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.

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

“…Notably, human GDF9 is latent, because it remains associated with its prodomain, whereas mouse GDF9 is particularly potent, because of its prodomain being readily displaced (3,12,13). By contrast, human BMP15 is recombinantly produced in an active form in mammalian cell culture, whereas mouse BMP15 is not expressed under the same conditions (4).…”

“…By contrast, human BMP15 is recombinantly produced in an active form in mammalian cell culture, whereas mouse BMP15 is not expressed under the same conditions (4). The species-specific differences in expression and activity of these oocyte-secreted growth factors is principally due to alterations in a small number of amino acids in the prodomains (BMP15) or mature domains (GDF9), which affect complex formation or stability, respectively (3,4,13). Hence, the relative levels of oocyte expression of BMP15 and GDF9 vary markedly across species, whereby the mono-ovular phenotype (i.e.…”

“…We chose to use a well established porcine model of low oocyte developmental competence, because this experimental model better approximates the complexities of human oocyte developmental competence than that of murine oocytes. This model uses oocytes from small antral follicles collected from abattoir-derived ovaries from gilts (peripubertal female pig) (13,(32)(33)(34). The methods used were as per recently described in detail (13,34).…”

“…This model uses oocytes from small antral follicles collected from abattoir-derived ovaries from gilts (peripubertal female pig) (13,(32)(33)(34). The methods used were as per recently described in detail (13,34). In brief, cumulus-oocyte complexes were aspirated from small antral follicles (2-4 mm) and matured in protein-free porcine oocyte maturation basic IVM medium (35), supplemented for the first 22 h of IVM with 1 mM dibutyryl-cAMP and 100 ng/ml amphiregulin (R&D Systems) and treated with either vehicle (control), 20 or 100 ng/ml pro-cumulin, 20 or 100 ng/ml mature cumulin, 100 ng/ml pro-GDF9, 100 ng/ml pro-BMP15, or 100 ng/ml of each of pro-GDF9 ϩ pro-BMP15.…”

“…Cumulus expansion was assessed at 22 h of IVM and scored as per standard criteria (36,37). At 22 h, cumulus-oocyte complexes were washed and matured for a further 22 h in porcine oocyte maturation without supplements or treatments (13,33,34). Thereafter, in vitro fertilization, in vitro embryo production, and assessment of blastocysts was performed as previously described (13).…”