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Endocrinology, doi:10.1210/en.2003-1642
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Endocrinology Vol. 145, No. 8 3686-3695
Copyright © 2004 by The Endocrine Society

Pregnancy-Associated Plasma Protein-A Production in Rat Granulosa Cells: Stimulation by Follicle-Stimulating Hormone and Inhibition by the Oocyte-Derived Bone Morphogenetic Protein-15

Motozumi Matsui, Barbara Sonntag, Seong Soo Hwang, Tara Byerly, Ariel Hourvitz, Eli Y. Adashi, Shunichi Shimasaki and Gregory F. Erickson

Department of Reproductive Medicine (M.M., S.S.H., T.B., S.S., G.F.E.), University of California, San Diego, La Jolla, California 92093; Department of Obstetrics and Gynecology (B.S.), University Clinic of Münster, Münster D-48129, Germany; Department of Obstetrics and Gynecology (A.H.), Sheba Medical Center, Tel Hashomer 52621, Israel; and Division of Reproductive Sciences (E.Y.A.), Department of Obstetrics and Gynecology, University of Utah Health Sciences Center, Salt Lake City, Utah 84112

Address all correspondence and requests for reprints to: Gregory F. Erickson, Ph.D., Department of Reproductive Medicine, University of California San Diego, La Jolla, California 92093-0674. E-mail: gerickson{at}ucsd.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Pregnancy-associated plasma protein-A (PAPP-A) is the major IGF binding protein-4 (IGFBP-4) protease in follicular fluid, consistent with its proposed role in folliculogenesis. Despite growing interest, almost nothing is known about how PAPP-A expression is regulated in any tissue. Here we show that FSH and oocytes regulate PAPP-A expression in granulosa cells (GCs). By in situ hybridization, ovary PAPP-A mRNA was markedly increased by pregnant mare serum gonadotropin treatment, and the message was localized to the membrana GCs but not cumulus GCs (CGCs) of dominant follicles. To explore the mechanism, we used primary cultures of rat GCs. Control (untreated) cells produced little or no PAPP-A spontaneously. Conversely, FSH markedly stimulated PAPP-A mRNA and protein in a dose- and time-dependent fashion. Interestingly, PAPP-A expression in isolated CGCs was also strongly induced by FSH, and the induction was inhibited by added oocytes. To investigate the nature of the inhibition, we tested the effect of oocyte-derived bone morphogenetic protein-15 (BMP-15). BMP-15 alone had no effect on basal levels of PAPP-A expression by cultures of membrana GCs or CGCs. However, BMP-15 markedly inhibited the FSH stimulation of PAPP-A production in a dose-dependent manner. The cleavage of IGFBP-4 by conditioned media from FSH-treated GCs was completely inhibited by anti-PAPP-A antibody, indicating the IGFBP-4 protease secreted by GCs is PAPP-A. These results demonstrate stimulatory and inhibitory roles for FSH and BMP-15, respectively, in regulating PAPP-A production by GCs. We propose that FSH and oocyte-derived BMP-15 form a controlling network that ensures the spatiotemporal pattern of GC PAPP-A expression in the dominant follicle.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
UNDERSTANDING THE MECHANISMS by which dominant follicles develop in the ovary is a major goal of reproductive research because of its vital role in female fertility. FSH is required for dominant follicle development, and no other ligand by itself can serve in this regulatory capacity (1, 2). Although FSH is essential, several growth factors produced by the follicle cells themselves are capable of modulating, either amplifying or attenuating, FSH action (3). The prevailing concept is that growth factors form a controlling network to ensure the proper spatial and temporal pattern of FSH-dependent gene expression in the granulosa cells (GCs) during folliculogenesis, for better or for worse (4). The importance of the growth factors is underscored by the fact that any impairment of FSH function may cause infertility. In rodents, two growth factors known to be involved in regulating FSH action are IGF-I and bone morphogenetic protein (BMP)-15. IGF-I produced by the GCs has an essential role in FSH signaling (5, 6), whereas BMP-15 produced by oocytes constrains FSH action (7). How or whether the IGF-I and BMP-15 actions are linked in controlling FSH action has yet to be answered.

With regard to IGF-I, the IGF binding proteins (IGFBPs) are important because they can inhibit the binding of IGF to the type II growth factor receptor (8). The IGFBP family is comprised of six high-affinity-binding proteins (IGFBP-1, -2, -3, -4, -5, -6) (9) and several low-affinity binders termed IGFBP-related proteins (10). In the ovary, there is mounting evidence that IGFBPs are involved in the control of follicle dominance and atresia (reviewed in Ref. 11, 12, 13, 14, 15). This was first emphasized when Ling et al. (16) discovered a novel FSH inhibitor in porcine follicular fluid that proved to be an IGFBP. Examination of the expression of the IGFBPs in mammalian ovaries revealed that normal folliculogenesis and luteogenesis are accompanied by striking changes in the spatiotemporal pattern of expression of the six IGFBPs (17, 18, 19, 20, 21). Particular interest has focused on IGFBP-4 because the gene is strongly expressed in atretic, but not healthy, follicles (20, 21, 22), and IGFBP-4 can inhibit FSH action in cultured GCs (23, 24, 25). Collectively, these findings have led to the proposal that follicle atresia may be governed, at least in part, through IGFBP-4.

Within this context, proteases can degrade IGFBP-1 to -6 into fragments that have greatly reduced affinity for the IGF-I and IGF-II, thereby increasing the concentration of free bioavailable IGF ligand (8, 26). Recently much attention has been focused on a novel IGFBP-4 protease, pregnancy-associated plasma protein (PAPP)-A (27, 28, 29). Mature PAPP-A is secreted as a homodimer of approximately 400 kDa composed of two 200-kDa disulfide-bound subunits (28). The placenta is a major site of PAPP-A synthesis (30); however, a relatively high level of PAPP-A has been found in ovarian follicular fluid (31, 32, 33) in which it functions as an IGFBP-4 protease (34). The observation that follicular fluid from healthy follicles had considerably higher levels of PAPP-A activity than did atretic follicles led to the hypothesis that PAPP-A expression may play a role in the dynamics of dominant follicle development (34). Experimentally, this concept has been supported by in situ hybridization studies in which PAPP-A mRNA was identified in GCs of healthy, but not atretic, follicles (35, 36).

Although much is known about the structural and functional relationships of PAPP-A, we know very little about how PAPP-A expression is regulated. In the human placenta, PAPP-A protein synthesis can be stimulated by cAMP (37) and progesterone (38, 39). In mouse ovaries, pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin have been shown to induce high levels of PAPP-A mRNA in developing follicles and corpora lutea, respectively (36). In the mouse follicle, PAPP-A mRNA was not identified in the cumulus GCs (CGCs), suggesting that the oocytes may function to negatively regulate PAPP-A gene expression (36).

In the present study, we show that the production of PAPP-A by rat GCs is directly stimulated and inhibited by FSH and oocyte-derived BMP-15, respectively. Our findings suggest a model in which FSH and the oocytes work in concert to regulate the spatiotemporal pattern of PAPP-A expression in the GCs during the growth and development of the dominant follicle.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
The University of California at San Diego Institutional Animal Care and Use Committee approved the animal protocols. Female rats (Harlan Sprague Dawley, Indianapolis, IN) were housed under controlled temperature and lighting (14-h light, 10-h darkness). For GC culture, immature (23 d old) rats were implanted with a silastic capsule containing 10 mg diethylstilbestrol (DES, Sigma, St. Louis, MO) and killed 4 d later by CO2 asphyxiation (40). For in situ hybridization and cumulus cell culture, 25-d-old female rats received a single ip injection of 10 IU PMSG (Sigma) in 0.1 ml saline and were killed 24 h later by CO2 asphyxiation. It should be noted that we performed our investigation in the immature rat ovary because more is known about its structure/function relationships than in most other species, making further studies profitable, and the addition of FSH to serum-free medium induces cytodifferentiation of primary cultures of GCs that parallels normal in vivo responses (41). For these reasons, the immature rat is well suited for the analysis of the physiological control mechanisms governing GC cytodifferentiation during folliculogenesis, including the control and consequences of PAPP-A expression.

In situ hybridization
In situ hybridization for PAPP-A was performed essentially as described (36). Ovaries (control and 24 h after PMSG injection) were fixed overnight in Bouin’s fixative at 4 C, embedded in paraffin, and sectioned at 10 µm. A partial mouse PAPP-A cDNA (553 bp), a PCR product (GenBank accession no. AF258461; nucleotides 1599–2151) generated from mouse ovaries with PAPP-A-specific primers (36), was cloned into pGEM-T Easy Vector (Promega, Madison, WI). After linearization of plasmids, antisense and sense [35S]-uridine 5-triphosphate-labeled cRNA probes were generated by in vitro transcription using SP6 and T7, respectively. Sections were hybridized for 16 h at 60 C with 100 µl of a hybridization solution containing 1 x 106 cpm of a labeled PAPP-A antisense or sense probe. After hybridization, sections were treated with ribonuclease (20 µg/ml RNase A for 30 min, at 37 C), gradually desalted [2x saline sodium citrate (SSC), 1x SSC, and 0.5x SSC] and then incubated with 0.1x SSC at 75 C for 40 min. Dehydrated slides were exposed to BioMax MR film (Eastman Kodak Co., Rochester, NY) for 3 d. Sections were coated with NTB-2 emulsion (Eastman Kodak), exposed for 2 wk at 4 C in a desiccated dark box, developed, and stained with hematoxylin and eosin.

Membrana granulosa cell culture
DES-primed membrana GCs (MGCs) were isolated from late preantral/early antral follicles (300–400 µm in diameter) and cultured as described previously (40). After the GCs had been expressed from the follicles, the oocyte-cumulus complexes were removed by pipetting and the remaining population of MGCs collected for culture. MGCs (2 x 105 viable cells) were pipetted into 96-well culture plates containing 200 µl (final volume) of tissue culture medium [McCoy’s 5a medium (Invitrogen, Carlsbad, CA) containing 100 U/ml penicillin, 100 mg/ml streptomycin sulfate, 2 mM L-glutamine, and 100 nM androstenedione (Sigma)]. MGCs were cultured for different periods of time (0–48 h) with and without the indicated concentrations of ovine FSH (National Institute of Diabetes and Digestive and Kidney Diseases-oFSH-20 provided by Dr. A. F. Parlow, National Hormone and Pituitary Program, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases) and/or recombinant human BMP-15 (42).

CGC culture
After 24 h of PMSG exposure, the ovaries were collected and placed into HEPES-buffered McCoy’s 5a medium containing 3 mg/ml BSA. The Graafian follicles (approximate 10 follicles/ovary) were punctured with a 27G needle to release oocyte cumulus complexes (OCCs). Isolated OCCs (free of MGCs) were collected by pipetting and rinsed twice in tissue culture medium. The CGCs were mechanically separated from the oocytes through repeated pipetting through a small bore pipette. The CGCs collected from 60 OCCs were pipetted into 96-well culture plates containing 200 µl of the tissue culture medium. They were cultured for 24 h (37 C in an atmosphere containing 5% CO2 in air) in the presence and absence of 10 ng/ml FSH, oocytes (n = 60), and/or 100 ng/ml BMP-15.

Western immunoblot analysis
The GC conditioned medium (CM) was concentrated 20 times using the Ultrafree-Filter unit (10,000 molecular weight cut-off, Millipore, Bedford, MA). SDS-PAGE was performed in the presence of a reducing agent (5% ß-mercaptoethanol). Samples were loaded onto 8–16% Tris-glycine polyacrylamide gel and electrophoresed at 100 V for 2 h. Proteins were transferred to a 0.2-µm polyvinylidene difluoride membrane for 2 h at 150 mA. Molecular weights were estimated by comparison with prestained standards. Immunoblotting was performed by treating polyvinylidene difluoride membranes with blocking buffer overnight at 4 C, followed by incubation with primary antibody for 2 h at 4 C. For PAPP-A detection, 1% BSA/TBST buffer (1% BSA in 20 mM Tris-HCl and 0.01% Tween 20) was used as the blocking buffer. A rabbit antihuman PAPP-A polyclonal antibody (Zymed, South San Francisco, CA, 1:7500 dilution in blocking buffer) was used for PAPP-A detection. For IGFBP-4 detection, 1% BSA + 5% dry milk/TBST (1% BSA and 5% dry milk in TBST) was used as the blocking buffer. The anti-IGFBP-4 antibody was polyclonal antibody Rb-8 raised against a synthetic peptide fragment containing amino acids 110–121 of rat IGFBP-4 (23). As such it recognizes the amino terminus fragment of IGFBP-4. It was used at a 1:30,000 dilution in blocking buffer. We chose to assay PAPP-A in CM rather that cell lysates because PAPP-A is a secreted protein and its physiological relevance is coupled to it, being secreted into the microenvironment.

Chemiluminescence and densitometric analysis
Membranes were washed three times with TBST buffer and then incubated for 90 min with horseradish peroxidase-conjugated goat antirabbit IgG (Calbiochem, La Jolla, CA, 1:15,000 dilution for PAPP-A, 1:20,000 dilution for IGFBP-4). The membranes were washed and incubated with enhanced chemiluminescence (Amersham Biosciences Corp., Piscataway, NJ) detection reagents for 1 min and exposed to Hyperfilm-enhanced chemiluminescence (Amersham Biosciences) for 1–5 min. Western blots were scanned with a color scanner, and relative densities were analyzed using National Institutes of Health image software.

IGFBP-4 protease assay
CM was centrifuged and concentrated 20 times with Ultrafree centrifugal filter device (30,000 relative molecular weight cut-off; Millipore) to remove IGFBP-4. CM was incubated with 4 ng IGFBP-4 purified from adult rat serum (43) together with 30 nM IGF-I for 16 h at 37 C. The IGF-I was added to the reaction mixture because PAPP-A is an IGF-dependent protease (27). Absence of IGFBP-4 in filtered CM was confirmed by Western immunoblotting for IGFBP-4 detection. To examine the IGFBP-4 proteolysis, intact IGFBP-4 (24 and 28 kDa) and IGFBP-4 fragment (17.5 kDa) were detected by Western immunoblotting analysis. To verify that IGFBP-4 protease in CM is PAPP-A, 25 µl of concentrated CM were incubated with nonspecific rabbit IgG (Calbiochem, 0.05, 0.1, and 0.5 µg) and PAPP-A polyclonal antibody (0.05, 0.1, and 0.5 µg). After a 3-h incubation at room temperature, rat IGFBP-4 (4 ng) and IGF-I (30 nM) were added and the reaction mixture incubated for 16 h (37 C). The samples were then subjected to Western immunoblotting analysis.

Semiquantitative RT-PCR
Semiquantitative RT-PCR was employed to measure PAPP-A mRNA levels. Total RNA from MGCs or CGCs was extracted by guanidium acid-isothiocyanate-phenol-chloroform methods using TRIZOL (Invitrogen) according to the manufacturer’s instructions. RNA concentration was determined by spectrophotometric analysis at 260 nm and RNA was stored at –70 C until RT-PCR. Total RNA (1 µg) was isolated from MGCs or CGCs obtained from 60 cumulus-oocytes. It was treated with 1 U of RQ1 RNase-free DNase (Promega) and reverse transcribed in the 50 µl reverse transcription reaction mixture containing SUPERSCRIPT II RNase H– reverse transcriptase (200 U), deoxynucleotide triphosphate (0.2 mM), and oligo (dT)12–18 primer (500 µg/ml) following the manufacturer’s protocol (Invitrogen). The 50-µl PCR mixture contained 2 µl cDNA product, MgCl2 (2.5 mM), deoxynucleotide triphosphate (0.2 mM), Taq polymerase (0.4 U) and 0.5 pmol/ml primers. Primers for PCR were as follows: PAPP-A: 5'-CAGAATGCACTGTTACCTGGA-3' (nt 1179–1199) and 5'-GCTGATCCCAATTCTCTTTCA-3' (nt 1346–1326); and ß-actin: 5'-ATCGTGGGCCGCCCTAGGCA-3' (nt 1343–1362) and 5'-TGGCCTTAGGGTTCAGAGGGG-3' (nt 1673–1653). Numbers in parentheses refer to the positions in the corresponding cDNA sequence. Gene and cDNA sequences were obtained from GenBank (accession no: PAPP-A, AF258461; ß-actin, V01217). All primers were custom ordered from Life Technologies, Inc. (Grand Island, NY). The samples were subjected to 30 cycles of amplification in a thermal cycler with 1 min denaturation at 94 C, 1 min primer annealing at 65 C for PAPP-A or 54 C for ß-actin, and 1 min primer extension at 72 C. To determine the optimal quantity of reverse transcripts needed for PCR and to verify that cDNA product was dependent on the amount of transcript used, varying quantities of transcript template and varying number of cycles (in the range of 25–40 cycles) were tested in the PCR.

PCR products were analyzed by 2% agarose gel electrophoresis and visualized by ethidium bromide staining. Densitometric analysis of the PCR products was performed with the NIH Image program (version 1.62). Signals corresponding to PAPP-A expression in the linear range of amplification were normalized relative to ß-actin for each sample.

Statistical analysis
Each experiment was repeated at least three times. Data were analyzed by ANOVA followed by Fisher’s protected least significant difference (StatView 5.0 software, SAS Institute Inc., Cary, NC). Differences were accepted as significant when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PMSG induces PAPP-A mRNA expression in rat MGCs in vivo
At present, nothing is known about PAPP-A expression in rat ovaries. To address this question, we first analyzed PAPP-A mRNA in control (untreated) and PMSG-treated immature ovaries by semiquantitative RT-PCR. As shown in Fig. 1Go, PAPP-A mRNA was essentially undetectable in control ovaries, whereas the message was markedly increased 24 h after stimulation with PMSG. The results also show that PMSG had a strong stimulatory effect on PAPP-A message levels in the MGCs but not the CGCs (Fig. 1Go).



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  		FIG. 1. PMSG induces PAPP-A mRNA expression in rat ovarian granulosa cells in vivo. Immature rats were injected with 10 IU PMSG and the ovaries collected 24 h later. The GCs (MGCs and CGCs) were released from control (untreated) and PMSG-primed follicles as described in Materials and Methods. Total RNA was extracted and subjected to semiquantitative RT-PCR. Upper panel, typical electrophoresis of PCR products. Lower panel, data (mean ± SEM) for the ratios of PCR products (PAPP-A/ß-actin) from three experiments. *, P < 0.01 when compared with control.

 
Temporal and spatial regulation of PAPP-A mRNA expression in rat ovaries
To characterize further the tissue-specific pattern of PAPP-A expression, we performed in situ hybridization. In control (untreated) immature rat ovaries, PAPP-A mRNA was very low or undetectable (Fig. 2Go, A and B). PAPP-A mRNA was strongly expressed in PMSG-treated ovaries, being limited to the GCs of healthy Graafian follicles (Fig. 2Go, C and D). It was abundant in the MGCs but was undetectable in the periantral GCs and CGCs (Fig. 2Go, E and F). There was no detectable PAPP-A mRNA in atretic follicles (Fig. 2Go, C and D). No signal above background was seen in ovary sections hybridized with the sense probe (Fig. 2Go, G and H). The above results lead us to propose a role for FSH and the oocytes in regulating PAPP-A expression in the GCs during dominant follicle development.



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  		FIG. 2. Spatiotemporal pattern of expression of PAPP-A mRNA in PMSG-treated rat ovaries. Bright-field (A, C, E, and G) and dark-field photomicrographs of the same sections (B, D, F, and H). PA, Periantral GCs; O, oocytes; HF, healthy follicle; AF, atretic follicle. A and B, Untreated control. C and D, PMSG treated. E and F, PMSG treated. G and H, PMSG treated, sense probe. Bars, 100 µm.

 
FSH stimulates PAPP-A production by granulosa cells in vitro
First, we investigated whether FSH could stimulate PAPP-A expression in GCs. We used the serum-free DES-primed rat GC model in which most of the cells are of the membrana type and they exhibit physiological responses to FSH in vitro (41). When these cells were cultured for 48 h in the absence of FSH, there was little or no PAPP-A mRNA or protein detected in the cells and conditioned medium, respectively (Fig. 3Go). In contrast, treatment with nanomolar concentrations of FSH caused a marked stimulation of the expression of both PAPP-A mRNA (Fig. 3AGo) and protein (Fig. 3BGo), and the FSH effects were dose dependent (ED50 = ~1.2 ng/ml). We also investigated the time course of FSH-induced PAPP-A production. When cells were treated with a saturating dose of FSH (10 ng/ml), PAPP-A mRNA expression began to increase during the first 12 h and peaked after 36 h of stimulation (Fig. 3CGo). Analysis of CD demonstrated that the levels of the 200 kDa PAPP-A proteins increased only slightly during the first 36 h of culture but increased sharply and significantly from 36 to 48 h (Fig. 3DGo). Thus, FSH can stimulate PAPP-A expression in MGCs.



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  		FIG. 3. FSH stimulates PAPP-A production by cultured GCs. DES-primed granulosa cells were cultured in serum-free medium with the indicated concentrations of FSH. After culture, PAPP-A mRNA in the cells and PAPP-A protein in the CM were measured by RT-PCR and Western immunoblotting, respectively. A and B, Dose-response experiment. PAPP-A mRNA (A) and protein (B) were assayed after 48 h culture. C and D, Time-course experiment. In A and C, a representative electrophoresis of PCR products is shown at the top; at the bottom, the relative amount of PAPP-A mRNA is expressed as PAPP-A/ß-actin ratio (mean ± SEM) from three experiments. Bars with different letters are statistically different (P < 0.05). In B and D, a representative Western immunoblot is shown at the top; at the bottom, data (mean ± SEM) for the 200-kDa molecular mass band analyzed by densitometry from four independent experiments are plotted as arbitrary units. Relative densities of PAPP-A signal in CM were compared with the human PAPP-A standard (hPAPP-A, 25 ng). *, P < 0.01 when compared with untreated control (0 ng/ml FSH).

 
FSH-induced PAPP-A expression in cumulus cells is inhibited by oocytes
Given that PMSG-stimulated CGCs do not express PAPP-A message in situ, we wished to determine whether the oocyte is involved in regulating PAPP-A expression. Isolated CGCs were cultured as controls (no additions) or with FSH ± oocytes; after culture, total cumulus RNA was subjected to RT-PCR analysis. As shown in Fig. 4Go, control (untreated) CGCs expressed little or no PAPP-A mRNA spontaneously during culture in serum-free medium alone. However, like MGCs, treatment with FSH had a marked stimulatory effect (up to 10-fold) on PAPP-A mRNA expression. Interestingly, the FSH-stimulation of PAPP-A message was significantly decreased when the CGCs were cocultured with isolated fully grown ooctyes (Fig. 4Go). Thus, the oocyte appears to negatively regulate the FSH-induction of PAPP-A expression in CGCs.



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  		FIG. 4. FSH-induced PAPP-A expression in cumulus cells is inhibited by oocytes. Isolated CGCs (collected from 60 cumulus-oocytes from PMSG-primed immature rats) were cultured for 24 h in 200 µl of serum-free medium with either FSH (10 ng/ml) alone or with FSH plus 60 cumulus-free oocytes. After culture, PAPP-A mRNA in the CGCs was analyzed by RT-PCR. Upper panel, Typical electrophoresis of PCR products. Lower panel, Ratios (mean ± SEM; n = 3 experiments) of PCR products (PAPP-A/ß-actin). Bars with different letters are statistically different (P < 0.05).

 
FSH-induced PAPP-A in MGCs is inhibited by BMP-15
The concept that oocyte-derived BMP-15 is capable of inhibiting FSH action in rat GCs has been established (7). To determine whether BMP-15 can regulate PAPP-A, we tested the effects of recombinant BMP-15 on PAPP-A expression in MGCs from DES-primed rats. As shown in Fig. 5Go, BMP-15 alone had no effect on the basal expression of PAPP-A mRNA and protein in control (untreated) cells. In contrast, a marked dose-dependent decrease in FSH-induced PAPP-A mRNA (Fig. 5AGo) and protein (Fig. 5BGo) expression was observed upon coincubation with BMP-15. BMP-15 (100 ng/ml) produced an 80 and 70% decrease in PAPP-A mRNA and protein, respectively. The inhibition was seen at low (1 ng/ml) and maximally effective (10 ng/ml) concentrations of FSH. Thus, the inhibitory function of oocyte-derived BMP-15 can regulate FSH-induced PAPP-A production.



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  		FIG. 5. FSH-induced PAPP-A in MGCs is inhibited by BMP-15. GCs from DES-primed immature rats were cultured for 48 h in serum-free medium with the indicated concentrations of FSH and/or BMP-15. After culture, PAPP-A mRNA was analyzed by RT-PCR, whereas PAPP-A protein in CM was analyzed by Western blotting. A, Upper panel, a typical electrophoresis of PCR products. Lower panel, Data (mean ± SEM; n = 3 experiments) of the ratios of PCR products (PAPP-A/ß-actin). *, P < 0.05 when compared with matched control (FSH treatment only). B, Upper panel, a typical Western immunoblot. Lower panel, Relative densities (mean ± SEM; n = 3 experiments) of the 200-kDa molecular mass band. Relative densities of the PAPP-A signal in conditioned medium were compared with that of the human PAPP-A standard (hPAPP-A, 25 ng). *, P < 0.05 when compared with matched controls.

 
BMP-15 inhibits FSH-induced PAPP-A gene expression in cumulus cells
We next investigated the possible role of BMP-15 in regulating PAPP-A expression in the CGCs. Oocyte-free CGCs were cultured in the presence of a saturating dose of FSH and/or BMP-15. As indicated in Fig. 6Go, BMP-15 alone had no effect on basal PAPP-A expression, but BMP-15 inhibited by approximately 50% the ability of FSH to stimulate PAPP-A mRNA expression. These observations are consistent with the involvement of oocyte-derived BMP-15 in suppressing PAPP-A gene expression in CGCs.



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  		FIG. 6. BMP-15 inhibits FSH-induced PAPP-A gene expression in cumulus cells. Isolated cumulus cells from PMSG-primed rats were cultured for 24 h in serum-free medium with FSH (10 ng/ml) and/or BMP-15 (100 ng/ml). After culture, total RNA was extracted and subjected to semiquantitative RT-PCR. Upper panel, Typical electrophoresis of PCR products. Lower panel, Ratios (mean ± SEM; n = 3 experiments) of PCR products (PAPP-A/ß-actin). Bars with different letters are statistically different (P < 0.05).

 
PAPP-A secreted by rat GCs exhibits IGFBP-4 protease activity
First, we investigated whether PAPP-A in GC CM has IGFBP-4 proteolytic activity. Incubation of IGFBP-4 with CM that contained immunoreactive PAPP-A product (Fig. 7AGo, upper panel) resulted in the degradation of intact IGFBP-4 as evidenced by the generation of IGFBP-4 fragments (Fig. 7AGo, lower panel).



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  		FIG. 7. PAPP-A secreted by rat GCs exhibits IGFBP-4 protease activity. GC CM was subjected to assay for protease activity of PAPP-A as described in Materials and Methods. A, DES-primed GCs were treated for 48 h with or without 10 ng/ml FSH after which the CM was concentrated and assayed for PAPP-A and degradation of IGFBP-4. Upper panel, Western blot analysis for PAPP-A. Lower panel, Western blot analysis of added intact IGFBP-4 (4 ng) incubated together with CM. Protease activity of PAPP-A is evidenced by a decrease in intact IGFBP-4 (24 and 28 kDa) and the appearance of a 17.5-kDa IGFBP-4 fragment. B, Immunoneutralization of PAPP-A in CM. CM was preincubated together with increasing amounts of either anti-PAPP-A antibody or control rabbit IgG. Immunoneutralized CM was incubated together with intact IGFBP-4 (4 ng) and analyzed for IGFBP-4 degradation by Western blotting.

 
To evaluate whether PAPP-A secreted by the GCs functions as an IGFBP-4 protease, we determined the effects of anti-PAPP-A antibody on the ability of conditioned media from FSH-stimulated cells to degrade added IGFBP-4. As shown in Fig. 7BGo, incubation with increasing amounts of control IgG protein (0.05–0.5 µg) had no significant affect on the ability of conditioned media to degrade added 24 kDa IGFBP-4 into its 17.5-kDa fragment. In contrast, a dose- dependent decrease in IGFBP-4 degradation was observed upon incubation of CM with anti-PAPP-A antibody (Fig. 7BGo). Here proteolysis of IGFBP-4 appeared to be completely blocked by treatment with 0.5 µg anti-PAPP-A antibody.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This work provides the first insight into the cellular mechanisms regulating PAPP-A expression in the mammalian ovary. We show that PAPP-A transcripts are strongly expressed in the PMSG-primed rat ovary, being present in the MGCs of healthy Graafian (dominant) follicles. The mechanism of PAPP-A induction in the MGCs is by FSH stimulation. Although the expression of PAPP-A is robust in the MGCs, it is absent in the CGCs juxtaposed to the oocyte. A principal finding is that oocytes can negatively regulate FSH-induced PAPP-A gene expression by a mechanism involving oocyte-derived BMP-15. The PAPP-A protein secreted by MGCs degrades IGFBP-4. Accordingly, PAPP-A produced by rat GCs is a functional IGFBP-4 protease. Collectively, these findings support a model in which MGCs in the dominant follicle respond to FSH stimulation with the synthesis and secretion of the IGFBP-4 protease, PAPP-A, which in turn acts to degrade IGFBP-4, a known inhibitor of FSH action in the ovary. This model predicts that PAPP-A may play a key role in regulating FSH-dependent dominant follicle development in the rat. It is noteworthy that recent results in domestic species also indicate that the acquisition of PAPP-A is a key component of the selection of follicles for dominance and potential ovulation (15, 33, 44, 45, 46, 47). Therefore, the present results on the regulation of PAPP-A in rat follicles complement nicely this previous work.

The present in situ hybridization data indicate that PAPP-A mRNA is expressed in the GCs of dominant follicles induced by PMSG. In contrast, it was not expressed in other follicles including the cohort of atretic Graafian follicles. The interpretation is that PAPP-A may be an observable differentiation marker for developing dominant follicles in rats. In support of this idea, a similar spatiotemporal pattern of ovary PAPP-A gene expression has been reported in normal cycling animals including mice (36), humans (35), sheep, cattle, pigs, and horses (33). Furthermore, the selective expression of PAPP-A protein in healthy Graafian and preovulatory follicles (31, 32, 48) argues that the PAPP-A mRNA expressed in dominant follicles is being translated. Evidence has also accumulated that assigns PAPP-A protein in follicular fluid a very important role in the proteolysis of IGFBP-4, and the expression of this protease activity correlates with selection of the dominant follicle (44, 47, 49). Collectively, this body of evidence supports the conclusion that PAPP-A synthesis is a characteristic feature of dominant follicles in mammals. It is notable that PAPP-A mRNA is expressed in all developing follicles in the rhesus monkey indicating that species differences do exist (50).

We have previously shown in the mouse that PMSG causes PAPP-A expression in the outer-layer GCs (the membrana or mural) but not in the inner periantral-layer GC and CGCs (36). In the present study, we confirm this finding using the immature rat model. These findings clearly show that the way in which the GCs respond to PMSG with PAPP-A gene expression depends on their position within the dominant follicle. The concept to emerge from these observations is that PAPP-A expression is regulated with great precision and that the process involves both stimulatory and inhibitory mechanisms within the GC system. In the rat, PMSG has been shown to exhibit equal FSH and LH bioactivity (51). Our finding that FSH is a potent inducer of PAPP-A gene expression in cultured rat GCs suggests that the FSH bioactivity in PMSG is principally responsible for the induction of PAPP-A mRNA in the MGC; however, further studies will be required to determine whether the LH bioactivity in PMSG plays a role in regulating PAPP-A expression.

In this context, a comparable situation is seen in the FSH induction of other molecular markers of GC cytodifferentiation including the expression of LH receptor (52), prolactin receptor (53), P450 aromatase (54), P450 side-chain cleavage (55), 3ß-hydroxy steroid dehydrogenase (56), and the inhibin-{alpha} and -ßA subunits (57, 58). Findings of this type have led to the idea that the FSH-induced program of cytodifferentiation is restricted to the MGCs. It is worth emphasizing that the program of FSH-dependent GC cytodifferentiation is more complex. For example, treatment with FSH in vivo causes the complete inhibition of IGF-I (59, 60) and BMP-6 mRNA expression (61) in MGCs, whereas the expression remains high in the cumulus and periantral cells. In contrast, FSH administration does not change the relatively high levels of expression of the mRNAs encoding GnRH receptor (62, 63), BMPRII (61), BMP-2 (61), and kit ligand (64) in the MGCs, but causes the loss of these transcripts in the cumulus and periantral GCs. Thus, the complexity of the FSH-induced program of cytodifferentiation is clearly evident; it is not restricted to a particular set of cells but rather occurs in the entire population of GCs of the dominant follicle; the direction of differentiation depends on their position within the granulosa system; and the process entails a composite of both gene activation and inhibition.

The theory that oocyte-secreted growth factors are involved in diversification of FSH responses has gained acceptance in recent years (65, 66, 67, 68, 69, 70, 71). In two key studies in this area, Eppig and coworkers (72, 73) provided clear evidence that oocytes are capable of determining the spatial pattern of FSH-induced LH receptors and kit ligand expression in mouse GCs. These findings (and others) have led to the following theory: all GCs (membrana, periantral, and cumulus) are competent to respond to FSH stimulation by expressing the fully differentiated state; however, oocyte controls operate to determine when and where this cytodifferentiation program is expressed within the GC system. If this theory applies to PAPP-A, then the oocyte-free CGCs should respond to FSH by expressing the PAPP-A gene. Indeed, we found that when CGCs are placed under oocyte-free conditions, then FSH causes them to express PAPP-A, a situation that clearly does not happen in situ. By comparison, we show that if oocytes are added back, then the FSH-induced PAPP-A gene activity is significantly suppressed. These observed differences in FSH-induced PAPP-A gene expression are clearly related to the presence or absence of oocytes.

Our in vitro experiments indicate that the oocyte growth factor, BMP-15, is involved in regulating PAPP-A expression in GCs. Two lines of evidence support this view. The first is that recombinant BMP-15 inhibits the ability of FSH to stimulate PAPP-A mRNA and protein production by cultured MGCs. The abrogation of PAPP-A production by these cells was nearly complete at the highest concentration of BMP-15 tested. The second is that the ability of FSH to induce PAPP-A gene expression in isolated CGCs is inhibited by BMP-15. Taken as a whole, these results support a model in which an oocyte-derived BMP-15 operates to establish the FSH-dependent spatiotemporal pattern of PAPP-A gene expression that occurs in the GC system in situ. The cellular mechanisms by which BMP-15 decreases PAPP-A expression remain to be determined.

It is noteworthy that PAPP-A gene expression by CGCs was only partially inhibited by concentrations of BMP-15 that nearly completely inhibited the induction of PAPP-A expression in MGCs. Realization that PAPP-A expression is completely inhibited in these cells in situ raises the possibility that other oocyte growth factors might regulate PAPP-A expression. To date, five oocyte growth factors have been identified: TGFß2 (74), growth differentiation factor (GDF)-9 (75, 76), BMP-15 (42, 77, 78), BMP-6 (61, 79), and fibroblast growth factor-8 (80) (for review, see Ref. 81). Two good candidates are GDF-9 and BMP-6. GDF-9 has been shown to inhibit FSH-stimulated steroidogenesis (82) and kit ligand expression (73) in cultured GCs. And BMP-6 can act to inhibit FSH action including stimulation of steroidogenic acute regulatory protein, P450 side-chain cleavage, LH receptor, and inhibin/activin subunits ({alpha}, ßA, and ßB) gene expression by suppressing adenylate cyclase activity in rat GCs (83). Further studies will be required to investigate whether other oocyte growth factors play a role regulating PAPP-A gene expression, particularly at the level of the cumulus and periantral GCs.

Finally, our finding that PAPP-A proteolysis of IGFBP-4 in CM was eliminated by anti-PAPP-A antibody fits the prediction that PAPP-A is the predominant, if not sole, IGF-dependent IGFBP-4 protease secreted by FSH-stimulated rat GCs. This result is consistent with the findings that PAPP-A protein accounts for all the activity of IGFBP-4 protease in CM collected from cultured human in vitro fertilization granulosa lutein cells (49). The likelihood that these findings are physiologically relevant comes from studies showing that PAPP-A accounts for all the activity of IGFBP-4 protease in follicular fluid of preovulatory and estrogen-dominant follicles in human (34), ovine, bovine, porcine, and equine ovaries (33). It remains possible that follicular PAPP-A plays other roles, such as degradation of IGFBP-5 (29, 84); however, future studies are required to test this hypothesis.

In summary, our results indicate that FSH and BMP-15 can have antagonistic roles in regulating PAPP-A expression. In rat GCs, FSH stimulates PAPP-A production, whereas BMP-15 antagonizes the FSH stimulation. This push-pull type of interaction in governing PAPP-A expression is believed to be important in generating the spatiotemporal pattern of PAPP-A gene expression in the GC system of dominant follicles during the follicular phase of the cycle. In this way, oocyte-derived BMP-15 would serve to modulate FSH-dependent functions (including PAPP-A expression) in both space and time. The fact that PAPP-A gene expression occurs in healthy but not atretic follicles is consistent with the concept that PAPP-A functions to destroy IGFBP-4 in follicular fluid for the purpose of ensuring access of the GCs to a local source of the survival and differentiation factor, IGF-I (85, 86, 87, 88). Further experiments will be needed to determine whether enhanced BMP-15 signaling could play a role in atresia and growth arrest of follicles by mechanisms involving the inhibition of FSH-induced PAPP-A expression.


   Footnotes
 
This work was supported by National Institutes of Health Grant U54 HD12303 as part of the Specialized Cooperative Centers Program in Reproduction Research.

Abbreviations: BMP, Bone morphogenetic protein; CGC, cumulus GC; CM, conditioned medium; DES, diethylstilbestrol; GC, granulosa cell; GDF, growth differentiation factor; IGFBP, IGF binding protein; MGC, membrana GC; OCC, oocyte cumulus complex; PAPP, pregnancy-associated plasma protein; PMSG, pregnant mare serum gonadotropin; SSC, saline sodium citrate; TBST, Tris-HCl and Tween 20.

Received December 2, 2003.

Accepted for publication April 6, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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