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Cumulin, an Oocyte-secreted Heterodimer of the Transforming Growth Factor-β Family, Is a Potent Activator of Granulosa Cells and Improves Oocyte Quality *

Cumulin, an Oocyte-secreted Heterodimer of the Transforming Growth Factor-β Family, Is a Potent... crossmark THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 39, pp. 24007–24020, September 25, 2015 © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Cumulin, an Oocyte-secreted Heterodimer of the Transforming Growth Factor- Family, Is a Potent Activator of Granulosa Cells and Improves Oocyte Quality Received for publication, June 26, 2015, and in revised form, August 6, 2015 Published, JBC Papers in Press, August 8, 2015, DOI 10.1074/jbc.M115.671487 ‡§ ‡¶  ‡ ‡‡ ‡ David G. Mottershead , Satoshi Sugimura , Sara L. Al-Musawi , Jing-Jie Li **, Dulama Richani , Melissa A. White , ‡ ‡ ‡§§ ‡ ¶¶ Georgia A. Martin , Andrew P. Trotta , Lesley J. Ritter , Junyan Shi , Thomas D. Mueller , Craig A. Harrison , ‡ ‡‡1 and Robert B. Gilchrist From the Robinson Research Institute and Discipline of Obstetrics and Gynaecology, School of Paediatrics and Reproductive §§ Health and the Australian Research Council Centre of Excellence in Nanoscale BioPhotonics, University of Adelaide, Adelaide, § ¶ South Australia 5005, Australia, Mottasis Oy Ltd., 00430 Helsinki, Finland, the Institute of Agriculture, Department of Biological Production, Tokyo University of Agriculture and Technology, Tokyo 183-0057, Japan, the Hudson Institute of Medical Research, Clayton, Victoria 3168, Australia, the **Center of Reproductive Medicine, the Sixth Affiliated Hospital, Sun Yat-sen University, ‡‡ 510655 Guangzhou, China, the Discipline of Obstetrics & Gynaecology, School of Women’s & Children’s Health, University of New ¶¶ South Wales, Sydney, New South Wales 2052, Australia, and the Department of Plant Physiology and Biophysics, Julius-von- Sachs Institute of the University Wuerzburg, D-97082 Wuerzburg, Germany Background: Cumulin is a newly identified heterodimeric member of the TGF- family. Results: Mature cumulin potently stimulates granulosa cell signaling and function, whereas pro-cumulin promotes oocyte quality. Conclusion: Formation of cumulin and its potent actions are likely to be central to oocyte paracrine signaling and mammalian fecundity. Significance: The discovery of cumulin provides unique opportunities to improve female fertility in mammals. Growth differentiation factor 9 (GDF9) and bone morphoge- activating both SMAD2/3 and SMAD1/5/8 signaling pathways netic protein 15 (BMP15) are oocyte-specific growth factors and promoting proliferation and expression of a set of genes with central roles in mammalian reproduction, regulating spe- associated with oocyte-regulated GC differentiation. Cumulin cies-specific fecundity, ovarian follicular somatic cell differenti- was more potent than pro-cumulin, pro-GDF9, pro-BMP15, or ation, and oocyte quality. In the human, GDF9 is produced in a the two combined on GC. However, on cumulus-oocyte com- latent form, the mechanism of activation being an open ques- plexes, pro-cumulin was more effective than all other growth tion. Here, we produced a range of recombinant GDF9 and factors at notably improving oocyte quality as assessed by sub- BMP15 variants, examined their in silico and physical interac- sequent day 7 embryo development. Our results support a tions and their effects on ovarian granulosa cells (GC) and model of activation for human GDF9 dependent on cumulin oocytes. We found that the potent synergistic actions of GDF9 formation through heterodimerization with BMP15. Oocyte-se- and BMP15 on GC can be attributed to the formation of a het- creted cumulin is likely to be a central regulator of fertility in erodimer, which we have termed cumulin. Structural modeling mono-ovular mammals. of cumulin revealed a dimerization interface identical to homodimeric GDF9 and BMP15, indicating likely formation of In this study, we consider the unique interaction of two a stable complex. This was confirmed by generation of recombi- members of the TGF- family: growth differentiation factor 9 nant heterodimeric complexes of pro/mature domains (pro-cu- (GDF9) and bone morphogenetic protein 15 (BMP15). These mulin) and covalent mature domains (cumulin). Both pro-cu- two closely related proteins are oocyte-secreted factors essen- mulin and cumulin exhibited highly potent bioactivity on GC, tial for fertility in mammals (1). The primary role of GDF9 and BMP15 is to act on the cells surrounding the oocyte, known as * This work was supported by the National Health and Medical Research granulosa cells (GC), which in turn supply the oocyte with the Council of Australia through Project Grants 627151, 1024358, and support necessary for future healthy embryo/fetal development 1062762, Development Grants 1017484 and 1076004, and Fellowships 1023210 and 1013533. This work was also supported by research grants (2). As with all members of the TGF- family, GDF9 and from Cook Medical, Sun Yat-sen University International Program Fund BMP15 are produced as pro-proteins, consisting of an N-ter- 985 Project (to J. J. L.) and from the Japanese Society for the Promotion of minal proregion and a receptor binding C-terminal mature Science Postdoctoral Fellowship for Research Abroad (to S. S.). R. B. G. is listed as an inventor on Patent US20140073052A1 owned by the University region. Activation of these factors requires proteolytic pro- of Adelaide. R. B. G. and D. G. M. are listed on a distribution of benefits cessing by a furin-like protease (3, 4). For most TGF- mem- agreement in connection with Patent US20140073052A1. To whom correspondence should be addressed: Robert B Gilchrist, Dis- cipline of Obstetrics & Gynaecology, School of Women’s & Children’s The abbreviations used are: GDF9, growth differentiation factor 9; ALK, Health, Royal Hospital for Women, University of New South Wales, Syd- activin receptor-like kinase; BMP15, bone morphogenetic protein 15; BMP- ney, Australia. Tel.: 61-2-93852562; Fax: 61-2-93852573; E-mail: RII, BMP receptor type II; GC, granulosa cell; IVM, oocyte in vitro maturation; r.gilchrist@unsw.edu.au. SMAD; Sma- and Mad-related proteins. This is an open access article under the CC BY license. SEPTEMBER 25, 2015 • VOLUME 290 • NUMBER 39 JOURNAL OF BIOLOGICAL CHEMISTRY 24007 Cumulin, a Novel Ovarian Growth Factor bers, the processed mature domains signal as covalently linked ways. Interestingly, only the promature heterodimer is capable homodimers, although covalent heterodimers are also known of improving oocyte quality, indicative of the crucial function (inhibin A and B (5)). GDF9 and BMP15 are unusual within the that the prodomain plays in the functioning of these proteins. TGF- family in that they (together with GDF3 and Lefty1/2) In deference to its potent activation of cumulus cells and to lack the cysteine residue involved in forming the mature region align with the existing nomenclature for the other major repro- intersubunit disulfide bond (6). Despite this, mature GDF9 and ductive TGF- superfamily heterodimer, inhibin, we have BMP15 are thought to maintain the canonical dimer architec- called this new heterodimer cumulin. ture of other TGF- ligands and signal as noncovalent dimers. Experimental Procedures As with all members of the TGF- family, GDF9 and BMP15 signal via cell surface receptors, which activate intracellular Expression Plasmids, Stable Cell Lines, and Protein Sma- and Mad-related (SMAD) proteins by phosphorylation of Production—Expression cassettes encoding wild type human critical residues, which subsequently regulate the transcription GDF9 or BMP15 or mouse GDF9 (mGDF9) were synthesized of various genes (1). The receptors are Ser/Thr kinases and are, (Genscript USA, Inc., Piscataway, NJ) incorporating the rat on the basis of distinct features in the cytoplasmic domain, serum albumin signal sequence at the 5 end followed by referred to as either type I (activated by the relevant type II sequences encoding for His and StrepII affinity tags at the N receptor) or type II (constitutively active) receptors. Following terminus of the respective proregion. The various human 418 356 prodomain displacement, BMP15 binds to complexes of BMP GDF9 and BMP15 Ser-Cys mutants (Ser in GDF9 and Ser receptor type II (BMPRII) and activin receptor-like kinase 6 in BMP15) used in this study were derived from expression (ALK6), thereby leading to activation of the SMAD1/5/8 intra- cassettes (Genscript USA, Inc., Piscataway, NJ) incorporating cellular pathway (7, 8). Mature GDF9 binds BMPRII and ALK5 the glutamate receptor GluR-D subunit signal sequence and signals through the SMAD2/3 pathway (9–11). (MRIICRQIVLLFSGFWGLAMG) at the 5 end followed by GDF9 and BMP15 are also functionally unusual within the sequences encoding for the FLAG tag (a design based on Kuusi- TGF- family, in that they show remarkable species differences nen et al. (25)) and His affinity tag at the N terminus of the in their activity. Notably, human GDF9 is latent, because it respective proregion (as used previously (8)). All the cDNA remains associated with its prodomain, whereas mouse GDF9 fragments were cloned into the pEF-IRES expression vector is particularly potent, because of its prodomain being readily (26). displaced (3, 12, 13). By contrast, human BMP15 is recombi- HEK293T cells were obtained from the American Type Cul- nantly produced in an active form in mammalian cell culture, ture Collection and cultured in DMEM (Invitrogen) containing whereas mouse BMP15 is not expressed under the same condi- 10% FCS, 2 mML-glutamine, 100 IU/ml penicillin (Sigma), and tions (4). The species-specific differences in expression and 100 g/ml streptomycin. The different expression plasmids activity of these oocyte-secreted growth factors is principally were transfected into HEK293T cells using Lipofectamine 2000 due to alterations in a small number of amino acids in the (Invitrogen) following the manufacturer’s instructions. Stable prodomains (BMP15) or mature domains (GDF9), which affect cell lines were established via puromycin selection. For produc- complex formation or stability, respectively (3, 4, 13). Hence, tion of cells producing pro-cumulin, HEK293T cells were co- the relative levels of oocyte expression of BMP15 and GDF9 transfected with both an expression plasmid for human GDF9 vary markedly across species, whereby the mono-ovular phe- of wild type sequence, incorporating no epitope tags, along with notype (i.e. low fecundity) is characterized by balanced the expression plasmid for wild type human BMP15 (His /Stre- BMP15:GDF9 ratios, whereas polyovular species (high pII-tagged), as described above. For production of cells produc- fecundity) express predominately GDF9 (14). As such, ing cumulin (as a covalently stabilized form), HEK293T cells GDF9, but not BMP15, is essential for fertility in the mouse were co-transfected with expression plasmids encoding human (15, 16), whereas both are required in mono-ovular species, GDF9 and BMP15 as the Ser-Cys mutant forms, as described such as sheep (17, 18). above. Interactions at the genetic level between these two proteins The mature regions of GDF9 (12), BMP15 (27) and the have been observed in both mice (16) and sheep (1, 18). Evi- BMP15 Ser-Cys mutant (8) were produced and purified as pre- dence for GDF9/BMP15 physical interactions have also been viously described. Cells producing pro-GDF9, pro-mGDF9, provided by co-immunoprecipitation results (19, 20), and a pro-BMP15, pro-cumulin, or covalent cumulin were grown to number of functional studies have demonstrated marked syn- near confluence, and growth medium was then replaced by pro- ergistic interactions between these two proteins (20–23). duction media containing DMEM/F12 GlutaMAX, 0.1 mg/ml Recently, Peng et al. (24) have provided evidence that het- BSA (Sigma), 25 IU/ml Fragmin (Pfizer), 100 units/ml penicil- erodimerization of GDF9 and BMP15 gives rise to a more lin, and 100 g/ml streptomycin (Sigma). After 48 h of incuba- potent ligand that signals solely via the SMAD2/3 pathway. tion, the conditioned culture medium was collected. In the current study, we hypothesized that human GDF9 and Chromatography—Production medium containing pro- BMP15 form a stable heterodimer that has potent actions on GDF9, pro-BMP15, or pro-cumulin was modified to include 10 mural GC, cumulus GC, and oocyte development. We provide mM imidazole, 500 l of protease inhibitor mixture (Thermo the first evidence of a purified BMP15-GDF9 heterodimer. We Scientific), 500 l of phosphatase inhibitor mixture (Calbi- find that both the promature form and a covalent mature form ochem, Alexandria, Australia) and subsequently adjusted to pH of the heterodimer are particularly potent and are dual pathway 7.4. Invitrogen nickel-nitrilotriacetic acid-agarose resin was activators, signaling via the SMAD2/3 and SMAD1/5/8 path- incubated with conditioned medium on a rotary agitator at 4 °C 24008 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 39 • SEPTEMBER 25, 2015 Cumulin, a Novel Ovarian Growth Factor for 2 h and centrifuged at 900 rpm for 5 min, and the superna- 24 h. The cells were treated with BMP15, GDF9, or cumulin tant was removed. The His-tagged target protein was eluted variants for 16 h. The medium was then aspirated, and the cells from the nickel-nitrilotriacetic acid-agarose using elution were solubilized in solubilization buffer (25 mM glycylglycine, buffer (PBS/500 mM imidazole) and stored in LoBind tubes pH 7.8, 15 mM MgSO ,4mM EGTA, 1% (v/v) Triton X-100, and (Eppendorf, Hamburg, Germany) at 80 °C. Cumulin (the 1mM dithiothreitol), and luciferase reporter activity was mea- covalently stabilized form) was produced and purified as above, sured as previously described (30). with the exception that the mature region was eluted from the RT-PCR—Mural GC were collected from 21–26-day-old IMAC resin via a 7 M urea wash. This material was then loaded C57BL/6  DBA F1 hybrid female mice 46 h after intraperito- onto a reverse phase HPLC column equilibrated with 0.1% neal administration of gonadotropin priming. Mice were main- TFA, and the different mature region forms (i.e. GDF9, cumulin tained in accordance with the Australian Code for the Care and and finally BMP15) were eluted via a linear gradient of acetoni- Use of Animals for Scientific Purposes and with the approval of trile. The fractions were stored in LoBind tubes at 80 °C. the University of New South Wales Animal Ethics Committee. Silver Staining, Western Blotting, and Protein Quanti- Mural GC (1 10 live cells per 24-plate well) were cultured for fication—Fractions from the cumulin reverse phase HPLC were 18 h in bicarbonate-buffered MEM (Gibco Life Technologies) prepared for electrophoresis by lyophilizing 5 l and resus- supplemented with 3 mg/ml BSA (Sigma-Aldrich). The cells pending in SDS-PAGE loading buffer DTT. All samples were were then treated for 6 h with fresh medium containing recom- heated at 95 °C for 5 min and then loaded onto a Mini binant human FSH (50 mIU/ml; Puregon; Organon, Oss, The PROTEAN TGX 4–15% gel (Bio-Rad). After 40 min at 150 V, Netherlands) plus the following treatments, as indicated: 100 the gels were used for silver staining or Western blotting. Silver ng/ml cumulin, 100 ng/ml pro-cumulin, 100 ng/ml pro-mature staining was carried out following the manufacturer’s instruc- GDF9, 100 ng/ml pro-mature BMP15, or 50 ng/ml each of pro- tions using a silver quest staining kit (Invitrogen). For Western mature GDF9  pro-mature BMP15 when added together. blotting, proteins were transferred to a Hybond-ECL mem- RNA extraction, reverse transcription, and quantitative PCR brane (GE Healthcare) for 75 min at 100 V. The membranes were performed as previously described (31) using their were blocked with 2% blocking reagent (supplied in an ECL reported primers; however, PCRs were performed on a Roche Advance kit; GE Healthcare) diluted in TBS containing 0.1% LightCycler 480, and results are expressed as raw gene expres- (v/v) Tween 20 for1hat room temperature. Subsequently the sion levels relative to the geometric means of two housekeeping membranes were incubated with anti-GDF9 antibody (mAb53 (CT) genes (Mrpl19 and Ppia) using the 2 method. The data (28)) or anti-BMP15 antibody (mAb28 (27)) diluted 1:5000 at are means  S.E. of four replicate experiments. 4 °C overnight, followed by incubation with goat anti-mouse Oocyte Quality Assay—The effects of treatments on oocyte TM IRDye 800 CW antibody diluted 1:10,000, for1hinthe dark. quality (oocyte developmental competence) were assessed by Fluorescence intensity was determined using an Odyssey infra- treating cumulus-oocyte complexes with the different growth red imaging system (Licor Bioscience, Lincoln, NE). The vari- factors during the oocyte in vitro maturation (IVM) phase, fol- ous purified forms of GDF9 or BMP15 were quantified via lowed by in vitro fertilization and embryo culture and assess- Western analysis relative to recombinant mGDF9 or hBMP15 ment of subsequent blastocyst yield and quality. We chose to standards, respectively (R&D Systems). use a well established porcine model of low oocyte develop- Granulosa Cell Bioassays—Bioactivities of pro-forms and/or mental competence, because this experimental model better mature forms of BMP15, GDF9, and cumulin were compared approximates the complexities of human oocyte developmen- using primary mouse mural GC and a human granulosa tumor 3 tal competence than that of murine oocytes. This model uses cell line (COV434). The mouse granulosa cell [ H]thymidine oocytes from small antral follicles collected from abattoir-de- incorporation bioassay was performed using standard proce- rived ovaries from gilts (peripubertal female pig) (13, 32–34). dures as previously described (28, 29). In brief, mural GC were The methods used were as per recently described in detail (13, collected from 129/Sv mice 44–46 h after gonadotropin prim- 34). In brief, cumulus-oocyte complexes were aspirated from ing. Cells were cultured at 37 °C in 5% CO in protein-free small antral follicles (2–4 mm) and matured in protein-free medium at 2 10 cells/ml with treatments for 18 h followed by porcine oocyte maturation basic IVM medium (35), supple- a further 6 h with 15.4 kBq [ H]thymidine (PerkinElmer Life mented for the first 22 h of IVM with 1 mM dibutyryl-cAMP and Sciences). Granulosa cell [ H]thymidine incorporation was 100 ng/ml amphiregulin (R&D Systems) and treated with either assessed using a Wallac Microbeta plate counter as an indicator vehicle (control), 20 or 100 ng/ml pro-cumulin, 20 or 100 ng/ml of cell DNA synthesis. Each treatment was performed in dupli- mature cumulin, 100 ng/ml pro-GDF9, 100 ng/ml pro-BMP15, cate, and the experiments were replicated three to six times. or 100 ng/ml of each of pro-GDF9  pro-BMP15. Cumulus The relative capacity of BMP15, GDF9, and cumulin to acti- expansion was assessed at 22 h of IVM and scored as per stan- vate SMAD signaling was assessed with luciferase assays using dard criteria (36, 37). At 22 h, cumulus-oocyte complexes were human COV434 cells (3, 30). In brief, cells were plated at 33 washed and matured for a further 22 h in porcine oocyte mat- 10 cells/well in 96-well plates in complete medium (DMEM uration without supplements or treatments (13, 33, 34). There- supplemented with 10% FCS) at 37 °C in 5% CO . After 24 h of incubation, cells were transfected (130 ng/well) with a BMP- after, in vitro fertilization, in vitro embryo production, and responsive reporter, BRE-luciferase, or an activin/TGF- assessment of blastocysts was performed as previously responsive reporter, A3-luciferase, using Lipofectamine 2000 described (13). Five replicate experiments were performed with (Invitrogen) according to the manufacturer’s instructions for 180 oocytes/replicate. SEPTEMBER 25, 2015 • VOLUME 290 • NUMBER 39 JOURNAL OF BIOLOGICAL CHEMISTRY 24009 Cumulin, a Novel Ovarian Growth Factor Molecular Modeling—To obtain three-dimensional struc- ture models for mature human GDF9, mature human BMP15, and cumulin, homology modeling was performed using either the software SWISS-Modeler or a manual approach. To iden- tify the most suitable structure template, the sequences of GDF9 and BMP15 were blasted against the protein sequences present in the Protein Data Bank, revealing that both oocyte factors exhibit the highest sequence homology (32–33% iden- tity) to either BMP2 or GDF5. For manual modeling the sequences of the mature region of GDF9 and BMP15 were then aligned to the amino acid sequence of the identified templates, i.e. BMP2 (Protein Data Bank entry 3BMP and 1REW (chains A and B) and 3BK3 (chains A and B)) and GDF5 (Protein Data Bank entry 1WAQ) using the software CLUSTALW. A model 327 430 of human GDF9 comprising residues Asn –Arg was then built manually by replacing amino acid residues in the BMP2 template (chains A and B of Protein Data Bank entry 3BK3) employing the tool ProteinDesign of software package FIGURE 1. Pro-forms of BMP15 and GDF9 synergize. Pro-hBMP15 syner- Quanta2008 (MIS Accelrys, San Diego, CA). A single amino gizes with pro-mouse GDF9 and also with pro-human GDF9, which is natu- acid insertion at the end of helix 1 found in GDF9 (when rally latent. Bioactivity was assessed by [ H]thymidine incorporation in pri- mary mouse mural GC. The various pro-proteins were produced in HEK-293T compared with the template BMP2) was rebuilt manually. For cells with N-terminal His tags attached to their prodomains allowing purifica- modeling of human BMP15 (comprising residues Asn tion by IMAC. Arg ), the same approach was applied but using the structure of human GDF5 (Protein Data Bank entry 1WAQ) as BMP15, bond was subsequently refined by a short energy minimization similar to GDF5, binds specifically the BMP receptor BMPR1b. run to obtain a thioether bond of correct length. Both initial models were subsequently refined first by perform- Statistical Analysis—For mRNA expression data, differences ing rotamer searches using the tool X-BUILT of the software between means were assessed by one-way analysis of variance Quanta2008 to identify side chain conformations not leading to followed by Tukey’s multiple comparison test. Similarly, effects van der Waals clashes. Then energy minimization runs were of treatments on embryo yield were assessed by one-way anal- performed employing only geometrical but no electrostat- ysis of variance on arc-sine transformed data followed by a least ic energy terms using Quanta2008 and the forcefield significant difference post hoc test. Cumulus expansion data CHARMm27. First, all backbone atoms were kept fixed, and were assessed by a Kruskal-Wallis one-way analysis of variance only side atoms were allowed to move but restrained by har- 1 2 on ranks followed by a Dunnett’s post hoc test. p 0.05 was monic positional restraints (5 kcal mol Å ) to minimize considered statistically significant. atom movements. After energy minimization of side chain atoms converged, these restraints were removed, and the posi- Results tions of the backbone atoms were refined by applying a strong 1 2 harmonic potential (25 kcal mol The various forms of GDF9, BMP15, and cumulin analyzed Å ). The final models of GDF9 and BMP15 exhibit good backbone and side chain geom- in this study were as follows: 1) pro-forms: noncovalent homo- etry. The structure models for both homodimeric factors are and heterodimers of the promature regions of BMP15 and highly similar, yielding a root mean square deviation of only GDF9 (the form in which these proteins are secreted from the 1.1Å for 164 C carbons. Only 40 residues, which are located cell); 2) mature forms: noncovalent homo- and heterodimers of either in the fingertips or the loop N-terminal of helix 1, the mature regions of BMP15 and GDF9 (the form in which exhibit differing conformations, which may be due to selecting these proteins bind to their signaling receptors); and 3) covalent different template structures for the models and does not forms: covalent homo- and heterodimers of the mature regions reflect inherent structural differences between GDF9 and of BMP15 and GDF9 (stabilized versions of the mature growth BMP15. factors). The model of cumulin was then built by superimposing the GDF9/BMP15 Synergism Is Mediated by Heterodimer (i.e. three-dimensional models of homodimeric GDF9 and BMP15 Cumulin) Formation—We have consistently observed GDF9 and selecting one chain of each structure to form a heterodi- and BMP15 interacting in a synergistic manner, whether in meric assembly. Because of the almost identical amino acid their pro-forms (i.e. as isolated pro/mature complexes; Fig. 1) composition in the dimer interface, the model of cumulin or as purified mature proteins (23, 38). The extracellular forma- required very minor minimization, which was performed by tion of a BMP15/GDF9 heterodimer, which we have termed rotamer searches for side chains with close contacts. The het- cumulin, could underlie this potent synergism. To directly test erodimeric noncovalent form of cumulin was then converted this possibility, we generated a covalent BMP15 homodimer (by 356 356 into the covalent form by exchanging the serine residues Ser replacing Ser with the corresponding cysteine found in other in BMP15 and Ser in GDF9 against cysteines and connecting TGF- proteins), which would be incapable of forming cumu- lin in the presence of wild type GDF9. both sulfur atoms to form the thioether bond. The disulfide 24010 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 39 • SEPTEMBER 25, 2015 Cumulin, a Novel Ovarian Growth Factor Covalent human BMP15 mature region homodimer (BMP15 ) was purified and tested for its ability to synergize S:C with human GDF9 (mature region homodimer) in a mural granulosa cell proliferation assay. At the low doses tested, GDF9, BMP15, and BMP15 did not promote proliferation; S:C however, the GDF9BMP15 combination showed potent syn- ergism (Fig. 2A). In contrast, this synergism was not observed with the GDF9BMP15 combination. Because we have pre- S:C viously shown that GDF9/BMP15 synergism is dependent upon Smad2/3 activation (23, 38), we examined the ability of our BMP15 variants to affect this response in COV434 cells. Although wild type BMP15 promoted GDF9-dependent Smad2/3 transcriptional activity in a dose-dependent manner, the BMP15 variant could not potentiate a GDF9 effect (Fig. S:C 2B). To ensure the BMP15 variant had bioactivity, we tested S:C its ability to stimulate the SMAD1/5/8 pathway in COV434 cells transfected with a BMP-responsive luciferase reporter. In this system, the BMP15 protein was equally potent as wild S:C type BMP15 (Fig. 2C). As expected, GDF9 did not activate the SMAD1/5/8 pathway. Together, these results indicate that extracellular heterodimerization of mature GDF9 and BMP15 is possible, because simple co-addition of GDF9BMP15, but not of GDF9BMP15 , resulted in potent synergism. S:C To further explore extracellular associations between GDF9 and BMP15, we mixed conditioned media from cells expressing untagged pro-GDF9 with conditioned media from cells expressing N-terminally poly-His-tagged pro-BMP15 and car- ried out IMAC purification of the pro-BMP15. Under the mild conditions used, the untagged GDF9 mature region was capa- ble of associating and co-purifying with pro-BMP15, as evi- denced by GDF9 immunoreactivity co-eluting off the IMAC resin (Fig. 3A). No GDF9 immunoreactivity was detected when BMP15 was purified alone (data not shown), and the untagged GDF9 did not bind to the IMAC resin in the absence of the poly-His-tagged BMP15 (Fig. 3B). It is clear from these results that, like the mature proteins, the pro forms of GDF9 and BMP15 are capable of associating extracellularly to form pro-cumulin. Intracellular Formation of Cumulin—GDF9 and BMP15 are co-expressed in the oocyte throughout folliculogenesis and so do not naturally exist in isolation (reviewed in Ref. 39). Thus, heterodimerization of these two TGF- family members to form cumulin is likely to occur during synthesis within the oocyte, as well as after secretion into the extracellular space. In the in vivo context, cumulin will be in a form where the mature regions are noncovalently associated and will most likely act as a pro/mature complex. To produce such a form (i.e. pro-cumu- lin), we co-expressed N-terminally poly-His-tagged pro- BMP15 with untagged pro-GDF9 and purified the noncovalent FIGURE 2. GDF9/BMP15 synergism is mediated by heterodimer forma- pro-cumulin complex by IMAC. Theoretically, this approach tion. The BMP15 (S356C) covalent homodimer (BMP15 ) does not synergize S:C could lead to the purification of a mixture of pro-BMP15 with GDF9, in contrast to wild type BMP15, where clear synergism is observed. homodimers and pro-cumulin. Quantification of the levels of Bioactivity was assessed by [ H]thymidine incorporation in primary mouse mural GC (A), Smad2/3-responsive luciferase reporter activity (B), or Smad1/ both the processed GDF9 and BMP15 mature regions following 5/8 luciferase reporter activity, in COV434 human GC (C). The various proteins IMAC purification indicated a 3:1 ratio of pro-BMP15 were produced in HEK-293T cells with N-terminal His tags attached to their homodimer:pro-cumulin heterodimer (data not shown). prodomains allowing purification by IMAC, followed by isolation of mature domains by reverse-phase HPLC. BMP15, HPLC-purified human BMP15 Pro-cumulin Is a Potent Activator of Granulosa Cells via Both mature region homodimer; GDF9, HPLC purified human GDF9 mature region SMAD Pathways—We next examined the potency of pro-cu- homodimer; BMP15 , HPLC-purified human BMP15 (Ser-Cys) mature region S:C covalent homodimer. mulin in ovarian GC bioassays. We compared pro-cumulin to SEPTEMBER 25, 2015 • VOLUME 290 • NUMBER 39 JOURNAL OF BIOLOGICAL CHEMISTRY 24011 Cumulin, a Novel Ovarian Growth Factor FIGURE 3. GDF9 associates and co-purifies with BMP15 to form pro- cumulin. Conditioned media containing untagged pro-GDF9 and N-termi- nally His -tagged pro-BMP15 were combined and subjected to IMAC chroma- tography. A, Western blot of resin load probed with Mab28 (specific for human BMP15), or the resin load, unbounded material, wash, and eluted peak fraction, probed with Mab53 (specific for GDF9). B, Western blot for IMAC chromatography of conditioned media containing only untagged pro-GDF9. Shown are the resin load, unbound material, wash, and eluate, probed with Mab53. pro-GDF9, pro-BMP15, and pro-GDF9  pro-BMP15, after quantitating each protein form relative to the level of the GDF9 mature region (data not shown). Pro-GDF9 alone was inactive in promoting GC DNA synthesis, as shown previously (3), as was pro-BMP15 alone (Fig. 4A). In contrast, pro-cumulin was exceptionally potent on primary mouse mural GC, stimulating a 3.8-fold increase in [ H]thymidine incorporation at the lowest dose tested (1 ng/ml; Fig. 4A). Pro-cumulin was notably more potent (6–12-fold) than pro-GDF9pro-BMP15 at all doses examined. To examine SMAD signaling by pro-cumulin, we conducted transcriptional reporter assays using the human granulosa cell line, COV434, as previously reported (3). Pro-cumulin dose- dependently activated SMAD2/3 signaling in GC, producing high levels of A3-Lux reporter activity at 50 and 100 ng/ml (Fig. 4B). Pro-GDF9  pro-BMP15 also dose-dependently activated SMAD2/3 but did not reach the same levels of stimulation as pro-cumulin. As expected, pro-GDF9 did not activate SMAD2/3, because it is latent (3, 12). Interestingly, pro-cumu- lin and pro-GDF9  pro-BMP15 promoted similar levels of BRE-luciferase activation (reflecting the level of SMAD1/5/8 phosphorylation) as pro-BMP15 (Fig. 4C). As expected, GDF9 did not activate the SMAD1/5/8 pathway (Fig. 4C), and BMP15 did not activate the SMAD2/3 pathway (Fig. 4B). Engineering Covalent Mature Cumulin—The pro-cumulin preparation described above and that previously produced by Peng et al. (24, 40) contain significant amounts of contaminat- FIGURE 4. Bioactivity and SMAD signaling of pro-cumulin. A–C, dose-re- ing pro-BMP15 homodimer. This was due to the almost equal sponse curves of the different pro-protein forms of GDF9, BMP15, and cumu- lin. Bioactivity was assessed by [ H]thymidine incorporation in primary propensity of monomeric BMP15 to homodimerize or het- mouse mural GC (A), Smad2/3-responsive luciferase reporter activity (B), or erodimerize (with GDF9) and the fact that the purification Smad1/5/8 luciferase reporter activity (C) in COV434 human GC. The various pro-proteins were produced in HEK-293T cells with N-terminal His tags strategies were directed against tagged BMP15 prodomains. To attached to their prodomains allowing purification by IMAC. overcome these limitations and to more conclusively examine the biological function of cumulin, we undertook to engineer a covalent form of cumulin, which could be fully purified from pressed the resultant cDNAs in HEK293T cells. We have used contaminating BMP15 and GDF9. To produce such a protein, this approach before to produce human covalent mature 418 356 we replaced Ser in GDF9 and Ser in BMP15 with the cor- BMP15 (8). Both the pro-BMP15 and pro-GDF9 mutants S:C S:C responding cysteine found in other TGF- proteins and co-ex- were produced with N-terminal poly-His tags; hence IMAC 24012 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 39 • SEPTEMBER 25, 2015 Cumulin, a Novel Ovarian Growth Factor purification of conditioned media from cells co-expressing these proteins theoretically could result in the co-purification of pro-GDF9 or pro-BMP15 homodimers, as well as the sought after pro-cumulin. We decided to target the GDF9 and BMP15 mature regions for purification to avoid any potentially mis- folded disulfide linked pro/mature complexes. This was accomplished by a two-step procedure of IMAC chromatog- raphy followed by reverse phase HPLC. This approach suc- ceeded in resolving three distinct covalent species: mature GDF9, mature cumulin, and mature BMP15 (Fig. 5A). This can be concluded because Western analysis of the fractions from the reverse phase HPLC purification indicated that fractions 31–32 contained GDF9, but no BMP15; fractions 33–34 contained both GDF9 and BMP15 (i.e. cumulin), whereas fractions 36 –39 contained only BMP15 (Fig. 5B). The silver-stained gels confirmed the presence of disulfide- linked dimers, as the reduced bands at 15–18 kDa (Fig. 5C, fractions 32–34 and 37) that migrated at 25–35 kDa without reduction (Fig. 5D, fractions 32–34 and 37). Covalent Mature Cumulin Is Potently Bioactive—Because purified mature cumulin has not previously been produced, it was important to determine its bioactivity, especially given its form as a stabilized disulfide linked covalent mature region het- erodimer. We initially tested the fractions across the reverse phase HPLC profile for bioactivity in the mural GC assay. The peak of bioactivity (Fig. 5E, fraction 34) coincided with the frac- tion containing the peak of covalent cumulin protein (Fig. 5, B–D, fraction 34). We next directly compared the bioactivity of covalent mature cumulin to the previously characterized noncovalent pro-cumulin on GC. Mature cumulin was highly potent in the range of different granulosa cell assays performed; in most cases, it was more potent than pro-cumulin (Fig. 6). In the pri- mary mouse mural granulosa cell assay of [ H]thymidine incor- poration, covalent mature cumulin had an ED of 0.6 ng/ml, whereas pro-cumulin had an ED of 4 ng/ml (Fig. 6A). Mature covalent cumulin was also more effective than pro-cumulin at promoting SMAD2 signaling in human COV434 cells, as assessed by A3-Lux activity (Fig. 6B), whereas the two cumulin forms were equipotent at promoting SMAD1/5/8 signaling (Fig. 6C). Together, our analysis of noncovalent pro-cumulin and covalent mature cumulin allows us to propose a model whereby cumulin activates both SMAD pathways but SMAD2/3 in particular (Fig. 7). One of the physiological roles of oocyte-secreted GDF9 and BMP15 is to direct granulosa cell differentiation toward the FIGURE 5. Engineering and bioactivity of covalent cumulin. Condi- cumulus cell phenotype (41), which can be characterized by tioned media from cells co-expressing GDF9 and BMP15, both harboring expression of genes required for cumulus cell mucification the appropriate Ser-Cys mutations, were subjected to IMAC chromatog- (Ptx3, Has2, Tnfaip6, and Ptgs2) (42). Pro-cumulin and mature raphy followed by reverse phase HPLC. A, absorbance profile at 280 nm from the HPLC purification. B, Western blot of HPLC fractions probed for covalent cumulin were notably more effective at promoting GDF9 with Mab53 or for BMP15 with Mab28. C and D, silver-stained SDS- mRNA expression of these genes in primary mouse mural GC PAGE gel of the HPLC fractions, run under reduced (C) or nonreduced (D) conditions. E, bioactivity of the fractions (0.25 l of each 1-ml fraction) than pro-GDF9, pro-BMP15, or both combined (Fig. 6D). across the HPLC profile, as measured by [ H]thymidine incorporation into Mature cumulin was more effective than pro-cumulin, partic- primary cultures of murine mural GC. ularly at promoting Ptx3 mRNA expression (p 0.05). Pro-cumulin, but Not Mature Covalent Cumulin, Is a Potent Stimulator of Oocyte Developmental Competence—To test our unstimulated ovarian follicles, treated them for 24 h with our human proteins in a model closer to the in vivo situation, we different proteins, and examined cumulus cell expansion and isolated porcine cumulus-oocyte complexes from small oocyte developmental competence (capacity of the oocyte to SEPTEMBER 25, 2015 • VOLUME 290 • NUMBER 39 JOURNAL OF BIOLOGICAL CHEMISTRY 24013 Cumulin, a Novel Ovarian Growth Factor FIGURE 6. The mature domain of covalent cumulin is more potent than pro-cumulin on GC. The pro-cumulin preparation was produced in HEK-293T cells with a N-terminal His tag in the prodomain allowing purification by IMAC. To generate purified mature cumulin, the IMAC product was subsequently subjected to reverse phase HPLC, generating a highly pure mature domain protein with fixed heterodimer architecture (mature covalent cumulin). Bioactivity was assessed by [ H]thymidine incorporation in primary mouse mural GC (A), Smad2/3-responsive luciferase reporter activity (B), and Smad1/5/8 luciferase reporter activity in COV434 human GC (C), and mouse primary mural GC mRNA expression of genes associated with cumulus cell differentiation (D), Ptx3, Tnfaip6, Has2, and Ptgs2 (all proteins at 100 ng/ml, except pro-GDF9  pro-BMP15, which were added at 50 ng/ml each). The bars with different letters indicate significant differences at p 0.05 within the same graph. support embryo development to day 7). With best practice IVM quality as assessed by a substantial improvement in capacity to and embryo production, this model of low oocyte developmen- support embryo development to the blastocyst (2.3-fold; 28- tal competence typically yields 10–20% blastocysts (13, 32). 63%; p 0.05; Fig. 8B) and hatched blastocyst stages (3.8-fold; Interestingly, when porcine cumulus-oocyte complexes are 5–19%; p 0.05; Fig. 8C). There was no significant difference in used as the target, rather than cells in a monolayer (either mural blastocyst rates when oocytes were treated with 20 or 100 ng/ml granulosa or COV434 cells), the most bioactive protein was pro-cumulin (63 and 55%, respectively) or mature cumulin (32 noncovalent pro-cumulin and not covalent mature cumulin and 39%, respectively). No other treatment improved oocyte (Fig. 8). For cumulus expansion, the only protein that caused a developmental competence, although pro-GDF9  pro- significant increase was pro-cumulin (Fig. 8A). Exposure of BMP15 tended to increase blastocyst and hatched blastocyst oocytes to 20 ng/ml pro-cumulin for 24 h improved oocyte rates (p 0.05; Fig. 8C). There was no effect of any treatments 24014 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 39 • SEPTEMBER 25, 2015 Cumulin, a Novel Ovarian Growth Factor FIGURE 7. Hypothesized model of human cumulin formation and signal- ing. BMP15 and GDF9 are co-expressed in the oocyte throughout most of oogenesis. a, during synthesis, the prodomains of BMP15 and GDF9 direct folding and dimerization of their respective mature domains. b, dimeric pre- cursors are cleaved by furin-like proteases, and then BMP15 and GDF9 are secreted from the oocyte noncovalently associated with their prodomains, forming their respective pro-forms. The pro-cumulin heterodimer is likely to assemble within the oocyte but can also form in the extracellular space (ECM). In isolation, human pro-GDF9 is latent, and activation is dependent upon heterodimerization with BMP15 to form cumulin. Following prodomain dis- placement (c), cumulin activates both SMAD2/3 and SMAD1/5 transcription factors (e), presumably through a receptor complex involving two BMPRII receptors, an ALK6 receptor and an ALK5/4 receptor, on cumulus GC and mural GC (d). Pro-cumulin and mature cumulin exhibit differing potencies on different granulosa cell lineages. FIGURE 8. Pro-cumulin, but not mature cumulin, is a potent stimulator of on subsequent blastocyst quality as assessed by blastocyst cell oocyte developmental competence. A porcine in vitro experimental model numbers (Fig. 8D). of low oocyte developmental competence was used to examine the effect of Molecular Modeling and Analysis of Cumulin Assembly—To the various growth factors on oocyte quality. Porcine cumulus-oocyte com- plexes were treated with vehicle, 20 ng/ml pro-cumulin, 20 ng/ml mature provide molecular insights into cumulin assembly, stability, covalent cumulin, 100 ng/ml pro-GDF9, 100 ng/ml pro-BMP15, or 100 ng/ml and potential receptor-binding properties, we performed ho- of each of pro-GDF9  pro-BMP15 for the first 22 h of oocyte in vitro matura- tion. A, morphological cumulus expansion was assessed and scored at 22 h mology modeling to obtain structural models of homo- and according to the Vanderhyden criteria. Effects of the treatments on oocyte heterodimeric forms of GDF9 and BMP15. Structure templates development competence were examined by assessing embryo develop- were selected on the basis of sequence homology to BMPs and ment on day 7. B and C, blastocyst rate (B) and hatching blastocyst rate (C) [hatching or hatched blastocysts], both expressed as a percentage of the GDFs with known structure in the Protein Data Bank. The number of cleaved embryos. D, average blastocyst cell number. All values are models of mature GDF9 (Fig. 9, A and B) and BMP15 (Fig. 10, A represented as means  S.E. from five replicate experiments. The bars with and B) exhibit the canonical butterfly-shaped architecture of different letters indicate significant differences at p 0.05 within the same graph. TGF- ligands. The structure of the monomeric subunits is SEPTEMBER 25, 2015 • VOLUME 290 • NUMBER 39 JOURNAL OF BIOLOGICAL CHEMISTRY 24015 Cumulin, a Novel Ovarian Growth Factor FIGURE 10. Homology model of mature BMP15. A, ribbon plot of human mature BMP15 showing the canonical dimer architecture. The two mono- FIGURE 9. Homology model of mature GDF9. A, ribbon plot of human mer subunits of the homodimer are colored in light and dark green. The matureGDF9highlightingthecanonicaldimerarchitecture.Thetwomono- potential binding epitopes for the type I (wrist) and type II (knuckle) recep- mer subunits of the homodimer are colored in red and orange. The poten- tors are indicated. The six cysteine residues (colored in yellow) forming the tial binding epitopes for the type I (wrist) and type II (knuckle) receptors characteristic cysteine knot motif, as well as the serine residues replacing are marked. The six cysteine residues forming the characteristic cysteine the cysteine residues, which are involved in the intermolecular disulfide knot motif, as well as the serine residues replacing the cysteine residues, bond found in other TGF- members, are shown as sticks. B,asin A but which are involved in the intermolecular disulfide bond found in other rotated around the x axis by 90°. C, detailed view of the BMP15 dimer TGF- members, are shown as sticks. B,asin A but rotated around the x axis interface showing that interfacial amino acid residues are highly con- by 90°. C, detail of the GDF9 dimer interface showing that interfacial amino served with those found in GDF9 (see also Fig. 9), suggesting that cumulin acid residues are highly conserved between GDF9 and BMP15 (see also formation (see Fig. 11) is very likely not impaired by steric or chemical Fig. 10), suggesting that heterodimer formation (see Fig. 11) is very likely restraints by differences in the dimer interface. not impaired by steric or chemical restraints resulting from differences in the dimer interface. to form cumulin (Fig. 11, A and B). Importantly, the cumulin defined by the cysteine knot, which results in three loops ema- model shows a dimerization interface (Fig. 11C) identical to nating from this central motif. The two loops running in paral- homodimeric GDF9 and BMP15 (Figs. 9C and 10C, respec- lel form -sheets, whereas the third loop, which emanates from tively), indicating that formation of the heterodimer is pos- the cysteine core in the opposite direction, folds into an -helix. sible and that the stability of such a complex is likely indis- The monomer architecture is also often compared with an open tinguishable from the homodimeric forms. Production of hand, with the -sheets forming fingers 1 and 2 and the -helix covalent cumulin also appears likely based upon our model- forming the wrist (Figs. 9, A and B, and 10, A and B). The dimer ing, because in silico exchange of the two serine residues 418 356 assembly can then be envisioned as the inner sides of the two (Ser in GDF9 and Ser in BMP15; Fig. 11B) locates both hands coming together at palm and wrist. sulfur atoms sufficiently close to form a thioether bond with- Unlike most TGF- ligands, GDF9 and BMP15 lack the out requiring conformational or large structural rearrange- cysteine residue that forms an intermolecular disulfide ments of the dimer architecture (Fig. 11D). bond, locking the dimer assembly (this cysteine is replaced Finally, analysis of our homology models indicates that the by an isosteric serine in both GDF9 and BMP15). This binding sites for type I receptors within the “wrist” regions are unique feature, together with the very high sequence conser- distinct in cumulin, relative to its homodimeric ancestors (Figs. vation across the GDF9 and BMP15 dimer interfaces (Figs. 11B,9B, and 10B). This is due to the fact that, in contrast to the 9C and 10C), enables these growth factors to heterodimerize type II receptor epitopes, the wrist or type I receptor-binding 24016 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 39 • SEPTEMBER 25, 2015 Cumulin, a Novel Ovarian Growth Factor FIGURE 11. Molecular modeling of mature cumulin. A, ribbon plot of the homology model of human mature cumulin showing the butterfly-shaped dimer architecture and with the monomer subunits colored in red and green. Structural features and the potential binding epitopes for the type I (wrist) and type II (knuckle) receptors are indicated. The six cysteine residues forming the characteristic cysteine knot motif are shown as sticks. B,asin A but rotated around the x axis by 90°. C, detail of the cumulin dimer interface showing that, despite lacking the intermolecular disulfide bond present in most other TGF- ligands, similar polar and hydrophobic interactions stabilize dimer assembly. D, production of covalent cumulin appears possible, because in silico exchange of the two 418 356 serine residues (Ser in GDF9 and Ser in BMP15) with cysteines locates both sulfur atoms sufficiently close to form a thioether bond without requiring conformational or large structural rearrangements of the dimer architecture. site is built from both monomer subunits. In cumulin, this been described by others as the action of two homodimers epitope therefore presents a chimera with unique amino acid (24). Our results do not support this contention. We deduce composition not found in either homodimeric growth factor this from the observation that the BMP15 covalent S:C ancestor. These differences may alter type I receptor affinity homodimer, which cannot heterodimerize with GDF9, does and/or specificity for cumulin and contribute to its enhanced not synergize with wild type GDF9, in contrast to wild type signaling capacity, particularly in comparison to latent human BMP15. The BMP15 mutant in all other aspects resembles S:C GDF9. wild type BMP15. Furthermore, both pro-cumulin and mature covalent cumulin elicited a more potent response Discussion than the synergistic effects of co-addition of wild type GDF9 In females, GDF9 and BMP15 are essentially only expressed and BMP15 homodimers. Using the novel covalent cumulin in oocytes where they are co-expressed throughout most of alleviates any concerns arising from studying solely a nonco- oogenesis and so do not naturally exist in isolation. Moreover, valent heterodimer, which may exist in an equilibrium given their high sequence homology, these key growth factors between homodimers and heterodimers, as we previously can be expected to interact with each other and hence should discussed (43). This leads us to hypothesize that the process generally be considered as acting together. There is ample evi- of heterodimerization is the mechanism of activation of the dence for interactions between GDF9 and BMP15 at genetic (1, otherwise latent GDF9 in the human. 16, 18), biochemical (19, 20), and functional levels (20–23). A Although inhibin A and B are the other major naturally putative BMP15-GDF9 heterodimer was first modeled by occurring TGF- heterodimers (5), it is possible to co-express McNatty et al. (1) in 2004. Most recently, Peng et al. (24) pro- other TGF- ligands and force their dimerization and in so duced GDF9-BMP15 heterodimers with potent bioactivity. We doing generate more biologically potent proteins. Within the have pointed out a number of potential methodological and BMP subgroup, BMP2/7 (44, 45), BMP2/6 (46), and BMP7/ interpretation deficiencies in that study (43), including that the GDF7 (47) heterodimers are all reported as more potent or as preparation contained other protein forms, and the only evi- exhibiting altered bioactivity, in comparison with a combina- dence presented for formation of a heterodimer was co-immu- tion of homodimers. There are a number of possible mecha- noprecipitation of the two proteins. In the current study, we nisms to account for greater potency of heterodimers. First, a have conducted detailed molecular modeling of heterodimer heterodimer may have a decreased binding affinity for extracel- assembly and succeeded in producing two heterodimeric lular BMP binding proteins, which commonly act as antago- forms: pro-cumulin and mature covalent cumulin. Both forms nists. Many of these antagonists act themselves as obligate or are particularly potent on GCs acting via both the SMAD2/3 nonobligate dimers and thus require a symmetrical homodimer and SMAD1/5/8 pathways and show differential actions on for maximal binding affinity. Another explanation is that mural GC versus cumulus GC. TGF- ligands tend to have a high affinity for either a type I or The co-addition of separately expressed GDF9 and BMP15 for a type II receptor, often leading to a strict sequential binding homodimers elicits a potent synergistic response on GC mechanism. A heterodimer, however, could combine a high (20 –23). The results of the current study provide strong evi- affinity binding site for a type I and a type II receptor within the dence that the basis for such synergism is formation of the cumulin heterodimer. Such GDF9/BMP15 synergism has one dimeric ligand, thereby greatly enhancing the overall affin- SEPTEMBER 25, 2015 • VOLUME 290 • NUMBER 39 JOURNAL OF BIOLOGICAL CHEMISTRY 24017 Cumulin, a Novel Ovarian Growth Factor ity to its cell surface receptors. Finally, because of its inherent erodimer activity are unknown. In contrast, covalent mature asymmetry, a heterodimeric ligand is more prone to recruit two cumulin does not contain any tags, and pro-cumulin incorpo- different type I receptors into the signaling complex. For exam- rates a tag only at the N terminus of the BMP15 prodomain. ple, the cumulin receptor complex (Fig. 7) is predicted to com- Hence, in all forms of GDF9, BMP15, or cumulin used in the prise two BMPRII receptors: one ALK4/5/7 receptor, which is current study, the amino acid sequence of the mature region is necessary for the activation of the SMAD2/3 pathway, and one identical to the wild type sequence (apart from the Ser-Cys ALK6 receptor, which is needed for SMAD1/5/8 signaling, as mutation to form covalent dimers). The lack of any modifica- originally hypothesized by McNatty et al. (1). tions to our GDF9 and BMP15 mature regions may also explain An open question prior to this study was whether cumulin why we are able to detect synergism between separately could only form inside the oocyte or whether it might also spon- expressed and purified pro-GDF9 and pro-BMP15 (current taneously assemble from homodimeric GDF9 and BMP15 pres- study), whereas others do not (24, 53). ent in the extracellular space. The latter seemed feasible An intriguing and important observation from the current because GDF9 and BMP15 naturally form transient dimers, study is that when cumulus-oocyte complexes were treated, whose dimer architecture is not fixed by an intermolecular only the pro-cumulin form improved oocyte quality, as assessed disulfide bond. Indeed, we observed that the two wild type by subsequent embryo yield, whereas mature covalent cumulin homodimeric proteins, separately produced and purified, syn- did not, despite being exceptionally potent on mural GC. ergize via a process of heterodimer formation. High resolution Clearly, pro-cumulin contains the prodomains of GDF9 and structure studies of different BMPs provide a possible explana- BMP15 as part of a complex, whereas mature cumulin is an tion for GDF9 and BMP15 disassembly and subsequent cumu- isolated mature region heterodimer (Fig. 3). These results are lin formation. Specifically, the structures show that the inter- entirely consistent with our recent observation that pro- face between the monomeric halves of TGF- proteins BMP15, but not mature domain BMP15 nor mature domain is considerably hydrated, which is unusual compared with mouse GDF9, enhances oocyte developmental competence the water-free hydrophobic core of soluble proteins (48–51). (54, 55). It is well established that prodomains of TGF- The localization of these conserved water molecules in the superfamily growth factors have important roles in protein dimerization interface around the central cysteine knot folding and regulation of bioactivity (56, 57). In particular, likely limits the stability of the dimer; however, in the case of interactions of the prodomains of cumulin with the special- covalent BMPs, a full disassembly of the dimer is prevented ized extracellular matrix of cumulus GC may facilitate pres- because of the covalent linkage via the intermolecular disul- entation of the heterodimeric mature domain to its recep- fide. However, because GDF9 and BMP15 lack this bond, tors. In support of these concepts, we have shown that both factors potentially undergo full disassembly and reas- pro-GDF9 binds strongly to heparin, suggesting that certain sembly, which, when both factors are simultaneously present heparan sulfate proteoglycans may act as co-receptors for pro- (usual situation in most mammals), would yield an equilib- GDF9, pro-BMP15, and pro-cumulin (58). Hence, the differen- rium-based mixture of homo- and heterodimers unless tial activities of pro-cumulin and mature cumulin on mural GC steric restraints specifically interfere with heterodimer for- versus cumulus GC may be attributed to differential expression mation. The latter seems highly unlikely, however, given the of heparan sulfate proteoglycans and/or type I or II receptors in fact that the interface residues are highly conserved between these GC. GDF9 and BMP15. Together, this evidence suggests that the Finally, the current study also has significant practical impli- formation of cumulin in vivo might, at least in part, occur cations for reproductive medicine and advanced breeding in postsecretion from the oocyte in the extracellular matrix of domestic animals. The development of purified and highly the granulosa and cumulus cells. If so, it seems plausible that potent pro-cumulin is likely to prove an important additive for the nature and composition of the extracellular matrix, oocyte IVM (59). IVM is an assisted reproductive technology which in turn is affected by in vivo and in vitro context, is that generates viable embryos using reduced or no exogenous likely to affect cumulin assembly and bioactivity. ovarian hormone stimulation of the patient. IVM is thus an Two remarkable features of cumulin bioactivity is its capac- important adjunct technology to in vitro fertilization. To make ity to activate both the SMAD2/3 and SMAD1/5/8 signaling IVM more clinically viable, including in fertility preservation in pathways, as well as its enhanced signaling potency, in particu- cancer survivors, an additive such as pro-cumulin is needed to lar when compared with the GDF9 and BMP15 homodimers. improve the efficiency of IVM (60). We and others have shown On isolated mural GCs, mature covalent cumulin was even that addition of “native” oocyte-secreted factors (e.g. pro- more potent than pro-cumulin, particularly in terms of GC pro- BMP15 or pro-GDF9) to IVM enhances oocyte quality, having liferation, which is consistent with its enhanced SMAD2/3 sig- profound effects on embryo development and fetal survival (13, naling (29). Dual SMAD pathway activation by cumulin is in 54, 55, 61–64). This study is the first report on the effects of disagreement with Peng et al. (24), who claimed that the GDF9- cumulin on oocyte quality. We carefully chose to use an estab- BMP15 heterodimer binds to, but does not activate, the ALK6 lished porcine model of low oocyte developmental competence receptor. The form of heterodimer that Peng et al. (24) pro- using oocytes from growing antral follicles to better recapitu- duced has an epitope tag (either FLAG or myc) incorporated late the human scenario. The addition of pro-cumulin led to a into the N terminus of the mature region. Although this major improvement in oocyte quality, more than doubling appears to not impede BMP15 homodimer bioactivity (52), the embryo yield, which, if translatable to human oocytes, would effects of the GDF9 (myc) and BMP15 (FLAG) tags on het- have a large impact on clinical IVM and oncofertility. 24018 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 39 • SEPTEMBER 25, 2015 Cumulin, a Novel Ovarian Growth Factor 13. Li, J. J., Sugimura, S., Mueller, T. D., White, M. A., Martin, G. A., Ritter, Author Contributions—D. G. M. and R. B. G. designed the study. L. J., Liang, X. Y., Gilchrist, R. B., and Mottershead, D. G. (2015) Modifi- D. G. M., C. A. H., and R. B. G. secured funding for the project. cations of human growth differentiation factor 9 to improve the genera- D. G. M. oversaw all aspects of recombinant protein design, produc- tion of embryos from low competence oocytes. Mol. Endocrinol. 29, tion, and purification, which were performed by J.-J. L., M. A. W., 40–52 G. A. M., A. P. T., J. S., and D. G. M. S. S. developed methodology for 14. Crawford, J. L., and McNatty, K. P. (2012) The ratio of growth differenti- and performed the porcine oocyte quality experiments. Additional ation factor 9: bone morphogenetic protein 15 mRNA expression is tightly co-regulated and differs between species over a wide range of ovulation experiments were performed and analyzed by the following authors: rates. Mol. Cell Endocrinol. 348, 339–343 D. R., RT-PCR; S. L. M., COV434 SMAD reporters; L. J. R., mouse 15. 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K., Gilchrist, erodimers assemble heteromeric type I receptor complexes to pattern the R. B., and Thompson, J. G. (2012) Metabolic differences in bovine cumulus dorsoventral axis. Nat. Cell Biol. 11, 637–643 oocyte complexes matured in vitro in the presence or absence of follicle 46. Valera, E., Isaacs, M. J., Kawakami, Y., Izpisua Belmonte, J. C., and Choe, S. stimulating hormone and bone morphogenetic protein 15. Biol. Reprod. (2010) BMP-2/6 heterodimer is more effective than BMP-2 or BMP-6 87, 81–88 24020 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 39 • SEPTEMBER 25, 2015 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry American Society for Biochemistry and Molecular Biology

Cumulin, an Oocyte-secreted Heterodimer of the Transforming Growth Factor-β Family, Is a Potent Activator of Granulosa Cells and Improves Oocyte Quality *

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American Society for Biochemistry and Molecular Biology
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Copyright © 2015 Elsevier Inc.
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0021-9258
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1083-351X
DOI
10.1074/jbc.m115.671487
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Abstract

crossmark THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 39, pp. 24007–24020, September 25, 2015 © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Cumulin, an Oocyte-secreted Heterodimer of the Transforming Growth Factor- Family, Is a Potent Activator of Granulosa Cells and Improves Oocyte Quality Received for publication, June 26, 2015, and in revised form, August 6, 2015 Published, JBC Papers in Press, August 8, 2015, DOI 10.1074/jbc.M115.671487 ‡§ ‡¶  ‡ ‡‡ ‡ David G. Mottershead , Satoshi Sugimura , Sara L. Al-Musawi , Jing-Jie Li **, Dulama Richani , Melissa A. White , ‡ ‡ ‡§§ ‡ ¶¶ Georgia A. Martin , Andrew P. Trotta , Lesley J. Ritter , Junyan Shi , Thomas D. Mueller , Craig A. Harrison , ‡ ‡‡1 and Robert B. Gilchrist From the Robinson Research Institute and Discipline of Obstetrics and Gynaecology, School of Paediatrics and Reproductive §§ Health and the Australian Research Council Centre of Excellence in Nanoscale BioPhotonics, University of Adelaide, Adelaide, § ¶ South Australia 5005, Australia, Mottasis Oy Ltd., 00430 Helsinki, Finland, the Institute of Agriculture, Department of Biological Production, Tokyo University of Agriculture and Technology, Tokyo 183-0057, Japan, the Hudson Institute of Medical Research, Clayton, Victoria 3168, Australia, the **Center of Reproductive Medicine, the Sixth Affiliated Hospital, Sun Yat-sen University, ‡‡ 510655 Guangzhou, China, the Discipline of Obstetrics & Gynaecology, School of Women’s & Children’s Health, University of New ¶¶ South Wales, Sydney, New South Wales 2052, Australia, and the Department of Plant Physiology and Biophysics, Julius-von- Sachs Institute of the University Wuerzburg, D-97082 Wuerzburg, Germany Background: Cumulin is a newly identified heterodimeric member of the TGF- family. Results: Mature cumulin potently stimulates granulosa cell signaling and function, whereas pro-cumulin promotes oocyte quality. Conclusion: Formation of cumulin and its potent actions are likely to be central to oocyte paracrine signaling and mammalian fecundity. Significance: The discovery of cumulin provides unique opportunities to improve female fertility in mammals. Growth differentiation factor 9 (GDF9) and bone morphoge- activating both SMAD2/3 and SMAD1/5/8 signaling pathways netic protein 15 (BMP15) are oocyte-specific growth factors and promoting proliferation and expression of a set of genes with central roles in mammalian reproduction, regulating spe- associated with oocyte-regulated GC differentiation. Cumulin cies-specific fecundity, ovarian follicular somatic cell differenti- was more potent than pro-cumulin, pro-GDF9, pro-BMP15, or ation, and oocyte quality. In the human, GDF9 is produced in a the two combined on GC. However, on cumulus-oocyte com- latent form, the mechanism of activation being an open ques- plexes, pro-cumulin was more effective than all other growth tion. Here, we produced a range of recombinant GDF9 and factors at notably improving oocyte quality as assessed by sub- BMP15 variants, examined their in silico and physical interac- sequent day 7 embryo development. Our results support a tions and their effects on ovarian granulosa cells (GC) and model of activation for human GDF9 dependent on cumulin oocytes. We found that the potent synergistic actions of GDF9 formation through heterodimerization with BMP15. Oocyte-se- and BMP15 on GC can be attributed to the formation of a het- creted cumulin is likely to be a central regulator of fertility in erodimer, which we have termed cumulin. Structural modeling mono-ovular mammals. of cumulin revealed a dimerization interface identical to homodimeric GDF9 and BMP15, indicating likely formation of In this study, we consider the unique interaction of two a stable complex. This was confirmed by generation of recombi- members of the TGF- family: growth differentiation factor 9 nant heterodimeric complexes of pro/mature domains (pro-cu- (GDF9) and bone morphogenetic protein 15 (BMP15). These mulin) and covalent mature domains (cumulin). Both pro-cu- two closely related proteins are oocyte-secreted factors essen- mulin and cumulin exhibited highly potent bioactivity on GC, tial for fertility in mammals (1). The primary role of GDF9 and BMP15 is to act on the cells surrounding the oocyte, known as * This work was supported by the National Health and Medical Research granulosa cells (GC), which in turn supply the oocyte with the Council of Australia through Project Grants 627151, 1024358, and support necessary for future healthy embryo/fetal development 1062762, Development Grants 1017484 and 1076004, and Fellowships 1023210 and 1013533. This work was also supported by research grants (2). As with all members of the TGF- family, GDF9 and from Cook Medical, Sun Yat-sen University International Program Fund BMP15 are produced as pro-proteins, consisting of an N-ter- 985 Project (to J. J. L.) and from the Japanese Society for the Promotion of minal proregion and a receptor binding C-terminal mature Science Postdoctoral Fellowship for Research Abroad (to S. S.). R. B. G. is listed as an inventor on Patent US20140073052A1 owned by the University region. Activation of these factors requires proteolytic pro- of Adelaide. R. B. G. and D. G. M. are listed on a distribution of benefits cessing by a furin-like protease (3, 4). For most TGF- mem- agreement in connection with Patent US20140073052A1. To whom correspondence should be addressed: Robert B Gilchrist, Dis- cipline of Obstetrics & Gynaecology, School of Women’s & Children’s The abbreviations used are: GDF9, growth differentiation factor 9; ALK, Health, Royal Hospital for Women, University of New South Wales, Syd- activin receptor-like kinase; BMP15, bone morphogenetic protein 15; BMP- ney, Australia. Tel.: 61-2-93852562; Fax: 61-2-93852573; E-mail: RII, BMP receptor type II; GC, granulosa cell; IVM, oocyte in vitro maturation; r.gilchrist@unsw.edu.au. SMAD; Sma- and Mad-related proteins. This is an open access article under the CC BY license. SEPTEMBER 25, 2015 • VOLUME 290 • NUMBER 39 JOURNAL OF BIOLOGICAL CHEMISTRY 24007 Cumulin, a Novel Ovarian Growth Factor bers, the processed mature domains signal as covalently linked ways. Interestingly, only the promature heterodimer is capable homodimers, although covalent heterodimers are also known of improving oocyte quality, indicative of the crucial function (inhibin A and B (5)). GDF9 and BMP15 are unusual within the that the prodomain plays in the functioning of these proteins. TGF- family in that they (together with GDF3 and Lefty1/2) In deference to its potent activation of cumulus cells and to lack the cysteine residue involved in forming the mature region align with the existing nomenclature for the other major repro- intersubunit disulfide bond (6). Despite this, mature GDF9 and ductive TGF- superfamily heterodimer, inhibin, we have BMP15 are thought to maintain the canonical dimer architec- called this new heterodimer cumulin. ture of other TGF- ligands and signal as noncovalent dimers. Experimental Procedures As with all members of the TGF- family, GDF9 and BMP15 signal via cell surface receptors, which activate intracellular Expression Plasmids, Stable Cell Lines, and Protein Sma- and Mad-related (SMAD) proteins by phosphorylation of Production—Expression cassettes encoding wild type human critical residues, which subsequently regulate the transcription GDF9 or BMP15 or mouse GDF9 (mGDF9) were synthesized of various genes (1). The receptors are Ser/Thr kinases and are, (Genscript USA, Inc., Piscataway, NJ) incorporating the rat on the basis of distinct features in the cytoplasmic domain, serum albumin signal sequence at the 5 end followed by referred to as either type I (activated by the relevant type II sequences encoding for His and StrepII affinity tags at the N receptor) or type II (constitutively active) receptors. Following terminus of the respective proregion. The various human 418 356 prodomain displacement, BMP15 binds to complexes of BMP GDF9 and BMP15 Ser-Cys mutants (Ser in GDF9 and Ser receptor type II (BMPRII) and activin receptor-like kinase 6 in BMP15) used in this study were derived from expression (ALK6), thereby leading to activation of the SMAD1/5/8 intra- cassettes (Genscript USA, Inc., Piscataway, NJ) incorporating cellular pathway (7, 8). Mature GDF9 binds BMPRII and ALK5 the glutamate receptor GluR-D subunit signal sequence and signals through the SMAD2/3 pathway (9–11). (MRIICRQIVLLFSGFWGLAMG) at the 5 end followed by GDF9 and BMP15 are also functionally unusual within the sequences encoding for the FLAG tag (a design based on Kuusi- TGF- family, in that they show remarkable species differences nen et al. (25)) and His affinity tag at the N terminus of the in their activity. Notably, human GDF9 is latent, because it respective proregion (as used previously (8)). All the cDNA remains associated with its prodomain, whereas mouse GDF9 fragments were cloned into the pEF-IRES expression vector is particularly potent, because of its prodomain being readily (26). displaced (3, 12, 13). By contrast, human BMP15 is recombi- HEK293T cells were obtained from the American Type Cul- nantly produced in an active form in mammalian cell culture, ture Collection and cultured in DMEM (Invitrogen) containing whereas mouse BMP15 is not expressed under the same condi- 10% FCS, 2 mML-glutamine, 100 IU/ml penicillin (Sigma), and tions (4). The species-specific differences in expression and 100 g/ml streptomycin. The different expression plasmids activity of these oocyte-secreted growth factors is principally were transfected into HEK293T cells using Lipofectamine 2000 due to alterations in a small number of amino acids in the (Invitrogen) following the manufacturer’s instructions. Stable prodomains (BMP15) or mature domains (GDF9), which affect cell lines were established via puromycin selection. For produc- complex formation or stability, respectively (3, 4, 13). Hence, tion of cells producing pro-cumulin, HEK293T cells were co- the relative levels of oocyte expression of BMP15 and GDF9 transfected with both an expression plasmid for human GDF9 vary markedly across species, whereby the mono-ovular phe- of wild type sequence, incorporating no epitope tags, along with notype (i.e. low fecundity) is characterized by balanced the expression plasmid for wild type human BMP15 (His /Stre- BMP15:GDF9 ratios, whereas polyovular species (high pII-tagged), as described above. For production of cells produc- fecundity) express predominately GDF9 (14). As such, ing cumulin (as a covalently stabilized form), HEK293T cells GDF9, but not BMP15, is essential for fertility in the mouse were co-transfected with expression plasmids encoding human (15, 16), whereas both are required in mono-ovular species, GDF9 and BMP15 as the Ser-Cys mutant forms, as described such as sheep (17, 18). above. Interactions at the genetic level between these two proteins The mature regions of GDF9 (12), BMP15 (27) and the have been observed in both mice (16) and sheep (1, 18). Evi- BMP15 Ser-Cys mutant (8) were produced and purified as pre- dence for GDF9/BMP15 physical interactions have also been viously described. Cells producing pro-GDF9, pro-mGDF9, provided by co-immunoprecipitation results (19, 20), and a pro-BMP15, pro-cumulin, or covalent cumulin were grown to number of functional studies have demonstrated marked syn- near confluence, and growth medium was then replaced by pro- ergistic interactions between these two proteins (20–23). duction media containing DMEM/F12 GlutaMAX, 0.1 mg/ml Recently, Peng et al. (24) have provided evidence that het- BSA (Sigma), 25 IU/ml Fragmin (Pfizer), 100 units/ml penicil- erodimerization of GDF9 and BMP15 gives rise to a more lin, and 100 g/ml streptomycin (Sigma). After 48 h of incuba- potent ligand that signals solely via the SMAD2/3 pathway. tion, the conditioned culture medium was collected. In the current study, we hypothesized that human GDF9 and Chromatography—Production medium containing pro- BMP15 form a stable heterodimer that has potent actions on GDF9, pro-BMP15, or pro-cumulin was modified to include 10 mural GC, cumulus GC, and oocyte development. We provide mM imidazole, 500 l of protease inhibitor mixture (Thermo the first evidence of a purified BMP15-GDF9 heterodimer. We Scientific), 500 l of phosphatase inhibitor mixture (Calbi- find that both the promature form and a covalent mature form ochem, Alexandria, Australia) and subsequently adjusted to pH of the heterodimer are particularly potent and are dual pathway 7.4. Invitrogen nickel-nitrilotriacetic acid-agarose resin was activators, signaling via the SMAD2/3 and SMAD1/5/8 path- incubated with conditioned medium on a rotary agitator at 4 °C 24008 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 39 • SEPTEMBER 25, 2015 Cumulin, a Novel Ovarian Growth Factor for 2 h and centrifuged at 900 rpm for 5 min, and the superna- 24 h. The cells were treated with BMP15, GDF9, or cumulin tant was removed. The His-tagged target protein was eluted variants for 16 h. The medium was then aspirated, and the cells from the nickel-nitrilotriacetic acid-agarose using elution were solubilized in solubilization buffer (25 mM glycylglycine, buffer (PBS/500 mM imidazole) and stored in LoBind tubes pH 7.8, 15 mM MgSO ,4mM EGTA, 1% (v/v) Triton X-100, and (Eppendorf, Hamburg, Germany) at 80 °C. Cumulin (the 1mM dithiothreitol), and luciferase reporter activity was mea- covalently stabilized form) was produced and purified as above, sured as previously described (30). with the exception that the mature region was eluted from the RT-PCR—Mural GC were collected from 21–26-day-old IMAC resin via a 7 M urea wash. This material was then loaded C57BL/6  DBA F1 hybrid female mice 46 h after intraperito- onto a reverse phase HPLC column equilibrated with 0.1% neal administration of gonadotropin priming. Mice were main- TFA, and the different mature region forms (i.e. GDF9, cumulin tained in accordance with the Australian Code for the Care and and finally BMP15) were eluted via a linear gradient of acetoni- Use of Animals for Scientific Purposes and with the approval of trile. The fractions were stored in LoBind tubes at 80 °C. the University of New South Wales Animal Ethics Committee. Silver Staining, Western Blotting, and Protein Quanti- Mural GC (1 10 live cells per 24-plate well) were cultured for fication—Fractions from the cumulin reverse phase HPLC were 18 h in bicarbonate-buffered MEM (Gibco Life Technologies) prepared for electrophoresis by lyophilizing 5 l and resus- supplemented with 3 mg/ml BSA (Sigma-Aldrich). The cells pending in SDS-PAGE loading buffer DTT. All samples were were then treated for 6 h with fresh medium containing recom- heated at 95 °C for 5 min and then loaded onto a Mini binant human FSH (50 mIU/ml; Puregon; Organon, Oss, The PROTEAN TGX 4–15% gel (Bio-Rad). After 40 min at 150 V, Netherlands) plus the following treatments, as indicated: 100 the gels were used for silver staining or Western blotting. Silver ng/ml cumulin, 100 ng/ml pro-cumulin, 100 ng/ml pro-mature staining was carried out following the manufacturer’s instruc- GDF9, 100 ng/ml pro-mature BMP15, or 50 ng/ml each of pro- tions using a silver quest staining kit (Invitrogen). For Western mature GDF9  pro-mature BMP15 when added together. blotting, proteins were transferred to a Hybond-ECL mem- RNA extraction, reverse transcription, and quantitative PCR brane (GE Healthcare) for 75 min at 100 V. The membranes were performed as previously described (31) using their were blocked with 2% blocking reagent (supplied in an ECL reported primers; however, PCRs were performed on a Roche Advance kit; GE Healthcare) diluted in TBS containing 0.1% LightCycler 480, and results are expressed as raw gene expres- (v/v) Tween 20 for1hat room temperature. Subsequently the sion levels relative to the geometric means of two housekeeping membranes were incubated with anti-GDF9 antibody (mAb53 (CT) genes (Mrpl19 and Ppia) using the 2 method. The data (28)) or anti-BMP15 antibody (mAb28 (27)) diluted 1:5000 at are means  S.E. of four replicate experiments. 4 °C overnight, followed by incubation with goat anti-mouse Oocyte Quality Assay—The effects of treatments on oocyte TM IRDye 800 CW antibody diluted 1:10,000, for1hinthe dark. quality (oocyte developmental competence) were assessed by Fluorescence intensity was determined using an Odyssey infra- treating cumulus-oocyte complexes with the different growth red imaging system (Licor Bioscience, Lincoln, NE). The vari- factors during the oocyte in vitro maturation (IVM) phase, fol- ous purified forms of GDF9 or BMP15 were quantified via lowed by in vitro fertilization and embryo culture and assess- Western analysis relative to recombinant mGDF9 or hBMP15 ment of subsequent blastocyst yield and quality. We chose to standards, respectively (R&D Systems). use a well established porcine model of low oocyte develop- Granulosa Cell Bioassays—Bioactivities of pro-forms and/or mental competence, because this experimental model better mature forms of BMP15, GDF9, and cumulin were compared approximates the complexities of human oocyte developmen- using primary mouse mural GC and a human granulosa tumor 3 tal competence than that of murine oocytes. This model uses cell line (COV434). The mouse granulosa cell [ H]thymidine oocytes from small antral follicles collected from abattoir-de- incorporation bioassay was performed using standard proce- rived ovaries from gilts (peripubertal female pig) (13, 32–34). dures as previously described (28, 29). In brief, mural GC were The methods used were as per recently described in detail (13, collected from 129/Sv mice 44–46 h after gonadotropin prim- 34). In brief, cumulus-oocyte complexes were aspirated from ing. Cells were cultured at 37 °C in 5% CO in protein-free small antral follicles (2–4 mm) and matured in protein-free medium at 2 10 cells/ml with treatments for 18 h followed by porcine oocyte maturation basic IVM medium (35), supple- a further 6 h with 15.4 kBq [ H]thymidine (PerkinElmer Life mented for the first 22 h of IVM with 1 mM dibutyryl-cAMP and Sciences). Granulosa cell [ H]thymidine incorporation was 100 ng/ml amphiregulin (R&D Systems) and treated with either assessed using a Wallac Microbeta plate counter as an indicator vehicle (control), 20 or 100 ng/ml pro-cumulin, 20 or 100 ng/ml of cell DNA synthesis. Each treatment was performed in dupli- mature cumulin, 100 ng/ml pro-GDF9, 100 ng/ml pro-BMP15, cate, and the experiments were replicated three to six times. or 100 ng/ml of each of pro-GDF9  pro-BMP15. Cumulus The relative capacity of BMP15, GDF9, and cumulin to acti- expansion was assessed at 22 h of IVM and scored as per stan- vate SMAD signaling was assessed with luciferase assays using dard criteria (36, 37). At 22 h, cumulus-oocyte complexes were human COV434 cells (3, 30). In brief, cells were plated at 33 washed and matured for a further 22 h in porcine oocyte mat- 10 cells/well in 96-well plates in complete medium (DMEM uration without supplements or treatments (13, 33, 34). There- supplemented with 10% FCS) at 37 °C in 5% CO . After 24 h of incubation, cells were transfected (130 ng/well) with a BMP- after, in vitro fertilization, in vitro embryo production, and responsive reporter, BRE-luciferase, or an activin/TGF- assessment of blastocysts was performed as previously responsive reporter, A3-luciferase, using Lipofectamine 2000 described (13). Five replicate experiments were performed with (Invitrogen) according to the manufacturer’s instructions for 180 oocytes/replicate. SEPTEMBER 25, 2015 • VOLUME 290 • NUMBER 39 JOURNAL OF BIOLOGICAL CHEMISTRY 24009 Cumulin, a Novel Ovarian Growth Factor Molecular Modeling—To obtain three-dimensional struc- ture models for mature human GDF9, mature human BMP15, and cumulin, homology modeling was performed using either the software SWISS-Modeler or a manual approach. To iden- tify the most suitable structure template, the sequences of GDF9 and BMP15 were blasted against the protein sequences present in the Protein Data Bank, revealing that both oocyte factors exhibit the highest sequence homology (32–33% iden- tity) to either BMP2 or GDF5. For manual modeling the sequences of the mature region of GDF9 and BMP15 were then aligned to the amino acid sequence of the identified templates, i.e. BMP2 (Protein Data Bank entry 3BMP and 1REW (chains A and B) and 3BK3 (chains A and B)) and GDF5 (Protein Data Bank entry 1WAQ) using the software CLUSTALW. A model 327 430 of human GDF9 comprising residues Asn –Arg was then built manually by replacing amino acid residues in the BMP2 template (chains A and B of Protein Data Bank entry 3BK3) employing the tool ProteinDesign of software package FIGURE 1. Pro-forms of BMP15 and GDF9 synergize. Pro-hBMP15 syner- Quanta2008 (MIS Accelrys, San Diego, CA). A single amino gizes with pro-mouse GDF9 and also with pro-human GDF9, which is natu- acid insertion at the end of helix 1 found in GDF9 (when rally latent. Bioactivity was assessed by [ H]thymidine incorporation in pri- mary mouse mural GC. The various pro-proteins were produced in HEK-293T compared with the template BMP2) was rebuilt manually. For cells with N-terminal His tags attached to their prodomains allowing purifica- modeling of human BMP15 (comprising residues Asn tion by IMAC. Arg ), the same approach was applied but using the structure of human GDF5 (Protein Data Bank entry 1WAQ) as BMP15, bond was subsequently refined by a short energy minimization similar to GDF5, binds specifically the BMP receptor BMPR1b. run to obtain a thioether bond of correct length. Both initial models were subsequently refined first by perform- Statistical Analysis—For mRNA expression data, differences ing rotamer searches using the tool X-BUILT of the software between means were assessed by one-way analysis of variance Quanta2008 to identify side chain conformations not leading to followed by Tukey’s multiple comparison test. Similarly, effects van der Waals clashes. Then energy minimization runs were of treatments on embryo yield were assessed by one-way anal- performed employing only geometrical but no electrostat- ysis of variance on arc-sine transformed data followed by a least ic energy terms using Quanta2008 and the forcefield significant difference post hoc test. Cumulus expansion data CHARMm27. First, all backbone atoms were kept fixed, and were assessed by a Kruskal-Wallis one-way analysis of variance only side atoms were allowed to move but restrained by har- 1 2 on ranks followed by a Dunnett’s post hoc test. p 0.05 was monic positional restraints (5 kcal mol Å ) to minimize considered statistically significant. atom movements. After energy minimization of side chain atoms converged, these restraints were removed, and the posi- Results tions of the backbone atoms were refined by applying a strong 1 2 harmonic potential (25 kcal mol The various forms of GDF9, BMP15, and cumulin analyzed Å ). The final models of GDF9 and BMP15 exhibit good backbone and side chain geom- in this study were as follows: 1) pro-forms: noncovalent homo- etry. The structure models for both homodimeric factors are and heterodimers of the promature regions of BMP15 and highly similar, yielding a root mean square deviation of only GDF9 (the form in which these proteins are secreted from the 1.1Å for 164 C carbons. Only 40 residues, which are located cell); 2) mature forms: noncovalent homo- and heterodimers of either in the fingertips or the loop N-terminal of helix 1, the mature regions of BMP15 and GDF9 (the form in which exhibit differing conformations, which may be due to selecting these proteins bind to their signaling receptors); and 3) covalent different template structures for the models and does not forms: covalent homo- and heterodimers of the mature regions reflect inherent structural differences between GDF9 and of BMP15 and GDF9 (stabilized versions of the mature growth BMP15. factors). The model of cumulin was then built by superimposing the GDF9/BMP15 Synergism Is Mediated by Heterodimer (i.e. three-dimensional models of homodimeric GDF9 and BMP15 Cumulin) Formation—We have consistently observed GDF9 and selecting one chain of each structure to form a heterodi- and BMP15 interacting in a synergistic manner, whether in meric assembly. Because of the almost identical amino acid their pro-forms (i.e. as isolated pro/mature complexes; Fig. 1) composition in the dimer interface, the model of cumulin or as purified mature proteins (23, 38). The extracellular forma- required very minor minimization, which was performed by tion of a BMP15/GDF9 heterodimer, which we have termed rotamer searches for side chains with close contacts. The het- cumulin, could underlie this potent synergism. To directly test erodimeric noncovalent form of cumulin was then converted this possibility, we generated a covalent BMP15 homodimer (by 356 356 into the covalent form by exchanging the serine residues Ser replacing Ser with the corresponding cysteine found in other in BMP15 and Ser in GDF9 against cysteines and connecting TGF- proteins), which would be incapable of forming cumu- lin in the presence of wild type GDF9. both sulfur atoms to form the thioether bond. The disulfide 24010 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 39 • SEPTEMBER 25, 2015 Cumulin, a Novel Ovarian Growth Factor Covalent human BMP15 mature region homodimer (BMP15 ) was purified and tested for its ability to synergize S:C with human GDF9 (mature region homodimer) in a mural granulosa cell proliferation assay. At the low doses tested, GDF9, BMP15, and BMP15 did not promote proliferation; S:C however, the GDF9BMP15 combination showed potent syn- ergism (Fig. 2A). In contrast, this synergism was not observed with the GDF9BMP15 combination. Because we have pre- S:C viously shown that GDF9/BMP15 synergism is dependent upon Smad2/3 activation (23, 38), we examined the ability of our BMP15 variants to affect this response in COV434 cells. Although wild type BMP15 promoted GDF9-dependent Smad2/3 transcriptional activity in a dose-dependent manner, the BMP15 variant could not potentiate a GDF9 effect (Fig. S:C 2B). To ensure the BMP15 variant had bioactivity, we tested S:C its ability to stimulate the SMAD1/5/8 pathway in COV434 cells transfected with a BMP-responsive luciferase reporter. In this system, the BMP15 protein was equally potent as wild S:C type BMP15 (Fig. 2C). As expected, GDF9 did not activate the SMAD1/5/8 pathway. Together, these results indicate that extracellular heterodimerization of mature GDF9 and BMP15 is possible, because simple co-addition of GDF9BMP15, but not of GDF9BMP15 , resulted in potent synergism. S:C To further explore extracellular associations between GDF9 and BMP15, we mixed conditioned media from cells expressing untagged pro-GDF9 with conditioned media from cells expressing N-terminally poly-His-tagged pro-BMP15 and car- ried out IMAC purification of the pro-BMP15. Under the mild conditions used, the untagged GDF9 mature region was capa- ble of associating and co-purifying with pro-BMP15, as evi- denced by GDF9 immunoreactivity co-eluting off the IMAC resin (Fig. 3A). No GDF9 immunoreactivity was detected when BMP15 was purified alone (data not shown), and the untagged GDF9 did not bind to the IMAC resin in the absence of the poly-His-tagged BMP15 (Fig. 3B). It is clear from these results that, like the mature proteins, the pro forms of GDF9 and BMP15 are capable of associating extracellularly to form pro-cumulin. Intracellular Formation of Cumulin—GDF9 and BMP15 are co-expressed in the oocyte throughout folliculogenesis and so do not naturally exist in isolation (reviewed in Ref. 39). Thus, heterodimerization of these two TGF- family members to form cumulin is likely to occur during synthesis within the oocyte, as well as after secretion into the extracellular space. In the in vivo context, cumulin will be in a form where the mature regions are noncovalently associated and will most likely act as a pro/mature complex. To produce such a form (i.e. pro-cumu- lin), we co-expressed N-terminally poly-His-tagged pro- BMP15 with untagged pro-GDF9 and purified the noncovalent FIGURE 2. GDF9/BMP15 synergism is mediated by heterodimer forma- pro-cumulin complex by IMAC. Theoretically, this approach tion. The BMP15 (S356C) covalent homodimer (BMP15 ) does not synergize S:C could lead to the purification of a mixture of pro-BMP15 with GDF9, in contrast to wild type BMP15, where clear synergism is observed. homodimers and pro-cumulin. Quantification of the levels of Bioactivity was assessed by [ H]thymidine incorporation in primary mouse mural GC (A), Smad2/3-responsive luciferase reporter activity (B), or Smad1/ both the processed GDF9 and BMP15 mature regions following 5/8 luciferase reporter activity, in COV434 human GC (C). The various proteins IMAC purification indicated a 3:1 ratio of pro-BMP15 were produced in HEK-293T cells with N-terminal His tags attached to their homodimer:pro-cumulin heterodimer (data not shown). prodomains allowing purification by IMAC, followed by isolation of mature domains by reverse-phase HPLC. BMP15, HPLC-purified human BMP15 Pro-cumulin Is a Potent Activator of Granulosa Cells via Both mature region homodimer; GDF9, HPLC purified human GDF9 mature region SMAD Pathways—We next examined the potency of pro-cu- homodimer; BMP15 , HPLC-purified human BMP15 (Ser-Cys) mature region S:C covalent homodimer. mulin in ovarian GC bioassays. We compared pro-cumulin to SEPTEMBER 25, 2015 • VOLUME 290 • NUMBER 39 JOURNAL OF BIOLOGICAL CHEMISTRY 24011 Cumulin, a Novel Ovarian Growth Factor FIGURE 3. GDF9 associates and co-purifies with BMP15 to form pro- cumulin. Conditioned media containing untagged pro-GDF9 and N-termi- nally His -tagged pro-BMP15 were combined and subjected to IMAC chroma- tography. A, Western blot of resin load probed with Mab28 (specific for human BMP15), or the resin load, unbounded material, wash, and eluted peak fraction, probed with Mab53 (specific for GDF9). B, Western blot for IMAC chromatography of conditioned media containing only untagged pro-GDF9. Shown are the resin load, unbound material, wash, and eluate, probed with Mab53. pro-GDF9, pro-BMP15, and pro-GDF9  pro-BMP15, after quantitating each protein form relative to the level of the GDF9 mature region (data not shown). Pro-GDF9 alone was inactive in promoting GC DNA synthesis, as shown previously (3), as was pro-BMP15 alone (Fig. 4A). In contrast, pro-cumulin was exceptionally potent on primary mouse mural GC, stimulating a 3.8-fold increase in [ H]thymidine incorporation at the lowest dose tested (1 ng/ml; Fig. 4A). Pro-cumulin was notably more potent (6–12-fold) than pro-GDF9pro-BMP15 at all doses examined. To examine SMAD signaling by pro-cumulin, we conducted transcriptional reporter assays using the human granulosa cell line, COV434, as previously reported (3). Pro-cumulin dose- dependently activated SMAD2/3 signaling in GC, producing high levels of A3-Lux reporter activity at 50 and 100 ng/ml (Fig. 4B). Pro-GDF9  pro-BMP15 also dose-dependently activated SMAD2/3 but did not reach the same levels of stimulation as pro-cumulin. As expected, pro-GDF9 did not activate SMAD2/3, because it is latent (3, 12). Interestingly, pro-cumu- lin and pro-GDF9  pro-BMP15 promoted similar levels of BRE-luciferase activation (reflecting the level of SMAD1/5/8 phosphorylation) as pro-BMP15 (Fig. 4C). As expected, GDF9 did not activate the SMAD1/5/8 pathway (Fig. 4C), and BMP15 did not activate the SMAD2/3 pathway (Fig. 4B). Engineering Covalent Mature Cumulin—The pro-cumulin preparation described above and that previously produced by Peng et al. (24, 40) contain significant amounts of contaminat- FIGURE 4. Bioactivity and SMAD signaling of pro-cumulin. A–C, dose-re- ing pro-BMP15 homodimer. This was due to the almost equal sponse curves of the different pro-protein forms of GDF9, BMP15, and cumu- lin. Bioactivity was assessed by [ H]thymidine incorporation in primary propensity of monomeric BMP15 to homodimerize or het- mouse mural GC (A), Smad2/3-responsive luciferase reporter activity (B), or erodimerize (with GDF9) and the fact that the purification Smad1/5/8 luciferase reporter activity (C) in COV434 human GC. The various pro-proteins were produced in HEK-293T cells with N-terminal His tags strategies were directed against tagged BMP15 prodomains. To attached to their prodomains allowing purification by IMAC. overcome these limitations and to more conclusively examine the biological function of cumulin, we undertook to engineer a covalent form of cumulin, which could be fully purified from pressed the resultant cDNAs in HEK293T cells. We have used contaminating BMP15 and GDF9. To produce such a protein, this approach before to produce human covalent mature 418 356 we replaced Ser in GDF9 and Ser in BMP15 with the cor- BMP15 (8). Both the pro-BMP15 and pro-GDF9 mutants S:C S:C responding cysteine found in other TGF- proteins and co-ex- were produced with N-terminal poly-His tags; hence IMAC 24012 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 39 • SEPTEMBER 25, 2015 Cumulin, a Novel Ovarian Growth Factor purification of conditioned media from cells co-expressing these proteins theoretically could result in the co-purification of pro-GDF9 or pro-BMP15 homodimers, as well as the sought after pro-cumulin. We decided to target the GDF9 and BMP15 mature regions for purification to avoid any potentially mis- folded disulfide linked pro/mature complexes. This was accomplished by a two-step procedure of IMAC chromatog- raphy followed by reverse phase HPLC. This approach suc- ceeded in resolving three distinct covalent species: mature GDF9, mature cumulin, and mature BMP15 (Fig. 5A). This can be concluded because Western analysis of the fractions from the reverse phase HPLC purification indicated that fractions 31–32 contained GDF9, but no BMP15; fractions 33–34 contained both GDF9 and BMP15 (i.e. cumulin), whereas fractions 36 –39 contained only BMP15 (Fig. 5B). The silver-stained gels confirmed the presence of disulfide- linked dimers, as the reduced bands at 15–18 kDa (Fig. 5C, fractions 32–34 and 37) that migrated at 25–35 kDa without reduction (Fig. 5D, fractions 32–34 and 37). Covalent Mature Cumulin Is Potently Bioactive—Because purified mature cumulin has not previously been produced, it was important to determine its bioactivity, especially given its form as a stabilized disulfide linked covalent mature region het- erodimer. We initially tested the fractions across the reverse phase HPLC profile for bioactivity in the mural GC assay. The peak of bioactivity (Fig. 5E, fraction 34) coincided with the frac- tion containing the peak of covalent cumulin protein (Fig. 5, B–D, fraction 34). We next directly compared the bioactivity of covalent mature cumulin to the previously characterized noncovalent pro-cumulin on GC. Mature cumulin was highly potent in the range of different granulosa cell assays performed; in most cases, it was more potent than pro-cumulin (Fig. 6). In the pri- mary mouse mural granulosa cell assay of [ H]thymidine incor- poration, covalent mature cumulin had an ED of 0.6 ng/ml, whereas pro-cumulin had an ED of 4 ng/ml (Fig. 6A). Mature covalent cumulin was also more effective than pro-cumulin at promoting SMAD2 signaling in human COV434 cells, as assessed by A3-Lux activity (Fig. 6B), whereas the two cumulin forms were equipotent at promoting SMAD1/5/8 signaling (Fig. 6C). Together, our analysis of noncovalent pro-cumulin and covalent mature cumulin allows us to propose a model whereby cumulin activates both SMAD pathways but SMAD2/3 in particular (Fig. 7). One of the physiological roles of oocyte-secreted GDF9 and BMP15 is to direct granulosa cell differentiation toward the FIGURE 5. Engineering and bioactivity of covalent cumulin. Condi- cumulus cell phenotype (41), which can be characterized by tioned media from cells co-expressing GDF9 and BMP15, both harboring expression of genes required for cumulus cell mucification the appropriate Ser-Cys mutations, were subjected to IMAC chromatog- (Ptx3, Has2, Tnfaip6, and Ptgs2) (42). Pro-cumulin and mature raphy followed by reverse phase HPLC. A, absorbance profile at 280 nm from the HPLC purification. B, Western blot of HPLC fractions probed for covalent cumulin were notably more effective at promoting GDF9 with Mab53 or for BMP15 with Mab28. C and D, silver-stained SDS- mRNA expression of these genes in primary mouse mural GC PAGE gel of the HPLC fractions, run under reduced (C) or nonreduced (D) conditions. E, bioactivity of the fractions (0.25 l of each 1-ml fraction) than pro-GDF9, pro-BMP15, or both combined (Fig. 6D). across the HPLC profile, as measured by [ H]thymidine incorporation into Mature cumulin was more effective than pro-cumulin, partic- primary cultures of murine mural GC. ularly at promoting Ptx3 mRNA expression (p 0.05). Pro-cumulin, but Not Mature Covalent Cumulin, Is a Potent Stimulator of Oocyte Developmental Competence—To test our unstimulated ovarian follicles, treated them for 24 h with our human proteins in a model closer to the in vivo situation, we different proteins, and examined cumulus cell expansion and isolated porcine cumulus-oocyte complexes from small oocyte developmental competence (capacity of the oocyte to SEPTEMBER 25, 2015 • VOLUME 290 • NUMBER 39 JOURNAL OF BIOLOGICAL CHEMISTRY 24013 Cumulin, a Novel Ovarian Growth Factor FIGURE 6. The mature domain of covalent cumulin is more potent than pro-cumulin on GC. The pro-cumulin preparation was produced in HEK-293T cells with a N-terminal His tag in the prodomain allowing purification by IMAC. To generate purified mature cumulin, the IMAC product was subsequently subjected to reverse phase HPLC, generating a highly pure mature domain protein with fixed heterodimer architecture (mature covalent cumulin). Bioactivity was assessed by [ H]thymidine incorporation in primary mouse mural GC (A), Smad2/3-responsive luciferase reporter activity (B), and Smad1/5/8 luciferase reporter activity in COV434 human GC (C), and mouse primary mural GC mRNA expression of genes associated with cumulus cell differentiation (D), Ptx3, Tnfaip6, Has2, and Ptgs2 (all proteins at 100 ng/ml, except pro-GDF9  pro-BMP15, which were added at 50 ng/ml each). The bars with different letters indicate significant differences at p 0.05 within the same graph. support embryo development to day 7). With best practice IVM quality as assessed by a substantial improvement in capacity to and embryo production, this model of low oocyte developmen- support embryo development to the blastocyst (2.3-fold; 28- tal competence typically yields 10–20% blastocysts (13, 32). 63%; p 0.05; Fig. 8B) and hatched blastocyst stages (3.8-fold; Interestingly, when porcine cumulus-oocyte complexes are 5–19%; p 0.05; Fig. 8C). There was no significant difference in used as the target, rather than cells in a monolayer (either mural blastocyst rates when oocytes were treated with 20 or 100 ng/ml granulosa or COV434 cells), the most bioactive protein was pro-cumulin (63 and 55%, respectively) or mature cumulin (32 noncovalent pro-cumulin and not covalent mature cumulin and 39%, respectively). No other treatment improved oocyte (Fig. 8). For cumulus expansion, the only protein that caused a developmental competence, although pro-GDF9  pro- significant increase was pro-cumulin (Fig. 8A). Exposure of BMP15 tended to increase blastocyst and hatched blastocyst oocytes to 20 ng/ml pro-cumulin for 24 h improved oocyte rates (p 0.05; Fig. 8C). There was no effect of any treatments 24014 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 39 • SEPTEMBER 25, 2015 Cumulin, a Novel Ovarian Growth Factor FIGURE 7. Hypothesized model of human cumulin formation and signal- ing. BMP15 and GDF9 are co-expressed in the oocyte throughout most of oogenesis. a, during synthesis, the prodomains of BMP15 and GDF9 direct folding and dimerization of their respective mature domains. b, dimeric pre- cursors are cleaved by furin-like proteases, and then BMP15 and GDF9 are secreted from the oocyte noncovalently associated with their prodomains, forming their respective pro-forms. The pro-cumulin heterodimer is likely to assemble within the oocyte but can also form in the extracellular space (ECM). In isolation, human pro-GDF9 is latent, and activation is dependent upon heterodimerization with BMP15 to form cumulin. Following prodomain dis- placement (c), cumulin activates both SMAD2/3 and SMAD1/5 transcription factors (e), presumably through a receptor complex involving two BMPRII receptors, an ALK6 receptor and an ALK5/4 receptor, on cumulus GC and mural GC (d). Pro-cumulin and mature cumulin exhibit differing potencies on different granulosa cell lineages. FIGURE 8. Pro-cumulin, but not mature cumulin, is a potent stimulator of on subsequent blastocyst quality as assessed by blastocyst cell oocyte developmental competence. A porcine in vitro experimental model numbers (Fig. 8D). of low oocyte developmental competence was used to examine the effect of Molecular Modeling and Analysis of Cumulin Assembly—To the various growth factors on oocyte quality. Porcine cumulus-oocyte com- plexes were treated with vehicle, 20 ng/ml pro-cumulin, 20 ng/ml mature provide molecular insights into cumulin assembly, stability, covalent cumulin, 100 ng/ml pro-GDF9, 100 ng/ml pro-BMP15, or 100 ng/ml and potential receptor-binding properties, we performed ho- of each of pro-GDF9  pro-BMP15 for the first 22 h of oocyte in vitro matura- tion. A, morphological cumulus expansion was assessed and scored at 22 h mology modeling to obtain structural models of homo- and according to the Vanderhyden criteria. Effects of the treatments on oocyte heterodimeric forms of GDF9 and BMP15. Structure templates development competence were examined by assessing embryo develop- were selected on the basis of sequence homology to BMPs and ment on day 7. B and C, blastocyst rate (B) and hatching blastocyst rate (C) [hatching or hatched blastocysts], both expressed as a percentage of the GDFs with known structure in the Protein Data Bank. The number of cleaved embryos. D, average blastocyst cell number. All values are models of mature GDF9 (Fig. 9, A and B) and BMP15 (Fig. 10, A represented as means  S.E. from five replicate experiments. The bars with and B) exhibit the canonical butterfly-shaped architecture of different letters indicate significant differences at p 0.05 within the same graph. TGF- ligands. The structure of the monomeric subunits is SEPTEMBER 25, 2015 • VOLUME 290 • NUMBER 39 JOURNAL OF BIOLOGICAL CHEMISTRY 24015 Cumulin, a Novel Ovarian Growth Factor FIGURE 10. Homology model of mature BMP15. A, ribbon plot of human mature BMP15 showing the canonical dimer architecture. The two mono- FIGURE 9. Homology model of mature GDF9. A, ribbon plot of human mer subunits of the homodimer are colored in light and dark green. The matureGDF9highlightingthecanonicaldimerarchitecture.Thetwomono- potential binding epitopes for the type I (wrist) and type II (knuckle) recep- mer subunits of the homodimer are colored in red and orange. The poten- tors are indicated. The six cysteine residues (colored in yellow) forming the tial binding epitopes for the type I (wrist) and type II (knuckle) receptors characteristic cysteine knot motif, as well as the serine residues replacing are marked. The six cysteine residues forming the characteristic cysteine the cysteine residues, which are involved in the intermolecular disulfide knot motif, as well as the serine residues replacing the cysteine residues, bond found in other TGF- members, are shown as sticks. B,asin A but which are involved in the intermolecular disulfide bond found in other rotated around the x axis by 90°. C, detailed view of the BMP15 dimer TGF- members, are shown as sticks. B,asin A but rotated around the x axis interface showing that interfacial amino acid residues are highly con- by 90°. C, detail of the GDF9 dimer interface showing that interfacial amino served with those found in GDF9 (see also Fig. 9), suggesting that cumulin acid residues are highly conserved between GDF9 and BMP15 (see also formation (see Fig. 11) is very likely not impaired by steric or chemical Fig. 10), suggesting that heterodimer formation (see Fig. 11) is very likely restraints by differences in the dimer interface. not impaired by steric or chemical restraints resulting from differences in the dimer interface. to form cumulin (Fig. 11, A and B). Importantly, the cumulin defined by the cysteine knot, which results in three loops ema- model shows a dimerization interface (Fig. 11C) identical to nating from this central motif. The two loops running in paral- homodimeric GDF9 and BMP15 (Figs. 9C and 10C, respec- lel form -sheets, whereas the third loop, which emanates from tively), indicating that formation of the heterodimer is pos- the cysteine core in the opposite direction, folds into an -helix. sible and that the stability of such a complex is likely indis- The monomer architecture is also often compared with an open tinguishable from the homodimeric forms. Production of hand, with the -sheets forming fingers 1 and 2 and the -helix covalent cumulin also appears likely based upon our model- forming the wrist (Figs. 9, A and B, and 10, A and B). The dimer ing, because in silico exchange of the two serine residues 418 356 assembly can then be envisioned as the inner sides of the two (Ser in GDF9 and Ser in BMP15; Fig. 11B) locates both hands coming together at palm and wrist. sulfur atoms sufficiently close to form a thioether bond with- Unlike most TGF- ligands, GDF9 and BMP15 lack the out requiring conformational or large structural rearrange- cysteine residue that forms an intermolecular disulfide ments of the dimer architecture (Fig. 11D). bond, locking the dimer assembly (this cysteine is replaced Finally, analysis of our homology models indicates that the by an isosteric serine in both GDF9 and BMP15). This binding sites for type I receptors within the “wrist” regions are unique feature, together with the very high sequence conser- distinct in cumulin, relative to its homodimeric ancestors (Figs. vation across the GDF9 and BMP15 dimer interfaces (Figs. 11B,9B, and 10B). This is due to the fact that, in contrast to the 9C and 10C), enables these growth factors to heterodimerize type II receptor epitopes, the wrist or type I receptor-binding 24016 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 39 • SEPTEMBER 25, 2015 Cumulin, a Novel Ovarian Growth Factor FIGURE 11. Molecular modeling of mature cumulin. A, ribbon plot of the homology model of human mature cumulin showing the butterfly-shaped dimer architecture and with the monomer subunits colored in red and green. Structural features and the potential binding epitopes for the type I (wrist) and type II (knuckle) receptors are indicated. The six cysteine residues forming the characteristic cysteine knot motif are shown as sticks. B,asin A but rotated around the x axis by 90°. C, detail of the cumulin dimer interface showing that, despite lacking the intermolecular disulfide bond present in most other TGF- ligands, similar polar and hydrophobic interactions stabilize dimer assembly. D, production of covalent cumulin appears possible, because in silico exchange of the two 418 356 serine residues (Ser in GDF9 and Ser in BMP15) with cysteines locates both sulfur atoms sufficiently close to form a thioether bond without requiring conformational or large structural rearrangements of the dimer architecture. site is built from both monomer subunits. In cumulin, this been described by others as the action of two homodimers epitope therefore presents a chimera with unique amino acid (24). Our results do not support this contention. We deduce composition not found in either homodimeric growth factor this from the observation that the BMP15 covalent S:C ancestor. These differences may alter type I receptor affinity homodimer, which cannot heterodimerize with GDF9, does and/or specificity for cumulin and contribute to its enhanced not synergize with wild type GDF9, in contrast to wild type signaling capacity, particularly in comparison to latent human BMP15. The BMP15 mutant in all other aspects resembles S:C GDF9. wild type BMP15. Furthermore, both pro-cumulin and mature covalent cumulin elicited a more potent response Discussion than the synergistic effects of co-addition of wild type GDF9 In females, GDF9 and BMP15 are essentially only expressed and BMP15 homodimers. Using the novel covalent cumulin in oocytes where they are co-expressed throughout most of alleviates any concerns arising from studying solely a nonco- oogenesis and so do not naturally exist in isolation. Moreover, valent heterodimer, which may exist in an equilibrium given their high sequence homology, these key growth factors between homodimers and heterodimers, as we previously can be expected to interact with each other and hence should discussed (43). This leads us to hypothesize that the process generally be considered as acting together. There is ample evi- of heterodimerization is the mechanism of activation of the dence for interactions between GDF9 and BMP15 at genetic (1, otherwise latent GDF9 in the human. 16, 18), biochemical (19, 20), and functional levels (20–23). A Although inhibin A and B are the other major naturally putative BMP15-GDF9 heterodimer was first modeled by occurring TGF- heterodimers (5), it is possible to co-express McNatty et al. (1) in 2004. Most recently, Peng et al. (24) pro- other TGF- ligands and force their dimerization and in so duced GDF9-BMP15 heterodimers with potent bioactivity. We doing generate more biologically potent proteins. Within the have pointed out a number of potential methodological and BMP subgroup, BMP2/7 (44, 45), BMP2/6 (46), and BMP7/ interpretation deficiencies in that study (43), including that the GDF7 (47) heterodimers are all reported as more potent or as preparation contained other protein forms, and the only evi- exhibiting altered bioactivity, in comparison with a combina- dence presented for formation of a heterodimer was co-immu- tion of homodimers. There are a number of possible mecha- noprecipitation of the two proteins. In the current study, we nisms to account for greater potency of heterodimers. First, a have conducted detailed molecular modeling of heterodimer heterodimer may have a decreased binding affinity for extracel- assembly and succeeded in producing two heterodimeric lular BMP binding proteins, which commonly act as antago- forms: pro-cumulin and mature covalent cumulin. Both forms nists. Many of these antagonists act themselves as obligate or are particularly potent on GCs acting via both the SMAD2/3 nonobligate dimers and thus require a symmetrical homodimer and SMAD1/5/8 pathways and show differential actions on for maximal binding affinity. Another explanation is that mural GC versus cumulus GC. TGF- ligands tend to have a high affinity for either a type I or The co-addition of separately expressed GDF9 and BMP15 for a type II receptor, often leading to a strict sequential binding homodimers elicits a potent synergistic response on GC mechanism. A heterodimer, however, could combine a high (20 –23). The results of the current study provide strong evi- affinity binding site for a type I and a type II receptor within the dence that the basis for such synergism is formation of the cumulin heterodimer. Such GDF9/BMP15 synergism has one dimeric ligand, thereby greatly enhancing the overall affin- SEPTEMBER 25, 2015 • VOLUME 290 • NUMBER 39 JOURNAL OF BIOLOGICAL CHEMISTRY 24017 Cumulin, a Novel Ovarian Growth Factor ity to its cell surface receptors. Finally, because of its inherent erodimer activity are unknown. In contrast, covalent mature asymmetry, a heterodimeric ligand is more prone to recruit two cumulin does not contain any tags, and pro-cumulin incorpo- different type I receptors into the signaling complex. For exam- rates a tag only at the N terminus of the BMP15 prodomain. ple, the cumulin receptor complex (Fig. 7) is predicted to com- Hence, in all forms of GDF9, BMP15, or cumulin used in the prise two BMPRII receptors: one ALK4/5/7 receptor, which is current study, the amino acid sequence of the mature region is necessary for the activation of the SMAD2/3 pathway, and one identical to the wild type sequence (apart from the Ser-Cys ALK6 receptor, which is needed for SMAD1/5/8 signaling, as mutation to form covalent dimers). The lack of any modifica- originally hypothesized by McNatty et al. (1). tions to our GDF9 and BMP15 mature regions may also explain An open question prior to this study was whether cumulin why we are able to detect synergism between separately could only form inside the oocyte or whether it might also spon- expressed and purified pro-GDF9 and pro-BMP15 (current taneously assemble from homodimeric GDF9 and BMP15 pres- study), whereas others do not (24, 53). ent in the extracellular space. The latter seemed feasible An intriguing and important observation from the current because GDF9 and BMP15 naturally form transient dimers, study is that when cumulus-oocyte complexes were treated, whose dimer architecture is not fixed by an intermolecular only the pro-cumulin form improved oocyte quality, as assessed disulfide bond. Indeed, we observed that the two wild type by subsequent embryo yield, whereas mature covalent cumulin homodimeric proteins, separately produced and purified, syn- did not, despite being exceptionally potent on mural GC. ergize via a process of heterodimer formation. High resolution Clearly, pro-cumulin contains the prodomains of GDF9 and structure studies of different BMPs provide a possible explana- BMP15 as part of a complex, whereas mature cumulin is an tion for GDF9 and BMP15 disassembly and subsequent cumu- isolated mature region heterodimer (Fig. 3). These results are lin formation. Specifically, the structures show that the inter- entirely consistent with our recent observation that pro- face between the monomeric halves of TGF- proteins BMP15, but not mature domain BMP15 nor mature domain is considerably hydrated, which is unusual compared with mouse GDF9, enhances oocyte developmental competence the water-free hydrophobic core of soluble proteins (48–51). (54, 55). It is well established that prodomains of TGF- The localization of these conserved water molecules in the superfamily growth factors have important roles in protein dimerization interface around the central cysteine knot folding and regulation of bioactivity (56, 57). In particular, likely limits the stability of the dimer; however, in the case of interactions of the prodomains of cumulin with the special- covalent BMPs, a full disassembly of the dimer is prevented ized extracellular matrix of cumulus GC may facilitate pres- because of the covalent linkage via the intermolecular disul- entation of the heterodimeric mature domain to its recep- fide. However, because GDF9 and BMP15 lack this bond, tors. In support of these concepts, we have shown that both factors potentially undergo full disassembly and reas- pro-GDF9 binds strongly to heparin, suggesting that certain sembly, which, when both factors are simultaneously present heparan sulfate proteoglycans may act as co-receptors for pro- (usual situation in most mammals), would yield an equilib- GDF9, pro-BMP15, and pro-cumulin (58). Hence, the differen- rium-based mixture of homo- and heterodimers unless tial activities of pro-cumulin and mature cumulin on mural GC steric restraints specifically interfere with heterodimer for- versus cumulus GC may be attributed to differential expression mation. The latter seems highly unlikely, however, given the of heparan sulfate proteoglycans and/or type I or II receptors in fact that the interface residues are highly conserved between these GC. GDF9 and BMP15. Together, this evidence suggests that the Finally, the current study also has significant practical impli- formation of cumulin in vivo might, at least in part, occur cations for reproductive medicine and advanced breeding in postsecretion from the oocyte in the extracellular matrix of domestic animals. The development of purified and highly the granulosa and cumulus cells. If so, it seems plausible that potent pro-cumulin is likely to prove an important additive for the nature and composition of the extracellular matrix, oocyte IVM (59). IVM is an assisted reproductive technology which in turn is affected by in vivo and in vitro context, is that generates viable embryos using reduced or no exogenous likely to affect cumulin assembly and bioactivity. ovarian hormone stimulation of the patient. IVM is thus an Two remarkable features of cumulin bioactivity is its capac- important adjunct technology to in vitro fertilization. To make ity to activate both the SMAD2/3 and SMAD1/5/8 signaling IVM more clinically viable, including in fertility preservation in pathways, as well as its enhanced signaling potency, in particu- cancer survivors, an additive such as pro-cumulin is needed to lar when compared with the GDF9 and BMP15 homodimers. improve the efficiency of IVM (60). We and others have shown On isolated mural GCs, mature covalent cumulin was even that addition of “native” oocyte-secreted factors (e.g. pro- more potent than pro-cumulin, particularly in terms of GC pro- BMP15 or pro-GDF9) to IVM enhances oocyte quality, having liferation, which is consistent with its enhanced SMAD2/3 sig- profound effects on embryo development and fetal survival (13, naling (29). Dual SMAD pathway activation by cumulin is in 54, 55, 61–64). This study is the first report on the effects of disagreement with Peng et al. (24), who claimed that the GDF9- cumulin on oocyte quality. We carefully chose to use an estab- BMP15 heterodimer binds to, but does not activate, the ALK6 lished porcine model of low oocyte developmental competence receptor. The form of heterodimer that Peng et al. (24) pro- using oocytes from growing antral follicles to better recapitu- duced has an epitope tag (either FLAG or myc) incorporated late the human scenario. The addition of pro-cumulin led to a into the N terminus of the mature region. Although this major improvement in oocyte quality, more than doubling appears to not impede BMP15 homodimer bioactivity (52), the embryo yield, which, if translatable to human oocytes, would effects of the GDF9 (myc) and BMP15 (FLAG) tags on het- have a large impact on clinical IVM and oncofertility. 24018 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 39 • SEPTEMBER 25, 2015 Cumulin, a Novel Ovarian Growth Factor 13. Li, J. J., Sugimura, S., Mueller, T. D., White, M. A., Martin, G. A., Ritter, Author Contributions—D. G. M. and R. B. G. designed the study. L. J., Liang, X. Y., Gilchrist, R. B., and Mottershead, D. G. (2015) Modifi- D. G. M., C. A. H., and R. B. G. secured funding for the project. cations of human growth differentiation factor 9 to improve the genera- D. G. M. oversaw all aspects of recombinant protein design, produc- tion of embryos from low competence oocytes. Mol. Endocrinol. 29, tion, and purification, which were performed by J.-J. L., M. A. W., 40–52 G. A. M., A. P. T., J. S., and D. G. M. S. S. developed methodology for 14. Crawford, J. L., and McNatty, K. P. (2012) The ratio of growth differenti- and performed the porcine oocyte quality experiments. Additional ation factor 9: bone morphogenetic protein 15 mRNA expression is tightly co-regulated and differs between species over a wide range of ovulation experiments were performed and analyzed by the following authors: rates. Mol. Cell Endocrinol. 348, 339–343 D. R., RT-PCR; S. L. M., COV434 SMAD reporters; L. J. R., mouse 15. 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Journal

Journal of Biological ChemistryAmerican Society for Biochemistry and Molecular Biology

Published: Sep 25, 2015

Keywords: bone morphogenetic protein (BMP); protein assembly; protein secretion; SMAD transcription factor; transforming growth factor beta (TGF-β); IVM; cumulin; cumulus granulosa cell; growth differentiation factor; oocyte quality

References