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

Abstract

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are oocyte-specific growth factors with central roles in mammalian reproduction, regulating species-specific fecundity, ovarian follicular somatic cell differentiation, and oocyte quality. In the human, GDF9 is produced in a latent form, the mechanism of activation being an open question. Here, we produced a range of recombinant GDF9 and BMP15 variants, examined their in silico and physical interactions and their effects on ovarian granulosa cells (GC) and oocytes. We found that the potent synergistic actions of GDF9 and BMP15 on GC can be attributed to the formation of a heterodimer, which we have termed cumulin. Structural modeling of cumulin revealed a dimerization interface identical to homodimeric GDF9 and BMP15, indicating likely formation of a stable complex. This was confirmed by generation of recombinant heterodimeric complexes of pro/mature domains (pro-cumulin) and covalent mature domains (cumulin). Both pro-cumulin and cumulin exhibited highly potent bioactivity on GC, activating both SMAD2/3 and SMAD1/5/8 signaling pathways and promoting proliferation and expression of a set of genes associated with oocyte-regulated GC differentiation. Cumulin was more potent than pro-cumulin, pro-GDF9, pro-BMP15, or the two combined on GC. However, on cumulus-oocyte complexes, pro-cumulin was more effective than all other growth factors at notably improving oocyte quality as assessed by subsequent day 7 embryo development. Our results support a model of activation for human GDF9 dependent on cumulin formation through heterodimerization with BMP15. Oocyte-secreted cumulin is likely to be a central regulator of fertility in mono-ovular mammals.

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

FIGURE 1.

Pro-forms of BMP15 and GDF9 synergize. Pro-hBMP15 synergizes with pro-mouse GDF9 and also with pro-human GDF9, which is naturally latent. Bioactivity was assessed by [³H]thymidine incorporation in primary mouse mural GC. The various pro-proteins were produced in HEK-293T cells with N-terminal His tags attached to their prodomains allowing purification by IMAC.

FIGURE 2.

GDF9/BMP15 synergism is mediated by heterodimer formation. The BMP15 (S356C) covalent homodimer (BMP15_S:C) does not synergize with GDF9, in contrast to wild type BMP15, where clear synergism is observed. Bioactivity was assessed by [³H]thymidine incorporation in primary mouse mural GC (A), Smad2/3-responsive luciferase reporter activity (B), or Smad1/5/8 luciferase reporter activity, in COV434 human GC (C). The various proteins were produced in HEK-293T cells with N-terminal His tags attached to their prodomains allowing purification by IMAC, followed by isolation of mature domains by reverse-phase HPLC. BMP15, HPLC-purified human BMP15 mature region homodimer; GDF9, HPLC purified human GDF9 mature region homodimer; BMP15_S:C, HPLC-purified human BMP15 (Ser-Cys) mature region covalent homodimer.

FIGURE 3.

GDF9 associates and co-purifies with BMP15 to form pro-cumulin. Conditioned media containing untagged pro-GDF9 and N-terminally His₈-tagged pro-BMP15 were combined and subjected to IMAC chromatography. 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.

FIGURE 4.

Bioactivity and SMAD signaling of pro-cumulin. A–C, dose-response curves of the different pro-protein forms of GDF9, BMP15, and cumulin. Bioactivity was assessed by [³H]thymidine incorporation in primary mouse mural GC (A), Smad2/3-responsive luciferase reporter activity (B), or 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 attached to their prodomains allowing purification by IMAC.

FIGURE 5.

Engineering and bioactivity of covalent cumulin. Conditioned media from cells co-expressing GDF9 and BMP15, both harboring the appropriate Ser-Cys mutations, were subjected to IMAC chromatography followed by reverse phase HPLC. A, absorbance profile at 280 nm from the HPLC purification. B, Western blot of HPLC fractions probed for GDF9 with Mab53 or for BMP15 with Mab28. C and D, silver-stained SDS-PAGE gel of the HPLC fractions, run under reduced (C) or nonreduced (D) conditions. E, bioactivity of the fractions (0.25 μl of each 1-ml fraction) across the HPLC profile, as measured by [³H]thymidine incorporation into primary cultures of murine mural GC.

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 [³H]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.

FIGURE 7.

Hypothesized model of human cumulin formation and signaling. 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 precursors 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 displacement (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 oocyte developmental competence. A porcine in vitro experimental model of low oocyte developmental competence was used to examine the effect of the various growth factors on oocyte quality. Porcine cumulus-oocyte complexes were treated with vehicle, 20 ng/ml pro-cumulin, 20 ng/ml mature covalent cumulin, 100 ng/ml pro-GDF9, 100 ng/ml pro-BMP15, or 100 ng/ml of each of pro-GDF9 + pro-BMP15 for the first 22 h of oocyte in vitro maturation. A, morphological cumulus expansion was assessed and scored at 22 h according to the Vanderhyden criteria. Effects of the treatments on oocyte development competence were examined by assessing embryo development 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 number of cleaved embryos. D, average blastocyst cell number. All values are represented as means ± S.E. from five replicate experiments. The bars with different letters indicate significant differences at p < 0.05 within the same graph.

FIGURE 9.

Homology model of mature GDF9. A, ribbon plot of human mature GDF9 highlighting the canonical dimer architecture. The two monomer subunits of the homodimer are colored in red and orange. The potential binding epitopes for the type I (wrist) and type II (knuckle) receptors are marked. The six cysteine residues forming the characteristic cysteine knot motif, as well as the serine residues replacing the cysteine residues, which are involved in the intermolecular disulfide bond found in other TGF-β members, are shown as sticks. B, as in A but rotated around the x axis by 90°. C, detail of the GDF9 dimer interface showing that interfacial amino acid residues are highly conserved between GDF9 and BMP15 (see also Fig. 10), suggesting that heterodimer formation (see Fig. 11) is very likely not impaired by steric or chemical restraints resulting from differences in the dimer interface.

FIGURE 10.

Homology model of mature BMP15. A, ribbon plot of human mature BMP15 showing the canonical dimer architecture. The two monomer subunits of the homodimer are colored in light and dark green. The potential binding epitopes for the type I (wrist) and type II (knuckle) receptors are indicated. The six cysteine residues (colored in yellow) forming the characteristic cysteine knot motif, as well as the serine residues replacing the cysteine residues, which are involved in the intermolecular disulfide bond found in other TGF-β members, are shown as sticks. B, as in A but rotated around the x axis by 90°. C, detailed view of the BMP15 dimer interface showing that interfacial amino acid residues are highly conserved with those found in GDF9 (see also Fig. 9), suggesting that cumulin formation (see Fig. 11) is very likely not impaired by steric or chemical restraints by differences in the dimer interface.

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, as in 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-β ligands, similar polar and hydrophobic interactions stabilize dimer assembly. D, production of covalent cumulin appears possible, because in silico exchange of the two serine residues (Ser⁴¹⁸ in GDF9 and Ser³⁵⁶ 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.