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Originally published In Press as doi:10.1074/jbc.M403212200 on May 6, 2004

J. Biol. Chem., Vol. 279, Issue 31, 32281-32286, July 30, 2004
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Growth Differentiation Factor 9 Regulates Expression of the Bone Morphogenetic Protein Antagonist Gremlin*

Stephanie A. Pangas{ddagger}§, Carolina J. Jorgez¶, and Martin M. Matzuk{ddagger}§||**

From the Departments of {ddagger}Pathology, §Molecular and Cellular Biology, and ||Molecular and Human Genetics and Program in Developmental Biology, Baylor College of Medicine, Houston, Texas 77030

Received for publication, March 23, 2004 , and in revised form, May 4, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Growth differentiation factor 9 (GDF9) is an oocyte-expressed member of the transforming growth factor {beta} (TGF-{beta}) superfamily and is required for normal ovarian follicle development and female fertility. GDF9 acts as a paracrine factor and affects granulosa cell physiology. Only a few genes regulated by GDF9 are known. Our microarray analysis has identified gremlin as one of the genes up-regulated by GDF9 in cultures of granulosa cells. Gremlin is a known member of the DAN family of bone morphogenetic protein (BMP) antagonists, but its expression and function in the ovary are unknown. We have investigated the regulation of gremlin in mouse granulosa cells by GDF9 as well as other members of the TGF-{beta} superfamily. GDF9 and BMP4 induce gremlin, but TGF-{beta} does not. In addition, in cultures of granulosa cells, gremlin negatively regulates BMP4 signaling but not GDF9 activity. The expression of gremlin in the ovary was also examined by in situ hybridization. A distinct change in gremlin mRNA compartmentalization occurs during follicle development and ovulation, indicating a highly regulated expression pattern during folliculogenesis. We propose that gremlin modulates the cross-talk between GDF9 and BMP signaling that is necessary during follicle development because both ligands use components of the same signaling pathway.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The bone morphogenetic proteins (BMPs)1 and growth differentiation factors (GDFs) constitute the largest subgroup of the TGF-{beta} superfamily of secreted growth factors (1). Many of the ligands are expressed throughout embryogenesis as well as in adult tissues, reflecting the diversity of their biological activities. BMPs were first characterized as growth factors that cause de novo bone synthesis in non-skeletal sites (2, 3). Subsequent work has demonstrated that they are multifunctional proteins involved in mesoderm formation, limb development, branching morphogenesis, germ cell development, cardiac development, and female fertility, among other functions (1, 4). BMPs are secreted dimeric proteins that bind to membrane-bound receptor serine-threonine kinases (5). The type I and type II receptors, in turn, phosphorylate cytoplasmic SMAD proteins that act as transcription factors to regulated down-stream target genes (6).

GDF9 is a TGF-{beta}-related protein necessary for female fertility (7, 8). Although Gdf9 knock-out mice arrest at the primary follicle stage, in vitro studies have shown that GDF9 signaling is also critical during ovulation. During ovulation, GDF9 induces cumulus cell expansion, an inflammation-like process that involves the mucification of the cells surrounding the oocyte. This process binds the cumulus cells to the oocyte, protecting the oocyte during follicular extrusion and assisting in sperm binding and fertilization. Cumulus cell expansion partly involves the production of hyaluronic acid, prostaglandin E2 (PGE2), pentraxin 3, and tumor necrosis factor-induced protein 6 (9–13), all of which have been demonstrated to be regulated by GDF9 in vitro (10, 14, 15).

Ovarian follicles produce a number of TGF-{beta}-related proteins in addition to GDF9 during folliculogenesis: BMP6 and BMP15 are co-expressed in the oocyte with GDF9 (16); anti-Mullerian hormone, TGF-{beta}s, activins, and inhibins are produced from the granulosa cells; and BMP4, BMP7, and TGF-{beta}s are synthesized from the thecal cells (8, 17–20). In addition, granulosa cells express most of the cell surface binding receptors, including BMPR2, ACVR2, ACVR2B, TGFBR2, and AMHR2. GDF9, BMP15, BMP4, and BMP7 all use BMPR2 as a binding receptor (21–23). The type I receptor ALK6 has also been shown to mediate signaling of those ligands as well (21, 22, 24). It is presently unclear how granulosa cells discriminate between signals from multiple ligands that bind to the same receptor complexes.

Several high-affinity binding proteins antagonize BMP signaling, including follistatin, noggin, chordin/SOG, and members of the DAN family, including DAN, cerberus, and gremlin (25–27). Their primary mode of inhibition occurs by binding to the ligand, which prevents the association with the receptor complex (28, 29). The extracellular antagonists are often part of negative feedback loops and thus are directly up-regulated by the ligands that they antagonize (25). Although follistatin is highly expressed in the mouse ovary, the expression of other negative regulatory factors in the ovary is unknown. During a screen for GDF9-regulated genes, we discovered that gremlin was up-regulated in GDF9-treated preovulatory granulosa cells. During ovulation, gremlin expression is up-regulated in cumulus cells and down-regulated in mural granulosa cells, and our in vitro data demonstrate that, although both GDF9 and BMP4 induce gremlin, gremlin selectively blocks BMP signaling and not GDF9 signaling. We hypothesize that during ovulation this distinction drives mural granulosa cells toward eventual luteinization, whereas cumulus cells undergo cell expansion through GDF9 signaling.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Animals—All experimental animals were maintained in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All experiments used 19–21-day-old CD1 female mice (Baylor College of Medicine). Mice were injected intraperitoneally for the indicated times with 5 IU of PMSG (Gestyl) (Diosynth Biotechnology, Oss, Holland) to stimulate follicular growth. After 48 h, the mice were injected intraperitoneally with 5 IU of hCG (Pregnyl) (Diosynth Biotechnology) for the indicated times to induce ovulation and luteinization.

Recombinant Mouse GDF9 Production and Recombinant Ligands— Recombinant mouse GDF9-conditioned medium was produced from stably transfected Chinese hamster ovary cells (14). Control-conditioned medium was produced from Chinese hamster ovary cells stably transfected with the parent plasmid. Cells were grown to confluence in Dulbecco's modified Eagle's medium-F12 (Invitrogen) containing 10% heat-inactivated fetal bovine serum (Sigma) and 1% penicillin/streptomycin (Invitrogen). All experimental GDF9 and control media were serum-free, except the medium used for microarrays (see below). For collection, the medium was replaced with Dulbecco's modified Eagle's medium-F12 containing 5% crystalline grade bovine serum albumin (Serologicals Protein Inc., Kankakee, IL) and collected every 72 h. Following final collection, the medium was concentrated 10-fold on a tangential flow concentrator (Millipore, Bedford, MA), and the amount of GDF9 was quantified by immunoblotting and comparison to a standard curve of purified Escherichia coli-produced recombinant standard. GDF9-containing medium and control-conditioned medium were treated identically throughout the process. Recombinant human BMP4 was a gift of Wyeth (Cambridge, MA). Recombinant mouse gremlin was purchased from R&D Systems (Minneapolis, MN). For experiments with cell cultures treated with gremlin, ligands were incubated for 30 min with recombinant gremlin before being added to cultures.

Cell Cultures—Granulosa cells were isolated from large antral follicles from CD1 mice that had been treated with PMSG for 48 h before collection as described (14). These cells represent a mixture of both mural and cumulus granulosa cells and are referred to generically as "granulosa cells" in the text. Animals were 21–23 days old at the time of cell collection. 4 x 105 cells/well were plated in 12-well tissue culture dishes in 0.5% heat-inactivated FBS, 10 µg/ml insulin, 5.5 µg/ml transferrin, 6.7 pg/ml selenium (ITS-A, Invitrogen), and 10 units/ml penicillin/streptomycin. Cells were treated immediately on harvesting, unless indicated in the text. For microarray experiments, cells were treated with control-conditioned medium or 200 ng/ml GDF9-conditioned medium that contained 1% FBS. All other experiments were performed with serum-free conditioned media.

Microarray Analysis—cDNA was prepared from total RNA using the SuperScript double-stranded cDNA synthesis kit (Invitrogen). Biotinylated antisense cRNA was prepared using the Enzo BioArray High Yield RNA labeling kit (Enzo Diagnostics, Farmingdale, NY). The labeled cRNA was separated from unincorporated ribonucleotides using a Chroma Spin-100 column (BD Biosciences) and subsequently precipitated. RNA collected from a single experiment was used for array analysis (n = 1). Array data were compared with array data from previous independent experiments that used the same treatment protocols. Previous microarrays (10) would not have identified gremlin as a target because it was not present on the GeneChips (Affymetrix, Mu11K and Mu19K) used in those experiments.

The Affymetrix oligonucleotide arrays (MG_U74Av2) containing oligonucleotide probes corresponding to ~12,500 full-length annotated genes and expressed sequence tag sequences were hybridized, washed, and scanned using Affymetrix equipment and protocols. Sample loading and variations in staining were standardized by scaling the average of the fluorescent intensities of all genes on an array to constant target intensity for all arrays used. Data analysis was conducted using Microarray Suite version 5.0 (Affymetrix) following user guidelines. The signal intensity for each gene was calculated as the average intensity difference, represented by [{Sigma}(PM – MM)/(number of probe pairs)], where PM denotes perfect match and MM denotes mismatch probes.

RNA Isolation and Northern Blot—RNA was extracted from cells using TRIzol reagent (Invitrogen) and quantified by spectrophotometry. 10 µg of total RNA was electrophoresed through a 1% agarose MOPS gel, transferred to nylon membrane, and probed with 32P-labeled DNA probes generated from random priming of cDNA for mouse gremlin or glyceraldehyde-3-phosphate dehydrogenase using Ready-to-go labeling beads (Amersham Biosciences). Blots were hybridized with 2 x 106 cpm/ml hybridization buffer (Church and Gilbert hybridization buffer: 0.5 M NaHPO4, 7% SDS, 1 mM EDTA) (30) with 100 µg/ml salmon sperm DNA at 65 °C overnight. Blots were washed in sequential washes of SSC, 0.1% SDS from 2x SSC to 0.1x SSC at 65 °C, exposed to film or to a PhosphorImager screen, and quantified on a PhosphorImager (Amersham Biosciences).

Quantitative Real Time RT-PCR—Real time RT-PCR was performed on the ABI Prism 7700 sequence detection system (ABI, Foster City, CA) using Assays-On-Demand (ABI) PCR primer and probe sets for mouse gremlin (GenBankTM accession no. NM011824; ABI assay ID, Mm00488615) and eukaryotic 18 S rRNA as the endogenous control. RNA was treated with DNase I (DNA-Free, Ambion, Austin, TX) prior to RT-PCR reactions. RT-PCR was performed using the TaqMan Universal RT-PCR Master Mix (ABI) in a 20-µl solution. The reaction conditions were 30 min hold at 48 °C, 10 min hold at 95 °C followed by 40 cycles of 15 s at 95 °C (denaturation), and then 1 min and 60 s (annealing/extension). Each sample was analyzed in duplicate from three independent experiments. Two non-template control (RNase-free water) samples were included on each plate for each primer-probe set. The amount of gremlin was normalized to the endogenous reference (18 S rRNA). The mean sample threshold cycle (CT) and mean endogenous control CT were calculated for each sample from duplicate wells. The mean CT of the endogenous control was then subtracted from the CT of gremlin to give the {Delta}CT. The {Delta}CT of each sample was then subtracted from the control (time 0, untreated) {Delta}CT, which served as the reference sample. The relative amount of target gene expression was calculated using the formula 2–({Delta}{Delta}CT). The average and standard errors were calculated for the triplicate measurements, and the relative amount of target gene expression for each sample was plotted in Excel.

In Situ Hybridization—In situ hybridization was performed as described previously (8, 31). Briefly, freshly dissected ovaries were fixed in 4% paraformaldehyde overnight and embedded in paraffin. Four-µmthick sections were cut and pretreated as described. A riboprobe was generated from a plasmid containing the full-length gremlin transcript. [{alpha}-35S]UTP-labeled antisense and sense probes were generated using the Riboprobe T7/T3/SP6 kits (Promega Corporation, Madison, WI) and hybridized to serial sections at 55 °C in 50% deionized formamide, 0.3 M NaCl, 20 mM Tris-HCl (pH 8.0), 5 mM EDTA (pH 8.0), 10 mM NaPO4 (pH 8.0), 10% dextran sulfate, 1x Denhardt's solution, and 0.5 mg/ml yeast RNA. All sense probes showed minimal to no background hybridization. High stringency washes were carried out in 2x SSC, 50% formamide, and 0.1x SSC at 65 °C. Dehydrated sections were dipped in NTB-2 emulsion (Eastman Kodak Co.) and exposed at 4 °C for variable times. After they were developed, slides were counterstained with Mayer's hematoxylin and mounted for photography.

PGE2 Assays—Granulosa cells were plated at a density of 8 x 104 cells/well in 48-well tissue culture dishes in Dulbecco's modified Eagle's medium-F12 containing 2% heat-inactivated FBS, 10 µg/ml insulin, 5.5 µg/ml transferrin, 6.7 pg/ml selenium (ITS-A, Invitrogen), and 10 units/ml penicillin/streptomycin (Invitrogen) and cultured overnight. The following morning, the cells were washed once with phosphate-buffered saline and then treated in medium containing 0.5% heat-inactivated FBS, 6.7 pg/ml selenium, penicillin/streptomycin, and the indicated ligands. Each treatment condition was carried out in duplicate or triplicate wells. Cell culture medium was collected after 12 h of treatment and stored at –20 °C until assayed. PGE2 was measured by enzyme-linked immunosorbent assay using the PGE2 enzyme-linked immunoassay Express Kit (Cayman Chemicals, Ann Arbor, MI). The detection limit of this assay is 36 pg/ml. The results were verified in a minimum of three independent experiments.

Statistical Analysis—Statistical analysis was performed using one-way analysis of variance followed by the Tukey-Kramer HSD test for multiple comparisons (version 5.1, JMP Software, Cary, NC). PGE2 data were analyzed by one-way analysis of variance followed by Dunnett's test for comparing treatment to control groups. p values smaller than 0.05 were considered statistically significant. Statistics were performed on no less than three independent experiments.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
GDF9 Induces the Expression of Gremlin in Vitro—In vivo, GDF9 promotes the development of preovulatory follicles by changes in granulosa cell gene expression, including hyaluronan synthase 2, cyclooxygenase 2, pentraxin 3, urokinase plasminogen activator, and luteinizing hormone receptor (14, 32). To identify additional targets of GDF9 signaling, microarray analysis (Affymetrix, murine genome set U74Av2) was used to identify genes and expressed sequence tags differentially regulated by recombinant GDF9 in cultures of preovulatory granulosa cells. Granulosa cells were isolated from large antral follicles of immature mice and cultured in the presence or absence of recombinant mouse GDF9. A number of preovulatory genes previously identified as regulated by GDF9 (10, 14) were confirmed by this assay (Table I). Pentraxin 3, an important regulator of cumulus cell-oocyte integrity and a down-stream gene of the GDF-9 signal transduction cascade (10), was up-regulated 5.3-fold. Also, hyaluronan synthase 2, the major hyaluronic acid synthase protein involved in cumulus expansion (14, 33), was up-regulated 8-fold.


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  		TABLE I
Genes in granulosa cells up-regulated by GDF9

 
In this array experiment, gremlin showed the highest level of up-regulation by GDF9 and was increased by 8.6-fold. To confirm this result, independent samples of RNA from granulosa cells treated with GDF9 were analyzed by Northern blot analysis and quantitative real time PCR. By Northern blot analysis, a low but detectable amount of gremlin mRNA was present in control-treated cells (Fig. 1A). GDF9 induced the expression of gremlin within 5 h of treatment (Fig. 1A). Additionally, when cells were treated with increasing concentrations of GDF9, a dose-dependent induction of gremlin mRNA was observed (Fig. 1B). Doses as low as 12.5 ng/ml were able to stimulate the expression of gremlin (Fig. 1B). At the highest concentration tested (100 ng/ml), GDF9 was able to increase the expression of gremlin by ~6–7-fold (Fig. 1B), consistent with the microarray results.



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  		FIG. 1.
Recombinant GDF9 induces gremlin expression in cultures of granulosa cells. A, Northern blot analysis for granulosa cells treated for 5 h with 100 ng/ml GDF9 shows an increase in gremlin mRNA compared with control-treated cells. Glyceraldehyde-3-phosphate dehydrogenase (Gapd) was used as an RNA loading control. B, relative gremlin expression was measured by quantitative real time PCR. The control level is taken as 1, and the data are expressed as -fold increase over control values. Treatment for 5 h with increasing doses of GDF9, from 0 to 100 ng/ml, causes a significant increase in gremlin expression versus the control-treated cells. Bars with different letters a–d indicate that the group means are statistically different (p < 0.05). The mean ± S.E. for three independent experiments is shown.

 
To test the timing of gremlin induction by GDF9, granulosa cells were treated for 20 h with or without GDF9. Gremlin expression significantly increased after 3 h of treatment and continued to be significantly different from control-treated cells until 8 h post-induction (Fig. 2). At the end of these experiments, 20 h post-ligand stimulation, however, the level of gremlin in GDF9-treated samples was no longer different from the control-treated cells.



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  		FIG. 2.
Gremlin induction by GDF9 is temporally restricted in granulosa cells. Relative gremlin expression was measured by quantitative real time PCR during a 20-h time course for control and GDF9-treated (100 ng/ml) granulosa cells. mRNA was measured for cells collected at time 0, 3, 5, 8, and 20 h post-ligand stimulation. Data for both control-treated and GDF9-treated (100 ng/ml) cells were normalized to gremlin levels at the time of collection (0 h). Data are shown as the -fold increase of GDF9-treated cells over control-treated cells at each time point. A significant increase in gremlin expression was observed after 3 h and until 8 h in the GDF9-treated cells. Bars with different letters a and b indicate that the group means are statistically different (p < 0.05). The mean ± S.E. for three independent experiments is shown.

 
Expression of Gremlin during Follicle Development in Mouse Ovaries—The expression of gremlin in the mouse ovary has not been described previously. To determine the cellular localization of gremlin during folliculogenesis, we performed in situ hybridization on ovaries from immature mice following superovulation with PMSG and hCG (Fig. 3). In unstimulated wild type mouse ovaries, gremlin is not expressed in type 3a (primary) follicles. However, gremlin localizes to granulosa cells of follicles of type 4 follicles (secondary) to later stages. Gremlin is not expressed in thecal cells and oocytes of any stage. 48 h after injection with PMSG, gremlin is up-regulated in mural as well as cumulus granulosa cells. Following an injection of hCG, a change in the expression pattern was apparent. By 5 h after hCG treatment, expression of gremlin was restricted to the layer of granulosa cells immediately adjacent to the antrum, as well as up-regulated in cumulus cells. Thus, the expression of gremlin in early follicles and cumulus granulosa cells was consistent with the known expression pattern of GDF9 in the adjacent oocytes during folliculogenesis.



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  		FIG. 3.
Gremlin expression during ovarian follicle development following superovulation. Sections from 21-day-old control (uninjected) (A, D, G, J), 48-h PMSG-treated (B, E, H, K), and 48-h PMSG plus 5 h hCG-treated (C, F, I, L) mouse ovaries were hybridized with an 35S-labeled antisense gremlin probe. Silver grains in the dark field images (A–C and G–I) represent the positive signal. Ovarian sections were counterstained with hematoxylin to visualize the tissue and imaged under bright field (D–F and J–L). A, D, G, J, gremlin is expressed in granulosa cells of wild-type untreated mice after follicular stage 4. G and J, the same ovaries as A and D at a higher magnification. B, E, H, K, gremlin is expressed strongly in mural granulosa cells and cumulus granulosa cells following 48 h of treatment with PMSG. H and K, the same sections as B and E at a higher magnification. C, F, I, L, gremlin is down-regulated in mural granulosa cells (Gr) but maintained in cumulus cells (Cu). PrF, preantral follicle; AnF, antral follicle; Cu, cumulus cell; Gr, mural granulosa cell; Th, thecal cell; Oo, oocyte. Original magnification in A–F is 31.25x and in G–L is 62.6x.

 
Gremlin Induction Is Limited to GDF9 and BMP—Additional members of the TGF-{beta} superfamily were tested for the ability to stimulate gremlin expression in cultures of granulosa cells. BMP4, like GDF9, was able to increase the levels of gremlin in granulosa cells (Fig. 4); however, the level of gremlin in TGF-{beta}1-treated granulosa cells was not different from control-treated cells (Fig. 4).



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  		FIG. 4.
GDF9 and BMP4 but not TGF-{beta} induce gremlin expression in granulosa cells. Relative gremlin expression was measured by quantitative real time PCR. The control-treated sample level was set at 1, and data are expressed as -fold increase over control-treated cells. Granulosa cells treated with GDF9 (100 ng/ml) and BMP4 (100 ng/ml) showed a statistically significant induction of gremlin compared with control-treated cells at 5 h of treatment. TGF-{beta}1 (5 ng/ml) did not show a significant induction of gremlin. Bars with different letters a and b indicate that the group means are statistically different (p < 0.05). The mean ± S.E. for three independent experiments is shown.

 
Gremlin Blocks Downstream Effects of BMP4 but Not GDF9 Activity—Gremlin is a known inhibitor of the BMPs, but its effects on other members of the TGF-{beta} superfamily protein are unknown. Gremlin expression by granulosa cells could potentially inhibit BMP4 signaling from the thecal cells, or the BMP15 and GDF9 signaling from the oocyte, or both. To determine the effect of gremlin on GDF9 and BMP4 signaling, GDF9 and BMP4 were preincubated with recombinant gremlin prior to the addition to granulosa cells, and the activity of these ligands was measured by their ability to increase PGE2 levels. PGE2 is up-regulated following GDF9 stimulation (Fig. 5) (15) and is also induced in these cells by BMP4 (Fig. 5). When BMP4 was preincubated with recombinant gremlin prior to the addition to the culture medium, PGE2 induction by BMP4 was consistently inhibited in a dose-dependent manner. A 20-fold excess of gremlin (1 µg/ml) to BMP4 (50 ng/ml) was able to completely block BMP4-induced PGE2 accumulation. In contrast, no consistent decrease in GDF9 activity, as measured by PGE2 accumulation, was seen when cells were co-cultured with GDF9 (50 ng/ml) and gremlin, even at the levels that completely blocked BMP4 activity (1 µg/ml).



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  		FIG. 5.
Recombinant gremlin suppresses BMP4 but not GDF9 activity. Granulosa cells were treated with or without gremlin and with or without GDF9 or BMP for 12 h and then assayed by enzyme-linked immunosorbent assay for PGE2 accumulation in the media. Control-treated cells treated with and without gremlin (1 µg/ml) showed no difference in PGE2 levels. GDF9 (50 ng/ml) induces PGE2 levels, but GDF9-treated cells with increasing doses of gremlin (0, 0.05, 0.5, 1.0 µg/ml) show no changes in PGE2 levels. BMP4 (50 ng/ml) with the addition of increasing doses of gremlin (0, 0.05, 0.5, 1.0 µg/ml) showed a decrease in PGE2 accumulation over that of BMP4 treatment alone. The highest dose of gremlin is able to block BMP4-induced PGE accumulation. The error bars in 0.05 and 0.5 ng/ml gremlin with BMP are too small to be seen on this scale. Asterisks indicate that the treatment was significantly different from control values (p < 0.05). The data are presented as mean ± S.E. of three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
These experiments provide evidence that GDF9 induces the expression of gremlin in granulosa cells, similar to BMP4. In cultured granulosa cells derived from large antral follicles, gremlin is expressed at low levels. After 5 h of treatment with either GDF9 or BMP4, gremlin was up-regulated by 5–7-fold over control levels. Gremlin is a member of the DAN family of BMP antagonists (34), and this is the first report of the expression pattern in the mouse ovary and specifically within the granulosa cell compartment. Furthermore, we show in a functional assay measuring PGE2 accumulation that gremlin inhibits BMP4 but not GDF9 activity. This finding suggests that a conserved mechanism of BMP antagonism by negative regulatory factors during folliculogenesis is similar to other developmental processes, for example, during limb development (35, 36) and lung morphogenesis (37).

The inability of gremlin to inhibit GDF9 activity is a surprising finding. The extracellular BMP antagonists noggin, chordin, follistatin, and members of the DAN family have been shown to be up-regulated by the ligands that they inhibit (26). For example, in cultured osteoblasts, noggin is strongly induced by BMP2, BMP4, and BMP6 (and weakly by TGF-{beta}1), and in turn noggin inhibits the activity of the BMP ligands (38). Gremlin is up-regulated by BMP2 in cultured osteoblast cells and likewise attenuates BMP2 activity in vitro (39). In granulosa cells, however, BMP4 and GDF9 appear to be equally capable of increasing gremlin expression, but only BMP4-induced PGE2 synthesis is affected. Thus, gremlin may serve an autoregulatory function when induced by BMPs but may also act as a paracrine factor, with expression that is mediated by different ligands and that has different neutralization properties for the various TGF-{beta} superfamily ligands. This may be analogous to the broad effects known for follistatin, the activin antagonist. Follistatin is up-regulated by activin (40, 41) and GDF9 (10) but inhibits the activity of activin (42), BMP4 (43), BMP7 (44), BMP15 (45), and myostatin (46). Understanding which of the interactions is physiologically relevant will require specific knowledge about the spatial and temporal induction of the negative regulatory proteins and the different ligands that induce them during follicle development.

With the exception of follistatin (47), the in vivo significance of the extracellular BMP antagonists in reproductive tissues is unknown. The importance of BMP antagonism by these regulatory proteins has been well established by studies in other tissues (26). For example, gremlin antagonism of BMP is necessary to maintain a sonic hedgehog-fibroblast growth factor feedback loop necessary for proper limb patterning and outgrowth (35, 36). In gremlin null mice, limb development is disrupted, likely because of overactive BMP signaling (35). Unfortunately, these mice die 48 h after birth because of kidney agenesis (35) and thus cannot be used to examine postnatal follicle development. Fibroblast growth factors and BMPs are expressed in the rodent ovary (48, 49), suggesting that there may be a functional conservation in their physiological interactions, which may include the induction of regulatory proteins to limit ligand bioactivity or to create activity gradients. Thus, more data are needed on the expression and function of gremlin and other BMP antagonists in the reproductive system.

It is unclear how gremlin functions during ovarian follicle development, but the compartmentalization of gremlin expression changes dramatically as follicles mature. This suggests that the functional antagonism of BMP signaling may be necessary at multiple follicular stages. A model is presented in Fig. 6. In mice, GDF9 is not expressed in primordial follicles nor is it required for the transition from primordial to primary follicle (7, 8). The data presented here indicate that gremlin is not expressed at these stages either. The BMPs, however, have been implicated in the transition from primordial to primary follicles (50, 51), and the lack of gremlin expression may allow this to occur. During later folliculogenesis, GDF9 and BMP4/BMP7 presumably form inverse gradients because GDF9 is expressed from the oocyte, and BMP4 and BMP7 are expressed mostly from the thecal cell layer (see Fig. 6). Granulosa cells at this stage may be directed by GDF9 activity, and GDF9-induced gremlin may serve to restrict the BMP signal, perhaps to the outermost layers of granulosa or thecal cells or to the stroma. Five hours after an ovulatory dose of hCG, gremlin expression was shut off in the mural granulosa cells but maintained in the cumulus granulosa cells (i.e. those cells immediately adjacent to the oocyte). This has several implications. First, gremlin expression may be under the control, either directly or indirectly, of leutinizing hormone, the pituitary gonadotropin; following the preovulatory surges, GDF9 was able to direct or maintain expression of gremlin in the cumulus cells. Second, the lack of expression in mural granulosa cells and the maintenance of expression in cumulus cells may allow a BMP signal from the theca to act in the former but be blocked in the latter cells. High levels of BMP activity at this stage may be important for the onset of luteinization in mural granulosa cells. Presently, there are very few genes known to be direct targets of TGF-{beta} superfamily signaling pathways in granulosa cells at different stages. A more complete inventory of genes differentially controlled by GDF9 and the BMPs will be necessary for understanding the mechanisms of BMP/GDF signaling in folliculogenesis.



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  		FIG. 6.
Model for GDF9 antagonism of BMP through expression of gremlin. A, during primordial follicle recruitment, unopposed expression of BMP from interstitial or stromal cells may allow for the transition from the quiescent primordial stage to the primary follicle stage. B, GDF9 is expressed in growing oocytes after stage 3b and induces the expression of gremlin in the granulosa cells. The BMPs are expressed from the thecal cells. Action of BMP on granulosa cells is blocked by the expression of gremlin; however, BMP may act on thecal cells or stromal cells. C, during ovulation, the cumulus granulosa cells are physically separated from the mural granulosa cells by the antral cavity. Gremlin expression is maintained in the cumulus granulosa cells by oocyte-produced GDF9. Mural granulosa cell expression of gremlin is repressed following the gonadotropin surges. This allows BMP to act on the mural granulosa cells and, potentially, on thecal and interstitial or stroma cells.

 

   FOOTNOTES
 
* These studies were supported by National Institutes of Health Grants HD32067 and HD33438 (to M. M. M.) and a postdoctoral fellowship from the Center for Reproductive Biology (Training Grant HD007165 to S. A. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

** Stuart A. Wallace Chair and Professor. To whom correspondence should be addressed: Dept. of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Tel.: 713-798-6451; Fax: 713-798-5833; E-mail: mmatzuk{at}bcm.tmc.edu.

1 The abbreviations used are: BMP, bone morphogenetic protein; GDF, growth differentiation factor; TFG-{beta}, transforming growth factor {beta}; MOPS, 4-morpholinepropanesulfonic acid-formaldehyde; PGE2, prostaglandin E2; PMSG, pregnant mare serum gonadotropin; FBS, fetal bovine serum; RT, reverse transcription; hCG, human chorionic gonadotropin. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Laurie McKenzie for assistance with real time PCR assays.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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