Biological activity and in vivo half-life of pro-activin A in male rats.

Mature TGF-ß proteins are used in vivo to promote bone growth, combat obesity, reverse fibrosis and pulmonary arterial hypertension, and as potential rejuvenation factors. However, the serum half-life of this family of growth factors is short (∼5 min), limiting their therapeutic potential. Because TGF-ß proteins are normally secreted from cells with their prodomains attached, we considered whether these molecules could extend the in vivo half-life and activity of their respective growth factors. Using activin A as a model ligand, we initially modified the cleavage site between the pro- and mature domains to ensure complete processing of the activin A precursor. Co-immunoprecipitation studies confirmed mature activin A is secreted from cells in a non-covalent complex with its prodomain, however, the affinity of this interaction is not sufficient to suppress activin A in vitro biological activity. The plasma clearance profiles of purified pro- and mature activin A were determined over a 4 h period in adult male rats. Both activin forms demonstrated a two-phase decay, with the half-life of pro-activin A (t1/2 fast = 12.5 min, slow = 31.0 min) being greater than that of mature activin A (t1/2 fast = 5.5 min, slow = 20.3 min). Both pro- and mature activin A induced significant increases in serum follicle stimulating hormone levels after 4 h, but no differences were observed in the relative in vivo bioactivities of the two activin isoforms. Increased serum half-life of activin A in the presence of its prodomain identifies a new means to increase the therapeutic effectiveness of TGF-ß proteins.

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

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are oocyte-specific growth factors with central roles in mammalian reproduction, regulating species-specific fecundity, ovarian follicular somatic cell differentiation, and oocyte quality. In the human, GDF9 is produced in a latent form, the mechanism of activation being an open question. Here, we produced a range of recombinant GDF9 and BMP15 variants, examined their in silico and physical interactions and their effects on ovarian granulosa cells (GC) and oocytes. We found that the potent synergistic actions of GDF9 and BMP15 on GC can be attributed to the formation of a heterodimer, which we have termed cumulin. Structural modeling of cumulin revealed a dimerization interface identical to homodimeric GDF9 and BMP15, indicating likely formation of a stable complex. This was confirmed by generation of recombinant heterodimeric complexes of pro/mature domains (pro-cumulin) and covalent mature domains (cumulin). Both pro-cumulin and cumulin exhibited highly potent bioactivity on GC, activating both SMAD2/3 and SMAD1/5/8 signaling pathways and promoting proliferation and expression of a set of genes associated with oocyte-regulated GC differentiation. Cumulin was more potent than pro-cumulin, pro-GDF9, pro-BMP15, or the two combined on GC. However, on cumulus-oocyte complexes, pro-cumulin was more effective than all other growth factors at notably improving oocyte quality as assessed by subsequent day 7 embryo development. Our results support a model of activation for human GDF9 dependent on cumulin formation through heterodimerization with BMP15. Oocyte-secreted cumulin is likely to be a central regulator of fertility in mono-ovular mammals.

Development of novel activin-targeted therapeutics.

Soluble activin type II receptors (ActRIIA/ActRIIB), via binding to diverse TGF-ß proteins, can increase muscle and bone mass, correct anemia or protect against diet-induced obesity. While exciting, these multiple actions of soluble ActRIIA/IIB limit their therapeutic potential and highlight the need for new reagents that target specific ActRIIA/IIB ligands. Here, we modified the activin A and activin B prodomains, regions required for mature growth factor synthesis, to generate specific activin antagonists. Initially, the prodomains were fused to the Fc region of mouse IgG2A antibody and, subsequently, "fastener" residues (Lys(45), Tyr(96), His(97), and Ala(98); activin A numbering) that confer latency to other TGF-ß proteins were incorporated. For the activin A prodomain, these modifications generated a reagent that potently (IC(50) 5 nmol/l) and specifically inhibited activin A signaling in vitro, and activin A-induced muscle wasting in vivo. Interestingly, the modified activin B prodomain inhibited both activin A and B signaling in vitro (IC(50) ~2 nmol/l) and in vivo, suggesting it could serve as a general activin antagonist. Importantly, unlike soluble ActRIIA/IIB, the modified prodomains did not inhibit myostatin or GDF-11 activity. To underscore the therapeutic utility of specifically antagonising activin signaling, we demonstrate that the modified activin prodomains promote significant increases in muscle mass.

Aberrant GDF9 expression and activation are associated with common human ovarian disorders.

CONTEXT: Growth differentiation factor 9 (GDF9) is a central regulator of folliculogenesis and ovulation rate. Fourteen mutations in human (h) GDF9 have been reported in women with premature ovarian failure or polycystic ovarian syndrome as well as in mothers of dizygotic twins, implicating GDF9 in the etiology of these conditions. We sought to determine how these mutations alter the biological activity of hGDF9. OBJECTIVE: The objective of the study was to determine whether aberrant GDF9 expression or activation is associated with common ovarian disorders. DESIGN: Homology modeling was used to predict the location of individual mutations within structurally important regions of the pro domains and mature domains of hGDF9. Each hGDF9 variant was generated by site-directed mutagenesis, expressed from human embryonic kidney 293T cells and assessed as to whether it resulted in defective production or the enhanced activation of mature hGDF9 in an in vitro granulosa cell proliferation bioassay. RESULTS: Mutations observed in mothers of dizygotic twins (P103S and P374L) completely abrogated GDF9 expression, suggesting that women heterozygous for these mutations would have a 50% reduction in GDF9 levels. Comparable declines in GDF9 in ewes result in a 2-fold increase in ovulation rate and fecundity. Remarkably, three prodomain mutations associated with premature ovarian failure (S186Y, V216M, and T238A) all resulted in the activation of hGDF9. Mechanistically, these mutations reduced the affinity of the prodomain for mature hGDF9, allowing the growth factor to more readily access its signaling receptors. CONCLUSIONS: Together these findings indicate that alterations to hGDF9 synthesis and activity can contribute to the most common ovarian pathologies.

Species differences in the expression and activity of bone morphogenetic protein 15.

Oocyte-derived bone morphogenetic protein 15 (BMP15) regulates ovulation rate and female fertility in a species-specific manner, being important in humans and sheep and largely superfluous in mice. To understand these species differences, we have compared the expression and activity of human, murine, and ovine BMP15. In HEK293F cells, human BMP15 is highly expressed (120 ng/ml), ovine BMP15 is poorly expressed (15 ng/ml), and murine BMP15 is undetectable. Because BMP15 synthesis is dependent upon interactions between the N-terminal prodomain and the C-terminal mature domain, we used site-directed mutagenesis to identify four prodomain residues (Glu(46), Glu(47), Leu(49), and Glu(50)) that mediate the high expression of human BMP15. Substituting these residues into the prodomains of murine and ovine BMP15 led to significant increases in growth factor expression; however, maximal expression was achieved only when the entire human prodomain was linked to the mature domains of the other species. Using these chimeric constructs, we produced and purified murine and ovine BMP15 and showed that in a COV434 granulosa cell bioassay, these molecules displayed little activity relative to human BMP15 (EC(50) 0.2nM). Sequence analysis suggested that the disparity in activity could be due to species differences at the type I receptor binding interface. Indeed, murine BMP15 activity was restored when specific residues through this region (Pro(329)/Tyr(330)) were replaced with the corresponding residues (Arg(329)/Asp(330)) from human BMP15. The identified differences in the expression and activity of BMP15 likely underlie the relative importance of this growth factor between species.

In ovo temperature manipulation differentially influences limb musculoskeletal development in two lines of chick embryos selected for divergent growth rates.

Selective breeding has led to diverging phenotypic evolution in layer and broiler chickens through genomic and epigenetic modifications. Here we show that in ovo environmental manipulation differentially influences embryonic limb muscle phenotype in these two breeds. We demonstrate that raising incubation temperature from 37.5 to 38.5°C between embryonic days (ED) 4 and 7 increased motility and body mass in both layer and broiler embryos. In layers, this was accompanied by gastrocnemius muscle hypertrophy, increased fibre and nuclei numbers and a higher nuclei to fibre ratio (ED18), preceded by increased hindlimb Myf5 (ED5-8), Pax7 (ED5-10), BMP4 (ED6-9) and IGF-I (ED9-10, ED18) mRNAs. In broilers, the same temperature treatment led to reduced gastrocnemius cross-sectional area with fewer fibres and nuclei and an unchanged fibre to nuclei ratio (ED18). This was preceded by a delay in the peak of hindlimb Myf5 expression, increased Pax7 (ED5, ED7-10) and BMP4 (ED6-8) but reduced IGF-I (ED8-10) mRNAs. Rather than promoting myogenesis as in layer embryos, the temperature treatment promoted gastrocnemius intramuscular fat deposition in broilers (ED18) preceded by increased hindlimb PPARγ mRNA (ED7-10). The treatment increased tibia/tarsus bone length as well as femur cross-sectional area in both breeds, but femur length and bone to cartilage ratio in the femur and tibia/tarsus were only increased in treated layers (ED18). We conclude that in ovo temperature manipulation differentially affected the molecular regulation of hindlimb myogenic, adipogenic and growth factor expression in broiler and layer embryos, leading to differential changes in muscle phenotype. The underlying interactive mechanisms between genes and the environment need further investigation.

Prodomains regulate the synthesis, extracellular localisation and activity of TGF-ß superfamily ligands.

All transforming growth factor-ß (TGF-ß) ligands are synthesised as precursor molecules consisting of a signal peptide, an N-terminal prodomain and a C-terminal mature domain. During synthesis, prodomains interact non-covalently with mature domains, maintaining the molecules in a conformation competent for dimerisation. Dimeric precursors are cleaved by proprotein convertases, and TGF-ß ligands are secreted from the cell non-covalently associated with their prodomains. Extracellularly, prodomains localise TGF-ß ligands within the vicinity of their target cells via interactions with extracellular matrix proteins, including fibrillin and perlecan. For some family members (TGF-ß1, TGF-ß2, TGF-ß3, myostatin, GDF-11 and BMP-10), prodomains bind with high enough affinity to suppress biological activity. The subsequent mechanism of activation of these latent TGF-ß ligands varies according to cell type and context, but all activating mechanisms directly target prodomains. Thus, prodomains control many aspects of TGF-ß superfamily biology, and alterations in prodomain function are often associated with disease.

Muscle specific differences in the regulation of myogenic differentiation in chickens genetically selected for divergent growth rates.

With the human population predicted to reach 9 billion by 2050, increasing food supplies while maintaining adequate standards of animal welfare has become a global priority. In the poultry industry, broilers are genetically selected for greater pectoral but not leg muscularity yield leading to leg disorders and thereby welfare issues. It is known that the pectoralis major of broilers contains more muscle fibres of larger diameters than egg-layers but little is known about the leg gastrocnemius muscle cellular characteristics. As muscle fibre numbers are set by hatch, the molecular regulation of myogenesis was investigated in pectoral (selected) and gastrocnemius (unselected) muscles of chick embryos to help explain diverging post-hatch phenotypes. Results showed that broilers were more active from embryonic day (ED) 8 and heavier from ED12 to 18 than layers. The pectoral muscle of broilers exhibited increased myoblast proliferation on ED15 (raised myonuclei, MyoD and PCNA) followed by increased differentiation from ED16 (raised myogenin, IGF-I) leading to increased muscle fibre hyperplasia and mass by ED18 compared to layers. In the gastrocnemius muscle of broilers, cell proliferation was also raised up to ED15 accompanied by increased PCNA, MyoD and IGF-I mRNAs. However, from ED16, myogenin and IGF-I mRNAs were similar to that of layers and PCNA was reduced leading to similar fibre area, nuclei numbers and muscle mass at ED18. We conclude that genetic selection for enhanced post-hatch pectoral muscle growth has altered the temporal expression of IGF-I and thereby myogenin transcription affecting cellular characteristics and mass by hatch in a muscle specific manner. These observations should help develop intervention strategies aimed at improving leg muscle strength and thereby animal welfare to meet growing consumer demand.

Bone morphogenetic protein-6 enhances gonadotrophin-dependent progesterone and inhibin secretion and expression of mRNA transcripts encoding gonadotrophin receptors and inhibin/activin subunits in chicken granulosa cells.

The aims were to examine ovarian expression of bone morphogenetic protein (BMP) ligands/receptor mRNAs in the chicken and to test the hypothesis that theca-derived BMP(s) modulates granulosa cell function in a paracrine manner. RT-PCR revealed expression of multiple BMPs in granulosa and theca cells from pre hierarchical and preovulatory follicles with greater expression in theca cells; both cell types expressed BMP receptors-IA, -IB and -II consistent with tissue responsiveness. Preovulatory granulosa cells (F1, F2 and F3/4) were cultured with BMP-6 (expressed by theca but not granulosa) in the presence/absence of LH, FSH or 8-Br-cAMP. BMP-6 increased 'basal' and gonadotrophin-induced inhibin-A and progesterone secretion by each cell type but did not enhance the effect of 8-Br-cAMP. This indicates that the observed synergism between BMP-6 and gonadotrophin might involve BMP-induced up-regulation of gonadotrophin receptors. In support of this, BMP-6 alone increased LH-receptor (LHR) mRNA in F1 cells and FSH-receptor (FSHR) mRNA in F1, F2 and F3/4 cells. BMP-6 also enhanced LH/FSH-induced LHR transcript amount in each cell type but did not raise FSHR transcript amounts above those induced by BMP-6 alone. To further explore BMP-6 action on inhibin-A secretion, we quantified inhibin/activin subunits (alpha, beta(A), beta(B)) mRNAs. Consistent with its effect on inhibin-A secretion, BMP-6 enhanced 'basal' expression of alpha- and beta(A)-subunit mRNA in F1, F2 and F3/4 cells, and beta(B)-subunit mRNA in F3/4 cells. BMP-6 markedly enhanced FSH/LH-induced expression of alpha-subunit in all follicles and FSH-induced beta(A)-subunit in F2 and F3/4 follicles but not in F1 follicles. Neither BMP-6 alone, nor FSH/LH alone, affected 'basal' beta(B) mRNA abundance. However, co-treatment with gonadotrophin and BMP-6 greatly increased beta(B)-subunit expression, the response being lowest in F1 follicles and greatest in F3/4 follicles. Collectively, these results support the hypothesis that intraovarian BMPs of thecal origin have a paracrine role in modulating granulosa cell function in the chicken in a preovulatory stage-dependent manner.

Gonadotrophins modulate hormone secretion and steady-state mRNA levels for activin receptors (type I, IIA, IIB) and inhibin co-receptor (betaglycan) in granulosa and theca cells from chicken prehierarchical and preovulatory follicles.

Ovarian follicle development is regulated through endocrine and local mechanisms. Increasing evidence indicates roles for transforming growth factor beta superfamily members, including inhibins and activins. We recently identified divergent expression of mRNAs encoding activin receptors (ActR) and inhibin co-receptor betaglycan in chicken follicles at different stages of maturation. Here, we compare the actions of LH and FSH (0, 1, 10, 100 ng/ml) on levels of mRNA for ActRI, ActRIIA, ActRIIB and betaglycan in chicken granulosa and theca cells (GC and TC) from preovulatory (F1) and prehierarchical (6-8 mm) follicles. The expression of mRNAs for LH-R and FSH-R and production of inhibin A, oestradiol and progesterone were also quantified. FSH decreased ActRIIB and ActRI mRNA levels in 6-8 mm GC, whereas LH increased the mRNA levels. Both LH and FSH enhanced ActRIIA (5- and 8.5-fold) and betaglycan mRNA expression (2- and 3.5-fold) in 6-8 mm GC. In 6-8 mm TC, LH and FSH both increased the betaglycan mRNA level (7- and 3.5-fold respectively) but did not affect ActRI, ActRIIA and ActRIIB transcript levels. In F1 GC, both LH and FSH stimulated ActRI (2- and 2.4-fold), ActRIIB (3.2- and 2.7-fold) and betaglycan (7- and 4-fold) mRNA levels, while ActRIIA mRNA was unaffected. In F1 TC, LH and FSH reduced ActRIIA (35-50%) and increased (4.5- and 7.6-fold) betaglycan mRNA, but had no effect on ActRI and ActRIIB transcript levels. Results support the hypothesis that expression of ActR and betaglycan are differentially regulated by gonadotrophins during follicle maturation in the hen. This may represent an important mechanism for fine-tuning follicle responsiveness to local and systemic activins and inhibins.

Evidence for a reversal with age in the pattern of near-wall blood flow around aortic branches.

The changes that occur with age in the distribution of atherosclerotic lesions around arterial branch points challenge accepted theories relating disease to haemodynamic stresses. We investigated whether flow near branch points changes with age in a way that can account for the different lesion distributions. Flow around 20 branches from immature and mature aortas was investigated by examining the length:width ratio and orientation of endothelial nuclei; these properties depend on the magnitude and direction of near-wall flows, respectively. There were significant changes in the pattern of nuclear shape with age, consistent with a reversal in the pattern of shear around branches. In control regions away from branches, there were no such changes. The role of haemodynamic stresses in atherogenesis may require re-evaluation in the light of these results.