Modifications of human growth differentiation factor 9 to improve the generation of embryos from low competence oocytes

Abstract

Growth differentiation factor 9 (GDF9) is an oocyte-derived growth factor that plays a critical role in ovarian folliculogenesis and oocyte developmental competence and belongs to the TGF-β family of proteins. Recombinant human GDF9 (hGDF9) is secreted in a latent form, which in the case of the fully processed protein, has the proregion noncovalently associated with the mature region. In this study, we investigated a number of amino acid residues in the mature region of hGDF9 that are different from the corresponding residues in the mouse protein, which is not latent. We designed, expressed, and purified 4 forms of chimeric hGDF9 (M1-M4) that we found to be active in a granulosa cell bioassay. Using a porcine in vitro maturation model with inherent low developmental competence (yielding 10%-20% blastocysts), we tested the ability of the chimeric hGDF9 proteins to improve oocyte maturation and developmental competence. Interestingly, one of the chimeric proteins, M3, was able to significantly increase the level of embryo production using such low competence oocytes. Our molecular modeling studies suggest that in the case of hGDF9 the Gly(391)Arg mutation probably increases receptor binding affinity, thereby creating an active protein for granulosa cells in vitro. However, for an improvement in oocyte developmental competence, a second mutation (Ser(412)Pro), which potentially decreases the affinity of the mature region for the proregion, is also required.

Figure 1.. The mature region sequence alignment of wild-type mouse/human GDF9 and the chimeric GDF9 forms M1 to M4.

The 6 conserved cysteine residues forming the cystine knot are shown in yellow, and the amino acid residues different from the wild-type human GDF9 sequence are shown in blue. Numbering is based on the human GDF9 amino acid sequence.

Figure 2.. Silver-stained SDS-PAGE gels of mutant GDF9 (M1–M4) IMAC purification products: 1, molecular weight markers; 2, conditioned medium; 3, unbound material; 4, wash off no.

2; 5, elution no. 1; 6, elution no. 2; 7, elution no. 3; 8, elution no. 4. A, Proprotein form of GDF9. B, Processed proregion. C, Mature region.

Figure 3.. Bioactivity of the various human GDF9 forms on mouse mural granulosa cells.

Granulosa cells were cultured with increasing concentrations (12.5–200 ng/mL) of recombinant mGDF9, IMAC-purified wild-type human GDF9, or one of the human mutant GDF9 forms, M1 to M4. The incorporated [³H]thymidine was detected after a 24-hour incubation. Data points are the means ± SEM from 3 replicate experiments.

Figure 4.. Porcine cumulus cell expansion and competency related gene expression.

A, The CEI was evaluated after 22 hours of in vitro maturation with 200 ng/mL IMAC purified wild-type human GDF9 or one of the mutant human GDF9 forms, M1 to M4. Values represent the mean ± SEM of 5 replicates. After the CEI assessment, the expression levels of TNFαIP6 (B), HAS2 (C), and PTGS2 (D) were examined by real-time RT-PCR analysis. Values represent means ± SEM of 3 replicates. Different letters indicate significant differences at P < .05. Cont, control.

Figure 5.. Effect of human GDF9 forms (wild-type and M1–M4) during porcine oocyte maturation on subsequent oocyte developmental competence.

Porcine COCs with inherently low developmental competence were treated with vehicle, 200 ng/mL human wild-type GDF9 (WT), or one of the human mutant GDF9 forms (M1–M4) for the first 22 hours of IVM. The effects of treatments on oocyte development competence were examined by measuring the following points of embryo development: cleavage rate at day 2 (A), blastocyst rate expressed as a percentage of the number of cleaved embryos at day 7 (B), total blastocyst cell number on day 7 (C), and hatching blastocyst rate (number of hatching or hatched blastocysts/cleaved embryos) (D). All values are presented as means ± SEM from 5 replicate experiments. Different letters indicate significant differences at P < .05 within the same graph. Cont, control.

Figure 6.. 3D homology model of the proprotein complex of human GDF9.

The model was obtained using the crystal structure of pro-TGFβ1 as template. A, Ribbon plot of the homodimeric GDF9 colored in light/dark green (chain A) and blue/cyan (chain B). The mature region, which is released upon proteolytic processing of the proprotein at a conserved R-X-K/R-R site, is colored in light green and cyan. The prodomain engaging in a strait jacket–like interaction with the mature region, is marked in dark green and blue. The disulfide bonds of the characteristic cystine knot are shown as yellow sticks. Red spheres mark the Cα-carbon position of the mutations introduced to form the human-mouse GDF9 chimeras M1 to M4. B, Magnification around amino acid Ser⁴¹² located in the mature region of hGDF9 showing its close proximity to helix α1 of the prodomain. C, Exchange of Ser⁴¹² by proline, the equivalent residue in murine GDF9, possibly weakens this interaction and hence the pro/mature complex leading to a decreased latency for GDF9 chimeras M2 to M4.

Figure 7.. Homology model of the mature region of hGDF9 in complex with its type I and type II receptors.

A, Overview ribbon plot of the hGDF9 dimer with the monomeric subunits colored in green and cyan. The potential receptor binding sites are indicated by gray ribbon structures for the type I and type II receptor ectodomains; for better visibility, the type I and the type II receptors facing the front are shown as transparent structures. Disulfide bonds of the cystine knot are shown as yellow sticks; the position of the residues exchanged to form the human-mouse chimera are indicated by red spheres. B and C, The residue at position 391 is located within the potential type I receptor binding site. Docking of a type I receptor to hGDF9 suggests that the residue at 391 is near the receptor's β1β2-loop, which was shown to be important in ligand-type I receptor interactions. In human GDF9, a small glycine residue occupies position 391 (B), whereas in mouse GDF9 a large positively charged arginine requires more space (C) but is capable of forming additional hydrogen bonds with the type I receptor. D and E, A second mutation in the human-mouse GDF9 chimera M2 and M4 is the exchange of Lys⁴⁵⁰ to arginine. As for the former substitution, this exchange could also enable additional interactions due to the longer side chain and the bidentate polar head group.

Figure 8.. Alignment of 9 mammalian GDF9 mature region amino acid sequences.

The residues targeted in this study are highlighted in blue within the mouse sequence, and a cat-specific change in one of these residues is highlighted in green. The Cys residues involved in formation of the conserved “cystine knot” structural element are highlighted in yellow.