Bone Morphogenetic Protein 15 in the Pro-Mature Complex Form Enhances Bovine Oocyte Developmental Competence

Abstract

Developmental competence of in vitro matured (IVM) oocytes needs to be improved and this can potentially be achieved by adding recombinant bone morphogenetic protein 15 (BMP15) or growth differentiation factor (GDF9) to IVM. The aim of this study was to determine the effect of a purified pro-mature complex form of recombinant human BMP15 versus the commercially available bioactive forms of BMP15 and GDF9 (both isolated mature regions) during IVM on bovine embryo development and metabolic activity. Bovine cumulus oocyte complexes (COCs) were matured in vitro in control medium or treated with 100 ng/ml pro-mature BMP15, mature BMP15 or mature GDF9 +/− FSH. Metabolic measures of glucose uptake and lactate production from COCs and autofluorescence of NAD(P)H, FAD and GSH were measured in oocytes after IVM. Following in vitro fertilisation and embryo culture, day 8 blastocysts were stained for cell numbers. COCs matured in medium +/− FSH containing pro-mature BMP15 displayed significantly improved blastocyst development (57.7±3.9%, 43.5±4.2%) compared to controls (43.3±2.4%, 28.9±3.7%) and to mature GDF9+FSH (36.1±3.0%). The mature form of BMP15 produced intermediate levels of blastocyst development; not significantly different to control or pro-mature BMP15 levels. Pro-mature BMP15 increased intra-oocyte NAD(P)H, and reduced glutathione (GSH) levels were increased by both forms of BMP15 in the absence of FSH. Exogenous BMP15 in its pro-mature form during IVM provides a functional source of oocyte-secreted factors to improve bovine blastocyst development. This form of BMP15 may prove useful for improving cattle and human artificial reproductive technologies.

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

“…3). These results are entirely consistent with our recent observation that pro-BMP15, but not mature domain BMP15 nor mature domain mouse GDF9, enhances oocyte developmental competence (54,55). It is well established that prodomains of TGF-␤ superfamily growth factors have important roles in protein folding and regulation of bioactivity (56,57).…”

“…We and others have shown that addition of "native" oocyte-secreted factors (e.g. pro-BMP15 or pro-GDF9) to IVM enhances oocyte quality, having profound effects on embryo development and fetal survival (13,54,55,(61)(62)(63)(64). This study is the first report on the effects of cumulin on oocyte quality.…”

Promotion of EGF receptor signaling improves the quality of low developmental competence oocytes

“…In the present study we used human B15 and human G9 proteins in their full-length pro-mature forms, which importantly are the forms needed to enhance oocyte developmental competence, including in AREG-induced IVM (Hussein et al, 2006;Sudiman et al, 2014b;Sugimura et al, 2014). By contrast, the truncated mature domains of B15 and G9 do not enable EGF-stimulated expansion of COCs from small antral follicles and also do not appear to contribute to oocyte developmental competence (Sudiman et al, 2014a), the possible mechanisms of which are discussed elsewhere (Sudiman et al, 2014b). In addition, human B15 þG9 can activate not only SMAD1/5/8 and SMAD2/3, but also non-SMAD signaling pathways (Reader et al, 2014).…”

“…Compact COCs with greater than 3 layers of cumulus cells and a homogeneous ooplasm were cultured in porcine oocyte medium containing 3 mg/ml polyvinyl alcohol (POMþ PVA) (Yoshioka et al, 2008), and depending on the treatment: supplemented with or without standard porcine oocyte in vitro maturation (IVM) doses of gonadotropins (Gns) consisting of 10 IU/ml equine chorionic gonadotropin (Folligon, Intervet, Bendigo, Australia) and 10 IU/ml human chorionic gonadotropin (Organon, Sydney, Australia) or; 0-500 ng/ml recombinant human amphiregulin (AREG; R&D Systems, Minneapolis, MN, USA), in the presence or absence of 1 mmol/L dibutyryl cAMP (cAMP) (Funahashi et al, 1997); 100 ng/ ml recombinant human GDF9 (G9); 100 ng/ml recombinant human BMP15 (B15); or B15 þG9, for 22 h at 38.5°C in a highly humidified atmosphere of 5% CO 2 in air. After 22 h COCs were washed and then transferred into the same medium but without gonadotropins, AREG, B15, G9 or cAMP for an additional 22 h. G9 and B15 were produced in our laboratory as concentrated preparations of pro/mature-region complexes, produced in 293 T cells, as previously described (Pulkki et al, 2011(Pulkki et al, , 2012, and as described as supplements to oocyte IVM medium at the same doses as used here (Li et al, 2015;Sudiman et al, 2014b;Sugimura et al, 2014). Ten COCs were incubated in 100 ml of IVM medium, covered with paraffin oil in 35-mm Petri dishes.…”

Modifications of Human Growth Differentiation Factor 9 to Improve the Generation of Embryos From Low Competence Oocytes

“…Finally, the protein used for the above-mentioned granulosa cell studies consists of only the mature region of the protein, and we have recently shown that the purified mGDF9 mature region is not active in stimulating oocyte developmental competence (40). We have also shown for human BMP15 that the protein needs to be present as the purified pro-mature complex form to stimulate oocyte developmental competence (73). Hence, in our experiments, we have used the purified pro-mature complexes of hGDF9 and the related mutants.…”