Mutations in the bone morphogenetic protein 15 (BMP-15) gene cause female infertility in the monoovulatory human and sheep; however, in the polyovulatory mouse, loss-of-function of BMP-15 results only in reduced ovulation rate. To elucidate the cause of these species-specific differences, we investigated the functional role of BMP-15 in the mouse ovary. Here, we found that the functional mature form of BMP-15 is barely detectable in the mouse oocytes until just before ovulation, when it is markedly increased. Further, we found that BMP-15 induces cumulus expansion in mouse cumulus-oocyte complexes. The oocyte culture medium from immature mice primed with pregnant mare serum gonadotropin followed by human chorionic gonadotropin also stimulated cumulus expansion, and this activity was attenuated by BMP-15 antibody. Interestingly, the oocyte culture medium from mice treated with pregnant mare serum gonadotropin alone had no effect. Moreover, BMP-15 stimulated the expression of EGF-like growth factors in cumulus cells as well as a series of molecules downstream of EGF-like growth factor signaling, including cyclooxygenase 2, hyaluronan synthase 2, tumor necrosis factor-stimulated gene 6, and pentraxin 3, all of which are necessary for normal cumulus expansion. An antagonist of the EGF receptor completely abolished the effect of BMP-15 in inducing cumulus expansion. These results are consistent with the phenotype of BMP-15-null mice, which exhibit normal folliculogenesis but have defects in the ovulation process. The species-specific differences in the phenotypes caused by BMP-15 mutations may thus be attributed to the temporal variations in the production of the mature form of BMP-15.bone morphogenetic protein ͉ cumulus expansion enabling factor ͉ oocyte ͉ ovary ͉ ovulation ͉ folliculogenesis B one morphogenetic protein 15 (BMP-15) is an oocyte-specific growth factor that plays a crucial role in determining ovulation quota in mammals (1). Genetic studies have shown that mutations in the Bmp15 gene in ewes cause increased ovulation rates and fertility in heterozygotes, yet infertility in the homozygous carriers (2, 3). Studies by our laboratory have shown that these mutations are manifested through defects in the processing of the proproteins of the factors (4, 5). A recent study has shown that a mutation in the Bmp15 gene also causes infertility in humans (6), demonstrating a similar critical role of BMP-15 for fertility in women. Importantly, because this mutation occurs in the proregion of the BMP-15 protein, rather than in the functional mature region, the human BMP-15 mutation must be manifested by causing a defect in the posttranslational processing of the human BMP-15 proprotein, similar to the mutations in sheep (4, 5). However, in contrast to monoovulatory ewes and humans, BMP-15 does not seem to be necessary for folliculogenesis in the polyovulatory mouse (7). Specifically, BMP-15-null mice exhibit no obvious defects in folliculogenesis, completing all stages of follicle development and having multiple corp...

Whereas mutations in the bmp15 gene cause infertility in ewes and women due to defects in folliculogenesis, most defects in female mice lacking bone morphogenetic protein (BMP)-15 are confined to the ovulation process, supportive of the observation that functional mouse BMP-15 is barely detected in oocytes in vivo until after the LH surge. In addition, the mouse BMP-15 proprotein is not processed into the functional mature protein in transfected cells. However, a chimeric protein consisting of the human proregion, human cleavage site, and mouse mature region (termed hhmBMP-15) is processed and the mature protein secreted. To study the role of BMP-15 in folliculogenesis, we generated transgenic mice overexpressing hhmBMP-15, exclusively in oocytes during folliculogenesis and confirmed the overexpression of mouse BMP-15 mature protein. Immature transgenic mice exhibited accelerated follicle growth with decreased primary follicles and an increase in secondary follicles. Granulosa cells of immature mice displayed an increased mitotic index and decreased FSH receptor mRNA expression. Adult mice had normal litter sizes but an increased number of atretic antral follicles. Interestingly, aging mice exhibited an early onset of acyclicity marked by increased diestrus length and early occurrence of constant diestrus. These findings indicate the role of BMP-15 in vivo in promoting follicle growth and preventing follicle maturation, resulting in an early decline in the ovarian reserve of transgenic mice. Therefore, the lack of mouse BMP-15 during early folliculogenesis in the wild-type mice may be relevant to their polyovulatory nature as well as the preservation of ovarian function as the mice age.

Bone morphogenetic protein-15 (BMP-15) is an oocyte-secreted factor critical for the regulation of ovarian physiology. When recombinant human BMP-15 (rhBMP-15) produced in human embryonic kidney 293 cells was subjected to SDS-PAGE analysis, two mature protein forms corresponding to 16 kDa (P16) and 17 kDa (P17) were observed. Despite the physiological relevance and critical function of BMP-15 in female reproduction, little is known about the structure of rhBMP-15. Here, we have analyzed the structure of the rhBMP-15 mature proteins (P16 and P17) using state-of-the-art proteomics technology. Our findings are as follows: (1) the N-terminal amino acid of P16 and P17 is pyroglutamic acid; (2) the Ser residue at the sixth position of P16 is phosphorylated; (3) P17 is O-glycosylated at Thr 10 ; and (4) the C-terminal amino acid of P16 and P17 is truncated. These findings are the first knowledge of the structure of rhBMP-15 mature protein toward understanding the molecular basis of BMP-15 function and could provide an important contribution to the rapidly progressing research area involving oocyte-specific growth factors in modulation of female fertility.Keywords: bone morphogenetic protein; post-translational modification; mass spectrometry; phosphorylation; neutral loss scan; O-glycosylation; proteomics analysis; pyroglutamic acid Supplemental material: see www.proteinscience.org Much of the recent research in the field of ovarian physiology has been focused on characterizing the cellto-cell communication, especially between oocytes and follicular somatic cells (Eppig 2001). The current theory is that some oocyte factors act as morphogens to control follicle growth and differentiation (Erickson and Shimasaki 2000). Compelling evidence that this concept operates in vertebrates comes from the important work of Matzuk and co-workers (Dong et al. 1996;Carabatsos et al. 1998;Elvin et al. 2000), demonstrating that oocytederived growth and differentiation factor-9 (GDF-9), a member of the transforming growth factor-b (TGF-b) superfamily, is critical for normal folliculogenesis and female fertility.In 1998, another oocyte-specific growth factor, bone morphogenetic protein-15 (BMP-15) (Dube et al. 1998) or GDF-9B (Laitinen et al. 1998), was discovered as a new member of the TGF-b superfamily. The primary structure of mouse, human, and rat BMP-15 has high sequence homology with the corresponding sequences of Reprint requests to: Shunichi Shimasaki, Department of Reproductive Medicine, UCSD, 9500 Gilman Drive, La Jolla, CA 92093-0633, USA; e-mail: sshimasaki@ucsd.edu; fax: (858) 822-1482.Abbreviations: BMP-15, bone morphogenetic protein-15; ESI, electrospray ionization; GC, granulosa cell; G-CK, Golgi apparatus casein kinase; Hex, Hexose; LC, liquid chromatography; mAb, monoclonal antibody; MALDI-TOF, matrix-assisted laser-desorption/ionization time-of-flight; MS, mass spectrometry; MS/MS, tandem mass spectrometry; m/z, mass-to-charge ratio; NAc, N-acetyl group; NeuAc, N-acetylneuraminic acid; pyro-Glu, pyroglutamic aci...

Two highly homologous oocyte-secreted growth factors, bone morphogenetic protein (BMP)-15 and growth and differentiation factor (GDF)-9, are known to control folliculogenesis and ovulation through direct effects on granulosa cells in the developing follicles. Although much is known about the expression and biology of these proteins, the impact of posttranslational modifications of BMP-15 and GDF-9 is unknown. Here, we report that: 1) recombinant human (rh) BMP-15 and rhGDF-9 are phosphorylated; 2) the phosphorylation is essential for bioactivity; and 3) the dephosphorylated forms of rh-BMP-15 and rhGDF-9 can abolish the bioactivity of rhBMP-15, rhGDF-9, and rhBMP-7, but not rh activin A. These results indicate that the phosphorylation state of rhBMP-15 and rh-GDF-9 is a determinant of their agonistic and antagonistic activities. (Endocrinology 149: 812-817, 2008)