Bone Morphogenetic Protein System Research Articles

Regulation of ovarian folliculogenesis by IGF and BMP system in domestic animals

Involvement of insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) in ovarian folliculogenesis has been extensively studied during the last decade. In all mammalian species, IGF-I stimulates granulosa cell proliferation and steroidogenesis. The concentrations of IGF-I and -II do not vary during terminal follicular growth and atresia. In contrast, the levels of IGFBP-2 and -4, as well as IGFBP-5 in ruminants, dramatically decrease and increase during terminal follicular growth and atresia, respectively. These changes are responsible for an increase and a decrease in IGF bioavailability during follicular growth and atresia, respectively. They are partly explained by changes in ovarian expression. In particular, expression of IGFBP-2 mRNA decreases during follicular growth in ovine, bovine and porcine ovaries, and expression of IGFBP-5 mRNA dramatically increases in granulosa cells of bovine and ovine atretic follicles. Changes in IGFBP-2 and -4 levels are also due to changes in intrafollicular levels of specific proteases. Recently, we have shown that the pregnancy-associated plasma protein-A (PAPP-A) is responsible for the degradation of IGFBP-4 in preovulatory follicles of domestic animals. Expression of PAPP-A mRNA is restricted to the granulosa cell compartment, and is positively correlated to expression of aromatase and LH receptor. From recent evidence, the bone morphogenetic protein (BMP) family would also play a key role in ovarian physiology of domestic animals. In particular, we and others have recently shown that a non-conservative substitution (Q249R) in the bone morphogenetic protein-receptor type IB (BMPR-IB) coding sequence is fully associated with the hyperprolific phenotype of FecB B / FecB B Booroola ewes. BMP-4 and GDF-5, natural ligands of BMPR-IB, strongly inhibit secretion of progesterone by ovine granulosa cells in vitro, but granulosa cells from FecB B / FecB B ewes are less responsive than those from FecB +/ FecB + to the action of these peptides. It is suggested that in FecB B / FecB B ewes, Q249R substitution would impair the function of BMPR-IB, leading to a precocious differentiation of granulosa cells and of follicular maturation. Interestingly, recent findings have described mutations in BMP-15 gene associated with hyperprolific phenotypes in Inverdale and Hanna ewes, suggesting that the BMP pathway plays a crucial role in the control of ovulation rate.

A novel role for bone morphogenetic proteins in the synthesis of follicle-stimulating hormone.

FSH is produced in pituitary gonadotropes as an alpha/beta heterodimer, and synthesis of the beta-subunit is the rate-limiting step in overall FSH production. Synthesis of FSHbeta can be regulated by activin and inhibin, both of which are members of the transforming growth factor-beta superfamily. Bone morphogenetic proteins (BMPs) also belong to the transforming growth factor-beta family and are multifunctional growth factors involved in many aspects of tissue development and morphogenesis, including regulation of FSH action in the ovary. Here we report a novel function for BMP-7 and BMP-6 in regulating FSH synthesis in the pituitary. Using primary pituitary cell cultures derived from transgenic mice that carry the ovine FSHbeta promoter linked to a luciferase reporter gene (oFSHbetaLuc), BMP-7 or BMP-6 was found to stimulate oFSHbetaLuc expression by 6-fold. Transient expression of the oFSHbetaLuc in a transformed gonadotrope cell line, LbetaT2, was induced 4-fold by BMP-7 or BMP-6 treatment. More importantly, BMP-7 and BMP-6 increased endogenous FSH secretion by 10- and 14-fold, respectively, from LbetaT2 cells, demonstrating for the first time that a functional signaling BMP system is present in gonadotropes. Two bioneutralizing antibodies to BMP-7, which cross-react with BMP-6, but not with activin A, decreased basal oFSHbetaLuc expression and FSH secretion from transgenic mouse pituitary cultures by 83-88% and 47-48%, respectively, suggesting an autocrine or paracrine role for BMP-7 or BMP-6 in FSH synthesis. Neither bioneutralizing antibody to activin A or activin B decreased basal oFSHbetaLuc expression or mouse FSH secretion significantly. Dose-dependent inhibition of FSH synthesis by anti-BMP7 was also observed in rat and sheep pituitary cultures. These results combined with the fact that the messenger RNAs for BMP-7 and BMP-6 were detected in mouse pituitaries and LbetaT2 cells indicate that BMP-7 and/or BMP-6 can function as FSH stimulators and may be significant physiological factors maintaining basal FSH expression in vivo.

A Novel Role for Bone Morphogenetic Proteins in the Synthesis of Follicle-Stimulating Hormone

FSH is produced in pituitary gonadotropes as an α/β heterodimer, and synthesis of the β-subunit is the rate-limiting step in overall FSH production. Synthesis of FSHβ can be regulated by activin and inhibin, both of which are members of the transforming growth factor-β superfamily. Bone morphogenetic proteins (BMPs) also belong to the transforming growth factor-β family and are multifunctional growth factors involved in many aspects of tissue development and morphogenesis, including regulation of FSH action in the ovary. Here we report a novel function for BMP-7 and BMP-6 in regulating FSH synthesis in the pituitary. Using primary pituitary cell cultures derived from transgenic mice that carry the ovine FSHβ promoter linked to a luciferase reporter gene (oFSHβLuc), BMP-7 or BMP-6 was found to stimulate oFSHβLuc expression by 6-fold. Transient expression of the oFSHβLuc in a transformed gonadotrope cell line, LβT2, was induced 4-fold by BMP-7 or BMP-6 treatment. More importantly, BMP-7 and BMP-6 increased endogenous FSH secretion by 10- and 14-fold, respectively, from LβT2 cells, demonstrating for the first time that a functional signaling BMP system is present in gonadotropes. Two bioneutralizing antibodies to BMP-7, which cross-react with BMP-6, but not with activin A, decreased basal oFSHβLuc expression and FSH secretion from transgenic mouse pituitary cultures by 83–88% and 47–48%, respectively, suggesting an autocrine or paracrine role for BMP-7 or BMP-6 in FSH synthesis. Neither bioneutralizing antibody to activin A or activin B decreased basal oFSHβLuc expression or mouse FSH secretion significantly. Dose-dependent inhibition of FSH synthesis by anti-BMP7 was also observed in rat and sheep pituitary cultures. These results combined with the fact that the messenger RNAs for BMP-7 and BMP-6 were detected in mouse pituitaries and LβT2 cells indicate that BMP-7 and/or BMP-6 can function as FSH stimulators and may be significant physiological factors maintaining basal FSH expression in vivo.

Geometry of Carriers Controlling Phenotypic Expression in BMP-Induced Osteogenesis and Chondrogenesis

The effect of the geometry of extracellular matrices on bone morphogenetic protein (BMP)-induced osteogenesis has not been systematically studied. Geometry is crucially important for the scaffold in bone and joint tissue engineering. The purpose of this study was to elucidate principles of geometry of matrices in designing new scaffolds and matrices for use in reconstruction of bone and joints. More than ten biomaterials with different geometries, including a unique device of honeycomb-shaped hydroxyapatite, were combined with BMPs of recombinant (rhBMP-2) or natural bovine origin (S300 BMP cocktail) and implanted subcutaneously into 4-week-old Wistar-King rats. The implanted pellets were removed at 1-4 weeks and analyzed for bone and cartilage formation by histological and biochemical methods. BMP-induced bone and cartilage induction was highly dependent on the geometric properties of the carrier. Some carriers such as porous particles or blocks of hydroxyapatite induced osteogenesis directly, without detectable chondrogenesis, whereas other carriers such as fibrous glass membrane induced cartilage exclusively. Still other carriers induced mostly cartilage followed by bone formation. Solid particles of hydroxyapatite and fibrous glass membrane with too tight a meshwork did not induce bone or cartilage. The optimal pore size for bone-forming efficacy in porous blocks of hydroxyapatite was a diameter of 300-400 microm. In straight tunnel structures with various diameters in honeycomb-shaped hydroxyapatite, tunnels with smaller diameters (90-120 microm) induced cartilage followed by bone formation, whereas those with larger diameters (350 microm) induced bone formation directly within the tunnels. BMP carriers were classified into three types: bone-inducing, cartilage-inducing, and cartilage-bone-inducing. From the analysis of causative factors inducing osteogenesis and chondrogenesis in the BMP system, we concluded that the geometry of the carrier is crucially important and vasculature-inducing geometry should be considered in designing effective scaffolds for bone formation. We propose a classification of geometry of the artificial extracellular matrices that is useful for designing a scaffold for tissue engineering of bone and related tissues. Conventional requisites of the BMP carriers for clinical use have mainly concerned the affinities of carriers with cells and biomolecules and their mechanical strength. The vasculature-inducing geometry of carriers adds a new criterion in designing systems for effective bone and joint reconstruction. The geometries of porous structures-their sizes, continuity, and straightness as verified by hydroxyapatite in this study-will be applicable for other biomaterials for clinical reconstruction therapy.

A functional bone morphogenetic protein system in the ovary.

Bone morphogenetic proteins (BMPs) comprise a large group of polypeptides in the transforming growth factor beta superfamily with essential physiological functions in morphogenesis and organogenesis in both vertebrates and invertebrates. At present, the role of BMPs in the reproductive system of any species is poorly understood. Here, we have established the existence of a functional BMP system in the ovary, replete with ligand, receptor, and novel cellular functions. In situ hybridization histochemistry identified strong mRNA labeling for BMP-4 and -7 in the theca cells and BMP receptor types IA, IB, and II in the granulosa cells and oocytes of most follicles in ovaries of normal cycling rats. To explore the paracrine function of this BMP system, we examined the effects of recombinant BMP-4 and -7 on FSH (follicle-stimulating hormone)-induced rat granulosa cytodifferentiation in serum-free medium. Both BMP-4 and -7 regulated FSH action in positive and negative ways. Specifically, physiological concentrations of the BMPs enhanced and attenuated the stimulatory action of FSH on estradiol and progesterone production, respectively. These effects were dose- and time-dependent. Furthermore, the BMPs increased granulosa cell sensitivity to FSH. Thus, BMPs have now been identified as molecules that differentially regulate FSH-dependent estradiol and progesterone production in a way that reflects steroidogenesis during the normal estrous cycle. As such, it can be hypothesized that BMPs might be the long-sought "luteinization inhibitor" in Graafian follicles during their growth and development.