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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

Physiologie de la Reproduction et des Comportements (S.M., P.M.), Université F. Rabelais de Tours, 37380 Nouzilly, France; Department of Molecular and Structural Biology (M.T.O., C.O.), University of Aarhus, 8000 Aarhus C, Denmark; Department of Clinical Biochemistry Statens Seruminstitut (M.C.), 2300 Copenhagen S, Denmark; Endocrine Research Unit (C.A.C.), Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905; Laboratoire de Génétique Moléculaire (I.L., M.V.), Faculté de Pharmacie, 75006 Paris, France; Institut National de la Recherche Agronomique (G.T.-K.), Génétique Cellulaire, 31326 Castanet-Tolosan, France; and Department of Medicine (J.Z.), University Hospital, Zürich CH-8091, Switzerland

Address all correspondence and requests for reprints to: Philippe Monget, Physiologie de la Reproduction et des Comportements, UMR 6073 Institut National de la Recherche Agronomique-Centre National de la Recherche Scientifique-Université François Rabelais de Tours, 37380 Nouzilly, France. E-mail: monget{at}tours.inra.fr

	Abstract

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IGF binding protein-4 (IGFBP-4) proteolytic degradation is a common feature of preovulatory follicles from human, ovine, bovine, porcine, and equine ovary. In all these species, the protease is a zinc-dependent metalloprotease and its ability to degrade IGFBP-4 is IGF dependent. The human intrafollicular IGFBP-4-degrading protease has recently been identified as pregnancy-associated plasma protein-A (PAPP-A). The aim of this study was to investigate whether PAPP-A is also involved in IGFBP-4 degradation in ovine, bovine, porcine, and equine preovulatory follicles and to study the expression of PAPP-A mRNA in bovine and porcine granulosa cells from different classes of follicles. Immunoneutralization and immunoprecipitation with polyclonal antibodies raised against human PAPP-A inhibited IGFBP-4 proteolytic degradation in preovulatory follicular fluid from the four species studied. As previously reported for the intrafollicular proteolytic activity degrading IGFBP-4, recombinant human PAPP-A generated in vitro 17- and 10-kDa IGFBP-4-proteolytic fragments. Recombinant PAPP-A activity was also shown to be IGF dependent and was inhibited by heparin-binding domain-containing peptides. In all mammalian species studied, the PAPP-A sequences showed high degree of identity. Moreover, the PAPP-A gene was localized on porcine chromosome 1 (1q29–1q213), in agreement with the localization of human PAPP-A gene on human chromosome 9q33.1. In bovine and porcine ovaries, real-time quantitative RT-PCR showed that PAPP-A mRNA expression in granulosa cells was maximal in fully differentiated follicles and was positively correlated with expression of P450 aromatase and LH receptor mRNAs. Overall, these data show that PAPP-A is responsible for IGFBP-4 degradation in ovine, bovine, porcine, and equine preovulatory follicles. The high expression of PAPP-A mRNA in granulosa cells from large, differentiated follicles suggest that it is a new functional marker of follicular development.

	Introduction

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OVINE, BOVINE, PORCINE, equine, and human preovulatory ovarian follicles are characterized by a high proteolytic activity degrading IGF binding protein-4 (IGFBP-4). More precisely, Besnard et al. (1, 2) have shown that follicular growth and atresia are characterized by an increase and decrease, respectively, in intrafollicular proteolytic activity degrading IGFBP-4 in the ewe and sow. Chandrasekher et al. (3) have also shown the presence of a proteolytic activity degrading IGFBP-4 in follicular fluid from human dominant estrogenic but not atretic follicles. In all species studied, the intrafollicular protease degrading IGFBP-4 has been shown to belong to the metalloprotease superfamily, to be IGF dependent, and to be inhibited by heparin-binding domain-containing peptides (1, 2, 3, 4, 5, 6). These properties were also reported for IGFBP-4 proteases studied in numerous cell culture media (7). Although several studies have dealt with regulation of IGFBP-4 proteolytic degradation in ovarian follicular fluid or in conditioned media, the protease involved in IGFBP-4 degradation was unknown (4, 5, 6, 7, 8).

An IGFBP-4-specific protease produced by human fibroblasts and osteoblasts cells in culture was recently identified as pregnancy-associated plasma protein-A (PAPP-A) (9). PAPP-A is a large dimeric glycoprotein of 400 kDa. During pregnancy, it circulates in increasing concentrations as a 2:2 disulfide-bound complex of 500 kDa with the proform of eosinophil major basic protein (proMBP), denoted PAPP-A/proMBP (10, 11). Because proMBP is not visible in Coomassie-stained gels (10), this component was overlooked in earlier preparations of the protein (12, 13). As a consequence, preparations of polyclonal antibodies against PAPP-A isolated from pregnancy serum is in fact anti-PAPP-A/proMBP, also recognizing proMBP (10). Antigen recognized by such preparations was previously shown to be present in the human ovary (14, 15, 16, 17), and synthesis of both PAPP-A and proMBP mRNA in the human ovary has been demonstrated by RT-PCR (18). Using monoclonal antibodies specific for PAPP-A only, the presence of PAPP-A was recently confirmed in human preovulatory follicular fluid, and PAPP-A was identified as the protease responsible for IGFBP-4 degradation (19).

The aims of this study were to determine whether PAPP-A is involved in IGFBP-4 proteolytic degradation in ovine, bovine, porcine, and equine ovarian follicles, and to study mRNA expression in granulosa cells from different classes of follicles in comparison with mRNA expression of LH receptor and aromatase mRNAs.

	Materials and Methods

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Materials
Fluorogestone acetate sponges used to synchronize ovine estrous cycles and PMSG was obtained from Intervet (Angers, France). Altrenogest was obtained from Roussel-Uclaf (Romainville, France). Norgestomet implant was obtained from Intervet. Estrumate was obtained from Pitman-Moore (Meaux, France). Detomidine and prifinium bromide (Prifinial) were obtained from SmithKline & French (Courbevoie, France) and Vetoquinol (Lure, France), respectively. Mixtencilline, penicillin, and dihydrostreptomycin were obtained from Rh\|[ocirc ]\|ne-M\|[eacute]\|rieux (Lyon, France). IGF-I and IGF-II were generous gifts from Dr. H. H. Peter and Dr. A. Hinnen (Ciba-Geigy, Basel, Switzerland). Recombinant human IGFBP-4 was expressed in yeast and purified as previously described (20). Recombinant human PAPP-A (rhPAPP-A) was expressed in HEK 293 cells and purified as previously described (21). The 18-amino acid peptides, P3 (²¹⁵KKGFYKKKQCRPSKGRKR²³²) from human IGFBP-3 and P5 (²⁰¹RKGFYKRKQCRPSKGRKR²¹⁸) from human IGFBP-5, were a generous gift from R. S. Bar (Iowa City, IA) (22, 23). Synthetic peptide spanning the C-terminal region of human connective tissue growth factor (CTGF or IGFBP-related protein 2) CTGF^247–260 (EENIKKGKKCIRTP) was obtained from D. R. Brigstock (Columbus, OH) (24). The synthetic peptide containing the heparin-binding domain of human HIP (heparin/heparan sulfate-interacting protein) (CRPKAKAKAKAKDQTK) was kindly provided by Dr. D. D. Carson (Houston, TX) (25). The synthetic peptide containing a heparin-binding domain derived from the human p36 subunit of annexin II tetramer (KIRSEFKKKYGKSLYY) was obtained from Dr. D. M. Waisman (Calgary, Alberta, Canada) (26). Synthetic peptides derived from the heparin-binding domain of human vitronectin (VN), VN1 (³⁴¹APRPSLAKKQRFRHR³⁵⁵), VN3 (³⁵⁷RKGYRSQRGHSRGR³⁷⁰), and VN5 (³⁷¹NQNSRRPSRATWL³⁸³), were kindly provided by Dr. K. T. Preissner (Bad Nauheim, Germany) (27). Human IGFBP-4 C-terminal and N-terminal purified proteolytic fragments were kindly provided by St\|[auml ]\|ndker (Hannover, Germany) (28). Rabbit polyclonal antiserum against human IGFBP-4 was purchased from Ubi (Lake Placid, NY). Antirabbit IgG antibodies coupled to horseradish peroxidase were purchased from DAKO Corp. (Trappes, France). Rabbit polyclonal anti-PAPP-A/proMBP) (Overgaard M.T., C. Christiansen, and C. Oxvig, unpublished data) was raised against PAPP-A/proMBP purified from pregnancy serum (11). Protein G-plus/protein A-agarose was purchased from Oncogene Science, Inc. (Darmstadt, Germany). 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). PCR consumables used for RT-PCR/PCR was purchased from Promega Corp. (Madison, WI), except AmpliTAq polymerase from Perkin-Elmer Corp. (Meylan, France). SuperScript II Rnase H reverse transcriptase was purchased from Life Technologies, Inc. (Gaithersburg, MD) and random hexamers from Pharmacia (Uppsala, Sweden).

Animals and treatment
All procedures were approved by the agricultural and scientific research agencies (approval number A37801) and conducted in accordance with the guidelines for Care and Use of Agricultural Animals in Agricultural Research and Teaching.

Ewe. Four adult Romanov and Ile-de-France ewes were treated with progestogen (intravaginal fluorogestone acetate sponges, 40 mg) for 15 d to synchronize estrus. They were injected with 500 IU PMSG 30 h before sponge removal. Twenty-four hours after sponge removal, ovaries were collected by castration. Only large follicles (5–7 mm in diameter) were recovered. Preovulatory follicular fluid was aspired by puncture and individually stored at -20 C.

Sows. Six cyclic adult Pietrain x Large-White sows were synchronized by daily feeding of 20 mg Altrenogest for 15 d, as previously described (2). Ovaries were carefully dissected and collected by castration 24 h (beginning of follicular phase), 96 h (middle of follicular phase), and 120 h (end of follicular phase) after progestin withdrawal. Follicles were classified according to their size: small (3–4 mm), medium (5–6 mm), and large (7–8 mm) (see below). Between 10 and 15 follicles from each class were recovered.

Heifers. Estrus cycles were synchronized in 2 Holstein heifers with a Norgestomet implant for 10 d. Twelve days after detection of estrus (day 0 = day of estrus), the heifers were treated with two injections of 2000 IU PMSG and 2 injections of 25 mg im PGF₂ analog (Estrumate) and were killed at day 14. Follicles were carefully dissected and classified according to their size: small (4–6 mm), medium (10–12 mm), and large (15–20 mm) (see below). Approximately 5–10 follicles from each class were recovered.

Mares. Twelve cyclic Welsh pony mares were treated with 125 µg PGF₂ analog (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; Soci\|[eacute]\|t\|[eacute]\| Bernard, Nantes, France), as previously described (29). 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). 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 (60 cm long; Thiébaud Frères, Jouvernex Margencel, France), as previously described (30, 31). Before each puncture session, mares were sedated by a single injection of 0.2 ml detomidine iv [Domosédan, 1 mg/100 kg body weight (BW)]. Prifinium bromide (Prifinial) was injected (15 ml iv, 45 mg/100 kg BW) to ensure rectal relaxation. After puncture sessions, the mares were injected with antibiotics (20 ml im Mixtencilline; 1,600,000 IU penicillin/100 kg BW and 1.3 g dihydrostreptomycine/100 kg BW).

Classification of bovine and porcine follicles used for RNA extraction. In the heifer and the sow, follicles of different sizes were classified according to their quality. For this purpose, 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 (32). For each suspension, a smear of granulosa cells was performed on 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; follicles were judged normal (frequent mitosis, no pyknosis in granulosa cells) or atretic (no mitosis, numerous pyknotic bodies in granulosa cells). Furthermore, follicular fluids were analyzed individually by Western ligand blotting (WLB). In particular, as previously described (29, 33, 34, 35), ovine, porcine, bovine, and equine large follicles characterized by the presence of IGFBP-3; the absence of IGFBP-2, -4, and –5; and a high proteolytic activity degrading IGFBP-4 in follicular fluid were considered as preovulatory, whereas follicles characterized by high levels of IGFBP-2, -4, and -5 (heifers) were considered as early and late atretic follicles, respectively. The rest of granulosa cells of each bovine and porcine follicle was individually stored at -20 C before RNA extraction.

Degradation of IGFBP-4 by follicular fluid and by rhPAPP-A. Two microliters porcine or bovine preovulatory follicular fluid 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-4, with or without IGF-I for 20 h at 37 C (final volume, 10 µl). In some experiments, the synthetic peptides P3, P5, CTGF^247–260, VN, p36 subunit, or HIP were added to the incubation medium. At the end of the incubation, samples were analyzed by WLB or immunoblotting, as previously described (4, 5, 33).

Immunoneutralization and immunodepletion of IGFBP-4 proteolytic degradation. For immunoneutralization, 2 µl of preovulatory follicular fluid were incubated in TBS containing 0.1% BSA, 20 ng of IGFBP-4, and different dilutions of rabbit PAPP-A polyclonal antibody or nonspecific rabbit IgG or glycerol for 20 h at 37 C (final volume, 10 µl). At the end of the incubation, samples were analyzed by WLB. For immunodepletion, PAPP-A polyclonal antibody or nonspecific rabbit IgG was used in conjunction with protein G-plus/protein-A-agarose to immunoprecipitate IGFBP-4 protease activity. The supernatants were then studied for their ability to degrade [¹²⁵I]IGFBP-4, as previously described (19).

RNA extraction. Total RNAs from granulosa cells of individually dissected bovine and porcine follicles were prepared according to Chomczynsky and Sacchi (36) using acid guanidinium thiocyanate-phenol-chloroform extraction. Total RNAs were quantified by measuring the absorbance at 260 nm. Samples were stored at -70 C until used. RNAs from some follicles (particularly small and atretic follicles) were pooled according to their size and quality to obtain sufficient amounts to perform real-time RT-PCR experiments. RNAs were also extracted from granulosa cells of ovine and equine preovulatory follicles for RT-PCR and sequencing of cDNA fragments of PAPP-A in these species.

Isolation and sequencing of ovine, bovine, porcine, and equine PAPP-A cDNA fragments. A cDNA fragment from bovine PAPP-A was isolated by RT-PCR by using the upper primer U1 and the lower primer L1 derived from the nucleotide sequence of human PAPP-A (37) (Table 1). Then two other pairs of primers (U2-L2 and U3-L2) were chosen on the basis of both human and bovine sequence to amplify ovine, porcine, and equine cDNA (Table 1). Reverse transcription was performed during 1 h at 37 C in a total volume of 25 µl with 3 µg total ovarian RNAs as template, 1.5 mM dNTP, reverse transcriptase buffer 5x, 20 U Rnase inhibitor, 3.2 µM lower prime, and 200 U Moloney murine leukemia virus reverse transcriptase. The resulting cDNA was subjected to PCR. PCR was performed in a total volume of 50 µl with 1 U AmpliTaq polymerase, 2 mM MgCl₂, 0.2 mM dNTP, 0.3 µM each primer, AmpliTaq polymerase buffer 10x, and 5 µl of RT-PCR mixture. Samples were subjected to 30 rounds of PCR on a geneAmp PCR System 9700 (Perkin-Elmer Corp.). Denaturation, annealing, and extension were performed at 94 C for 1 min, 57 C for 1 min 30 sec, and 72 C for 1 min 30, respectively, on a geneAmpPCR System 9700. PCR products at the expected size were extracted from agarose using the gel extraction kit QIAquick (QIAGEN, Hilden, Germany). They were sequenced in both directions with several primers.

Real-time RT-PCR. Real-time quantitative PCR analyses for bovine and porcine PAPP-A, LH receptor, and P450 aromatase were performed on total RNAs from granulosa cells, using an ABI PRISM 7700 sequence detection system instrument and software (PE Applied Biosystems, Cortaboeuf, France). It was performed using specific primers presented in Table 2. Each transcript level of target genes (aromatase, LH receptor, and PAPP-A) was normalized on the basis of level of transcripts for the constitutive housekeeping gene product 36B4 coding for acidic ribosomal phosphoprotein (PO) (41) or the constitutive housekeeping gene coding for the TBP (TATA box-binding protein, a component of the DNA-binding protein complex TFIID) (42). They were measured to control the differences in RNA concentration between each sample. Bovine and porcine TBP and PO genes were partially sequenced to design the specific primers presented in Table 2. No difference in PO and TBP RNA levels was observed between healthy and atretic follicles: For each sample, the ratio PO mRNA expression level/TBP mRNA expression level = 1. So these two genes were considered efficient endogenous control genes.

Quantification of WLB. WLB was quantified by a PhosphorImager (Storm/ImageQuant, Molecular Dynamics, Inc., Sunnyvale, CA). Quantification was performed as previously described (1). 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-4 degradation by follicular fluid or rhPAPP-A was determined as the difference I_-20-I₃₇, where I_-20 is the IOD of IGFBP-4 band from samples not incubated, and I₃₇ is the IOD of 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 IGFBP-4 band from samples incubated at 37 C in the presence of PAPP-A polyclonal antibody or synthetic peptides.

Statistical analysis. Multiple comparisons of means of inhibition of IGFBP-4 proteolytic degradation without or with increasing concentrations of PAPP-A polyclonal antibodies or with increasing concentrations of peptides P3 and P5 were performed by ANOVA test followed by Tukey’s multiple comparison test. Means of inhibition of IGFBP-4 proteolytic degradation by peptides HIP, VN1, VN3, VN5, and CTGF were compared with 0 (absence of inhibition of IGFBP-4 degradation without peptides) by a paired t test. Comparisons with P values more than 0.05 were not considered significant. Simple correlation coefficient (Pearson) was calculated to assess relationship among PAPP-A, aromatase, and LH receptor gene expression (Prism, GraphPad Software, San Diego, CA). An r value with a value of P more than 0.05 was considered nonsignificant.

	Results

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Immunoneutralization and immunodepletion of IGFBP-4-degrading protease in preovulatory follicular fluid
As previously described and as shown in Fig. 1A (lane 2 vs. lane 1), ovine, bovine, porcine, and equine preovulatory follicular fluid contained IGFBP-4-degrading proteolytic activity (4, 5). Coincubation of follicular fluid with polyclonal antibody against human PAPP-A, but not rabbit IgG, inhibited in a dose-dependent manner IGFBP-4 proteolytic degradation in the four species (Fig. 1). Moreover, PAPP-A polyclonal antibodies were able to immunodeplete intrafollicular IGFBP-4 proteolytic activity from the four species as well (Fig. 2).

View larger version (26K): [in this window] [in a new window]   Figure 1. Inhibition of IGFBP-4 degradation in ovine, bovine, porcine, or equine preovulatory follicles by polyclonal antibodies against PAPP-A. A, Two microliters follicular fluid from ovine (a), bovine (b), porcine (c), and equine (d) preovulatory follicles were incubated for 20 h at 37 C with 20 ng IGFBP-4 (a–c: lanes 2–7; d: lanes 2–8) in the presence of increasing dilutions [1 (= 1.2 µg/tube) to 1/1000] of antibodies against PAPP-A (a–c: lanes 4–7; d: lanes 5–8), glycerol (G) (lane 3), or rabbit IgG (a–c: lane 7; d: lane 4) in a final volume of 10 µl. At the end of the incubation, samples were submitted to WLB. Lane 1, Samples stored at -20 C before WLB. Molecular mass, 44–42 kDa for the IGFBP-3 doublet, 24 kDa for IGFBP-4. Note the presence of endogenous IGFBP-3 that is not degraded during incubation. B, Quantitative analysis of WLB. Results are expressed as the mean ± SEM on three or four preovulatory follicles for each species, except for samples with anti-PAPP-A and IgG at dilution 1, n = 1; a significantly different from b. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

View larger version (38K): [in this window] [in a new window]   Figure 2. Immunodepletion of IGFBP-4 protease activity from ovine, equine, porcine, and bovine preovulatory follicular fluid with polyclonal antibodies against PAPP-A. Ovine, bovine, porcine, and equine follicular fluids were precleared with PAPP-A antibody (1/50) (lanes Ab) or nonspecific IgG (lanes IgG) complexed with protein G plus/protein A-agarose. Supernatants were recovered and incubated with [125I]IGFBP-4 in the presence (+) or the absence (-) of IGF-II at 37 C for 20 h as described in Materials and Methods. The large arrow indicates intact IGFBP-4. Small arrows indicate the proteolytic fragments of IGFBP-4.

View larger version (34K): [in this window] [in a new window]   Figure 3. Comparison of IGFBP-4 proteolytic degradation by preovulatory follicles and rhPAPP-A. Two microliters follicular fluid from porcine preovulatory follicles (lanes 1 and 2) or decreasing concentration of rhPAPP-A (lanes 3–5; 1/10 represents 0.3 ng/tube, i.e. 0.15 nM) were incubated for 20 h at 37 C with 150 ng of IGFBP-4 (lanes 2–5) without (lanes 1 and 2) or with (lanes 3–5) 50 ng of IGF-II in a final volume of 10 µl. Lane 1, Sample stored at -20 C. Lanes 6 and 7, Purified C- and N-terminal human IGFBP-4 proteolytic fragment, respectively. Samples were analyzed by immunoblotting using a specific polyclonal antibody raised against IGFBP-4. Molecular mass of IGFBP-4, 24 kDa. The arrows indicate the 17- and 10-kDa proteolytic fragments of IGFBP-4. Molecular mass was determined in preliminary experiments (data not shown).

View larger version (28K): [in this window] [in a new window]   Figure 4. Dose-dependent effect of the IGFBP-3 and IGFBP-5-derived heparin-binding peptide (P3 and P5) on IGFBP-4 degradation by rhPAPP-A. A, 0.2 ng of rhPAPP-A were incubated for 20 h at 37 C with IGFBP-4 (lanes 2–9) and increasing concentrations of P3 peptide (lanes 3–5), P5 peptide (lanes 6–8), and 8 µg of p36 subunit of annexin II tetramer-derived peptide (lane 9) in a final volume of 10 µl. Lane 1, Sample stored at -20 C. At the end of the incubation, samples were submitted to WLB. B, Quantitative analysis of WLB. Samples incubated with P3 () or P5 (). Results are obtained as the ± SEM on at least 3 experiments. *, P < 0.05; **, P < 0.01.

View larger version (11K): [in this window] [in a new window]   Figure 5. Effects of different heparin-binding peptides on IGFBP-4 degradation by rhPAPP-A. 0.2 ng of rhPAPP-A were incubated for 20 h at 37 C with IGFBP-4 (20 ng) and 10 µg of VN1 (VN343–355, VN3 (VN357–370), and VN5 (VN371–383) peptides, 5 µg of CTGF247–260 peptide, 5 µg of HIP, or 8 µg of p36 subunit of annexin II tetramer-derived peptide 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 ± SEM on three to five experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (in comparison with samples incubated without peptides).

View larger version (105K): [in this window] [in a new window]   Figure 6. Comparison of human (accession number X68280 (37 )), bovine, ovine, porcine, and equine PAPP-A nucleotide (B) and deduced amino acid (C) sequence. A, Schematic representation of the human PAPP-A region sequenced in the four species studied. The zinc-binding domain region is highlighted. Numbers indicated on the sequence refer to nucleic acids (na)/amino acids (aa). B, The nucleotide sequence 1–889 corresponds to the human sequence 1456–2344 (37 ). C, The amino acid sequence 1–296 corresponds to the human sequence 485–780. Conserved amino acids are dashed. The zinc-binding domain consensus sequence is indicated as HEXXHXXGXXH. The consensus sequence (Cs) is shown on the bottom line. %, Y/F; !, V/I; #, D/E.

Expression of mRNA of PAPP-A, aromatase, and LH receptor from bovine and porcine follicles of different classes
We have previously shown that intrafollicular proteolytic activity degrading IGFBP-4 increases during follicular growth to reach a maximum in preovulatory follicles (1, 2). So we have investigated whether this increase is associated with an increase in PAPP-A mRNA expression in granulosa cells during follicular growth and whether there is a correlation between expression of PAPP-A mRNA and expression of two well-known markers of follicular growth, aromatase, and LH receptor. Quantitative RT-PCR showed that expression of PAPP-A mRNA was highest in granulosa cells from bovine and porcine fully differentiated follicles in comparison with immature and atretic follicles. Moreover, expression of PAPP-A mRNA was closely correlated with expression of aromatase and LH receptor genes in both species (Fig. 7). Similar results were obtained by using either PO or TBP as endogenous RNA control (Table 3). The intrafollicular IGFBP-4 proteolytic degradation was also correlated with the level of PAPP-A RNAs (Fig. 7 and Table 3).

View larger version (27K): [in this window] [in a new window]   Figure 7. Expression of PAPP-A mRNA in granulosa cells of bovine and porcine follicles. Relationship between PAPP-A and aromatase mRNA expression level (A), PAPP-A and LH receptor mRNA expression level (B), PAPP-A and percent IGFBP-4 proteolytic degradation (C), and LH receptor mRNA and aromatase mRNA expression level (D), determined by real-time quantitative RT-PCR on granulosa cells from porcine and bovine individually dissected follicles. Endogenous gene control: TBP. Heifer: , healthy follicles 4–6 mm (n = 4); , healthy follicles 10–12 mm (n = 4); , healthy follicles 15–20 mm (n = 5); , early atretic follicles 4–6 mm (n = 3); , early atretic follicles 10–12 mm (n = 3); , early atretic follicles 15–20 mm (n = 3); +, late atretic follicles 4–6 mm (n = 3); x, late atretic follicles 10–12 mm (n = 3). Sow: , healthy follicles 3–4 mm (n = 5); , healthy follicles 5–6 mm (n = 7); , healthy follicles 7–8 mm (n = 7); , atretic follicles 3–4 mm (n = 6); , atretic follicles 5–6 mm (n = 5). In the present case, the level of expression of the target gene (PAPP-A, aromatase, LH receptor) was expressed as a ratio to the TBP housekeeping gene RNA level. The results are expressed in arbitrary units and are represented after logarithm conversion. Relationship among the variables was evaluated by simple correlation analysis with the nontransformed values (r, Pearson correlation coefficient).

	Discussion

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Overall, these results suggest that degradation of IGFBP-4 by PAPP-A in preovulatory follicles is a well-conserved mechanism in mammalian species. The coexpression of PAPP-A and aromatase and LH receptors in granulosa cells of preovulatory follicles suggest that PAPP-A could be considered a marker of follicular development. Of note, PAPP-A and proMBP mRNA expression in the ovary have been previously observed by Overgaard et al. (18). By in situ hybridization, Hourvitz et al. (43) have recently shown that PAPP-A was expressed in granulosa cells of healthy follicles from 5 mm antral to the preovulatory stage in human ovary. In vitro, FSH has been shown to stimulate IGFBP-4 degradation in rat and human granulosa cell culture (44, 45, 46). PAPP-A protein has also been recently detected in conditioned media from human granulosa derived from estrogen-dominant but not androgen-dominant follicles (47). Overall, these data suggest that PAPP-A expression in the ovary is restricted to granulosa cells of healthy dominant follicles. However, there is no evidence that PAPP-A mRNA expression is FSH dependent in the ovary. Progesterone could also be proposed as a modulator of PAPP-A secretion. Indeed, Sjöberg et al. (16) have found a positive correlation between intrafollicular PAPP-A level and progesterone concentration in human hyperstimulated follicles. In addition, PAPP-A mRNA and protein have been localized in corpus luteum and in luteinized granulosa cells in humans (16, 43). Further investigations are needed now to identify the factors regulating the expression of PAPP-A in the ovary.

In the present work, quantitative RT-PCR has shown that PAPP-A mRNA expression is detectable in some immature and atretic follicles in which no proteolytic activity degrading IGFBP-4 is observed, suggesting that the activity of residual PAPP-A is impaired. In particular, we have recently shown that in early atretic follicles, the decrease in IGF bioavailability, owing to the increase in IGFBP-2, may indirectly contribute to the decrease in IGFBP-4 degradation (4, 5). Moreover, in late atretic ovine and bovine follicles, the increase in peptides containing the heparin-binding domain from IGFBP-5 (following a dramatic increase in expression level) and from the C-terminal proteolytic fragment of IGFBP-3 (following an increase in proteolytic degradation) would strengthen the inhibition of IGFBP-4 degradation (4, 5). Further investigations are necessary to test whether proMBP, recently identified as a physiological inhibitor of PAPP-A activity, is involved in the modulation of intrafollicular IGFBP-4 degradation (21).

The intrafollicular degradation of IGFBP-4 by PAPP-A is closely conserved in preovulatory follicles of all the mammalian species studied. So one may hypothesize that degradation of IGFBP-4 has dramatic consequences on follicular maturation. Interestingly, very recent data have shown that as early as day 2 of the first follicular wave in cattle (at the beginning of luteal phase), IGFBP-4 proteolytic activity is higher in dominant in comparison with largest subordinate follicles, suggesting a role for the IGFBP-4 protease in the establishment of ovarian follicular dominance (48, 49). First, intrafollicular cleavage of IGFBP-4 could participate to the increase in bioavailability of IGFs that further stimulate granulosa cell proliferation and steroidogenesis (50, 51, 52). Moreover, it is possible that IGFBP-4 proteolytic fragments have an IGF-independent effect on follicular cells. PAPP-A could play a key role in ovarian follicles as well and particularly could participate in oocyte maturation. Finally, the increase in PAPP-A expression level in ovary coincides with dramatic proteolytic events associated with ovulation involving plasmin, matrix metalloproteases and tissue inhibitors of metalloproteases (53). It is possible that PAPP-A participates in ovulation by cleaving critical substrates.

We located the porcine PAPP-A gene to chromosome 1 (1q29–1q213). The human PAPP-A gene has been located to chromosome 9q33.1 by fluorescence in situ hybridization (54), and these two chromosomal regions have been shown to correspond to each other (55).

In summary, we have shown that PAPP-A is involved in IGFBP-4 degradation in ovine, bovine, porcine, and equine preovulatory follicles. For the first time, we have shown that PAPP-A mRNA is expressed by granulosa cells of these four mammals. In bovine and porcine ovary, PAPP-A expression is positively correlated with expression of both aromatase and LH receptors. Factors regulating the expression of PAPP-A mRNA in granulosa cells as well as biological consequences of PAPP-A expression and IGFBP-4 cleavage in preovulatory follicles remain to be determined.

	Acknowledgments

 
We acknowledge R. S. Bar for providing P3 and P5 peptides and D. R. Brigstock for the gift of CTGF-derived peptides. We are grateful to Dr. K. T. Preissner, Dr. D. D. Carson, and Dr. D. M. Waisman for providing the vitronectin peptides, the p36 subunit of annexin II tetramer peptide, and the HIP, respectively. We also wish to acknowledge Dr. L. Ständker for the gift of purified and recombinant N- and C-terminal fragments of human IGFBP-4. We are grateful to Dr. N. Gérard for providing equine follicular fluid. We thank Dr. D. Monniaux for helpful discussion and C. Pisselet and T. Delpuech for technical assistance. We are grateful to A. Beguey for the photographic work.

	Footnotes

 
This work was supported by Biotechnocentre/INRA grant and Grant 32-46808.96 from the Swiss National Science foundation. Sabine Mazerbourg was supported by a French fellowship from the "Ministère de l’éducation et de la recherche."

¹ Present address: Department of Gynecology and Obstetrics, Stanford University School of Medicine, Palo Alto, California 94305-5317.

Abbreviations: BW, Body weight; CTGF, connective tissue growth factor; HIP, heparin/heparan sulfate-interacting protein; IGFBP-4, IGF binding protein-4; ImpRH, INRA-Minnestota Porcine Radiation Map; IOD, integrated optical density; PAPP-A, pregnancy-associated plasma protein-A; PO, phosphoprotein; proMBP, proform of eosinophil major basic protein; rhPAPP-A, recombinant human PAPP-A; TBP, TATA box-binding protein; VN, vitronectin; WLB, Western ligand blotting.

Received May 23, 2001.

Accepted for publication July 8, 2001.

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