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Modifications of human growth differentiation factor 9 to improve the generation of embryos from low competence
Jing-Jie Li1, Satoshi Sugimura, Thomas D Mueller
1Center of Reproductive Medicine (J.-J.L., X.-Y.L.), the Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou 520655, China; Institute of Agriculture (S.S.), Department of Biological Production, Tokyo University of Agriculture and Technology, Tokyo 183-0057, Japan; Robinson Research Institute (J.-J.L., S.S., M.A.W., G.A.M., L.J.R., R.B.G., D.G.M.), School of Paediatrics and Reproductive Health, The University of Adelaide, Adelaide 5005, Australia; Discipline of Obstetrics and Gynaecology, School of Women's and Children's Health (R.B.G.), Royal Hospital for Women, University of New South Wales, Sydney, New South Wales 2031 Australia; and Department of Plant Physiology and Biophysics (T.D.M.), Julius-von-Sachs Institute of the University Wuerzburg, 97082 Wuerzburg, Germany.
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.
