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Ovary |
a INRA, UMR Physiologie de la Reproduction et des Comportements 6073, INRA/CNRS/Université, 37380 Nouzilly, France
b The Department of Medicine, University Hospital, Zürich, Switzerland
c Laboratoire d'Enzymologie et Biochimie des Protéines, INSERM EMI-U 00-10, Université François Rabelais, 37032 Tours Cedex, France
d Department of Molecular and Structural Biology, University of Aarhus, 8000 Aarhus C, Denmark
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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follicle, follicular development, growth factors, ovary
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During follicular growth, the decrease in IGFBP-2 levels is partly due to a decrease in mRNA expression by follicular cells in ovine, bovine, and porcine ovaries [17–20]. In addition, we have shown in ewes and sows that this decrease is also partly due to an increase in IGFBP-2 proteolytic degradation [2, 3]. The aim of the present work was 1) to confirm the presence of proteolytic activity that degrades IGFBP-2 in porcine preovulatory follicles and to test the presence of this activity in bovine preovulatory follicles, 2) to test the hypothesis that PAPP-A is involved in IGFBP-2 degradation in preovulatory follicles of both species, and 3) to characterize IGFBP-2 degradation by PAPP-A in vitro.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Altrenogest was obtained from Roussel-Uclaf (Romainville, France). Norgestomet implant was obtained from Intervet (Angers, France). Estrumate was obtained from Pitman-Moore (Meaux, France). IGF-I and IGF-II were generous gifts from Drs. H. H. Peter and A. Hinnen (Ciba-Geigy, Basel, Switzerland). Recombinant human IGFBP-4 was expressed in yeast and purified as previously described [21]. Recombinant bovine IGFBP-2 was from GroPep (Adelaide, Australia). Recombinant human PAPP-A (rhPAPP-A) was expressed in HEK 293 cells as previously described [22]. The 18-amino acid P5 peptide (201RKGFYKRKQCRPSKGRKR218) from human C-terminal domain of IGFBP-5 was purchased from Agrobio (Orléans, France). The 18-amino acid P3 peptide (215KKGFYKKKQCRPSKGRKR232) from human IGFBP-3 was a generous gift from R.S. Bar (Iowa City, IA). The synthetic peptide containing the heparin-binding domain of human heparin/heparan sulfate-interacting protein (HIP; CRPKAKAKAKAKDQTK) was kindly provided by Dr. D.D. Carson (Houston, TX). Synthetic peptides derived from the heparin-binding domain of human vitronectin, VN1 (341APRPSLAKKQRFRHR355) and VN3 (357RKGYRSQRGHSRGR370), were kindly provided by Dr. K.T. Preissner (Bad Nauheim, Germany). Rabbit polyclonal antiserum against human IGFBP-4 was purchased from Ubi (Lake Placid, NY), and rabbit polyclonal antiserum against bovine IGFBP-2 was generously donated by Dr. Jean Closset (University of Liège, Belgium). This antibody showed less than 1% cross-reactivity with IGFBP-1, IGFBP-3, IGFBP-4, and IGFBP-5 (data not shown). Anti-rabbit immunoglobulin G (IgG) antibodies coupled to horseradish peroxidase were purchased from DAKO (Trappes, France). Rabbit polyclonal anti-PAPP-A/proMBP was raised against PAPP-A/proMBP purified from pregnancy serum [13]. Nitrocellulose membranes were purchased from Schleicher & Schuell (Ecquevilly, France), and the enhanced chemiluminescence detection system for immunoblots was obtained from Amersham (Les Ulis, France).
Animals and Treatment
All procedures were approved by the French agricultural and scientific research agencies (approval A37801) and were conducted in accordance with the Guidelines for Care and Use of Agricultural Animals in Agricultural Research and Teaching.
Sows Six cyclic adult Pietrain x Large-White sows were synchronized by daily feeding of 20 mg of Altrenogest for 15 days as previously described [3]. Ovaries were carefully dissected and collected by ovariectomy 120 h after progestin withdrawal (the end of follicular phase, before the LH surge). Ten to 15 preovulatory follicles (7- to 8-mm diameter) per sow were recovered.
Heifers
Estrus cycles were synchronized in three Holstein heifers with a Norgestomet implant for 10 days as previously described [15]. Twelve days after detection of estrus (Day 0 = day of estrus), heifers were treated with two injections of 2000 IU eCG and two i.m. injections of 25 mg of prostaglandin F2
analogue (Estrumate) and were killed at Day 14, before the LH surge. Approximately 10 preovulatory follicles (15- to 20-mm diameter) per heifer were recovered.
Classification of bovine and porcine follicles To verify that recovered follicles were healthy, follicular fluids were aspired by puncture and individually stored at -20°C. Each follicle was then slit open in B2 medium, and a suspension of granulosa cells was prepared and stored at -70°C as described previously [23]. For each suspension, granulosa cells were smeared onto histological slides, fixed in methanol-formaldehyde-acetic acid (80:15:5), and subsequently stained with Feulgen. The quality of each follicle was assessed by microscopic examination of smears using classical histological criteria as previously described [23]. Furthermore, follicular fluids were analyzed individually by Western ligand blotting (WLB). In particular, as previously described [24–27], porcine and bovine large follicles were characterized by the presence of IGFBP-3; by the absence of IGFBP-2, IGFBP-4, and IGFBP-5; and by a high proteolytic activity that degrades IGFBP-4 in follicular fluid were considered as preovulatory.
Cleavage of IGFBP-2 and IGFBP-4 by follicular fluid and by rhPAPP-A Two microliters of porcine or bovine preovulatory follicular fluids or different concentrations of rhPAPP-A were incubated in a solution of 20 mM Tris (pH 7.6) containing 137 mM NaCl (TBS) and 0.1% BSA with IGFBP-2 or IGFBP-4, with or without IGF-I or IGF-II for 20 h at 37°C (final volume, 10 µl). In some experiments, the synthetic peptide P5 was added to the incubation medium. At the end of the incubation, samples were analyzed by WLB or immunoblotting.
Western ligand blotting IGF-II was iodinated by the iodogen method and purified by Sephadex G-50 chromatography using a 0.1 M ammonium acetate elution buffer. WLB was performed as previously described [6, 7, 25]. 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/L Tween-20, 200 mg/L protamine sulfate, 200 mg/L NaNO3, and 3.72 g/L EDTA (pH 7.4). Afterward, filters were washed with PBS containing 0.1% Tween-20, air-dried, and exposed to Hyperfilm MP (Amersham Corp., 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 1/10 000) or IGFBP-4 (final dilution 1/1000). Once washed in TBS containing 1% NFDM and 0.2% Tween-20, nitrocellulose filters were incubated for 1 h at 37°C with an anti-rabbit or anti-mouse IgG antibody coupled to horseradish peroxidase (final dilution 1/4000). After washing, the signal was revealed by chemiluminescence detection.
Immunoneutralization of IGFBP-2 proteolytic degradation For immunoneutralization, 2 µl of preovulatory follicular fluid were incubated in TBS containing 0.1% BSA, 75 ng IGFBP-2, and different dilutions of rabbit polyclonal antibody raised against PAPP-A, or nonspecific rabbit IgG, or glycerol for 20 h at 37°C (final volume, 10 µl), as previously described [15]. At the end of the incubation, samples were analyzed by WLB and immunoblotting.
Determination of the cleavage site in IGFBP-2 by PAPP-A Approximately 10 µg of bovine IGFBP-2 were incubated for 24 h in the presence of equimolar amounts of IGF-II and purified rhPAPP-A (approximately 0.1 µg immobilized to Protein G agarose; Life Technologies, Cergy Pontoise, France) with monoclonal antibody as previously described [28]. The entire reaction mixture, following removal of Protein G agarose-bound PAPP-A, was subjected directly to Edman degradation to determine all N-terminal sequences present [29].
Biotinylation of P5 Peptide by a Thiol-Specific Reagent
Specific labeling of the free sulfhydryl group of P5 peptide (RKGFYKRKQCRPSKGRKR) was performed by using PEO-maleimide-activated biotin from Pierce (Rockford, IL). Briefly, P5 peptide (450 µM) was incubated with PEO-maleimide-activated biotin (10 mM) for 20 h at 20°C under gentle agitation. Unlabeled P5 and excess chemical reagents were removed from the biotinylated P5 (Biot-P5) peptide and obtained by fractionation on a reverse phase chromatography (C18 Brownlee ODS32 column) using a 45-min linear (0%–60%) gradient of acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 1 ml/min. The homogeneity of purified Biot-P5 was checked by analytical reverse phase-high performance liquid chromatography (Brownlee C18 OD 300 column) using Spectacle software (ThermoQuest, Les Ulis, France) to analyze the elution profile [30]. Biot-P5 peptide was lyophilized before use (biotinylation yield = 72%). Of major interest, introduction of a long-chain, hydrophilic polyethylene oxide spacer arm (29.1 Å) between the substituted cysteinyl group of P5 and biotin leads to a high water solubility and avoids problems of steric hindrance during formation of the avidin/biotin complex.
BIAcore Analysis of the Interaction Between rhPAPP-A and P5 Peptide
Interaction between rhPAPP-A and P5 peptide was measured by surface plasmon resonance (SPR) using a BIAcore system (BIAcore, Uppsala, Sweden).
Immobilization of biotinylated P5 peptide to a streptavidin sensorchip Biot-P5 peptide was immobilized on a streptavidin-coated sensorchip (SA5) supplied by BIAcore. Before immobilization, the biosensor surface was washed three times by injecting 5 µl of 1 M NaCl and 50 mM NaOH as indicated by the manufacturer. Immobilization was performed as described [31] with a few modifications by using 10 mM Tris-HCl pH 7 as the running buffer. Biot-P5 peptide (0.2 mM in 10 mM Tris HCl, pH 7) was injected on streptavidin-coated dextran at a continuous flow rate of 5 µl/min. Injection was stopped when the biosensor surface was coupled to 500 response units (RUs).
Kinetic measurements of rhPAPP-A-P5 peptide interaction All experiments were performed at a flow rate of 5 µl/min in 10 mM Tris pH 7 as a running and diluting buffer at 23°C. Various concentrations of rhPAPP-A (from 0.3 to 5 nM) were injected on immobilized Biot-P5 peptide for 7 min (35 µl) and then washed out for 10 min before regeneration. The biosensor surface was regenerated by a 1-min injection of 0.5% SDS (5 µl); if necessary, a second injection was performed. A systematic control medium devoid of rhPAPP-A and diluted as rhPAPP-A medium was injected on the same surface for nonspecific binding subtraction. The specificity of the interaction was investigated using an anti-rhPAPP-A polyclonal antibody (6.6 nM in 10 mM Tris pH 7) that was injected just after the 10-min dissociation step.
Calculation of kinetic constants Kinetic data were analyzed using BIA Evaluation software (version 2.2.4). This BIAcore incorporated software allowed us to calculate kinetic constants (kon and koff) with standard deviations and a statistical validation test (chi-square). Before analysis, all binding curves were corrected for medium background and bulk refractive index by subtracting the reference curve obtained with the control medium.
Quantification of Western Ligand Binding
WLB was quantified with a PhosphorImager (Storm/Image Quant, Molecular Dynamics, Sunnyvale, CA). Quantification was performed as previously described [6]. Briefly, the amount of radiolabeled IGF-II bound to each IGFBP was expressed as the integrated optical density (IOD) of the corresponding band expressed in arbitrary units. The extent of IGFBP-2 and IGFBP-4 degradation by follicular fluid or rhPAPP-A was determined as the difference I-20-I37, where I-20 is the IOD of the IGFBP band from samples not incubated, and I37 is the IOD of the IGFBP band from samples incubated at 37°C. The percentage of IGFBP proteolysis inhibition was expressed as the ratio [(I-I37) x 100]:(I-20-I37), where I is the IOD of the IGFBP band from samples incubated at 37°C in the presence of PAPP-A polyclonal antibody or synthetic peptides.
Statistical Analysis
All experimental data are expressed as means ± SEM. Statistical comparisons of means were made using one-way ANOVA for the effects of concentrations of antibody against PAPP-A and P5 peptide on IGFBP-2 and IGFBP-4 levels quantified from the blots. One-way ANOVA followed by the Tukey or the Newmann-Keuls test was performed to test differences between different amounts of antibody against PAPP-A or P5 peptide. Means of inhibition of IGFBP-4 proteolytic degradation by peptides P3, HIP, VN1, and VN3 were compared with 0 (absence of inhibition of IGFBP-4 degradation without peptides) by a paired t-test. Comparisons with P > 0.05 were not considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In bovine and porcine species, immunoblotting revealed that only sera contained low levels of a 23-kDa proteolytic fragment (Fig. 1, lanes 2, 4, 6, 8, and 10), whereas two proteolytic fragments of 23 and 12 kDa were clearly seen in the corresponding preovulatory follicular fluids (Fig. 1, lanes 1, 3, 5, 7, and 9). In contrast, and as previously described, bovine and porcine preovulatory follicular fluids contained undetectable levels of intact IGFBP-2 assessed by WLB (Fig. 2A, lane 7). This suggests the presence of a proteolytic activity that degrades IGFBP-2 in preovulatory follicles, as shown previously in the sow [3].
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PAPP-A Is Involved in IGFBP-2 Proteolytic Degradation in Bovine and Porcine Preovulatory Follicular Fluids
Overnight incubation of bovine and porcine preovulatory follicular fluids with IGFBP-2 resulted in a slight proteolytic degradation and the appearance of bands corresponding to the 23- and 12-kDa proteolytic fragments in immunoblotting (Fig. 2A, lane 2). As for IGFBP-4, IGFBP-2 proteolytic degradation in bovine and porcine follicular fluids was maximal at pH 7.4–7.6, it was partially inhibited at pH 8.5, and it was completely inhibited at pH 6. IGFBP-2 proteolytic degradation in porcine follicular fluid was inhibited by EDTA and 1,10-phenanthroline, but clearly not by other protease inhibitors, as previously shown [3]. In bovine follicular fluid, IGFBP-2 degradation was also completely blocked by EDTA and 1,10-phenanthroline, and not significantly by serine protease inhibitors (data not shown). IGFBP-2 degradation as assessed by WLB and immunoblotting was enhanced in the presence of excess IGF-II (Fig. 2A, lane 3), and this enhancement effect was dose-dependent (Fig. 3). As for intrafollicular degradation of IGFBP-4, cleavage of IGFBP-2 by bovine and porcine preovulatory follicular fluids was inhibited by the presence of heparin-binding domain (HBD) peptides derived from IGFBP-5 (P5 peptide), as well as from IGFBP-3, vitronectin (VN), or heparin/heparan sulfate-interacting protein (HIP; see below). The cleavage of IGFBP-4 by PAPP-A was also shown to be enhanced by addition of IGF and inhibited by heparin-binding domain-derived peptides [6, 7, 15], suggesting involvement of PAPP-A in the intrafollicular cleavage of IGFBP-2 as well. Indeed, coincubation of follicular fluid with polyclonal antibody against human PAPP-A, but not rabbit IgG, inhibited IGFBP-2 proteolytic degradation in both species in a dose-dependent manner (Fig. 2A, lanes 4 and 5; Fig. 2B). In addition, PAPP-A polyclonal antibodies were able to immunodeplete intrafollicular IGFBP-2 proteolytic activity from both species as well (data not shown).
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Characterization of IGFBP-2 Proteolytic Cleavage by rhPAPP-A
Incubation of IGFBP-2 with rhPAPP-A generated 23- and 12-kDa proteolytic fragments in a dose-dependent manner, as occurs after incubation with follicular fluid (Fig. 4). The amount of cleavage was enhanced by addition of excess IGF-II. Addition of either IGF-I, IGF-II, or LR3-IGF-I resulted in a dose-dependent enhancement of IGFBP-2 cleavage, IGF-II being most efficient and LR3-IGF-I being less efficient than IGF-I (Fig. 5).
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IGFBP-2 was shown to be less sensitive than IGFBP-4 to proteolysis by rhPAPP-A. Indeed, in the presence of excess IGF-II, approximately 5-fold higher amounts of rhPAPP-A were necessary to obtain complete degradation of IGFBP-2 compared with that of IGFBP-4 (Fig. 4, B versus C). Moreover, after incubation with the same amount of PAPP-A, 50% of IGFBP-4 was degraded after 2 h of incubation, compared with 12 h for IGFBP-2 (Fig. 6).
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To determine where PAPP-A cleaves IGFBP-2, protein G agarose/antibody immobilized and purified rhPAPP-A was incubated with purified IGFBP-2 in the presence of IGF-II. By subjecting the entire reaction mixture to Edman degradation, only one IGFBP-2 N-terminal sequence was evident that was not present in the nonincubated reaction mixture: Met(166)-Gly-Lys-Gly-Gly-Lys. Hence, PAPP-A cleaves IGFBP-2 only at one site, between Gln165 and Met166.
Finally, as shown for IGFBP-4, P5 peptide dose-dependently inhibited IGFBP-2 cleavage by PAPP-A (Fig. 7). Similar inhibition was observed with heparin-binding peptides contained within IGFBP-3, VN, and HIP (Fig. 8). Degradation of IGFBP-2 by medium containing rhPAPP-A was sometimes accompanied by the appearance of two faint bands (Fig. 7, lane 8), the intensities of which were amplified by increasing amounts of P5 peptide. These bands likely correspond to alternative proteolytic fragments originating from cleavage by other proteases present in the medium that contains rhPAPP-A.
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Kinetic Analysis of rhPAPP-A/P5 Interaction
To assess whether the inhibition of PAPP-A proteolytic activity by the P5 peptide was due to a direct interaction with PAPP-A rather than with the substrate, a potential interaction between rhPAPP-A and the P5 peptide was investigated by surface plasmon resonance (SPR) with a BIAcore system using a sensorchip coupled with P5 peptide. The observed binding curve obtained after injection of 5 nM PAPP-A suggested a strong interaction (980 RU) and a slow rate of dissociation (800 RU after 10 min of dissociation; Fig. 9). Injection of polyclonal anti-PAPP-A just after the 10-min dissociation step gave rise to a stable signal (487 RU). This signal was clearly reduced (281 RU) when the polyclonal antibody was saturated with excess rhPAPP-A prior to the injection (data not shown), demonstrating specificity of the interaction between rhPAPP-A and P5 peptide. Injection of IGFBP-2 did not result in a stable, significant signal (data not shown). Overall, these results show that the P5 peptide specifically interacts with PAPP-A.
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By using a 1:1 binding model describing 1:1 binding between injected analyte (A) and immobilized ligand (B) (A + B 
AB), a good curve fitting was obtained. The model was fitted both globally across the data sets and to a single concentration. Binding rate constants demonstrating a strong affinity between rhPAPP-A and the P5 peptide are reported in Table 1 (KD value = 3.85 10-11 M).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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IGFBP-2 was shown to be less sensitive to cleavage by rhPAPP-A than IGFBP-4 in vitro. In contrast to IGFBP-4, bovine and porcine preovulatory follicular fluids induced only a partial proteolytic degradation of exogenous IGFBP-2 in the absence of exogenous IGFs, and this degradation was clearly enhanced in the presence of exogenous IGFs. These data suggest that IGFBP-2, like IGFBP-4, undergoes a conformational change after binding to IGFs that makes it more susceptible to cleavage, as previously described [32]. In vivo, proteolytic degradation of IGFBP-2 likely occurs only in preovulatory follicles that exhibit a high IGF bioavailability, whereas IGFBP-4 is already degraded in healthy growing follicles. In particular, we have previously shown in ewes that native IGFBP-4, assessed by WLB, was undetectable in follicles that contained more than 10 ng/ml estradiol [17]. In contrast, native IGFBP-2 was undetectable only in follicles that contained more than 100 ng/ml estradiol, suggesting that the disappearance of IGFBP-2 during follicular growth occurs later than that of IGFBP-4. Moreover, it is also possible that proteolytic degradation of IGFBP-2 also occurs in granulosa cells, resulting in secretion of IGFBP-2 fragments. Indeed, both IGFBP-2 and PAPP-A mRNA have been shown to be expressed in granulosa cells in sheep ([17] and unpublished results), cattle [15, 18], pigs [15, 19, 20] and humans [16, 33]. Finally, we and other researchers have also shown by in situ hybridization that follicular growth is accompanied by a decrease in follicular expression of IGFBP-2 but not IGFBP-4 (pig [19], cattle [18], sheep [17]). So, whereas the decrease in IGFBP-4 levels during follicular growth is essentially due to an increase in proteolytic degradation, the decrease in IGFBP-2 levels is likely due to a decrease in mRNA expression as well as to an increase in proteolytic degradation by PAPP-A, this degradation being maximal in preovulatory follicles that contain high levels of bioavailable IGFs.
Recently, Spicer et al. [4] claimed that bovine preovulatory follicles contained proteolytic activity that degrades IGFBP-4 but not IGFBP-2. The reason for the discrepancy with the present work is unknown. It may be due to the poor sensitivity of the radiolabeled IGFBP-2 used by these authors to the intrafollicular proteolytic activity of PAPP-A. In particular, in our hands, iodinated IGFBP-2, in contrast to native IGFBP-2, was not degraded by bovine or porcine preovulatory follicular fluid, and only poorly by rhPAPP-A. Of note, proteolytic degradation of exogenous IGFBP-2 in some follicular fluids was clearly visualized only by immunoblotting by using our very sensitive antibody raised against bovine IGFBP-2.
We have shown recently that addition of exogenous IGFBP-2 to follicular fluid from ovine, bovine, porcine, and equine preovulatory follicles was able to inhibit the IGFBP-4 degradation, suggesting that the increase in IGFBP-2 levels in early atretic follicles participates in the increase in IGFBP-4 levels [6, 7]. This inhibition is likely not due to a substrate competition between IGFBP-2 and IGFBP-4, because the latter is much more sensitive to the degradation by PAPP-A than the former. Rather, it might be due to the IGFBP-2-induced decrease in IGF bioavailability because this inhibition was reproduced by monoclonal antibodies raised against IGF-I and IGF-II [6, 7]. This suggests that in early atretic follicles, the decrease in PAPP-A expression [15] as well as the decrease in IGF bioavailability, due to the increase in IGFBP-2 mRNA expression, participates in the decrease in IGFBP-4 degradation.
We have shown here that addition of IGFs enhances cleavage of IGFBP-2 by preovulatory follicular fluid and by rhPAPP-A. This enhancing effect is likely due to a conformational change of the substrate after binding IGFs rather than a direct activation of PAPP-A activity, because LR3-IGF-I was less efficient than IGF-I or IGF-II. This is consistent with the mechanism for IGF enhancement of the IGFBP-4 cleavage by rhPAPP-A [28].
As for IGFBP-4 [6, 7], we have shown that cleavage of IGFBP-2 was inhibited by heparin-binding domain-containing peptides such as P5. Biacore analysis showed that the P5 peptide binds to PAPP-A with a high affinity, but not to IGFBP-2. Therefore, the inhibition is likely due to a direct interaction of the P5 peptide with PAPP-A rather than with IGFBP-2. So as previously suggested, the high increase in IGFBP-5 levels in late atretic ovine and bovine follicles might participate in the decrease in PAPP-A activity by direct interaction with PAPP-A via the heparin-binding domain encompassing the P5 peptide. We have shown that a peptide contained within the heparin-binding domain of vitronectin was also able to inhibit cleavage of IGFBP-4 [6, 7] and IGFBP-2 (present work) by rhPAPP-A. These results suggest that direct interaction of heparin-binding domain containing proteins, such as vitronectin and IGFBP-5, are able to modulate the proteolytic activity of PAPP-A in vivo.
We have shown here that PAPP-A cleaves IGFBP-2 within the central, poorly conserved region, which separates conserved, cysteine-rich, N- and C-terminal domains, between Gln165 and Met166. Our previous work has shown that human prostate kallikrein-2 (hK2) is able to cleave IGFBP-2 between Arg164 and Gln165, in agreement with the trypsin-like specificity of hK2 [34]. Two other cleavage sites have been identified in the IGFBP-2 sequence cleaved by a putative serine protease present in human milk, after Gly169 and Lys181 [35, 36].
The intrafollicular degradation of IGFBP-2 by PAPP-A seems to be a well-conserved mechanism in preovulatory follicles in mammalian species, suggesting that this degradation has dramatic consequences on follicular maturation. Intrafollicular cleavage of IGFBP-2 directly participates in the increase in bioavailability of IGFs that might further stimulate granulosa cell proliferation and steroidogenesis, as previously shown [37]. It is also possible that IGFBP-2 proteolytic fragments have IGF-independent effects on follicular cells, as was demonstrated for IGFBP-3 and IGFBP-5 fragments [38]. Recently, we showed that changes in intrafollicular IGFBP-2 levels, due to changes in mRNA expression and proteolytic degradation, are involved in the regulation of intrafollicular IGFBP-4 levels [6, 7]. It is interesting that recent data suggest that the regulation of intrafollicular IGFBP-4 proteolytic degradation is involved in the establishment of ovarian follicular dominance [39, 40]. So one might hypothesize that in growing healthy follicles, the decrease in IGFBP-2 levels, as well as the increase in PAPP-A activity, participates in the decrease in intact IGFBP-4 levels and the selection of the dominant follicle. In contrast, in early atretic follicles, the decrease in IGFBP-2 degradation, due to the decrease in PAPP-A expression, is partly responsible for the decrease in IGFBP-4 proteolytic degradation and the degeneration of the follicles of the cohort.
Overall, these data show that in bovine and porcine preovulatory follicles, PAPP-A is responsible for the IGF-dependent IGFBP-2 degradation. During follicular growth, the increase in IGFBP-2 cleavage by PAPP-A, as well as the decease in IGFBP-2 expression, are responsible for the decrease in IGFBP-2 levels and the increase in IGF bioavailability. In atretic follicles, the increase in IGFBP-2 and decrease in PAPP-A expression, as well as the inhibition of PAPP-A activity by heparin-binding domains present in IGFBP-5, and other proteins, might participate in the increase in IGFBP-2 levels and the decrease in IGF biaoavailability.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 33 2 47 42 77 43; monget{at}tours.inra.fr
3 Current address: Department of Gynecology and Obstetrics, Stanford University Medical Center, Stanford, CA 94305-5317
Received: 27 May 2002.
First decision: 18 June 2002.
Accepted: 12 July 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 S. E. Echternkamp, A. J. Roberts, D. D. Lunstra, T. Wise, and L. J. Spicer Ovarian follicular development in cattle selected for twin ovulations and births J Anim Sci, February 1, 2004; 82(2): 459 - 471. [Abstract] [Full Text] [PDF]
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R. Webb, P. C. Garnsworthy, J.-G. Gong, and D. G. Armstrong Control of follicular growth: Local interactions and nutritional influences J Anim Sci, January 1, 2004; 82(13_suppl): E63 - 74. [Abstract] [Full Text] [PDF]
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) and porcine (
) follicular fluids and increasing amounts of IGF-II in a final volume of 10 µl. Reaction mixtures were subjected to WLB at the end of incubation. Results are ± SEM of five bovine and five porcine follicular fluids. The increase in IGFBP-2 proteolytic degradation is significant for 12.5 ng and 50 ng IGF-II (P < 0.05 versus samples incubated without IGF-II)
) or absence (
).
