Functional and molecular characterization of naturally occurring mutations in the oocyte-secreted factors bone morphogenetic protein-15 and growth and differentiation factor-9

Bone morphogenetic protein-15 (BMP-15) and growth and differentiation factor-9 (GDF-9) are oocyte-secreted factors that are critical local regulators of ovarian physiology. Recent studies have identified a number of mutations in these genes that cause increased fertility and infertility in heterozygous or homozygous ewes carrying the mutations, respectively. Interestingly, heterozygous ewes with a mutation in both BMP-15 and GDF-9 exhibit higher fertility than those having mutation in only one of the genes. Here, we have produced recombinant human BMP-15 and GDF-9 that carry the mutations identified in those sheep, i.e. I31D and S99I in BMP-15 and S77F in GDF-9. We found that when individually expressed, both BMP-15 mutations had no effect on the processing, secretion, and dimerization of the mature proteins or on the biological activity of the molecules. However, when mutant BMP-15 was co-expressed with wild-type GDF-9, the secretion of BMP-15 and GDF-9 was significantly reduced, suggesting that the mechanisms by which the BMP-15 mutations affect sheep fertility occurs at the level of protein secretion rather than dimerization and biological activity. Moreover, when mutant GDF-9 was co-expressed with mutant BMP-15, the secretion levels of both proteins were significantly lower than those of cells co-expressing wildtype GDF-9 and mutant BMP-15, suggesting a possible mechanism for the extreme fertility observed in the compound heterozygous mutant sheep.