This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mazerbourg, S. Articles by Monget, P. Search for Related Content PubMed PubMed Citation Articles by Mazerbourg, S. Articles by Monget, P.

Insulin-Like Growth Factor Binding Protein-4 Proteolytic Degradation in Ovine Preovulatory Follicles: Studies of Underlying Mechanisms¹

Station INRA de Physiologie de la Reproduction des Mammifères Domestiques (S.M., P.M.), URA CNRS 1291, 37380 Nouzilly, France; The Department of Medicine (J.Z.), University Hospital, Zürich, Switzerland; The Department of Internal Medicine (R.S.B.), University of Iowa, Iowa City, Iowa 52246; The Department of Surgery (D.R.B.), Division of Pediatric Surgery, Children’s Hospital, Wexner Institute for Pediatric Research, Colombus, Ohio 43205; and Institut National de la Santé et de la Recherche Médicale (C.L., M.B.), Unité de recherches sur la régulation de la croissance (U-142), Hopital Saint Antoine 75012 Paris, France

Address all correspondence and requests for reprints to: P. Monget, Station INRA de Physiologie de la Reproduction des Mammifères Domestiques, URA CNRS 1291, 37380 Nouzilly, France. E-mail: monget{at}tours.inra.fr

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The regulation of insulin-like growth factor binding protein (IGFBP)-4 proteolytic degradation by insulin-like growth factors (IGFs) has been largely studied in vitro, but not in vivo. The aim of this study was to investigate the involvement of IGFs, IGFBP-2, IGFBP-3, and IGFBP-3 proteolytic fragments in the regulation of IGFBP-4 proteolytic activity in ovine ovarian follicles. Follicular fluid from preovulatory follicles contains proteolytic activity degrading exogenous IGFBP-4. The addition of an excess of IGF-I enhanced IGFBP-4 proteolytic degradation, whereas addition of IGFBP-2 or -3 or monoclonal antibodies against IGF-I and -II dose dependently inhibited IGFBP-4 proteolytic degradation. IGF-I and IGF-II, but not LongR3-IGF-I, reversed this inhibition in a dose-dependent manner. C-terminal, but not N-terminal, proteolytic fragments derived from IGFBP-3 (aa 161–264), as well as heparin-binding domain-containing peptides derived from the C-terminal domain of IGFBP-3 and -5 also induced the inhibition of IGFBP-4 proteolytic degradation. Other heparin-binding domain-containing peptides derived from the connective tissue growth factor (CTGF) and from proteins not related to IGFBP, heparan/heparin interacting protein (HIP) and vitronectin, but not from p36 subunit of annexin II tetramer, inhibited IGFBP-4 degradation. Furthermore, IGFBP-3, mutated on its heparin-binding domain, was not able to inhibit IGFBP-4 proteolytic degradation. So, in ovine preovulatory follicles, IGFBP-4 proteolytic degradation both 1) depends on IGFs, and 2) is inhibited by IGFBP-3 via its C-terminal heparin-binding domain as well as by heparin-binding domain containing peptides. These data suggest that in early atretic follicles, the increase in IGFBP-2 participates in the decrease in IGFBP-4 degradation. In late atretic follicles, the increase in the levels of C-terminal IGFBP-3 proteolytic fragments, generated by IGFBP-3 degradation, as well as the increase in IGFBP-5 expression would strengthen the inhibition of IGFBP-4 degradation. This inhibition might be partly mediated by direct interaction of IGFBP-4 proteinase(s) and heparin-binding domain within the C-terminal region from IGFBP-3 and -5.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
INSULIN-LIKE GROWTH FACTOR binding protein (IGFBP)-4 proteolytic degradation has been reported in a wide variety of in vitro models, including sheep and human fibroblasts (1, 2, 3, 4), nonsmall cell lung cancer cell lines (5), mouse (6) and human (7) osteoblasts, human decidual cells (8), human endometrial stromal cells (9), and rat granulosa cells (10). In these models, IGFBP-4 degradation was shown to be IGF dependent. Particulary, IGFs have been shown to enhance IGFBP-4 degradation in a receptor-independent manner in conditioned media of human endometrial stromal cells (9), human decidual cells (8), and human fibroblasts (1). Moreover, addition of IGFBP-3, -5, or -6 in MC3T3-E1 osteoblast-conditioned medium (6, 11), addition of IGFBP-3 in human dermal fibroblast-conditioned medium (4), addition of IGFBP-1, -2, or -3 in human follicular fluid (12), and addition of IGFBP-2, -3, -5, and -6 in rat granulosa cell-conditioned medium (10) led to an inhibition of IGFBP-4 degradation. The mechanism of IGF enhancement of IGFBP-4 proteolysis is unknown. In one hypothesis, it is proposed that a conformational change of IGFBP-4 upon binding to IGFs results in the enhancement of IGFBP-4 proteolytic degradation (8, 9). In an alternative hypothesis, Fowlkes et al. suggested that free IGFBP-3, -5, and -6 are able to directly inhibit IGFBP-4 protease(s) in MC3T3-E1 cells-conditioned medium by binding at the level of their C-terminal heparin binding domain (6, 11). In this latter model, saturation of IGFBP-3, -5, or -6 by IGFs would lead to the release and the activation of protease(s) responsible for IGFBP-4 degradation.

In vivo, in the sheep ovary, preovulatory follicles are characterized by a high IGF bioavailability, by high levels of IGFBP-3, likely coming from serum, and by a high IGFBP-4 proteolytic activity. In contrast, atretic follicles are characterized by a low IGF bioavailability, by a high increase in the expression of IGFBP-2 (early atresia) and -5 (late atresia), by an increase in IGFBP-3 proteolytic degradation (at least in small follicles), as well as by a low IGFBP-4 proteolytic activity (13, 14, 15). So, in the sheep ovary, there is a positive relationship between IGF bioavailability and IGFBP-4 degradation on one hand, and a negative relationship between IGFBP-3 proteolytic degradation, IGFBP-2 and -5 expression and IGFBP-4 degradation on the other hand. In this work, we have investigated the implication of IGFs, IGFBP-2, IGFBP-3 and IGFBP-3 proteolytic fragments on IGFBP-4 proteolytic degradation in ovine preovulatory follicles.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
Fluorogestone acetate sponges used to synchronise estrous cycles and PMSG were obtained from Intervet (Angers, France). FSH was obtained from Dr Y. Combarnous (Nouzilly, France). IGF-I and IGF-II were generous gifts from Drs. H. H. Peter and A. Hinnen (Ciba-Geigy, Basel, Switzerland). LongR3-IGF-I was obtained from GroPep Pty. Ltd. (Adelaide, Australia). Recombinant human IGFBP-4 and IGFBP-5 were expressed in yeast and purified as previously described (16). Purified glycosylated human IGFBP-3 was obtained from Dr. J. Closset (Liege, Belgium). Nonglycosylated human IGFBP-3 was obtained from Celtrix Pharmaceuticals, Inc. (Santa Clara, CA). IGFBP-3mHBD was generous gift of Dr. D. R. Powell (Houston, Texas). It is characterized by the replacement of the IGFBP-3 heparin-binding domain (HBD) sequence by a sequence derived from IGFBP-1 (²¹⁵KNGFYHSRQCETSMDGEA²³²). IGFBP-2 was obtained from Dr. J. C. Byatt (Monsanto, St. Louis, MO). The 18 amino-acids peptides, P1 (¹⁸³KNGFYHSRQCETSMDGEA²⁰⁰) synthesized from IGFBP-1, P3 (²¹⁵KKGFYKKKQCRPSKGRKR²³²) from IGFBP-3, P4 (¹⁸⁵RNGNFHPKQCHPALDGQR²⁰²) from IGFBP-4, P5 (²⁰¹RKGFYKRKQCRPSKGRKR²¹⁸) and PA5 (¹³⁰KAEAVKKDRRKK-LTQSKF¹⁴³) from IGFBP-5 were synthetized using the t-bag technique as previously described (17, 18). Synthetic peptides spanning the C-terminal region of human connective tissue growth factor (CTGF or IGFBP-related protein 2) CTGF^247–260 (EENIKKGKKCIRTP), CTGF^257–272 (IRTPKISKPIKFELSG), CTGF^259–275 (TPKISKPIKFELSGCTS), CTGF^274–283 (TSMKTYRAKF), CTGF^285–291 (GVCTDGR), CTGF^305–322 (FKCPDGEVM-KKNMMFIKT), CTGF^308–322 (PDGEVMKKNMMFIKT), CTGF^318–324 (MFIKTCA), Ser³²⁵CTGF^318–328 (MFIKTCASHYN), CTGF^326–349 (HYNCP-GDN-DIFESLYYRKMYGDMA), CTGF^339–349(LYYRKMYGDMA) were synthesized and received as a cleaved PepSet (19). The synthetic peptide containing the heparin-binding domain of HIP (heparin/heparan sulfate-interacting protein) (CRPKAKAKAKAKDQTK) was kindly provided by Dr. D. D. Carson (Houston, TX). The synthetic peptide containing an heparin-binding domain derived from the p36 subunit of annexin II tetramer (KIRSEFKKKYGKSLYY) was obtained from Dr. D. M. Waisman (Calgary, Canada). Synthetic peptides derived from the heparin-binding domain of vitronectin, VN^341–355 (APRPSLAKKQRFRHR), VN^357–370(RKGYRSQRGHSRGR) and VN^371–383 (NQNSRRPSRATWL), were kindly provided by Dr. K. T. Preissner (Bad Nauheim, Germany). Heparin (ammonium salt from porcine intestinal mucosa) and aprotinin were purchased from Sigma (L’Issle d’Abeau Chesnes, France). Heparin-Sepharose CL6B beads were obtained from Pharmacia & Upjohn (Uppsala, Sweden). EDTA was purchased from Prolabo (Fontenay-sous- bois, France). Rabbit polyclonal antiserum against human IGFBP-2 was obtained from Dr. Schwander (Basel, Switzerland) and rabbit polyclonal antiserum against IGFBP-4 was purchased from Ubi (Lake Placid, NY). Mouse monoclonal antiserum against human IGFBP-3 was purchased from UCB (Braine L’Alleud, Belgium). Mouse monoclonal antibody against IGF-I was a gift from Dr. J. Closset (Liège, Belgium), and mouse monoclonal against IGF-II was obtained from Amano Pharmaceutical Co. (Nagoya, Japan). Antirabbit and antimouse IgG antibodies coupled to horseradish peroxidase were purchased respectively from Diagnostic Pasteur (Marnes la coquette, France) and DAKO Corp. (Trappes, France). Nitrocellulose membranes were purchased from Schleicher & Schuell, Inc. (Ecquevilly, France) and the enhanced chemiluminescence detection system for immunoblots was obtained from Amersham Pharmacia Biotech (Les Ulis, France).

Collection of follicular fluid
Adult Romanov or Ile-de-France ewes were treated with progestagen (intravaginal fluorogestone acetate sponges, 40 mg) for 15 days to synchronize estrus. They were injected with 40 mg of FSH or 500 UI of PMSG 24 h before the sponge removal. Twenty-four hours after sponge removal, the ovaries were collected by castration. Follicular fluids from 5- to 7-mm diameter follicles were aspirated by puncture and individually stored at -20 C. The follicles characterized by the presence of IGFBP-3 and by the absence of IGFBP-2, -4, and -5 were considered as preovulatory, whereas follicles characterized by high levels of IGFBP-2 or IGFBP-2, -4, and -5 were considered as, respectively, early or late atretic follicles, as previously described (13, 14, 15)

Studies of IGFBP-4 proteolysis in preovulatory follicular fluid
Two microliters of follicular fluid were incubated, in a solution of 20 mM Tris (pH 7.6) containing 137 mM NaCl (TBS) and 0.1% BSA, with 15 or 20 ng of IGFBP-4, with or without exogenous IGFBP-3, IGFBP-3mHBD, IGFBP-2, monoclonal antibodies against IGF-I or IGF-II, synthetic peptides, proteolytic fragments of IGFBP-3, and in the presence or absence of exogenous IGF-I, IGF-II or LongR3-IGF-I for 20 h at 37 C (final volume, 10 µl). At the end of the incubation, samples were analyzed by Western ligand blotting (WLB) or immunoblotting (see below).

In other experiments, to test the ability of exogenous IGF-I to enhance intrafollicular degradation of IGFBP-4, follicular fluids from preovulatory and atretic follicles were incubated with IGFBP-4 for 30 min (preovulatory and early atretic follicles) and for 2 h (late atretic follicles), in the presence or absence of IGF-I.

IGFBP-3 proteolytic degradation and fragments separation
Twenty micrograms of nonglycosylated human IGFBP-3 (E. coli) were incubated with 0.2 µg plasmin (1:270 molar ratio) in 80 µl 20 mM Tris-HCl, pH 7.5, 0.15 M NaCl for 45 min at 37 C. The reaction was stopped by addition of 0.5 mM final Tosyl-lysine chloromethylketone (TLCK). The fragments were separated as already described (20). Briefly, the reaction mixture was diluted in 1:2 with 10% acetonitrile, 0.1% Trifluoro acetic acid (TFA), and loaded onto a C₈ RP 300 Aquapore Brownlee column. Peptides were eluted by 10–40% acetonitrile linear gradient. Elution peaks monitored by absorbance at 210 nm were identified by microsequencing. The collected tubes were dried under vacuum and stored at -20 C. Peptides used within this study correspond to 1–95 and 161–264 IGFBP-3 fragments (21).

Binding of intrafollicular IGFBP-3 or recombinant IGFBP-3 to heparin-Sepharose beads
We incubated 2.5 µl of follicular fluid from ovine preovulatory, early atretic or late atretic follicles in TBS/BSA with or without 10 µl of heparin-Sepharose beads (2 mg/10 µl in TBS) in the absence or presence of 200 ng IGF-I and/or 500 ng IGFBP-2 (final volume, 20 µl). After an overnight incubation at 37 C, the samples were centrifuged for 1 min at 15,000 x g. The pellets of heparin-Sepharose beads were rinced three times with TBS/BSA. We then added 10 µl of Laemmli sample buffer to both the supernatants and pellets before WLB. The same experiment was performed with 20 ng of nonglycosylated IGFBP-3 incubated in TBS/BSA with 5 mM EDTA, 10 µM Aprotinine with or without heparin-Sepharose beads in the presence or absence of 200 ng IGF-I (final volume, 20 µl).

IGFBP-4 proteolytic degradation by plasmin
To test the involvement of IGF-I in IGFBP-4 degradation by plasmin in vitro, 20 ng of IGFBP-4 were incubated in TBS/Tween 20 with 15 ng of plasmin, with or without IGF-I, for 5 min, 30 min, 2 h, or 5 h at 37 C (final volume, 10 µl). The reaction was stopped by addition of Laemmli sample buffer. The samples were then submitted to WLB.

Western ligand blotting (WLB)
IGF-II was iodinated by the iodogen method and purified by Sephadex G-50 chromatography by using a 0.1 M ammonium acetate elution buffer. WLB was performed according to the method of Hossenlopp et al. (22) modified by Monget et al. (13). Samples were submitted to electrophoresis on a 12% SDS-polyacrylamide gel under nonreducing conditions. The proteins were then electrotransferred onto nitrocellulose filters (0.2 µm pore size) overnight at 4 C. Filters were treated successively with PBS (0.01 M; pH 7.4) containing 0.1% Nodinet P-40, 0.5% gelatin, and 0.1% Tween-20, then incubated overnight at 4 C with [¹²⁵I]IGF-II in a solution containing 0.03 M NaH₂PO₄, 500 µl/liter Tween-20, 200 mg/liter protamine sulfate, 200 mg/liter NaNO₃, and 3.72 g/liter EDTA (pH 7.4). Afterward, filters were washed with PBS containing 0.1% Tween-20, air-dried, and exposed to Hyperfilm MP (Amersham Pharmacia Biotech, Arlington Heights, IL) with an intensifying screen at -70 C or to a phosphor screen for quantification.

Immunoblotting
After electrophoresis and electrotransfer of proteins, as described for WLB, nitrocellulose filters were treated for 2 h at room temperature with TBS containing 10% nonfat dry milk (NFDM) and 0.2% Tween-20 to saturate nonspecific sites. Thereafter, filters were incubated for 1 h at 37 C in TBS containing 5% NFDM, 0.05% Tween-20, and antibodies against IGFBP-2 (final dilution 1/1000), IGFBP3 (final dilution 1/2000) or IGFBP-4 (final dilution 1/1000). Once washed in TBS containing 1% NFDM and 0.2% Tween-20, nitrocellulose filters were incubated for 1h at 37 C with an antirabbit or antimouse IgG antibody coupled to horseradish peroxidase (final dilution 1/2000). After washing, the signal was revealed by chemiluminescence detection.

IGF-binding activity assay
Specific IGF-binding activity was assayed by incubating IGFBPs (10 ng of IGFBP-2, 10 ng of IGFBP-4, 20 ng of IGFBP-3) or monoclonal antibodies against IGF-I (0.02 µl) or IGF-II (0.05 µl) with [¹²⁵I]IGF-I or [¹²⁵I]IGF-II (20 000 cpm/tube) in the presence or absence of unlabeled IGF-I or IGF-II (12 pg to100 ng) overnight at 4 C in assay buffer (0.03 M NaH₂PO₄, 500 µl/liter Tween-20, 200 mg/liter protamine sulfate, 200 mg/liter NaNO₃, and 3.72 g/l EDTA, pH 7.4, final volume 0.5 ml). After incubation, free and bound [¹²⁵I]IGF-I or [¹²⁵I]IGF-II were separated using albumin-coated charcoal (0.5% charcoal, 2 mg/ml BSA) as previously described (13). Characteristics of IGFBP-2, -3, -4, IGFBP-3mHBD and for monoclonal antibodies against IGF-I and II (K_d, number of IGF binding sites) are shown in Table 1.

Statistical analysis
Data are presented as the mean ± SE. Statistical comparisons of means were performed by Student’s t test. Comparisons with P > 0.05 were not considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
IGF-dependence of IGFBP-4 proteolytic degradation
As previously described (15), incubation of 2 µl of follicular fluid from preovulatory follicles with 15 ng or 20 ng of IGFBP-4 for 20 h at 37 C led to a complete disappearance of IGFBP-4 as assessed by WLB (Fig. 1A, lane 2 vs. 1), and the generation of a 19-kDa fragment that failed to bind IGFs, detected by immunoblotting (Fig. 1B). Preliminary kinetic experiments showed that in these conditions approximately 40% of IGFBP-4 was degraded after 30 min of incubation. Therefore, we tested the effect of exogenous IGF-I on IGFBP-4 degradation by incubating preovulatory follicles with IGFBP-4 and IGF-I for 1 h. In these conditions, addition of 200 ng of exogenous IGF-I led to an increase of IGFBP-4 degradation (Fig. 2A; Fig. 2B, P < 0.01, n = 10). When the same experiment was performed on early and late atretic follicles, IGF-I did not enhance IGFBP-4 degradation whatever the duration of incubation (data not shown, P > 0.3).

View larger version (31K): [in this window] [in a new window]   Figure 1. Inhibition of IGFBP-4 degradation in preovulatory follicles by IGFBP-2, -3, and monoclonal antibodies raised against IGF-I and IGF-II. A, 2 µl of follicular fluid from ovine preovulatory follicles were incubated for 20 h at 37 C with 15 ng IGFBP-4 (lanes 2, 4, 5, 7–12) and 160 ng IGFBP-2 (lanes 3–5), 20 ng IGFBP-3 (lanes 6–8) or 2 µl monoclonal antibodies against IGF-I and IGF-II (lanes 10 and 11) in a final volume of 10 µl. We added 200 ng of IGF-I or IGF-I and -II to the incubation medium corresponding to the lanes 5, 8, and 11. Lanes 1, 3, 6: Samples stored at -20 C before WLB. Lane 9: Sample incubated with glycerol (antibodies diluent). At the end of the incubation, samples were submitted to WLB as described in Materials and Methods. B, 2 µl of follicular fluid from ovine preovulatory follicles were incubated for 20 h at 37 C with 100 ng IGFBP-4 (lanes 2 and 3) and 160 ng IGFBP-2 (lane 3) in a final volume of 10 µl. Lane 1: Sample stored at -20 C before Immunoblotting. At the end of the incubation, samples were analyzed by Immunoblotting using a specific polyclonal antibody raised against hIGFBP-4 as described in Materials and Methods.

View larger version (20K): [in this window] [in a new window]   Figure 2. Effects of IGF-I on IGFBP-4 degradation. A, 2 µl of follicular fluid from ovine preovulatory follicles were incubated for 30 min at 37 C with IGFBP-4 (lanes 2 and 3) in the absence (lane 2) or presence (lane 3) of 200 ng of IGF-I in a final volume of 10 µl. Lane 1: Sample stored at -20 C before WLB. At the end of the incubation, samples were submitted to WLB as described in Materials and Methods. B, Quantitative analysis of the WLB. , Samples incubated without IGF-I. , Samples incubated with IGF-I. Results are expressed as the mean ± SE of data obtained on 10 preovulatory follicles. **, P < 0.01.

View larger version (19K): [in this window] [in a new window]   Figure 3. Quantitative analysis of the inhibition of IGFBP-4 degradation in preovulatory follicles by IGFBP-2, -3, and antibodies raised against IGF-I and IGF-II. Two microliters of follicular fluid from ovine preovulatory follicles were incubated for 20 h at 37 C with IGFBP-4 (20 ng) and IGFBP-2 (160 ng) or IGFBP-3 (20 ng) or monoclonal antibodies raised against IGF-I and IGF-II (2 µl) in the absence () or presence () of an excess of IGF-I (200 ng) in a final volume of 10 µl. At the end of the incubation, samples were submitted to WLB and quantitative analysis was performed as described in Materials and Methods. Results are expressed as the mean ± SE of data obtained on 4 preovulatory follicles for IGFBP-2 and -3 and on 6 preovulatory follicles for the antibodies. *, P < 0.05; **, P < 0.01.

View larger version (15K): [in this window] [in a new window]   Figure 4. Dose-dependent effects of IGFBP-2 and -3 on IGFBP-4 degradation. Two microliters of follicular fluid from ovine preovulatory follicles were incubated for 20 h at 37 C with IGFBP-4 and increasing concentration of IGFBP-2 (•) or IGFBP-3 () in a final volume of 10 µl. Samples were submitted to WLB and quantitative analysis was performed as described in Materials and Methods. Results are expressed as the mean ± SE of two to three independent experiments and on two to three preovulatory follicles.

View larger version (44K): [in this window] [in a new window]   Figure 5. Dose-dependent effect of IGF-II on IGFBP-2-induced inhibition of IGFBP-4 degradation. Two microliters of follicular fluid from ovine preovulatory follicles were incubated for 20 h at 37 C with IGFBP-4 (lanes 2–8), IGFBP-2 (lanes 3–8) and decreasing concentrations of IGF-II (lanes 4–8). Lane 1: Sample stored at -20 C. At the end of the incubation, samples were submitted to WLB as described in Materials and Methods. Similar results were obtained with IGF-I (data not shown).

View larger version (18K): [in this window] [in a new window]   Figure 6. Dose-dependent effect of IGF-I, IGF-II and LongR3-IGF-I on IGFBP-2-induced inhibition of IGFBP-4 degradation. Two microliters of follicular fluid from ovine preovulatory follicle were incubated for 20 h at 37 C with IGFBP-4 (15 ng), IGFBP-2 (160 ng) and increasing concentration of IGF-I (•), IGF-II () or LongR3 IGF-I (x) in a final volume of 10 µl. At the end of the incubation, samples were submitted to WLB and quantitative analysis was performed as described in Materials and Methods. In this figure, the inhibition of IGFBP-4 degradation by 160 ng of IGFBP-2 in the presence of IGFs was expressed in comparison with inhibition of IGFBP-4 degradation in the absence of IGFs (taken as 100%). Experimental data were expressed as the mean ± SE of two to three independent experiments and on two to three follicles.

View larger version (16K): [in this window] [in a new window]   Figure 7. Dose-dependent effect of N- and C-terminal proteolytic fragments of IGFBP-3 on IGFBP-4 degradation. A, 2 µl of follicular fluid from ovine preovulatory follicles were incubated for 20 h at 37 C with IGFBP-4 (lanes 2–8) and increasing concentration of the IGFBP-3 C-terminal proteolytic fragment (residues 161–264) (lane 3, 8.4 ng; lane 4, 33.6 ng; lanes 5 and 6, 67.2 ng) or the IGFBP-3 N-terminal proteolytic fragment (lane 8, 28.4 ng) in the absence (lanes 2–5, 8) or presence (lane 6) of 10 µg of heparin in a final volume of 10 µl. Lane 7: Sample incubated with 10 µg of heparin alone. Lane 1: sample stored at -20 C. At the end of the incubation, samples were submitted to WLB as described in Materials and Methods. B, Quantitative analysis of the WLB. •, IGFBP-3 C-terminal fragment; , IGFBP-3 N-terminal fragment. Experimental data are expressed as the mean ± SE of three to eight independent experiments and on three to eight follicles.

View larger version (29K): [in this window] [in a new window]   Figure 8. Dose-dependent effect of the IGFBP-5-derived heparin-binding peptide (P5) on IGFBP-4 degradation. Two microliters of follicular fluid from ovine preovulatory follicles were incubated for 20 h at 37 C with IGFBP-4 (lanes 2–15) and decreasing concentrations of P5 peptide (lanes 4–15) in the presence of 10 µg of heparin (lanes 7, 11, 14) or 200 ng of IGF-I (8 12 15 ) in a final volume of 10 µl. Lane 3: Sample incubated with 10 µg of heparin alone. Lane 1: Sample stored at -20 C. At the end of the incubation, samples were submitted to WLB as described in Materials and Methods.

View larger version (18K): [in this window] [in a new window]   Figure 9. Dose-dependent effect of P3, P5, CTGF247–260, HIP and VN341–355 peptides on IGFBP-4 degradation. Two microliters of follicular fluid from ovine preovulatory follicles were incubated for 20 h at 37 C with IGFBP-4 (20 ng) and increasing concentration of P3 (•), P5 (), CTGF247–260 (x), HIP () or VN341–355 () peptides in a final volume of 10 µl. At the end of the incubation, samples were submitted to WLB and quantitative analysis was performed as described in Materials and Methods. Experimental data are expressed as the mean ± SE of two to seven independent experiments and on two to seven follicles.

View larger version (26K): [in this window] [in a new window]   Figure 10. Inhibition of IGFBP-4 degradation in preovulatory follicles by wild- type nonglycosylated IGFBP-3 (NG IGFBP-3) or nonglycosylated IGFBP-3 mutated on its heparin-binding domain (NG IGFBP-3 mHBD) A, 2 µl of follicular fluid from ovine preovulatory follicles were incubated for 20 h at 37 C with 20 ng IGFBP-4 (lanes 2–6) and 50 ng NG IGFBP-3 (lanes 3 and 4) or 50 ng NG IGFBP-3 mHBD (lanes 5 and 6) in a final volume of 10 µl. We added 200 to 500 ng of IGF-I to the incubation medium corresponding to the lanes 4 and 6. Lane 1: Sample stored at -20 C before WLB. At the end of the incubation, samples were submitted to WLB as described in Materials and Methods. B, Quantitative analysis of the WLB. , Samples incubated without IGF-I. , Samples incubated with IGF-I. Results are expressed as the mean ± SE of data obtained on six preovulatory follicles. a vs. b, P < 0.0001; a vs. c, P = 0.0005; c vs. d, means were not significantly different.

View larger version (16K): [in this window] [in a new window]   Figure 11. Binding of intrafollicular IGFBP-3 to heparin-Sepharose beads. We incubated 2.5 µl of follicular fluid from ovine preovulatory, early atretic, or late atretic follicles with heparin-Sepharose beads with or without 200 ng of IGF-I and 500 ng of IGFBP-2 in a final volume of 20 µl. After an overnight incubation at 37 C, the samples were centrifuged for 1 min at 15,000 x g. Then, pellets and supernatants were submitted to WLB as described in Materials and Methods. Experimental data are expressed as the mean ± SE of five to six independent experiments and on five to six follicles. Means were not significantly different.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Recently, Fowlkes et al. have shown that the C-terminal fragment of IGFBP-3, as well as peptides derived from the C-terminal heparin-binding domain of IGFBP-3, -5, or -6, are able to inhibit IGFBP-4 degradation in conditioned medium of murine bone cells (6, 11). In the present work, we have tested whether heparin-binding domains from IGFBP-3 and -5 were able to inhibit the IGFBP-4-degrading protease(s) in ovine follicles. Firstly, the C-terminal, but not the N-terminal, IGFBP-3 fragment as well as peptides derived from the C-terminal heparin-binding domains from IGFBP-3 (P3) and -5 (P5) dose dependently inhibited IGFBP-4 proteolysis, this inhibition being not clearly reversed by IGFs. A heparin-binding peptide derived from CTGF (CTGF^247–260) also dose dependently inhibited IGFBP-4 degradation. In the same way, heparin binding domain-containing peptides from HIP and vitronectin (VN^341–355, VN^357–370), but not from p36 subunit of annexin II tetramer, were able to inhibit the IGFBP-4 degradation. The ability of these peptides to inhibit IGFBP-4 degradation was not strictly correlated with their ability to bind heparin. Indeed, the peptide derived from the p36 subunit of annexin II tetramer, although able to bind heparin with high affinity (23), was not able to inhibit IGFBP-4 degradation. Furthermore, the vitronectin VN^341–355 peptide was more efficient than VN^357–370 for inhibition of IGFBP-4 degradation, whereas the latter has a higher affinity for heparin than the former (24). Overall, these data suggest some specificity in the inhibition of IGFBP-4 degradation by the IGFBP-3 and -5 C-terminal-containing heparin-binding domain. Interestingly, the P5 peptide, as well as the vitronectin peptide (VN^357–370), were recently shown to directly modulate plasminogen activation to plasmin (24, 25). Furthermore, IGFBP-3 was shown to bind plasminogen via its heparin-binding domain (26). Finally, in ovine follicle, IGFBP-3 mutated in the heparin-binding region (IGFBP-3mHBD) only slightly inhibited IGFBP-4 degradation in comparison with wild-type IGFBP-3. So, from these results, one could suggest that in ovine late atretic follicles, in vivo, the high proteolytic degradation of IGFBP-3, likely generating the 161–264 C-terminal fragment, as well as the high IGFBP-5 expression would participate in the inhibition of IGFBP-4 degradation by directly interacting with the IGFBP-4-degrading protease(s).

Our results suggest two mechanisms of regulation of IGFBP-4 proteolytic degradation in ovine preovulatory follicles. Firstly, the IGF dependence is suggested by the fact that, in the presence of intrafollicular endogenous IGFBP-3 (likely complexed to endogenous ALS and IGF-I) and exogenous added IGFBP-4, IGF-I enhances, whereas monoclonal antibodies against IGF-I and -II inhibit IGFBP-4 degradation. In these conditions, IGF dependence may be due to conformational changes of IGFBP-4 upon binding to its ligand allowing the protease to acceed to the cleavage site, as suggest by others (8, 9, 27). Arguing against this hypothesis is the fact that, in our in vitro conditions, IGFBP-4 proteolytic degradation by plasmin was not enhanced by the addition of IGF-I (data not shown). One can note that the intensity of IGF-enhancement of IGFBP-4 degradation in follicular fluid is low (54.5% of degradation vs. 40% without exogenous IGF-I, and only 10% of inhibition of degradation with monoclonal antibodies) in comparison with similar in vitro experiments (>90% of degradation of IGFBP-4 after addition of IGF-I (3, 7) and is observed only by incubating follicular fluid with IGFBP-4 during 30 min. This is likely due to the fact that in preovulatory follicles, levels of endogenous IGFs (approximately 1 µg/ml of both IGF-I and -II) as well as the IGFs/IGFBPs ratio are much higher than in most conditioned-cell culture medium in vitro. We suspect that this relatively large excess in endogenous IGFs could "mask" the effect of exogenous added IGFs. Secondly, the inhibition of IGFBP-4 degradation by IGFBP-heparin-binding domains is supported by the strong inhibition of IGFBP-4 proteolytic degradation by the IGFBP-3 C-terminal proteolytic fragment (75%) and by heparin-binding domain-containing peptides derived from IGFBP-3, -5, -RP-1 or other proteins different from IGFBPs (60–75%). Furthermore, IGFBP-3 mutated on its heparin-binding domain only slighly (10%, i.e. the level of inhibition by the antibodies against IGF-I and -II) inhibited the IGFBP-4 proteolytic degradation in comparison with intact IGFBP-3. Based on the experiment with mutated IGFBP-3, the reversion of IGFBP-3 effect by IGFs may be due to the ability of ligand to impair the interaction of IGFBP-3 with IGFBP-4 protease by masking its heparin-binding domain, as suggest by Fowlkes et al. (6). However, in our conditions, IGF-I had no effect on the ability of recombinant IGFBP-3 [contrasting with data obtained by Arai et al. (28)] or endogenous IGFBP-3 from preovulatory follicular fluid to bind to heparin-Sepharose beads. To explain this discrepancy in our experimental conditions, one could speculate that the type of interaction of IGFBP-3 heparin-binding domain with intrafollicular IGFBP-4 protease is different (affinity, conformational change . . .) than with heparin, rendering IGF-I able to modulate the interaction of IGFBP-3 with protease but not with heparin. Of note, Campbell et al. (26) have shown that the binding of IGFBP-3 to plasminogen was not impaired by IGF-I.

Anyway, addition of exogenous IGFBP-3 to preovulatory follicular fluid is only experimental because such an increase in levels of intrafollicular free IGFBP-3 does not occur in vivo and is not representative of a role of intrafollicular endogenous IGFBP-3 on IGFBP-4 degradation. In particular, we have shown that there was no difference in the ability of endogenous (native) IGFBP-3 from healthy preovulatory follicles in comparison with atretic follicles (characterized by high levels of IGFBP-2 and -5 and low IGF bioavailability) to bind to heparin-Sepharose beads. Furthermore, Cwyfan Hughes et al. (29) have shown by chromatography that in human atretic follicles, there was no more IGFBP-3 within the small (likely IGF-free) complex, susceptible to interact with IGFBP-4 protease, compared with dominant follicles, suggesting that endogenous native IGFBP-3 does not play a major role in the modulation of IGFBP-4 degradation in vivo. Moreover, the inhibition of IGFBP-4 degradation by IGFBP-2 may be due to both sequestration of IGFs and direct interaction with protease. In particular, IGFBP-2 has been shown to bind heparin, although further studies are needed to identify its heparin-binding domain (17, 28, 30). One may hypothesize that the lesser efficiency of IGFBP-2 to inhibit IGFBP-4 degradation, in comparison with IGFBP-3, is partly due to its lower affinity to heparin (17).

Finally, as an alternative hypothesis to explain the IGF dependence of IGFBP-4 degradation, Conover et al. (3) proposed that IGFs would directly activate IGFBP-4 proteinase(s). Of note is the recent cloning of a protein called L56 protease, containing an IGFBP-related N-terminal domain and a C-terminal domain related to serine proteases (31, 32, 33). Experiments are necessary to test the ability of IGFs to modulate the activity of L56 protease.

IGF-I and IGF-II have been shown to play a pivotal role in proliferation and differentiation of granulosa cells, synergizing with gonadotropins (34, 35). Moreover, IGFBP-4 has been shown to inhibit IGF action in a variety of tissues (36, 37, 38). In particular, protease-resistant but not -sensitive form of IGFBP-4 inhibits IGF actions (39, 40, 41). In the ovary, it has been shown that IGFBP-4, but not IGFBP-4 proteolytic fragments, inhibits IGF-I-induced oestradiol production by human granulosa cells in vitro (42, 43). So, changes in intrafollicular IGFBP-4 levels during growth and atresia of ovarian follicles might participate in the change in intrafollicular IGF-bioavailability, as suggested by in vitro experiments (13).

In conclusion, we have shown that in vivo, particularly in ovine ovarian follicles, IGFBP-4 proteolytic degradation is both IGF-dependent and inhibited by IGFBP-3 C-terminal domain as well as by heparin-binding domain-containing peptides. These data suggest that in early atretic follicles, the increase in IGFBP-2 expression participates in the decrease in IGFBP-4 degradation, the mechanism remaining to be elucidated. Furthermore, in late atretic follicles, the increase in the levels of C-terminal IGFBP-3 proteolytic fragments, generated by IGFBP-3 degradation, as well as the increase in IGFBP-5 expression, would strengthen the inhibition of IGFBP-4 degradation. This inhibition might be partly mediated by direct interaction of the heparin-binding domain within the C-terminal region from IGFBP-3 and -5 with IGFBP-4 proteinase(s).

	Acknowledgments

 
We acknowledge Drs. K. T. Preissner, D. D. Carson, and D. M. Waisman for providing the vitronectin peptides, the p36 subunit of annexin II tetramer peptide and the HIP, respectively. We wish also acknowledge Dr. J. Closset for donating glycosylated hIGFBP-3 and monoclonal antibody against IGF-I. We thank Dr. D. R. Powell for providing IGFBP-3mHBD. We also thank Dr. D. Monniaux for helpful discussion and C. Pisselet for technical assistance. We are grateful to A. Beguey for the photographic work.

	Footnotes

 
¹ This work was supported by Grant 32-46808.96 from the Swiss National Science Foundation.

Received December 30, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Kirk Neely E, Rosenfeld RG 1992 Insulin-like growth factors (IGFs) reduce IGF-binding protein-4 (IGFBP-4) concentration and stimulate IGFBP-3 independently of IGF receptors in human fibroblasts and epidermal cells. Endocrinology 130:985–993[Abstract]
2. Fowlkes J, Freemark M 1992 Evidence for a novel insulin-like growth factor (IGF)-dependent protease regulating IGF-binding protein-4 in dermal fibroblasts. Endocrinology 131:2071–2076[Abstract]
3. Conover CA, Kiefer MC, Zapf J 1993 Posttranslational regulation of insulin-like growth factor binding protein 4 in normal and transformed human fibroblasts. J Clin Invest 91:1129–1137
4. Donnelly MJ, Holly JMP 1996 The role of IGFBP-3 in the regulation of IGFBP-4 proteolysis. J Endocrinol 149:R1–R7
5. Noll K, Wegmann BR, Havemann K, Jaques G 1996 Insulin-like growth factors stimulate the release of Insulin-like growth factor binding protein-3 (IGFBP-3) and degradation of IGFBP-4 in nonsmall cell lung cancer cell lines. J Clin Endocrinol Metab 81:2653–2662[Abstract]
6. Fowlkes JL, Serra DM, Rosenberg CK, Thrailkill KM 1995 Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) functions as an IGF-reversible inhibitor of IGFBP-4 proteolysis. J Biol Chem 270:27481–27488[Abstract/Free Full Text]
7. Durham SK, Kiefer MC, Lawrence Riggs B, Conover CA 1994 Regulation of Insulin-like growth factor binding protein 4 by a specific insulin-like growth factor binding protein 4 proteinase in normal human osteoblast-like cells: implications in bone cell physiology. J Bone Miner Res 9:111–117[Medline]
8. Myers SE, To Cheung P, Handwerger S, Chernausek SD 1993 Insulin-like growth factor-I (IGF-I) enhanced proteolysis of IGF-binding protein-4 in conditioned medium from primary cultures of human decidua: independence from IGF receptor binding. Endocrinology 133:1525–1531[Abstract]
9. Irwin JC, Dsupin BA, Guidice LC 1995 Regulation of insulin-like growth factor-binding protein-4 in human endometrial stromal cell cultures:evidence for ligand-induced proteolysis. J Clin Endocrinol Metab 80:619–626[Abstract]
10. Murakami Y, Murakami K, Shimasaki S, Erickson GFInsulin-like growth factor binding proteins (IGFBPs) reversibly inhibit granulosa-derived IGFBP- 4 protease activity. Proceeding of the 4th International Symposium on Insulin-Like Growth Factors, Tokyo, 1997, p 126 (Abstract B53)
11. Fowlkes JL, Thrailkill KM, George-Nascimento C, Rosenberg CK, Serra DM 1997 Heparin-binding, highly basic regions within the thyroglobulin type-1 repeat of insulin-like growth factor (IGF)-binding protein-3 (IGFBPs) -3, -5, and -6 inhibit IGFBP-4 degradation. Endocrinology 138:2280–2285[Abstract/Free Full Text]
12. Cwyfan Hughes SC, Mason HD, Franks S, Holly JMP 1997 Modulation of Insulin-like growth factor binding proteins by follicle size in the human ovary. J Endocrinol 154:35–43[Abstract]
13. Monget P, Monniaux D, Pisselet C, Durand P 1993 Changes in insulin-like growth factor-I (IGF-I), IGF-II, and their binding proteins during growth and atresia of ovine ovarian follicles. Endocrinology 132:1438–1446[Abstract]
14. Besnard N, Pisselet C, Monniaux D, Locatelli A, Benne F, Gasser F, Hatey F, Monget P 1996 Expression of insulin-like growth factor-binding protein-2, -4, and -5 messenger ribonucleic acids in the ovine ovary: localization and changes during growth and atresia of antral follicles. Biol Reprod 55:1356–1367[Abstract]
15. Besnard N, Pisselet C, Zapf J, Hornebeck W, Monniaux D, Monget P 1996 Proteolytic activity is involved in changes in intrafollicular insulin-like growth factor-binding protein levels during growth and atresia of ovine ovarian follicles. Endocrinology 137:1599–1607[Abstract]
16. Kiefer MC, Schmid C, Waldvogel M, Schläpfer I, Futo E, Masiarz FR, Green K, Barr PJ, Zapf J 1992 Characterization of recombinant human Insulin-like growth factor binding proteins 4, 5, and 6 produced in yeast. J Biol Chem 267:12692–12699[Abstract/Free Full Text]
17. Booth BA, Boes M, Andress DL, Dake BL, Kiefer MC, Maack C, Linhardt RJ, Bar K, Cadwell EEO, Weiler J, Bar RS 1995 IGFBP-3 and IGFBP-5 association with endothelial cells: role of C-terminal heparin-binding domain. Growth Regul 5:1–17[Medline]
18. Booth BA, Boes M, Dake BL, Linhardt RJ, Cadwell EEO, Weiler JM, Bar RS 1996 Structure-fonction relationships in the heparin-binding C-terminal region of insulin-like growth factor binding protein-3. Growth Regul 6:206–213[Medline]
19. Brigstock DR, Steffen CL, Kim GY, Vegunta RK, Diehl JR, Harding PA 1997 Purification and characterization of novel heparin-binding growth factors in uterine secretory fluids. J Biol Chem 272:20275–20282[Abstract/Free Full Text]
20. Lalou C, Lassarre C, Binoux M 1996 A proteolytic fragment of insulin-like growth factor (IGF) binding protein-3 that fails to bind IGFs inhibits the mitogenic effects of IGF-I and insulin. Endocrinology 137:3206–3212[Abstract]
21. Lalou C, Sawamura S, Segovia B, Binoux M 1997 Proteolytic fragments of insulin-like growth factor binding protein-3: N-terminal sequences and relationships between structure and biological activity. C R Acad Sci Paris 320:621–628
22. Hossenlopp P, Seurin D, Segovia-Quinson B, Hardouin S, Binoux M 1986 Analysis of serum insulin-like growth factors binding proteins using western blotting: use of the method for titration of the binding proteins and competitive binding studies. Anal Biochem 154:138–143[CrossRef][Medline]
23. Kassam G, Manro A, Louie P, Braat CE, Fitzpatrick SL, Waisman DM 1997 Characterization of the heparin binding properties of annexin II tetramer. J Biol Chem 272:15093–15100[Abstract/Free Full Text]
24. Kost C, Stüber W, Ehrlich HJ, Pannekoek H, Preissner KT 1992 Mapping of binding sites for heparin, plasminogen activator inhibitor-1, and plasminogen to vitronectin’s heparin-binding region reveals a novel vitronectin-dependent feedback mechanism for the control of plasmin formation. J Biol Chem 267:12098–12105[Abstract/Free Full Text]
25. Campbell PG, Andress DL 1997 Plasmin degradation of insulin-like growth factor-binding protein-5 (IGFBP-5): regulation by IGFBP-5-(201–218). Am J Physiol 273:E996–E1004
26. Campbell PG, Durham SK, Suwanichkul A, Hayes JD, Powell DR 1998 Plasminogen binds the heparin-binding domain of Insulin-like Growth Factor-Binding Protein-3. Am J Physiol 275 (Endocrinology Metab 38):E321–E331
27. Iwashita M, Kudo Y, Takeda Y 1998 Effect of follicle stimulating hormone and insulin-like growth factors on proteolysis of insulin-like growth factor-binding protein-4 in human granulosa cells. Mol Hum Reprod 4:401–405[Abstract/Free Full Text]
28. Arai T, Busby W, Clemmons DR 1996 Binding of insulin-like growth factor (IGF) I or II to IGF-binding protein-2 enables it to bind to heparin and extracellular matrix. Endocrinology 137:4571–4575[Abstract]
29. Cwyfan Hughes SC, Mason HD, Franks S, Holly JMP 1997 The insulin-like growth factors (IGFs) in follicular fluid are predominantly bound in the ternary complex. J Endocrinol 155:R1–R4
30. Russo VC, Bach LA, Fosang AJ, Baker NL, Werther GA 1997 Insulin-like growth factors-binding protein-2 binds to cell surface proteoglycans in the rat brain olfactory bulb. Endocrinology 138:4858–4867[Abstract/Free Full Text]
31. Zumbrunn J, Trueb B 1996 Primary structure of a putative serine protease specific for IGF-binding proteins. FEBS Lett 398:187–192[CrossRef][Medline]
32. Zumbrunn J, Trueb B 1997 Localization of the gene for a serine protease with IGF-binding domain (PRSS11) to human chromosome 10q25.3-q26.2. Genomics 45:461–462[CrossRef][Medline]
33. Hou J, Conover CA, Smeekens SP Selective cleavage of insulin-like growth factor binding protein-5 by a novel human stress response pathway serine protease: identification, expression, and functional characterization. Program of the 80th Annual Meeting of The Endocrine Society, New Orleans, Louisiana, 1998 (Abstract ORO6–5)
34. Guidice LC 1992 Insulin-like growth factors and ovarian follicular development. Endocr Rev 13:641–669[CrossRef][Medline]
35. Monget P, Besnard N, Huet C, Pisselet C, Benne F, Gasser F, Hatey F, Mulsant P, Tomanek M, Monniaux D 1996 Role of the IGF system in the sheep ovary. Front Endocrinol 19:85–99
36. Mohan S, Bautista CM, Wergedal J, Baylink DJ 1989 Isolation of an inhibitory insulin-like growth factor (IGF) binding protein from bone cell-conditioned medium: a potential local regulator of IGF action. Proc Natl Acad Sci USA 86:8338–8342[Abstract/Free Full Text]
37. Duan C, Clemmons D R 1998 Differential expression and biological effects of the insulin-like growth factor-binding protein-4 and -5 in vascular smooth muscle cells. J Biol Chem 273:16836–16842[Abstract/Free Full Text]
38. Damon SE, Maddison L, Ware JL, Plymate SR 1998 Overexpression of an inhibitory insulin-like growth factor-binding protein (IGFBP), IGFBP-4, delays onset of prostate tumor formation. Endocrinology 139:3456–3464[Abstract/Free Full Text]
39. Conover CA, Durham SK, Zapf J, Masiarz FR, Kiefer MC 1995 Cleavage analysis of insulin-like growth factor (IGF)-dependent IGF-binding protein-4 proteolysis and expression of protease-resistant IGF-binding protein-4 mutants. J Biol Chem 270:4395–4400[Abstract/Free Full Text]
40. Chernausek SD, Smith CE, Duffin KL, Busby WH, Wright G, Clemmons DR 1995 Proteolytic cleavage of insulin-like growth factor binding protein 4 (IGFBP-4). J Biol Chem 270:11377–11382[Abstract/Free Full Text]
41. Rees C, Clemmons DR, Horvitz GD, Clarke JB, Busby WH 1998 A protease-resistant form of insulin-like growth factor (IGF) binding protein 4 inhibits IGF-I actions. Endocrinology 139:4182–4188[Abstract/Free Full Text]
42. Iwashita M, Kudo Y, Yoshimura Y, Adashi T, Katayama E, Takeda Y 1996 Physiological role of insulin-like growth factor-binding protein-4 in human folliculogenesis. Horm Res [Suppl 1] 46:31–36
43. Mason HD, Cwyfan-Hughes S, Holly JMP, Franks S 1998 Potent inhibition of human ovarian steroidogenesis by insulin-like growth factor binding protein-4 (IGFBP-4). J Clin Endocrinol Metab 83:284–286[Abstract/Free Full Text]

This article has been cited by other articles:

				L. J. Spicer Proteolytic Degradation of Insulin-Like Growth Factor Binding Proteins by Ovarian Follicles: A Control Mechanism for Selection of Dominant Follicles Biol Reprod, May 1, 2004; 70(5): 1223 - 1230. [Abstract] [Full Text] [PDF]

				A. J. Roberts and S. E. Echternkamp Insulin-like growth factor binding proteins in granulosa and thecal cells from bovine ovarian follicles at different stages of development J Anim Sci, November 1, 2003; 81(11): 2826 - 2839. [Abstract] [Full Text] [PDF]

				P. Monget, S. Mazerbourg, T. Delpuech, M.-C. Maurel, S. Maniere, J. Zapf, G. Lalmanach, C. Oxvig, and M. T. Overgaard Pregnancy-Associated Plasma Protein-A Is Involved in Insulin-Like Growth Factor Binding Protein-2 (IGFBP-2) Proteolytic Degradation in Bovine and Porcine Preovulatory Follicles: Identification of Cleavage Site and Characterization of IGFBP-2 Degradation Biol Reprod, January 1, 2003; 68(1): 77 - 86. [Abstract] [Full Text] [PDF]

				W. A. Price, B. M. Moats-Staats, and A. D. Stiles Pro- and Anti-inflammatory Cytokines Regulate Insulin-like Growth Factor Binding Protein Production by Fetal Rat Lung Fibroblasts Am. J. Respir. Cell Mol. Biol., March 1, 2002; 26(3): 283 - 289. [Abstract] [Full Text] [PDF]

				S. Mazerbourg, M. T. Overgaard, C. Oxvig, M. Christiansen, C. A. Conover, I. Laurendeau, M. Vidaud, G. Tosser-Klopp, J. Zapf, and P. Monget Pregnancy-Associated Plasma Protein-A (PAPP-A) in Ovine, Bovine, Porcine, and Equine Ovarian Follicles: Involvement in IGF Binding Protein-4 Proteolytic Degradation and mRNA Expression During Follicular Development Endocrinology, December 1, 2001; 142(12): 5243 - 5253. [Abstract] [Full Text] [PDF]

				G.M. Rivera, Y.A. Chandrasekher, A.C.O. Evans, L.C. Giudice, and J.E. Fortune A Potential Role for Insulin-Like Growth Factor Binding Protein-4 Proteolysis in the Establishment of Ovarian Follicular Dominance in Cattle Biol Reprod, July 1, 2001; 65(1): 102 - 111. [Abstract] [Full Text] [PDF]

				G.M. Rivera and J.E. Fortune Development of Codominant Follicles in Cattle Is Associated with a Follicle-Stimulating Hormone-Dependent Insulin-Like Growth Factor Binding Protein-4 Protease Biol Reprod, July 1, 2001; 65(1): 112 - 118. [Abstract] [Full Text] [PDF]

				L. C. Giudice Insulin-like Growth Factor Family in Graafian Follicle Development and Function Reproductive Sciences, January 1, 2001; 8(1_suppl): S26 - S29. [Abstract] [PDF]

D. Monniaux, P. Monget, C. Pisselet, J. Fontaine, and J.M. Elsen Consequences