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 transforming growth factor-β (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 pro-region non-covalently associated with the mature region. In this study we investigated a number of amino acid residues in the mature region of hGDF9 which are different from the corresponding residues in the mouse protein, which is not latent. We designed, expressed and purified four forms of chimeric hGDF9 (M1-M4) which we found to be active in a granulosa cell bioassay. Utilizing a porcine in vitro maturation (IVM) 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 utilizing such low competence oocytes. Our molecular modelling studies suggest that in the case of hGDF9 the Gly(391)Arg mutation likely 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 affinity of the mature region for the pro-region, is also required.