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Endocrinology Vol. 149, No. 3 1026-1030
Copyright © 2008 by The Endocrine Society

The Cooperative Effect of Growth and Differentiation Factor-9 and Bone Morphogenetic Protein (BMP)-15 on Granulosa Cell Function Is Modulated Primarily through BMP Receptor II

Sara J. Edwards, Karen L. Reader, Stan Lun, Andrea Western, Steve Lawrence, Kenneth P. McNatty and Jennifer L. Juengel

AgResearch (S.J.E.), Invermay Agricultural Centre, Mosgiel 9053, New Zealand; AgResearch (K.L.R., S.L., A.W., S.L., K.P.M., J.L.J.), Wallaceville Animal Research Centre, Upper Hutt 5140, New Zealand; and School of Biological Sciences (K.P.M.), Victoria University of Wellington, Wellington 6140, New Zealand

Address all correspondence and requests for reprints to: Jenny Juengel, AgResearch, Invermay Agricultural Centre, Puddle Alley, Private Bag 50034, Mosgiel 9053, New Zealand. E-mail: jenny.juengel{at}agresearch.co.nz.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Growth and differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15, GDF9B) are oocyte-derived proteins essential for the growth and function of ovarian follicles. Moreover, ovine (o) GDF9 and oBMP15 cooperate to increase both 3H-thymidine incorporation and {alpha}-inhibin production and to inhibit progesterone production by rat or ovine granulosa cells. Although the receptors through which these proteins act individually have been determined, the receptor(s) involved in mediating the cooperative effects of GDF9 and BMP15 is (are) unknown. In this study, the effects of the extracellular domains of the types I and II TGFβ receptors on 3H-thymidine incorporation by rat granulosa cells stimulated by oGDF9 and oBMP15 were investigated. Stimulation of 3H-thymidine incorporation was completely blocked by the BMP receptor II (BMPRII) extracellular domain but unaffected by any other type II or any type I receptor. These results suggest that the initial interaction of oGDF9 and oBMP15 is with BMPRII and that a type I receptor is either recruited or already associated with BMPRII to mediate the cooperative effects of these growth factors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MEMBERS OF THE TGFβ superfamily of proteins and their receptors have been shown to regulate ovarian follicular development (1). Two of these proteins, namely growth and differentiation factor (GDF)-9 and bone morphogenetic protein (BMP)-15, are produced by the oocyte and have been shown to be essential for follicular development in rodents, sheep, and humans (1, 2, 3, 4) and to regulate ovulation rate in sheep (5, 6, 7, 8). From in vitro studies, we have established that ovine (o) GDF9 and oBMP15 cooperate to increase 3H-thymidine incorporation by rat or ovine granulosa cells up to 3-fold greater than that of each growth factor alone (9, 10). Furthermore, FSH-stimulated progesterone production was markedly inhibited, whereas {alpha}-inhibin production was substantially increased by treatment when both growth factors were added together, compared with each growth factor alone. Indeed, in rat granulosa cells oGDF9 or oBMP15 alone caused little or no stimulatory or inhibitory effects on thymidine incorporation or progesterone or inhibin production.

The TGFβ superfamily of ligands act on target cells via specific type I and/or type II receptors. After ligand binding, receptor-activated Sma and mothers against decapentaplegic (Smad) proteins are phosphorylated by the type I receptor and activate two different signaling pathways, according to which type I receptor is involved. Generally, the evidence suggests that BMPs bind to activin receptor-like kinase (ALK)-2, ALK3, or ALK6 leading to phosphorylation of Smads 1, 5, and 8, whereas activin and TGFβ bind to ALK1, ALK4, or ALK5 leading to phosphorylation of Smads 2 and 3 (11). BMP15 has been shown to activate the Smad 1/5/8 pathway through BMPRII and ALK6 (12), with BMPRII having the greatest effect on BMP15 suppression of FSH-induced progesterone production. In sheep, a mutation in ALK6 is responsible for precocious follicular maturation and ovulation rates in excess of those observed for wild-type ewes (13, 14, 15). Moreover, sheep heterozygous for an inactivating mutation in BMP15 and heterozygous for the aforementioned mutation in ALK6 have additive or greater than additive increases in ovulation rate (16) consistent with the view that BMP15 and ALK6 share a common pathway. In contrast, GDF9 is considered to act through a different type I receptor, ALK5 (17, 18), but the same type II receptor as BMP15, namely BMPRII (19). Thus, GDF9 acting via ALK5 and BMPRII leads to activation of Smad 2 (18, 20) and Smad 3 (17). However, the combination of receptors that are involved in the cooperative effects of GDF9 and BMP15 are unknown. Therefore, the aims of the current study were to determine the potential receptor combinations through which the cooperative effects of oGDF9 and oBMP15 are mediated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Granulosa cell collection
Ovaries were collected from 23- to 26-d-old Sprague Dawley rats (University of Otago, Dunedin, New Zealand) approximately 46 h after ip administration of 20 IU equine chorionic gonadotrophin (Intervet Ltd., Auckland, New Zealand). The ovaries were dissected from the surrounding tissue and rinsed twice in Leibovitz L-15 media (Invitrogen, Auckland, New Zealand) containing 0.1% (wt/vol) BSA (ICPbio Ltd., Auckland, New Zealand), 100 U/ml penicillin, and 100 µg/ml streptomycin. Granulosa cells were collected by syringe aspiration and suspended in Leibovitz L-15 media. Oocyte-cumulus cell complexes, isolated oocytes, and follicular debris were visualized using a dissecting microscope, and most, if not all, were removed from the granulosa cells using a glass pipette. The remaining cells were washed once in 5 ml Leibovitz L-15 media and recovered by centrifugation at 300 x g for 5 min at room temperature. All animal protocols were carried out in accordance with the 1999 Animal Welfare Act (Part 6) of New Zealand and were approved by the Wallaceville Animal Ethics Committee.

Culture of granulosa cells for determination of 3H-thymidine incorporation
Granulosa cells were washed in 5 ml M199 (Earle’s; Sigma, Auckland, New Zealand) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM GlutaMAX-1 (Invitrogen), 0.3 mg/ml polyvinyl alcohol (Sigma), and 0.23 mM sodium pyruvate (Sigma) and resuspended in the same media (supplemented M199). Cell viability was determined using trypan blue exclusion. Granulosa cells were seeded at 20,000 viable cells in a total volume of 125 µl supplemented M199 per well and incubated at 37 C, 5% CO2. After 18 h, 0.4 µCi methyl-3H-thymidine (PerkinElmer, Boston, MA; 20 Ci/ml) was added to each well and the cells incubated for a further 6 h, after which cells were harvested with a cell harvester onto a thin filter mat. Incorporation of 3H-thymidine was determined using a Wallac Trilux MicroBeta 1450 liquid scintillation counter (Biolab, Auckland, New Zealand).

For all assays, treatments were applied at least in triplicate with a minimum of three independent pools (range of three to seven) of granulosa cells being tested, except for the BMP receptor II (BMPRII) dose-response experiment which was carried out on two independent pools.

Treatments
The recombinant receptor proteins that were used consist of a human receptor extracellular domain (ECD) fused to the Fc region of human IgG via a polypeptide linker (receptor ECD/Fc chimera proteins). As a control, a recombinant human homodimeric IgG Fc protein was used. Human BMP4, human activin A, IgG, and all receptor ECD/Fc chimera proteins were purchased from R&D Systems (via Pharmaco, New Zealand). The recombinant oGDF9 and oBMP15 proteins were prepared in-house and quantitated as described previously (10). oGDF9 was used at 16 ng/ml and oBMP15 at 1.24 ng/ml in a final concentration of 8% (vol/vol) 293H-conditioned media. Ligand and receptor proteins were added to the culture media and incubated at 37 C, 5% CO2 for 1–2 h before cell seeding. The cells in the control experiments were either treated with supplemented M199 (BMP4 and activin A treatments) or 8% conditioned media from untransfected 293H cells (oGDF9 and oBMP15 treatments).

Statistical analysis
The raw data were log transformed to stabilize their variance and then analyzed with a mixed model using PROC MIXED of SAS (SAS Institute, Cary, NC). Five of the 2165 observations were omitted as outliers (extremely high or low residuals). The treatment was fitted as a fixed effect, whereas date, plate within date, and pool within date and plate were initially fitted as random effects. The plate-within-date term was found to explain none of the variance and was dropped from the final model (and pool was nested within date only). Tests of specific differences (e.g. a treatment vs. its control) were made by comparing their respective least squares means.

SDS-PAGE immunoblotting
Culture supernatants containing 4 ng oGDF9 or 0.3 ng oBMP15 per lane in sodium dodecyl sulfate sample buffer were run under reducing conditions in 15% polyacrylamide gels before transferring to nitrocellulose (21, 22), followed by immunoblotting using standard procedures. For lanes containing both oGDF9 and oBMP15, these proteins were mixed together and incubated for 24 h with rotation before loading 2 ng oGDF9 and 0.15 ng oBMP15 per lane. Monoclonal antibodies raised against oGDF9 or oBMP15 peptides were supplied by Dr. Nigel Groome (Oxford Brookes University, Headington, Oxford, UK) and used for protein detection. Characterization of the GDF9 antibody has been previously published (8). The BMP15 antibody was raised against the peptide SEVPGPSREHDGPES(C) as previously described (10). These antibodies were used at 1 µg/ml in 5% nonfat bovine milk powder in Tris-buffered saline.

Cross-linking
A noncleavable cross-linker, bis-sulfosuccinimidyl suberate (Pierce, Rockford, IL) was added to culture supernatants at a final concentration of 2 mM, incubated at room temperature for 30 min, before SDS PAGE immunoblotting.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The cooperative effect of oGDF9 and oBMP15 on stimulation of granulosa cell proliferation is not directly modulated by the ECD of a type I receptor
To examine interactions between ligands and receptors, we measured the effect of receptor-ECD/Fc chimera proteins on 3H-thymidine incorporation into rat granulosa cells stimulated by oGDF9 and oBMP15. The addition of 16 ng/ml oGDF9 and 1.24 ng/ml oBMP15 together with 5 µg/ml control IgG caused a significant (6-fold; P < 0.0001) increase in 3H-thymidine incorporation, compared with cells treated with control media (Fig. 1Go, top panel). This stimulation was not significantly affected by substituting type I receptor-ECD/Fc chimera proteins (at 5 µg/ml) for IgG (Fig. 1Go, top panel). A similar pattern of results was seen at a dose of 1 µg/ml receptor-ECD/Fc chimera protein (data not shown). Cells treated with BMP4 and IgG showed a significant (P < 0.0001) increase in thymidine incorporation, compared with untreated cells. ALK3 and ALK6 (both at 5 µg/ml) significantly inhibited thymidine incorporation (Fig. 1Go, bottom panel; P < 0.0001, P < 0.001, respectively), although complete inhibition by these receptor ECDs was not achieved. Thymidine incorporation stimulated by activin A was not significantly affected by the addition of the ALK4 ECD.


Figure 1
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  		FIG. 1. The effect of type I receptors on oGDF9/oBMP15-stimulated 3H-thymidine incorporation into rat granulosa cells. Top panel, Receptor-ECD/Fc chimera proteins (5 µg/ml) were tested in the presence of oGDF9 (16 ng/ml) and oBMP15 (1.24 ng/ml) Bottom panels, As controls, the effects of selected receptor-ECD/Fc chimera proteins on 3H-thymidine incorporation into rat granulosa cells were tested in the presence of BMP4 (30 ng/ml) or activin A (10 ng/ml). Results are expressed relative to cells treated with control media (set at 1.0, dotted line). *, P < 0.001, compared with appropriate IgG control; **, P < 0.0001, compared with appropriate IgG control.

 
The cooperative effects of oGDF9 and oBMP15 on stimulation of granulosa cell proliferation is blocked by the ECD of BMPRII
The addition of 16 ng/ml oGDF9 and 1.24 ng/ml oBMP15 together with 1 µg/ml IgG caused a significant (4-fold; P < 0.0001) increase in thymidine incorporation, compared with cells treated with control media (Fig. 2Go, top panel). Substitution of IgG with 1 µg/ml BMPRII ECD/Fc completely blocked the effect of oGDF9 and oBMP15 on thymidine incorporation. No other type II receptor ECD/Fc had a significant affect. Furthermore, the inhibition of thymidine incorporation occurred in a dose-dependent manner (Fig. 2Go, middle panel) with complete blocking occurring at a dose of 0.5 µg/ml BMPRII ECD/Fc. By comparison, the increase in thymidine incorporation caused by BMP4 was not affected by the addition of BMPRII ECD/Fc, whereas activin A-induced thymidine incorporation was significantly inhibited (P < 0.0001) by the addition of each of the activin type II receptor ECD/Fcs.


Figure 2
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  		FIG. 2. The effect of type II receptors on oGDF9/oBMP15-stimulated 3H-thymidine incorporation into rat granulosa cells. Top panel, Receptor-ECD/Fc chimera proteins (1 µg/ml) were tested in the presence of oGDF9 (16 ng/ml) and oBMP15 (1.24 ng/ml). Middle panel, Dose-dependent effects of BMPRII-ECD/Fc chimera protein on oGDF9- (16 ng/ml) and oBMP15 (1.24 ng/ml)-stimulated 3H-thymidine incorporation into rat granulosa cells. Bottom panel, As controls, the effects of selected receptor-ECD/Fc chimera proteins on 3H-thymidine incorporation into rat granulosa cells of BMP4 (30 ng/ml) or activin A (10 ng/ml) were tested. Results are expressed relative to cells treated with control media (set at 1.0, dotted line). **, P < 0.0001, compared with appropriate IgG control.

 
Molecular forms of oGDF9 and oBMP15
The majority of the oGDF9 produced in-house was detected in monomeric and dimeric forms by immunoblot (Fig. 3Go). After cross-linking, there was a reduction in the monomeric form and an increase in dimers. In addition, higher-molecular-weight forms (e.g. multimers) were found, which may include the proregion. The oBMP15 produced was detected only in the monomeric form. Dimers were not detected, even after cross-linking, although larger molecular-weight multimeric forms were present. When combined, oGDF9 and oBMP15 showed no evidence of heterodimer formation although both proteins were found in larger multimers after cross-linking.


Figure 3
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  		FIG. 3. Detection of molecular forms of oGDF9 and oBMP15. Immunoblotting was carried out on media containing oGDF9 (4 ng per lane) or oBMP15 (0.3 ng per lane) or both (2 ng oGDF9 and 0.15 ng oBMP15 per lane) using monoclonal antibodies raised against oGDF9 or oBMP15 peptides. Samples were cross-linked as indicated. Molecular weight markers are indicated on the right.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Based on the observation that neither TGFβ nor activin has affinity for any type I receptor alone, whereas they are both able to bind type II receptors, it has been proposed that TGFβ and activin first bind to their type II receptor, leading to the recruitment of a type I receptor (4, 23). For BMP ligands, the mechanism of ligand-receptor interaction appears to be different, more complex, and perhaps dependent on the BMP and the origins of the material being studied (24). BMP receptors are found in homomeric and heteromeric complexes, even in the absence of ligand (25). Furthermore, BMPRII does not readily bind BMP ligands in the absence of a type I receptor, whereas ALK3 and ALK6 are able to bind BMP4 in the absence of BMPRII (26, 27). Therefore, it has been proposed that the ligand binds first to type I receptor and then recruits BMPRII (4, 28). It has also been demonstrated that the affinity of BMP ligands for BMPRII is dramatically increased when BMPRII is overexpressed together with a type I receptor (29, 30), suggesting an alternative mechanism. These data suggest that high-affinity ligand binding may occur only to preexisting type I/type II receptor complexes. Nohe et al. (28) proposed that, at least for BMP2, both mechanisms exist, each activating a different signaling pathway: either the Smad pathway when ligand binds to a preformed receptor complex, or the p38 MAPK pathway when BMPRII is recruited into a complex after ligand binding.

In our assays, BMP4 stimulation of thymidine incorporation was inhibited by the ALK3 and ALK6 but not the BMPRII ECD. This provides further support for the initial BMP4 ligand/receptor interaction being with the type I rather than the type II receptor.

BMP15 has been shown to bind to ALK6 with high affinity, although BMPRII is the most potent inhibitor of BMP15 bioactivity (12), despite binding the ligand with low affinity. Hence, BMP15 signaling was proposed to be mediated by binding first to ALK6 and then recruiting BMPRII to the complex (12). GDF9 also binds BMPRII, and the BMPRII ECD blocks stimulation of rat granulosa cell proliferation by GDF9 (19). ALK5 also appears to mediate GDF9 signaling leading to the phosphorylation of Smads 2 and 3 (17).

The combined effect of oGDF9 and oBMP15 on rat granulosa cells is greater than the effect of either growth factor alone (10). In the current study, the cooperative effect of oGDF9 and oBMP15 was completely blocked by the BMPRII ECD but unaffected by the ECDs of any other type II receptor or any of the type I receptors. One possible explanation for these findings is that oGDF9 and oBMP15 are physically interacting with each other before binding to BMPRII and are able to increase signaling through the receptor in this heteromeric form. However, our immunoblot results show no evidence that the ligands exist in such a form. Instead, our results suggest that the initial interaction is between one or more forms of oGDF9 and oBMP15 separately and BMPRII. Thereafter a type I receptor is either recruited to the complex or is already present in complex with BMPRII. The identity of the interacting type I receptor(s) is unknown at present, but ALK6 and/or ALK5 would appear to be obvious candidates and warrant further examination. In addition, whether GDF9 and BMP15 interact with their respective receptor complexes independent of each other or whether a complex of receptor and ligands comprised of BMPRII, ALK6, ALK5, GDF9, and BMP15 might exist are unknown.

It is possible that the recombinant forms of BMP15 and GDF9 are not representative of the forms found in follicular fluid or other biological fluids. However, our preliminary data in studies of ovine follicular fluid have been unable to demonstrate dimeric or heterodimeric forms of the mature region of BMP15 or indeed in freshly harvested oocytes (31 and our unpublished data). Although unlikely, it is also possible that dimeric forms of the mature region of BMP15 or heterodimeric forms of the mature regions of BMP15/GDF9 do exist and are present in culture media or biological fluids but are below the detection of our current assays, do not react to the cross-linking agents used in this study, and/or are not recognized by our antibodies. Nevertheless, all the current evidence suggests that the predominant form of mature BMP15 is monomeric, suggesting that this might be the biologically active moiety. However, it is important to note that higher-molecular-weight complexes, potentially consisting of dimers of the mature region and other proteins, were observed after cross-linking. Thus, the biologically active form of BMP15 remains obscure.

In summary, the cooperative effects of oGDF9 and oBMP15 on granulosa cell proliferation were completely blocked by the ECD of BMPRII. No effect of the ECD of any of the other known type II receptors or any of the known type I receptors on the cooperative effects of GDF9 and BMP15 was observed. Thus, the GDF9/BMP15 complex seems to act more like a TGF-β/activin subfamily member rather than a BMP subfamily member, even though it is acting through BMPRII. Given that the soluble ECD of BMPRII is not known to be able to bind any other members of the TGF-β superfamily, the ECD of BMPRII may be useful as a specific antagonist to block GDF9 and BMP15 bioactivity.


   Acknowledgments
 
We thank Ken Dodds for statistical analysis and Mika Laitinen and Olli Ritvos for preparations of GDF9 and BMP15.


   Footnotes
 
This work was supported by funding from New Zealand Foundation for Research, Science, and Technology; Ovita Ltd., and Meat and Wool New Zealand.

Disclosure Statement: AgResearch Ltd. has ownership interest in PCT/NZ01/00073, PCT/NZ0100113, PCT/NZ03/00109, and PCT/NZ05/000313. J.L.J. and/or K.P.M. are inventors on these patent applications. Both J.L.J. and K.P.M. have assigned their ownership in these patent applications to AgResearch Ltd.

First Published Online December 6, 2007

Abbreviations: ALK, Activin receptor-like kinase; BMP, bone morphogenetic protein; BMPRII, BMP receptor II; ECD, extracellular domain; GDF, growth and differentiation factor; o, ovine; Smad, Sma and mothers against decapentaplegic.

Received September 26, 2007.

Accepted for publication November 26, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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C. Wang and S. K. Roy
Expression of Bone Morphogenetic Protein Receptor (BMPR) during Perinatal Ovary Development and Primordial Follicle Formation in the Hamster: Possible Regulation by FSH
Endocrinology, April 1, 2009; 150(4): 1886 - 1896.
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