Functional and Molecular Characterization of Naturally Occurring Mutations in the Oocyte-secreted Factors Bone Morphogenetic Protein-15 and Growth and Differentiation Factor-9

Abstract

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.Rapidly advancing recent research demonstrating the importance of the oocyte-secreted factors, BMP-15 1 and GDF-9, in the regulation of ovarian function has had an immediate impact on our understanding of female fertility (1, 2). These growth factors exhibit a high degree of structural homology with a notable similarity that they both lack the fourth of seven conserved cystine residues that are characteristic features of the TGF-␤ superfamily (3-5). Our recent studies have shown that, although not covalently bound, both BMP-15 and GDF-9 do indeed exist as homodimers (6). Additionally, when BMP-15 and GDF-9 are co-expressed, BMP-15/GDF-9 heterodimers are formed; however, the physiological role of the heterodimer has yet to be established.The sheep model has proven to be a particularly valuable tool in elucidating the role of these factors in determining the quota of oocytes ovulated in a given ovarian cycle, primarily because of the identification of naturally occurring mutations in the bmp15 and gdf9 genes that have profound effects on sheep fertility (7-9). Direct implication of the role of BMP-15 in sheep reproduction was provided when it was shown that two strains of highly prolific sheep, Inverdale and Hanna, carried causal point mutations in the bmp15 gene (7). Interestingly, female sheep that are heterozygous carriers of the mutations exhibit increased fertility because of an increase in their ovulation quota, whereas female sheep that are homozygous carriers of the mutations are infertile with an ovarian phenotype that resembles that of GDF-9 knock-out mice ...

Growth and differentiation factor 9 promotes oocyte growth at the primary but not the early secondary stage in three-dimensional follicle culture

“…This effect is consistent with multiple previous studies in which GDF9 has been shown to stimulate the proliferation of isolated granulosa cells in vitro [26][27][28]. However, no significant further growth of the granulosa cell compartment is seen after 24 h, whereas the oocyte in the primary follicles continues to grow through day 3.…”

“…3). While follicle growth was observed at all doses tested, subsequent experiments were performed with GDF9 500 ng/ml as this represented the lowest dose with the most sustained response and was within the range of doses reported in the literature [28,[52][53][54]. We then cultured isolated primary follicles (50-89 μm in diameter, median diameter 77.9 μm at the start of culture) and early secondary follicles (90-120 μm in diameter, median diameter 101.4 μm at the start of culture) in the presence or absence of GDF9 at 500 ng/ml in serum-free media (Fig.…”

“…In addition, neither of these studies evaluated the effects of GDF9 on the granulosa cell and oocyte compartments independently. While it has been shown that GDF9 can promote growth of isolated granulosa cells in culture [26][27][28], studies in GDF9 null mice suggest that it acts to inhibit, rather than promote, growth of the oocyte [29]. The effect of GDF9 alone on the oocyte and granulosa cells during the earliest stages of preantral follicle growth, therefore, remains unclear.…”

Application of PCR-RFLP technique to determine Booroola gene polymorphism in the Sangsari sheep breed of Iran

“…The fact is that no association was found between those breeds, suggesting that other major gene(s) effects, other than the FecB gene, may be responsible for the high prolificacy of those groups of sheep breeds (1,3,9,25,28,29).…”

“…It has also been recognized that the Booroola phenotype is correlated to the bone morphogenic receptor-IB (BMPR-IB) (2,3). To identify this point mutation, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), using a short-acting enzyme of AvaΠ that cuts the 6/6 ACC locus, was utilized and the results indicated that noncarrier animals are free of this locus (22)(23)(24)(25)(26). In some countries, such as New Zealand, India, the Philippines, Indonesia, Poland, Iceland, France, and Ireland, mutation of the FecB gene has been studied in sheep of high prolificacy (4).…”