins such as connective tissue growth factor, vitronectin and heparin-interacting protein, previously shown to inhibit PAPP-A. Finally, the proteolytic activity was very low in subordinate follicles, was high in both early (25 mm diameter) and late (35 mm diameter) dominant follicles, and was slightly lower in preovulatory follicles recovered 35 h after human chorionic gonadotropin (hCG) treatment.Overall, these data show that in the equine ovary, the selection of dominant follicles is associated with an increase of the proteolytic degradation of IGFBP-2 by PAPP-A, as for IGFBP-4, and potentially other protease(s), probably contributing to the increase in IGF bioavailability. In atretic subordinate follicles, the decrease in the proteolytic degradation of IGFBP-2, probably due in part to a direct inhibition by peptides containing heparin-binding domains, contributes to the increase in IGFBP-2 levels and the decrease in IGF bioavailability. The expression of PAPP-A and IGFBP-2 mRNA during folliculogenesis remain to be investigated in the mare.

Seasonal effects on the response of ovarian follicles to IGF1 in mares

“…Taken together, these findings suggest that, in contrast to the ovulatory season (Gerard & Monget 1998, a decrease in follicular IGFBP2 levels in dominant follicles does not occur during the transitional period. A tendency for greater mean expression levels of PAPPA, an IGFBP protease (Gerard et al 2004), in follicles 25-34 mm than in follicles 15-24 mm during the ovulatory period but not during the transitional period in this study are consistent with that conclusion. The lack of an effect of IGF1 injection on follicular IGFBP2 levels is in agreement with results by Ginther et al (2004cGinther et al ( , 2005 who showed that a much higher dose of IGF1 than the one used in the present study is needed to induce any detectable changes in follicular levels of IGFBP2.…”

“…Follicular availability of IGF1 is thought to be regulated primarily by IGF-binding proteins (IGFBPs) which render IGF1 inactive (Mazerbourg et al 2003). In ovulatory mares, levels of IGFBPs, most notably IGFBP2 and IGFBP5, decrease in the dominant follicle and this is attributable, at least in part, to the proteolytic activity of pregnancy-associated plasma protein-A (PAPPA, Gerard & Monget 1998, Gerard et al 2004.…”

IGFBP-2 - taking the lead in growth, metabolism and cancer

“…Although formation of the IGFBP-2/IGF-I or -II complex might show an acquired resistance to proteolysis in some cases, a major function of IGFBP proteases is to cleave IGFBP and release IGFs from the IGFBP/IGF complex, an event likely to enhance local IGF action (Gerard et al 2004;Qin et al 2006). This has been exhaustively described in cancer and cancer cell models where proteolysis of IGFBP-2 might be regarded as an important post-translational event contributing to increased tumour aggressiveness.…”

Stanniocalcin-2 Inhibits Mammalian Growth by Proteolytic Inhibition of the Insulin-like Growth Factor Axis

“…One IGF-releasing enzyme is the large metalloproteinase, pregnancy-associated plasma protein-A (PAPP-A, pappalysin-1, EC 3.4.24.79), a member of the metzincin superfamily of metalloproteinases (6), that is able to cleave IGFBP-4 (7), IGFBP-5 (8), and IGFBP-2 (9). PAPP-A functions intimately with IGFBP-4 (8, 10 -15), and highly specific IGF-dependent (8) proteolytic cleavage by surface-tethered PAPP-A releases bioactive IGF from the inactive IGF⅐IGFBP-4 complex in close proximity to the receptor (16).…”