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Regular article |
a Station INRA de Physiologie de la Reproduction des Mammifères Domestiques, URA CNRS 1291, 37380 Nouzilly, France
b Department of Medicine, University Hospital, Zürich, Switzerland
c Department of Internal Medicine, University of Iowa, Iowa City, IA 52246
d Department of Surgery, Division of Pediatric Surgery, Children's Hospital, Wexner Institute for Pediatric Research, Columbus, Ohio 43205
ABSTRACT
We recently showed that insulin-like growth factor-binding protein-4 (IGFBP-4) proteolytic degradation in ovine preovulatory ovarian follicles is IGF-dependent and regulated by the heparin-binding domain (HBD) from IGFBP-3 and from connective tissue growth factor (CTGF), heparan/heparin-interacting protein (HIP), and vitronectin. The present study investigated regulation of IGFBP-4 proteolytic degradation in porcine, bovine, and equine ovarian preovulatory follicles. Follicular fluid from such preovulatory follicles contains proteolytic activity, degrading exogenous IGFBP-4. An excess of IGF-I enhanced IGFBP-4 degradation. In contrast, IGFBP-2 or -3 or monoclonal antibodies against IGF-I or -II dose-dependently inhibited IGFBP-4 degradation, and IGF-I or -II reversed this inhibition in a dose-dependent manner. Heparin-binding peptides derived from the C-terminal domain of IGFBP-3 or -5 inhibited IGFBP-4 degradation. Other heparin-binding peptides derived from CTGF, HIP, and vitronectin also inhibited IGFBP-4 degradation, except in porcine follicles. Finally, IGFBP-3 that was mutated in its HBD was less effective at inhibiting IGFBP-4 degradation. Thus, in bovine, porcine, and equine preovulatory follicles, IGFBP-4 proteolytic degradation both depends on IGFs and is inhibited by peptides containing HBD. Overall, these results suggest that during terminal development of follicles to the preovulatory stage in domestic animal species, the increase in IGF bioavailability might enhance IGFBP-4 degradation. In contrast, in atretic follicles, the decrease in IGF bioavailability, resulting partly from the increase in IGFBP-2 (sow, heifer, mare) and IGFBP-5 (heifer) expression would participate in the decrease of IGFBP-4 degradation. In bovine atretic follicles, IGFBP-5 would also strengthen the inhibition of IGFBP-4 degradation by direct interaction of its HBD with the protease. The involvement of other HBD-containing proteins in the modulation of intrafollicular proteases degrading IGFBP-4 remains to be investigated.
follicle, growth factors, ovary
INTRODUCTION
Evidence has accumulated that the insulin-like growth factor (IGF) system plays an essential role in ovarian function [1, 2]. In follicular fluid, IGF-I and -II are bound to high-affinity IGF-binding proteins (IGFBPs) that regulate IGF bioavailability at the level of the target cells [3, 4]. Changes in the levels of IGFBPs in follicular fluid correlate with functional changes of the ovarian follicles. In particular, Western ligand blotting has shown that follicular growth is accompanied by a slight increase in the intensity of the 44–42 kDa IGFBP-3 doublet in sow [5] and ewe [6] and by a decrease in the level of IGFBPs of less than 40 kDa, as reported in ewe (IGFBP-2, -4, and -5) [6], sow (IGFBP-2 and -4) [7, 8], heifer (IGFBP-2, -4, and -5) [9, 10], mare (IGFBP-2 and -4) [11], and in women (IGFBP-2 and -4) [12]. In contrast, atresia is primarily associated with a strong increase in intrafollicular levels of IGFBP-2 and -4 (in all species) and -5 (in ewe and heifer) and a slight decrease in IGFBP-3 levels (in ewe) [6, 8, 9, 11, 12]. These changes in IGFBP levels result partly from changes in specific intrafollicular proteolytic activity. In particular, in the ewe and sow, Besnard et al. [13, 14] showed that follicular growth and atresia are characterized by, respectively, an increase or decrease in intrafollicular proteolytic activity degrading IGFBP-2, -4, and -5. Chandrasekher et al. [15] showed the presence of a proteolytic activity degrading IGFBP-4 in follicular fluid from human dominant estrogenic, but not in atretic follicles [15]. In a preliminary study, these authors also reported the presence of proteolytic activity degrading IGFBP-4 in follicular fluid from bovine dominant follicle [16]. To our knowledge, no data are available in the mare.
All these mammalian species have a positive relationship between IGF bioavailability and IGFBP-4 degradation. Moreover, sheep have a negative relationship between IGFBP-3 proteolytic degradation and IGFBP-5 levels on one hand and IGFBP-4 degradation on the other [6, 13]. Recently, we showed in the ewe that in cell-free experimental conditions, intrafollicular IGFBP-4 proteolytic degradation was enhanced by addition of IGF-I and inhibited by blocking endogenous IGFs after the addition of IGFBP-2 (mimicking atresia), IGFBP-3, or monoclonal antibodies raised against IGF-I and -II. Furthermore, in this species, IGFBP-4 proteolytic degradation was inhibited by C-terminal, but not by N-terminal, IGFBP-3 fragments and by peptides that contain heparin-binding domain (HBD) from IGFBP-3, IGFBP-5, vitronectin, connective tissue growth factor (CTGF), and heparan/heparin-interacting protein (HIP) [17]. Hence, in sheep, the increase in IGF bioavailability among growing follicles might participate in the increase in IGFBP-4 degradation, whereas the decrease in IGF bioavailability, resulting from the increased levels of IGFBP-2, and the increase in HBDs contained within IGFBP-3 and -5 C-terminal regions might be partly involved in the decrease in IGFBP-4 degradation.
In the present work, we characterized proteolytic activity degrading IGFBP-4 in follicular fluid from porcine, bovine, and, for the first time to our knowledge, equine preovulatory follicles. Moreover, we investigated if, as observed in sheep, IGFBP-4 proteolytic degradation in preovulatory follicles is modulated by IGFs, IGFBP-2 (whose levels increase in atretic follicles in all these species), IGFBP-3, or peptides that contain HBDs.
MATERIALS AND METHODS
Materials
Altrenogest was obtained from Roussel-Uclaf (Romainville, France). Norgestomet implant was obtained from Intervet (Angers, France). Estrumate was obtained from Pitman-Moore (Meaux, France). Détomidine and prifinium bromide (Prifinial) were obtained from Smith Kline & French (Courbevoie, France) and Vetoquinol (Lure, France), respectively. Mixtencilline, penicillin, and dihydrostreptomycine were obtained from Rhône-Mérieux (Lyon, France). Porcine FSH was obtained from Dr. Y. Combarnous (Nouzilly, France), and the IGF-I and -II were generous gifts from Drs. H. H. Peter and A. Hinnen (Ciba-Geigy, Basel, Switzerland). The LongR3-IGF-I was obtained from GroPep (Adelaide, Australia). Recombinant human IGFBP-4 was expressed in yeast and purified as described elsewhere [18]. Nonglycosylated human IGFBP-3 was obtained from Celtrix Pharmaceuticals (Santa Clara, CA). The IGFBP-3mHBD was the generous gift of Dr. D. R. Powell (Houston, TX) and characterized by replacement of the IGFBP-3 HBD sequence by a sequence derived from IGFBP-1 (215KNGFYHSRQCETSMDGEA232) [19]. The IGFBP-2 was obtained from A. F. Schützdeller Biochemicals (Tübingen, Germany). The 18-amino-acid peptides P1 (183KNGFYHSRQCETSMDGEA200), which was synthesized from IGFBP-1; P3 (215KKGFYKKKQCRPSKGRKR232), synthesized from IGFBP-3; P4 (185RNGNFHPKQCHPALDGQR202), synthesized from IGFBP-4; P5 (201RKGFYKRKQCRPSKGRKR218) and PA5 (130KAEAVKKDRRKKLTQSKF143), both synthesized from IGFBP-5, were obtained using the t-bag technique as described elsewhere [20, 21]. Synthetic peptide spanning the C-terminal region of human CTGF (or IGFBP-related protein 2), CTGF247–260 (EENIKKGKKCIRTP), was synthesized and received as a cleaved PepSet [22]. The synthetic peptide containing the HBD of HIP (CRPKAKAKAKAKDQTK) was kindly provided by Dr. D.D. Carson (Houston, TX). The synthetic peptide containing an HBD derived from the p36 subunit of annexin II tetramer (KIRSEFKKKYGKSLYY) was obtained from Dr. D.M. Waisman (Calgary, Canada). Synthetic peptides derived from the HBD of vitronectin, VN1 (341APRPSLAKKQRFRHR355), VN3 (357RKGYRSQRGHSRGR370), VN5 (371NQNSRRPSRATWL383), and vitronectin multimeries were kindly provided by Dr. K.T. Preissner (Bad Nauheim, Germany). 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). Rabbit polyclonal antiserum against IGFBP-4 was purchased from Ubi (Lake Placid, NY). Antirabbit immunoglobulin (Ig) G antibodies coupled to horseradish peroxidase were purchased from Dako (Trappes, France). Nitrocellulose membranes were purchased from Schleicher and 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 agricultural agency and scientific research agency (approval number A37801) and were conducted in accordance with the Guidelines for Care and Use of Agricultural Animals in Agricultural Research and Teaching.
Sows Two cyclic adult Pietrain x Large-White sows were synchronized by daily feeding of 20 mg of Altrenogest for 15 days, as described elsewhere [14]. Female sows were slaughtered 96 h after progestin withdrawal (i.e., at the end of the follicular phase). Follicular fluid from large follicles (>7 mm) were aspirated by puncture and individually stored at -20°C. A mean of 15 preovulatory follicles was recovered from each sow.
Mares
As described elsewhere [11], 12 cyclic Welsh pony mares were treated with 125 µg of prostaglandin F2
analogue (Estrumate) during the midluteal phase to induce luteolysis. Ovarian activity was then assessed by routine transrectal ultrasonic imaging (Aloka 210 with a 5-MHz linear probe; Société Bernard, Nantes, France). Follicle diameter was estimated by averaging two cross-sectional measures of follicles, and follicular morphology was judged by the presence or absence of echogenic dots in follicular antrum. One healthy growing follicle per animal was punctured at the end of the follicular stage (33–35 mm) in each experiment. Follicular fluid was aspirated by transvaginal ultrasound-guided follicular puncture with a 7.5-MHz sectorial probe (Kretz, Soframed, Truchtersheim, France) coupled to a sterile, single-lumen needle (length, 60 cm; Thiébaud Frères, Jouvernex Margencel, France), as described elsewhere [23, 24]. Follicular fluid samples were then immediately aliquoted and stored at -20°C until analysis. Before each puncture session, mares were sedated by a single injection of 0.2 ml détomidine i.v. (Domosédan, 1 mg/100 kg body weight [BW]). Prifinium bromide (Prifinial) was injected (15 ml i.v., 45 mg/100 kg BW) to ensure rectal relaxation. After puncture sessions, the mares were injected with an antibiotic (Mixtencilline, 20 ml i.m., 1 600 000 IU penicillin/100 kg BW, and 1.3 g dihydrostreptomycine/100 kg BW).
Heifers
Estrus cycles were synchronized in 12 Charolais heifers with a Norgestomet implant for 10 days. Beginning on Day 4 (Day 0 = day of estrus), each heifer was treated with a decreasing-dose regimen of twice-daily i.m. injections of purified porcine FSH for 4 days (5, 5, 4, 4, 2, 2, 1, and 1 mg/injection, total dose = 24 mg). Luteolysis was induced at Day 6 with 15 mg of prostaglandin F2 analogue (Estrumate) given i.m. Heifers were slaughtered 24 h after the end of FSH treatment. Follicular fluid from each follicle (diameter, >6 mm) was collected by puncture and centrifuged at 300 x g for 5 min. The supernatant was then stored at -20°C.
Follicular Fluid and Classification
Porcine, bovine, and equine follicles characterized by the presence of IGFBP-3 and the absence of IGFBP-2, -4, and -5 were considered as being preovulatory, whereas follicles characterized by high levels of IGFBP-2 or of IGFBP-2, -4, and -5 (heifers) were considered as being early and late atretic follicles, respectively [5, 7, 9, 11, 14]. The status of follicles was confirmed in heifers and mares by measuring estradiol concentration in follicular fluid (data not shown). Five porcine, seven bovine, and 12 equine preovulatory follicles were used for the following experiments.
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 20 ng of IGFBP-4, with or without exogenous IGFBP-2, -3, -3mHBD, monoclonal antibodies against IGF-I or -II, synthetic peptides, and in the presence or absence of exogenous IGF-I, -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 (described later). In other experiments, to test the ability of exogenous IGF-I to enhance intrafollicular degradation of IGFBP-4, follicular fluid from preovulatory follicles was incubated with IGFBP-4 for 1 h in the presence or absence of IGF-I.
Western Ligand Blotting
The IGF-II was iodinated by the iodogen method and purified by Sephadex G-50 chromatography using a 0.1 M ammonium acetate elution buffer. Western ligand blotting was performed according to the method of Hossenlopp et al. [25] as modified by Monget et al. [6]. Samples were electrophoresed on a 12% SDS-polyacrylamide gel under nonreducing conditions. The proteins were then electrotransferred onto nitrocellulose filters (pore size, 0.2 µm) 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 and 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, 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 v/v to saturate nonspecific sites. Thereafter, filters were incubated for 1 h at 37°C in TBS containing 5% NFDM w/v, 0.05% Tween-20, and antibodies against 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 antirabbit IgG antibody coupled to horseradish peroxidase (final dilution, 1:2000). After washing, the signal was revealed by chemiluminescence detection.
Quantification of WLB
Quantification of WLB was performed by a PhosphoImager (Storm/Image Quant; Molecular Dynamics, Sunnyvale, CA). Quantification was performed as described elsewhere [13]. Briefly, the amount of radiolabeled IGF-II bound to each IGFBP was expressed as the integrated optical density (IOD) of the corresponding band in arbitrary units. The extent of IGFBP-4 degradation by follicular fluid was determined as the difference I-20-I37, where I-20 is the IOD of the IGFBP-4 band from samples not incubated and I37 is the IOD of the IGFBP-4 band from samples incubated at 37°C. The percentage of IGFBP-4 proteolysis inhibition was expressed as the ratio [(I-I37) x 100]/(I-20-I37), where I is the IOD of the IGFBP-4 band from samples incubated at 37°C in the presence of IGFBP-2, -3, -3mHBD, monoclonal antibodies against IGF-I and -II, or synthetic peptides.
Statistical Analysis
Data are presented as the mean ± SEM. Statistical comparisons of means of IGFBP-4 proteolytic degradation, with or without addition of IGF-I, as well as statistical comparisons of means of inhibition of IGFBP-4 proteolytic degradation by different peptides that contain HBDs were performed by Student's t-test. Statistical comparisons of means of the inhibition of IGFBP-4 proteolytic degradation by antibodies raised against IGF-I and -II in porcine preovulatory follicles were performed using Student's t-test with Welch's correction. Comparisons with P > 0.05 were not considered to be significant.
RESULTS
IGFBP-4 Proteolytic Degradation in Porcine, Bovine, and Equine Preovulatory Follicles
As previously described in the ewe [13, 17], incubation of 2 µl of follicular fluid from bovine, porcine, and equine preovulatory follicles with 20 ng of IGFBP-4 for 20 h at 37°C led to complete disappearance of IGFBP-4 as assessed by WLB (Fig. 1A, lane 2 vs. 1) and to generation of 19- and 15-kDa fragments that failed to bind IGFs as assessed by immunoblotting (Fig. 2). As in ovine [13] and porcine [14] follicles, IGFBP-4 proteolytic degradation in bovine and equine follicles was maximal at a pH of 7.4–7.6, was inhibited by EDTA and 1–10 phenanthroline but not clearly by other protease inhibitors, and was minimal in atretic follicles (data not shown). Thus, in several domestic animal species, IGFBP-4 is degraded by metallodependent proteases in preovulatory, but not in immature, follicles.
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IGF-Dependence of IGFBP-4 Proteolytic Degradation
We tested the effect of exogenous IGF-I on IGFBP-4 degradation by incubating preovulatory follicular fluid with IGFBP-4 and IGF-I for 1 h. In these conditions, addition of 200 ng of exogenous IGF-I led to enhancement of IGFBP-4 degradation (Fig. 3, A and B).
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When incubation of follicular fluid from bovine, porcine, and equine preovulatory follicles was performed for 20 h, addition of increasing amounts of IGFBP-2 or -3 or addition of antibodies against IGF-I or -II to the incubation medium led to dose-dependent inhibition of IGFBP-4 proteolytic degradation (Figs. 1, A and B, and 4). Inhibition of IGFBP-4 proteolytic degradation by IGFBP-2 was confirmed by immunoblotting (Fig. 2). Addition of increasing concentrations of IGF-I and -II dose-dependently reversed the inhibitory effects of IGFBP-2, -3, and antibodies against IGF-I and -II on IGFBP-4 degradation (Fig. 5, A and B). In the same conditions, LongR3-IGF-I led to a weak reversion of IGFBP-4 proteolysis (Fig. 5, A [b, lanes 7–9] and B). All these results suggest a role of IGF bioavailability in the regulation of IGFBP-4 degradation in follicular fluid from these species.
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Effects of Synthetic Peptides on Intrafollicular IGFBP-4 Proteolytic Degradation
Recently, in ovine preovulatory follicles, we showed that C-terminal domain derived from IGFBP-3 as well as heparin-binding peptides from IGFBP-3, -5, vitronectin, and CTGF could inhibit IGFBP-4 proteolytic degradation. In follicular fluid from the sow, heifer, and mare, heparin-binding peptides derived from the C-terminal region of IGFBP-3 (P3) and -5 (P5) could also inhibit, in a dose-dependent manner, IGFBP-4 proteolytic degradation (Fig. 6). Furthermore, among the other heparin-binding peptides tested, HIP peptide, vitronectin-derived peptides VN3 and VN1 (containing the consensus HBD XBBXBX), and CTGF247–260 at 5 µg/10 µl could inhibit IGFBP-4 degradation in bovine and equine preovulatory follicular fluid (Fig. 7). In contrast, in porcine preovulatory follicular fluid, none of these peptides at the concentration used in these conditions could inhibit IGFBP-4 proteolytic degradation (Fig. 7). Peptides derived from the vitronectin (VN5), vitronectin multimeries, and peptides P1, P4, and PA5 had no effect on IGFBP-4 proteolytic degradation, but a weak effect of the p36 subunit of annexin tetramer II was observed in the mare (Fig. 7).
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Effects of IGFBP-3mHBD on Intrafollicular IGFBP-4 Proteolytic Degradation
To assess whether the IGFBP-4 degradation induced by IGFBP-3 was mediated by its carboxy-terminal HBD, we tested the ability of a full-length IGFBP-3 protein mutated in the HBD region (IGFBP-3mHBD: 215KNGFYHSRQCETSMDGEA232) to inhibit IGFBP-4 proteolytic degradation. In contrast to native IGFBP-3, IGFBP-3mHBD only slightly inhibited IGFBP-4 degradation in porcine, bovine, and equine preovulatory follicles (Fig. 8, A and B).
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DISCUSSION
From our previous results and the present data, preovulatory follicles from all domestic animals studied contain a metallodependent proteolytic activity degrading IGFBP-4 [17]. To our knowledge, this is the first time such an activity has been described in equine preovulatory follicles. Moreover, several observations strongly suggest an IGF-dependent regulation of IGFBP-4 proteolytic degradation in porcine, bovine, and equine follicular fluid. First, in these species, addition of IGF-I to follicular fluid from preovulatory follicles enhanced IGFBP-4 degradation. Second, addition of IGFBP-2, -3, and monoclonal antibodies against IGF-I and -II dose-dependently inhibited IGFBP-4 degradation. This inhibition was dose-dependently reversed by IGF-I and -II but only poorly reversed by LongR3-IGF-I. These results are similar to those that we recently obtained with ovine preovulatory follicular fluid [17]. Similarly, Cwyfan Hughes et al. [26] showed that IGFBP-1, -2, and -3 inhibited IGFBP-4 degradation in human preovulatory follicles in vivo. In several in vitro models as well, addition of IGFBP-2, -3, -5, and/or -6 inhibited IGFBP-4 degradation, which was an effect reversed by IGFs [27–30]. All these results suggest that in growing follicles of mammalian species, the increase in IGF bioavailability participates in the increase in IGFBP-4 proteolytic degradation. In contrast, the decrease in IGF bioavailability in atretic follicles, resulting in part from an increase in IGFBP-2 and -5 (in heifer) levels, may be involved in the decrease in IGFBP-4 degradation.
Recently, we showed that HBD from IGFBP-3 and -5 could inhibit the IGFBP-4-degrading proteases in ovine follicles [17]. Similarly, Fowlkes et al. [28, 29] showed that the C-terminal fragment of IGFBP-3, as well as peptides derived from the C-terminal HBD of IGFBP-3, -5, or -6, could inhibit IGFBP-4 degradation in conditioned medium of murine bone cells. In the present work, we showed that peptides containing an HBD from IGFBP-3 (P3) and -5 (P5) could inhibit, in a dose-dependent manner, IGFBP-4-degrading proteases in porcine, bovine, and equine follicles. Except in porcine follicular fluid, peptides that contain an HBD from HIP, vitronectin (VN1, VN3), and CTGF (CTGF247–260) inhibited IGFBP-4 degradation in bovine and equine follicular fluid. The specificity of these peptides was confirmed by the absence of inhibition induced by the peptides P1, P4, or PA5, which cannot bind heparin [21]. Moreover, the ability of basic rich-peptides to inhibit intrafollicular IGFBP-4 degradation was not 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 [31], was not able to inhibit IGFBP-4 degradation in bovine and porcine follicles and had only weak effects in equine follicles. Overall, as in ovine ovary, these data suggest some specificity in the inhibition of intrafollicular IGFBP-4 degradation by heparin-binding peptides. Interestingly, the P5 peptide and the vitronectin peptide (VN357–370) have recently been shown to directly modulate plasminogen activation to plasmin [32, 33]. Finally, in porcine, bovine, and equine follicles, IGFBP-3 mutated in its heparin-binding region (IGFBP-3mHBD) only slightly inhibited IGFBP-4 degradation compared with wild-type IGFBP-3. Of note, IGFBP-3 mutant had an IGF-binding affinity equivalent to that of the intact IGFBP-3 (data not shown) [17].
As in ovine follicular fluid, our results suggest two mechanisms of regulation for IGFBP-4 proteolytic degradation in bovine, porcine, and equine preovulatory follicles. First, IGF dependence is suggested, because in the presence of only endogenous IGFBP-3 (likely of seric origin and complexed to endogenous ALS and IGF-I) and exogenous added IGFBP-4, IGF-I enhanced, whereas monoclonal antibodies against IGF-I and -II inhibited, IGFBP-4 degradation. In these conditions, IGF dependence could result from conformational changes of IGFBP-4 on binding to its ligand, leading to the protease being able to accede to the cleavage site, as suggested by others [34–36]. Second, inhibition of IGFBP-4 degradation by IGFBP-heparin-binding peptides is supported by the strong inhibition of IGFBP-4 proteolytic degradation induced by heparin-binding peptides derived from IGFBP-3, -5, CTGF, or other proteins different from IGFBP (vitronectin, HIP). Furthermore, IGFBP-3 mutated on its HBD only slightly inhibited the IGFBP-4 proteolytic degradation compared with intact IGFBP-3. Based on this latter experiment, reversion of the IGFBP-3 effect by IGF may result from the ability of ligand to impair the interaction of IGFBP-3 with IGFBP-4 protease by masking its HBD, as suggested by Fowlkes et al. [28, 29]. Of note in the present study, addition of exogenous IGFBP-3 to porcine, bovine, and equine preovulatory follicular fluid is "nonphysiological," in that such an increase in levels of intrafollicular free IGFBP-3 does not occur in vivo. In contrast, the inhibition of intrafollicular IGFBP-4 proteolytic degradation by IGFBP-2 is of physiological relevance, because IGFBP-2 levels increase in porcine, bovine, and equine atretic follicles. In these conditions, inhibition of proteolytic degradation may result from both sequestration of IGFs and direct interaction with protease. In particular, IGFBP-2 can bind heparin, although further studies are needed to identify its HBD [20, 37, 38]. One may hypothesize that the lesser efficiency of IGFBP-2 for inhibiting IGFBP-4 degradation compared with the efficiency of IGFBP-3 results partly from its lower affinity for heparin [20].
As stated above, inhibition of IGFBP-4 degradation by IGFBP-2 is of physiological relevance in all these species. In contrast, inhibition of IGFBP-4 degradation by P5 peptide is physiologically relevant only in bovine and ovine follicles. Indeed, late atresia is characterized by a high increase in intrafollicular levels of IGFBP-5 in bovine and ovine but not in porcine and equine species. One could hypothesize that proteins different from IGFBP-5, but containing similar HBDs, could modulate in vivo intrafollicular IGFBP-4 degradation in these species. In favor of this hypothesis is that HIP and vitronectin modulate the coagulation proteolytic cascade via their HBD [32, 39]. Moreover, both proteins and CTGF are ubiquitously expressed. For example, a high expression of HIP and CTGF has recently been found in rat testis and porcine ovarian follicles, respectively [40, 41].
In the present work, one can note some species differences in the regulation of intrafollicular IGFBP-4 degradation. In particular, porcine preovulatory follicles appeared to contain higher IGFBP-4 proteolytic activity than bovine, equine, and ovine follicles: nearly 50% of exogenous, added IGFBP-4 was degraded after 1 h of incubation, compared with 25% in bovine follicles, 10% in equine follicles, and 40% in ovine follicles [17]. Furthermore, P3 and P5 peptides as well as IGFBP-2 and -3 inhibited IGFBP-4 degradation in porcine follicular fluid to a lesser extent than in bovine or equine follicular fluids. Finally, in porcine follicular fluid, none of the HBD-containing peptides at the concentrations used could inhibit the IGFBP-4 proteolytic degradation. This latter difference could be explained by a difference in concentration of proteases and/or in their efficiency (less for endogenous protease inhibitors). It could also result from a difference in the nature of the zinc-dependent metalloprotease responsible for degradation of IGFBP-4 in these species [13, 14].
In conclusion, we have shown that in bovine, porcine, and equine ovarian follicles, IGFBP-4 proteolytic degradation is IGF-dependent and inhibited by IGFBP-3 and -5 C-terminal domain as well as by peptides containing an HBD (Fig. 9). These data suggest that in these three species, as in sheep, the increase in IGFBP-2 expression in atretic follicles participates in the decrease in IGFBP-4 degradation, with the specific mechanism remaining to be elucidated (Fig. 9-2). Furthermore, in bovine late atretic follicles, the increase in IGFBP-5 expression might strengthen the inhibition of IGFBP-4 degradation, with this inhibition being partly mediated by direct interaction of the C-terminal HBD of IGFBP-5 with IGFBP-4 proteinases (Fig. 9-3). Involvement of other proteins containing HBDs, such as CTGF, HIP, or vitronectin, in modulation of intrafollicular IGFBP-4 degradation as well as the consequence of this degradation on IGF-dependent or independent biological effects on granulosa cells remain to be investigated.
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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 the nonglycosylated hIGFBP-3 and monoclonal antibody against IGF-I. We thank Dr. D.R. Powell for providing the IGFBP-3mHBD. We also thank N. Gérard and B. Oussaïd for providing the equine and bovine follicular fluid, respectively. We thank Dr. D. Monniaux for helpful discussions and C. Pisselet for technical assistance, and we are grateful to A. Beguey for the photographic work.
FOOTNOTES
First decision: 3 December 1999.
1 Supported by Biotechnocentre/INRA and grant 32-46808.96 from the Swiss National Science foundation. 
2 Correspondence. FAX: 33 2 47 42 77 43; monget{at}tours.inra.fr
Accepted: March 8, 2000.
Received: October 26, 1999.
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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]
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 J. A. Kricker, C. L. Towne, S. M. Firth, A. C. Herington, and Z. Upton Structural and Functional Evidence for the Interaction of Insulin-Like Growth Factors (IGFs) and IGF Binding Proteins with Vitronectin Endocrinology, July 1, 2003; 144(7): 2807 - 2815. [Abstract] [Full Text] [PDF]
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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]
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 S. M. Firth and R. C. Baxter Cellular Actions of the Insulin-Like Growth Factor Binding Proteins Endocr. Rev., December 1, 2002; 23(6): 824 - 854. [Abstract] [Full Text] [PDF]
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 X. Wu, A. Blanck, G. Norstedt, L. Sahlin, and A. Flores-Morales Identification of genes with higher expression in human uterine leiomyomas than in the corresponding myometrium Mol. Hum. Reprod., March 1, 2002; 8(3): 246 - 254. [Abstract] [Full Text] [PDF]
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T. Nam, A. Moralez, and D. Clemmons Vitronectin Binding to IGF Binding Protein-5 (IGFBP-5) Alters IGFBP-5 Modulation of IGF-I Actions Endocrinology, January 1, 2002; 143(1): 30 - 36. [Abstract] [Full Text] [PDF]
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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]
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O.J. Ginther, M.A. Beg, D.R. Bergfelt, F.X. Donadeu, and K. Kot Follicle Selection in Monovular Species Biol Reprod, September 1, 2001; 65(3): 638 - 647. [Abstract] [Full Text] [PDF]
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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]
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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]
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