BMPR-II mRNA Research Articles

Is iron deficiency caused by BMPR2 mutations or dysfunction in pulmonary arterial hypertension patients?

Iron deficiency is common in idiopathic and heritable pulmonary arterial hypertension patients (I/HPAH). A previous report suggested a dysregulation of the iron hormone hepcidin, which is controlled by BMP/SMAD signaling involving the bone morphogenetic protein receptor 2 (BMPR-II). Pathogenic variants in the BMPR2 gene are the most common cause of HPAH. Their effect on patients'hepcidin levels has not been investigated. The aim of this study was to assess whether iron metabolism and regulation of the iron regulatory hormone hepcidin was disturbed in I/HPAH patients with and without a pathogenic variant in the gene BMPR2 compared to healthy controls. In this explorative, cross-sectional study hepcidin serum levels were quantified by enzyme-linked immunosorbent assay. We measured iron status, inflammatory parameters and hepcidin modifying proteins such as IL6, erythropoietin, and BMP2, BMP6 in addition to BMPR-II protein and mRNA levels. Clinical routine parameters were correlated with hepcidin levels. In total 109 I/HPAH patients and controls, separated into three groups, 23 BMPR2 variant-carriers, 56 BMPR2 noncarriers and 30 healthy controls were enrolled. Of these, 84% had iron deficiency requiring iron supplementation. Hepcidin levels were not different between groups and corresponded to the degree of iron deficiency. The levels of IL6, erythropoietin, BMP2, or BMP6 showed no correlation with hepcidin expression. Hence, iron homeostasis and hepcidin regulation was largely independent from these parameters. I/HPAH patients had a physiologically normal iron regulation and no false elevation of hepcidin levels. Iron deficiency was prevalent albeit independent of pathogenic variants in the BMPR2 gene.

Regulatory role of dihydrotestosterone on BMP-6 receptors in granular cells of sheep antral follicles.

Bone morphogenetic protein-6 (BMP-6) and dihydrotestosterone (DHT) affect steroid synthesis in follicles and regulate cell proliferation in the ovaries of female animals. However, little is known about granular cells (GCs) in sheep. We identified the key BMP-6 receptors, activin receptor-like kinase(ALK-6), and bone morphogenetic protein receptor type 2 (BMPRII) in sheep follicles using immunohistochemistry (IHC) and immunofluorescence (IF). Both ALK-6 and BMPRII were expressed in the GC layer, GC membranes, and cytoplasm. We evaluated ALK-6 and BMPRII expression at the follicular development stage using quantitative real-time PCR and western blotting to detect sheep GCs from large, medium, and small follicles (diameters of≥5, 2-5, and≤2mm, respectively). The mRNA abundance and protein expression of ALK-6 and BMPRII were significantly higher in GCs from large follicles compared to those in GCs from small follicles (P<0.05) and were the lowest in GCs from medium follicles. To assess whether DHT affects ALK-6 and BMPRII expression in sheep GCs, we cultured GCs from large follicles in vitro then incubated them with DHT (10-11, 10-9, 10-7 M). We found that 10-7-M DHT significantly inhibited ALK-6 and BMPRII mRNA and protein (P<0.05). We further explored whether DHT regulates ALK-6 and BMPRII through the nuclear androgen receptor (AR) pathway and found that 10-6-M flutamide, a non-selective androgen inhibitor, partially relieved the inhibitory effect of 10-7-M DHT on ALK-6 and BMPRII expression. Thus, GCs in sheep antral follicles differentially expressed ALK-6 and BMPRII at various stages, indicating that BMP-6 plays different roles to some extent during the development of antral follicles, and that high concentrations of DHT can inhibit the expression of ALK-6 and BMPRII via the androgen receptor pathway in sheep GCs. The present study aimed to determine the expression of the main BMP-6-related main receptors, namely, ALK-6 and BMPRII, during the development of GCs in sheep antral follicles and a potential mechanism of DHT regulation in sheep GCs. Our findings lay a foundation for the further exploration of the effects of ovarian BMP-6 expression on follicular development.

T1 TNFα mediates ectodomain shedding of BMPR-II: a mechanism for inflammation as a trigger for pulmonary arterial hypertension

BackgroundMutations in BMP type II receptor (BMPR-II) account for 70% of heritable pulmonary arterial hypertension (PAH) cases, but low penetrance (~20%) in mutation carriers implies a ‘second hit’ is required...

BMPRIB and BMPRII mRNA expression levels in goat ovarian follicles and the in vitro effects of BMP-15 on preantral follicle development

This study evaluated the levels of bone morphogenetic protein receptors BMPRIB and BMPRII mRNA in goat follicles and the effects of bone morphogenetic protein-15 (BMP-15) on the in vitro development of cultured preantral follicles. Real-time polymerase chain reaction (PCR) was used to analyze the levels of BMPRIB and BMPRII mRNA in caprine preantral follicles and in small and large antral follicles. Preantral follicles (≥150 μm) were also isolated from goat ovaries and cultured for 18 days in α-MEM(+) supplemented with or without BMP-15 (10, 50, or 100 ng/ml). At the end of culture, some follicles were fixed for ultrastructural evaluation. Real-time PCR showed a reduction in BMPRII mRNA levels from the primary to secondary follicles. Higher levels of BMPRIB mRNA were observed in granulosa/theca cells from large antral follicles compared with small antral follicles. Moreover, BMPRII mRNA was expressed to a greater extent in cumulus-oocyte complexes from large antral follicles than in their respective granulosa/theca cells. In culture, 50 ng/ml BMP-15 positively influenced antral cavity formation and follicle growth after 18 days and also maintained follicular integrity. Thus, BMPRIB and BMPRII mRNAs are present in all follicular categories. BMP-15 (50 ng/ml) stimulates growth, antrum formation and the ultrastructural integrity of isolated caprine preantral follicles after 18 days of culture.

MRNA Expression of Bone Morphogenetic Proteins and Their Receptors in Human Renal Cell Carcinoma

Introduction: Bone morphogenetic proteins (BMPs) have been studied in several cancers, but only limited information is available about renal cell carcinomas (RCCs). We determined the expression of mRNA of several BMP ligands and BMP receptors (BMPRs) in healthy kidney tissue and RCCs, and data were compared to clinicopathological parameters. Material and Methods: Sixty-four samples of RCCs and healthy renal tissues were prospectively examined. The expression of BMP2, BMP4, BMP6, BMP7, BMPRIA, BMPRIB and BMPRII mRNA was determined using semiquantitative reverse transcriptase-polymerase chain reaction. Results: The expression levels of different BMP ligands and BMPRs were considerably higher in RCCs than in normal kidney tissue. BMP ligands showed elevated expression in clear-cell RCCs, whereas all three BMPRs showed higher expression levels in non-clear-cell RCCs. In clear-cell RCCs, the expression levels of BMP2 progressively increased and expression levels of BMP6, BMP7 and BMPRIB were lost with higher tumor stage. Conclusions: All BMPs and their receptors have stronger expression levels in RCC. The expression level of BMP2 is strongly elevated in kidney cancer.

Transfection of truncated bone morphogenetic protein receptor-II into oral squamous cell carcinoma cell line Tca8113 and inhibitory effect on proliferation and inductive effect on apoptosis

Bone morphogenetic proteins (BMPs), one of the crucial regulators in embryonic development and bone formation, have been implicated in epithelium-derived tumors. Previous results showed the involvement of overexpression of BMP 2, 4, 5 in the carcinogenesis of oral epithelia. The ability of BMP receptor-II mutant to modify the malignant phenotype of oral squamous cell carcinoma cell line Tca8113 by blocking the BMP signal transduction pathway has been proposed. In this study, a negative truncated mutant of the BMP receptor-II (tBMPR-II) was transfected into Tca8113 cells. The effects were evaluated though RT-PCR, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, BrdU staining, cell cyclin assay, TdT-mediated dUTP nick end labeling (TUNEL) staining, and cell cycle protein detection. Overexpression of tBMPR-II gene transfection truncates the expression of BMPR-II mRNA expression, but not BMP 2, 4, 5. tBMPR-II resulted in a remarkable inhibition of cell proliferation and viability compared with control Tca8113. The inhibitory effects were partly attributed to the induction of apoptosis and cell cycle arrest in G(0) /G(1) accompanied by downregulation of the intracellular cell cycle proteins of cyclin D1 and cyclin-dependent kinases 4, as well as the upregulation of p27 and p57. Loss of BMP signals correlates tightly with suppression of cell proliferation, induction of apoptosis, and benign transformation of Tca8113 cells phenotype.

Retinoic acid inhibits osteogenic differentiation of mouse embryonic palate mesenchymal cells

All-trans-retinoic acid (ATRA), a known teratogenic factor affecting the development of cleft palate, has been shown to adversely affect craniofacial development. In the present study, we evaluated the effects of ATRA on the osteo-/adipogenic differentiation of mouse embryonic palate mesenchymal (MEPM) cells, which served as a valid model system for investigating the mechanisms regulating osteogenesis during palatogenesis. MEPM cells were derived from gestational day 13 C57BL/6N mouse embryos and induced to differentiate in the presence or absence of ATRA in either osteogenic medium (OM) or control medium (CM). Alkaline phosphatase (ALP) activity assays, von Kossa staining, and RT-PCR assays confirmed that MEPM cells underwent osteogenic differentiation when cultured in OM. Although ATRA induced ALP activity and lipid accumulation in MEPM cells, it failed to induce matrix mineralization and osteoblastic gene expression. BMPR-IB and Smad5 mRNA levels increased significantly in cells cultured in OM and declined following treatment with ATRA, whereas the expression of the BMPR-IA mRNA was up-regulated by ATRA. In conclusion, our results suggested that ATRA and the BMP signaling pathway cooperate to inhibit osteogenesis and promote adipogenesis of MEPM cells.

Bone Morphogenetic Protein (BMP) and Activin Type II Receptors Balance BMP9 Signals Mediated by Activin Receptor-like Kinase-1 in Human Pulmonary Artery Endothelial Cells

Mutations in transforming growth factor-beta (TGF-beta) receptor superfamily members underlie conditions characterized by vascular dysplasia. Mutations in endoglin and activin-like kinase receptor 1 (ALK1) cause hereditary hemorrhagic telangiectasia, whereas bone morphogenetic protein type II receptor (BMPR-II) mutations underlie familial pulmonary arterial hypertension. To understand the functional roles of these receptors, we examined their relative contributions to BMP signaling in human pulmonary artery endothelial cells (HPAECs). BMP9 potently and selectively induced Smad1/5 phosphorylation and Id gene expression in HPAECs. Contrary to expectations, BMP9 also stimulated Smad2 activation. Furthermore, BMP9 induced the expression of interleukin 8 and E-selectin. Using small interfering RNA, we demonstrate that the type I receptor, ALK1, is essential for these responses. However, small interfering RNA and inhibitor studies showed no involvement of ALK5 or endoglin. We further demonstrate that, of the candidate type II receptors, BMPR-II predominantly mediated IL-8 and E-selectin induction and mitogenic inhibition by BMP9. Conversely, activin receptor type II (ActR-II) contributed more to BMP9-mediated Smad2 activation. Only abolition of both type II receptors significantly reduced the Smad1/5 and Id responses. Both ALK1 and BMPR-II contributed to growth inhibition of HPAECs, whereas ActR-II was not involved. Taken together, our findings demonstrate the critical role of type II receptors in balancing BMP9 signaling via ALK1 and emphasize the essential role for BMPR-II in a subset of BMP9 responses (interleukin 8, E-selectin, and proliferation). This differential signaling may contribute to the contrasting pathologies of hereditary hemorrhagic telangiectasia and pulmonary arterial hypertension.

Expression of Bone Morphogenetic Protein Receptor (BMPR) during Perinatal Ovary Development and Primordial Follicle Formation in the Hamster: Possible Regulation by FSH

To understand whether bone morphogenetic protein plays any role in the formation of primordial follicles in the hamster, we examined the temporal and spatial expression of bone morphogenetic protein receptor (BMPR) mRNA and protein in embryonic (E) 13 through postnatal day (P) 15 ovarian cells and a possible regulation by FSH during the formation of primordial follicles on P8. BMPRIA and BMPRII mRNA levels were significantly higher than that of BMPR1B throughout ovary development. BMPRIA and BMPRII mRNA levels increased significantly on E14 and declined by P5 through P6. Whereas BMPRII mRNA increased again by P7, BMPRIA mRNA levels increased through P8 concurrent with primordial follicle formation. In contrast, BMPRIB mRNA levels increased greater than 10-fold on P7-9, with a further 3-fold increase by P10. BMPR proteins were low in the somatic cells and oocytes on E13 but increased progressively during postnatal development. BMPR expression in somatic cells increased markedly on P8. Whereas BMPRII expression declined by P10 and remained steady thereafter, BMPRIA protein expression fluctuated until P15 when it became low and steady. Overall, BMPRIB immunoreactivity also declined by P10 and then remained low in the interstitial cells through P15. FSH antiserum treatment on E12 significantly attenuated receptor mRNA and protein levels by P8, but equine chorionic gonadotropin replacement on P1 reversed the inhibition. Furthermore, FSH in vitro up-regulated BMPR levels in P4 ovaries. This unique pattern of BMPR expression in the oocytes and somatic cells during perinatal ovary development suggests that BMP may play a regulatory role in primordial follicle formation. Furthermore, FSH may regulate BMP action by modulating the expression of its receptors.

Retinoic acid inhibits osteogenic differentiation of rat bone marrow stromal cells

It has been well established that all-trans-retinoic acid (ATRA) influences bone metabolism when given in the treatment or prevention of cancer. However, the molecular mechanisms underlying this are unknown. In the present study, we investigated the effect of ATRA on differentiation of rat bone marrow stromal cells (BMSCs). BMSCs were harvested from rats and induced to differentiate in the presence or absence of ATRA in either osteogenic (OM) or control medium (CM). BMSCs underwent osteogenic differentiation, showed alkaline phosphatase (ALP) activity, a high level of matrix mineralization, and expressed osteonectin when cultured in OM. Although ATRA induced ALP activity, it failed to induce matrix mineralization and osteonectin, decrease mineralization in OM, and induce lipid accumulation in BMSCs. Moreover, while ATRA induced the expression of BMP-RIA, both BMP-RII and Smad5 mRNA were induced by OM and ATRA. Thus, ATRA inhibited osteogenesis and promoted adipogenesis of BMSCs. BMP signaling cooperated with ATRA in the differentiation of BMSCs.

Stage-specific expression of bone morphogenetic protein type I and type II receptor genes: Effects of follicle-stimulating hormone on ovine antral follicles

We investigated the mRNA expression patterns of receptor genes for bone morphogenetic proteins-15 (BMP15) and growth differentiation factor-9 (GDF-9) in granulosa cells of sheep treated with FSH. The effects of FSH and estradiol (E2) on the regulation of BMPRII, BMPRIB and ALK-5 in ovine granulosa cells were also examined. Ovaries were collected on day 16 of the estrous cycle and granulose cells were harvested from follicles of two sizes (3–5 and >5 mm in diameter). For in vitro studies, granulosa cells were obtained from follicles of 3–5 mm in diameter and cultured in serum-free McCoy's 5A medium supplemented with different doses of FSH (0, 1, 5, 10 ng/ml) or a combination of 5 ng/ml FSH with 1 ng/ml E2. Expression of BMPRII, BMPRIB and ALK-5 mRNA was estimated by quantitative real-time PCR. Our results demonstrated that BMPRII, BMPRIB and ALK-5 expression was significantly higher in the granulosa cells of large follicles than of small follicles. Treatment of granulose cells with FSH (1–10 ng/ml) alone down-regulated the expression of BMPRIB ( P < 0.05). BMPRII and ALK-5 mRNA expression was not significantly different at an FSH concentration of 5 ng/ml compared to control. A further increase in FSH (10 ng/ml) down-regulated the expression of BMPRII and ALK-5 ( P < 0.05). The combination of FSH (5 ng/ml) and E2 (1 ng/ml) up-regulated the expression of BMPRII, BMPRIB and ALK-5 in granulose cells ( P < 0.05). Therefore, the present study establishes the expression levels of the receptor genes of BMP15 and GDF-9 and suggests that the expression of BMPRII, BMPRIB and ALK-5 may be regulated by FSH and E2 in ovine granulosa cells.

Expression of bone morphogenetic proteins 4 and 7 and their receptors IA, IB, and II in human ovaries from fetuses and adults

To investigate the immunocytochemical and mRNA expression of bone morphogenetic proteins 4 (BMP-4) and 7 (BMP-7) and their three receptors (BMPR-IA, BMPR-IB, and BMPR-II) in ovaries from human adults and fetuses. Immunocytochemical and in situ hybridization study. Major tertiary care and referral academic centers. Sixteen adolescents/adults aged 13-38 years and 31 women undergoing second and third trimester pregnancy terminations. None. Immunocytochemistry and in situ hybridization on paraffin sections of human ovaries from fetuses and adults. The expression of the proteins for BMP-4 and BMP-7 and their receptors was detected in oogonia/oocytes and stroma cells from both sources (fetuses and women/adolescents). BMP-7 and the three receptors were identified in all of these granulosa cells, and BMP-4 was detected only in the granulosa cells of women/adolescents. Transcripts for all five ligands were identified in stroma cells of all samples and in fetal oogonia/oocytes. BMPR-IA and BMPR-IB mRNA expression was also identified in oocytes from women/adolescents. BMP-7 and BMPR-IA mRNA staining was detected in fetal granulosa cells. This is the first report of the expression of BMP-4 and BMP-7 and their receptors in human ovaries from fetuses as well as adults. However, to elucidate whether indeed BMP-4 or BMP-7 are involved in the initiation of human primordial follicular growth, they should be added to the culture medium.

Hepatocyte growth factor up-regulates the expression of the bone morphogenetic protein (BMP) receptors, BMPR-IB and BMPR-II, in human prostate cancer cells.

Hepatocyte growth factor (HGF) plays multiple roles in cancer, by acting as a motility, invasion and angiogenesis stimulating factor, which promotes metastasis and tumour growth. Bone morphogenetic proteins (BMPs) are members of the TGF-beta superfamily. The effects of BMPs are mediated by two subgroups of receptors, type I and type II. Recent studies have shown that some BMPs, via their signaling pathways, affect the growth of prostate cancer cells. BMPR-IB and BMPR-II have been reported to be expressed at low levels in prostate cancer. However, little is known about the crosstalk between HGF and BMP pathways. In this study, prostate cancer cells (PC-3 and DU-145) were exposed to HGF at different concentrations (1-75 ng/ml) for 18 h, or were treated with HGF at 40 ng/ml over various time periods (up to 24 h). The effect of HGF on BMP receptor expression was further investigated in a nude mouse PC-3 xenograft model. Mice were treated with either HGF, the HGF antagonist NK4, or a combination of both. The expression of BMPR-IB and BMPR-II mRNA was up-regulated by HGF, as shown by both conventional PCR and quantitative PCR. An elevation of BMPR-IB and BMPR-II at the protein level was confirmed by both Western blot analysis and immunocytochemical staining. In a murine prostate tumour model, infusion of recombinant HGF resulted in an increase in the levels of both BMPR-IB and BMPR-II transcript in prostate tumours. Concomitant delivery of NK4, an HGF antagonist, prevented this effect. In conclusion, HGF up-regulates the expression of the bone morphogenetic protein receptors, BMPR-IB and BMPR-II, in prostate cancer cells, both in vitro and in vivo. This may have important implications in the development of bone metastasis in prostate cancer.

Expression of Bmp ligands and receptors in the developing Xenopus retina

Bone morphogenetic proteins (BMPs) act repeatedly in the development of nervous system tissues. While BMP signaling is critical for the early growth and patterning of the eye, we are interested in possible later functions of BMPs in the morphological development of retinal neurons and formation of synaptic connections. Therefore, we conducted an in situ hybridization analysis of the mRNA expression for the ligands Bmp2, -4 and 7 and the type Ia, Ib and II receptors (BmprIa, BmprIb and BmprII) during development of the retina of Xenopus laevis. Bmp4 mRNA is expressed in the dorsal retina and Bmp7 in the distal peripheral retina during the period of cell differentiation, while Bmp2 is not present in the eye. The type I receptors are expressed predominantly ventrally, from the optic vesicle stage until at least stage 35/36, after most cells have differentiated and many synaptic connections have formed. BmprII mRNA, however, is distributed evenly across the dorsoventral axis, with highest expression in retinal ganglion cell and inner nuclear layers.

Hormonal regulation of expression of growth differentiation factor-9 receptor type I and II genes in the bovine ovarian follicle

Growth differentiation factor-9 (GDF-9) and bone morphogenetic proteins (BMPs) are crucial factors in follicular growth and development. GDF-9 and BMPs initiate signaling by assembling type I (ALK-3, ALK-5 and ALK-6) and type II (BMPRII) receptors. However, the mechanism regulating the expression of these receptors in the process of bovine follicle development is still unknown. The aim of the present study was to clarify the involvement of receptor systems for GDF-9 and BMPs in follicular selection by examining the effects of FSH and estradiol-17beta (E2) on the regulation of BMPRII, ALK-3, ALK-5 and ALK-6 mRNA expression in bovine granulosa cells (GCs). To observe mRNA expression during follicular development, follicles were obtained from heifers and classified into two groups: pre-selection follicles (PRFs) (an average of 7.7 mm follicles with low E2) and post-selection follicles (POFs) (an average of 15 mm follicles with high E2). Theca layer cells (TCs) and GCs were harvested from aspirated follicles. For in vitro studies, GCs were obtained from bovine follicles of 4-7 mm diameter and cultured in Dulbecco's modified Eagle's/F12 (DMEM/F-12) medium with 10% fetal calf serum for 24 h. The medium was then replaced with serum-free DMEM/F-12 supplemented with different doses of E2 (1, 10, 100 ng/ml) or FSH (1, 5, 10 ng/ml) or combinations of 1 ng/ml of E2 with different FSH doses. Total RNA was extracted and the mRNA expression of BMPRII, ALK-3, ALK-5 and ALK-6 was estimated by the quantitative real-time PCR method using a LightCycler. BMPRII and ALK-5 expression was significantly higher in the GCs of POFs than in those of PRFs, whereas ALK-3 expression was significantly lower in the GCs of POFs than in those of PRFs. There was no difference in ALK-6 expression in GCs between PRFs and POFs. The expression of BMPRII, ALK-5, ALK-3 and ALK-6 genes in the TCs was not significantly different between PRFs and POFs. Treatment of GCs with E2 alone increased BMPRII mRNA expression at a concentration of 100 ng/ml and ALK-5 mRNA expression at 10 ng/ml. BMPRII and ALK-5 mRNA levels were up-regulated by the combination of E2 (1 ng/ml) and FSH (5 ng/ml). On the other hand, FSH alone down-regulated the expression of BMPRII and ALK-5 in GCs. The results of the present study provide the first evidence that FSH and E2 regulate the expression of BMPRII and ALK-5 genes in bovine GCs. Thus, our data suggest that the GDF-9/BMPRII/ALK-5 system may be critically involved in the process of selection of bovine follicles.

BMP4 inhibits proliferation and promotes myocyte differentiation of lung fibroblasts via Smad1 and JNK pathways

Fibroblast proliferation, differentiation, and migration contribute to the characteristic pulmonary vascular remodeling seen in primary pulmonary hypertension (PPH). The identification of mutations in the bone morphogenetic protein type II receptor (BMPRII) in PPH have led us to question what role BMPRII and its ligands play in pulmonary vascular remodeling. Thus, to further understand the functional significance of BMPRII in the pulmonary vasculature, we examined the expression of TGF-beta superfamily receptors in human fetal lung fibroblasts (HFL) and investigated the role of BMP4 on cell cycle regulation, fibroblast proliferation, and differentiation. Furthermore, signaling pathways involved in these processes were examined. HFL expressed BMPRI and BMPRII mRNA and demonstrated specific I(125)-BMP4 binding sites. BMP4 inhibited [(3)H]thymidine incorporation and proliferation of HFL; protein expression was increased for the cell cycle inhibitor p21 and reduced for the positive regulators cyclin D and cdk2 by BMP4. BMP4 induced differentiation of HFL into a smooth muscle cell phenotype since protein expression of alpha-smooth muscle actin and smooth muscle myosin was increased. Furthermore, p38(MAPK), ERK1/2, JNK, and Smad1 were phosphorylated by BMP4. Using specific MAPK inhibitors, a dominant negative Smad1 construct, and Smad1 siRNA, we found that the antiproliferative and prodifferentiation effects of BMP4 were Smad1 dependent with JNK also contributing to differentiation. Because failure of Smad phosphorylation is a major feature of BMPRII mutations, these results imply that BMPRII mutations may promote the expansion of fibroblasts resistant to the antiproliferative, prodifferentiation effects of BMPs and suggest a mechanism for the vascular obliteration seen in familial PPH.

Expression of bone morphogenetic protein2 (BMP2), BMP4 and BMP receptors in the bovine ovary but absence of effects of BMP2 and BMP4 during IVM on bovine oocyte nuclear maturation and subsequent embryo development

Bone morphogenetic proteins (BMPs) have been implicated in the regulation of ovarian follicular development and are promising candidates to apply in IVM and IVF protocols. We investigated the expression of BMP2, BMP4 and BMP receptors in bovine ovaries and the effects of BMP2 and BMP4 during oocyte maturation on bovine IVM. Reverse transcription polymerase chain reaction studies with antral follicles showed the expression of BMPR-IA, BMPR-IB, ActR-IA, ActR-IIB, BMPR-II and BMP4 mRNA in all follicular compartments, while BMP2 mRNA was generally restricted to theca and cumulus tissue. Immunohistochemistry demonstrated the presence of BMPR-II in oocytes and granulosa cells of preantral follicles but only in oocytes of antral follicles. The immunostaining of BMP2 and BMP4 was limited to theca interna and approximately 25% of oocytes of antral follicles. Exogenously added BMP2 or BMP4 to IVM medium did not affect oocyte nuclear maturation, cumulus cell expansion, nor blastocyst formation following IVF. It is concluded that a BMP-signaling system, consisting of BMP2, BMP4, type II and I receptors, is present in bovine antral follicles and that this system plays a role in development and functioning of these follicles rather than in final oocyte maturation and cumulus expansion.

247.Molecular basis of oocyte - paracrine signalling that promotes mouse granulosa cell proliferation

Oocytes regulate follicle growth and development by secreting paracrine growth factors that act on granulosa cells (GC). We have recently determined that growth differentiation factor-9 (GDF-9) accounts for ~50% of the total mitogenic activity of oocytes, the remaining portion is as yet uncharacterised. This study was conducted to identify the receptor/signalling system utilised by oocytes to promote GC proliferation. We used an established oocyte-secreted mitogen bioassay, where denuded oocytes are co-cultured with primed-mouse mural GC. In this system, oocytes, GDF-9, TGF-b1 and activin-A all promoted GC DNA synthesis in a dose-dependent manner, but bone-morphogenetic protein-6 (BMP-6) and BMP-7 did not. The type-II receptor for GDF-9 is BMPRII and using real-time RT-PCR, cumulus cells (CC) and mural GC were found to express equivalent levels of BMPRII mRNA. We tested the capacity of the receptor ectodomain (ECD) to neutralise oocyte-stimulated mural GC proliferation. The BMPRII ECD antagonised both oocyte and GDF-9 bioactivity in a dose-dependent manner, completely abolishing activity of both mitogens at 1 ug/mL. The BMPRII ECD did not antagonise TGF-β and partially antagonised activin-A bioactivity, demonstrating its specificity. The TGFβR-II ECD, activin R-II ECD and activin R-IIB ECD all failed to neutralise oocyte- or GDF-9-stimulated GC DNA synthesis, whereas they did antagonise the activity of their respective ligands. The BMPRII ECD also completely antagonised oocyte-stimulated CC DNA synthesis. Using this oocyte-factor bioassay with mural GC transfected with Smad luciferase reporter constructs, we found that oocytes, GDF-9 and TGF-β (but not BMP-6) activated the Smad2/3 pathway. Consistent with this, oocytes and GDF-9 led to phosphorylation of GC Smad2 molecules as detected by Western blot. Conversely the Smad1/5/8 pathway was activated by BMP-6, but not by GDF-9, TGF-β nor surprisingly by oocytes. This study provides evidence that BMPRII is a key receptor for transmitting the paracrine actions of oocytes in GC. However, oocyte-secreted factors do not activate the BMP intracellular signalling pathway but rather the TGF-β/activin intracellular pathway.

Sequence analysis of bone morphogenetic protein receptor type II mRNA from ascitic and nonascitic commercial broilers

Ascites syndrome, also known as pulmonary hypertension syndrome (PHS), is a common metabolic disorder in rapidly growing meat-type chickens. Environmental factors, such as cold, altitude, and diet, play significant roles in development of the disease, but there is also an important genetic component to PHS susceptibility. The human disease familial primary pulmonary hypertension (FPPH) is similar to PHS in broilers both genetically and physiologically. Several recent studies have shown that mutations in the bone morphogenetic protein receptor type II (BMPR2) gene are a cause of FPPH in humans. To determine whether mutations in the chicken BMPR2 gene play a similar role in PHS susceptibility, BMPR-II mRNA from ascitic and nonascitic commercial broilers were sequenced and compared with the published Leghorn chicken BMPR-II mRNA sequence. Fourteen single nucleotide polymorphisms (SNP) were identified in the commercial broiler BMPR-II mRNA. No mutations unique to ascites-susceptible broilers were present in the coding, 5' untranslated or 3' untranslated regions of BMPR-II mRNA. The twelve SNP present within the coding region of BMPR-II mRNA were synonymous substitutions and did not alter the BMPR-II protein sequence. In addition, analysis of BMPR2 gene expression by reverse transcriptase-PCR indicated that there were no differences in BMPR-II mRNA levels in ascitic and nonascitic birds. Therefore, it appears unlikely that mutations in the BMPR2 gene were responsible for susceptibility to PHS in these commercial broilers.

BMPs and BMPRs in chicken ovary and effects of BMP-4 and -7 on granulosa cell proliferation and progesterone production in vitro.

Bone morphogenetic proteins (BMPs) and their receptors (BMPRs) are now known to have important roles in mammalian ovarian folliculogenesis. This study determined the expression of the mRNA encoding for BMPs and their receptors in the chicken ovary and explored possible roles for them. The expression of the mRNA for BMP-2, -4, -6, -7, and BMPR-IA, -IB, and -II was determined and quantified by a semiquantitative RT-PCR. The mRNAs for all the BMPs and receptors determined were present in both the granulosa (G) and theca (T) cells of the F1, F2, and F3 follicles. All BMP mRNAs increased in G cells with follicular development, whereas only BMP-7 mRNA had this trend in the T cells. BMP-2, -4, and -6 mRNAs in T were similar between follicles. BMPR-IA mRNA was similar in F2G and F3G but lower in F1G. BMPR-IB mRNA was similar in G of all follicles, and BMPR-II mRNA increased with development. In the T, each receptor subtype showed equal distribution between follicles. mRNA levels for BMPR-IB and -II were higher in G than in T, suggesting that the G is a major target for BMPs. BMP-4 and -7 stimulated basal, IGF-I-, and gonadotropin-stimulated progesterone production by cultured G cells, with differential responses between cells from the F1 and F3/4. This suggests involvement in follicular differentiation. BMP-4 and -7 reversed the inhibitory effects of transforming growth factor (TGF)-alpha on basal and gonadotropin-stimulated G cell progesterone production, with greater effect in the F1 than in the F3/4. This effect suggests an important role for BMPs interacting with TGF-alpha in modulating the effects of gonadotropins and IGF-I on follicular differentiation. Finally, BMP-7 stimulated G cell proliferation, but BMP-4 inhibited TGF-alpha+ IGF-I- and/or FSH-stimulated G cell proliferation, suggesting a role in the control of follicular growth during development. These effects of BMP-4 and -7 on the G cell function showed relationships with the expression levels of the BMPs and the BMPR-II.