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Abstracts from VI International Symposium on Avian EndocrinologyMarch 31 - April 5, 1996 Chateau Lake Louise, Alberta Continued Stem Cell Factor and its ReceptorM. Sakurai National Institute of Animal Health, Tsukuba, Ibaraki 305,
Japan Stem cell factor (SCF) is a pleiotropic cytokine that plays essential roles in various aspects of development including hematopoiesis, germ cell growth, and melanocyte formation. The cell-surface receptor for SCF is the Kit protein-tyrosine kinase. Until now, the biological roles of SCF have been extensively examined in the mouse and human. To gain insight into the possible roles of SCF in chicken development, we have cloned cDNAs for chicken SCF (chSCF) and for chicken Kit protein (chKit), and characterized the proteins. SCF is first produced as a transmembrane protein, and then processed to a soluble SCF which functions as a cytokine. Recombinant soluble chSCF was produced by using the baculovirus vector, and shown to have an ability to induce the outgrowth of neurites from chicken dorsal root ganglia cultured in vitro. chKit was produced in Cos cells transfected with an expression vector, and shown to be activated by chSCF, but not by mouse SCF. These functional features of the chicken proteins, together with the tissue distribution of their mRNAs, suggest that the chSCF/chKit signal transduction system plays developmental roles analogous to those in mammalians. Nuclear Hormone Receptors and Cytokine Membrane Receptors in the Control of Chicken Hematopoietic Differentiation and LeukemogenesisJ. Samarut Laboratoire de Biologie Moléculaire et Cellulaire, CNRS
UMR49, INRA LA913, Ecole Normale Supérieure de Lyon, 46 Allée
d'Italie, 69364 Lyon Cedex 07, France The avian erythroblastosis virus, AEV, induces an acute erythroleukemia in chickens and blocks the differentiation of erythrocytic progenitor cells at the BFUE/CFUE transition. This virus encodes two oncoproteins, respectively vErbA and vErbB, which cooperate for the leukemogenic transformation. The vErbA protein is an altered form of the nuclear receptor for the thyroid hormone T3, cErbAa. Because of its altered structure the protein behaves as a transdominant inhibitor of the normal receptor for T3, but also of the receptors for retinoic acid (RA). vErbA is responsible for the block of differentiation by inhibiting the expression of erythrocytic genes normally regulated by T3 and RA. The other oncoprotein, vErbB is an altered form of the membrane receptor for EGF and TGFa. The normal cErbB receptor is required in erythrocytic progenitors for the control of cell divisions. vErbB works as an overactivated receptor and then delivers in the cells a constitutive and amplified mitogenic signal. In normal erythrocytic progenitors, cErbA and cErbB together then control cell differentiation and proliferation. Expression of altered forms of these two proteins through AEV preverts these two signalling pathways. This virus therefore provides a helpful model to investigate how signalling pathways from nuclear and membrane receptors cooperate in the control of cell differentiation and oncogenesis. Potential Roles for Tumor Necrosis Factor-Alpha in the Development of the Chick Embryo LensM.a. Wride, E.J. Sanders Department of Physiology, University of Alberta, Edmonton, AB
T6G 2H7 Canada The chick embryo lens expresses TNFa and TNF receptors. TNFa immunoreactivity can be detected in lens fibres at ED 4 and this persists until ED 6. From ED 8 onwards, TNFa becomes restricted to the epithelium, annular pad and degenerating nuclei of fibres (detected using TUNEL, which detects DNA fragmentation). TNFR1 is first detected in the fibres, but not in the epithelium, at ED 4. TNFR2 immunoreactivity is not significantly detected in the lens at this stage. TNFR1 is associated with the lens epithelium and the peripheral fibre cells at ED 8 and with cell nuclei. TNFR2 is also expressed at ED 8, in a similar pattern, but it is not associated with cell nuclei. The cell death inhibiting protein bcl2 is also expressed in the developing lens epithelium, and its expression is downregulated in the lens fibres concomitant with the onset of lens fibre cell nuclei degeneration. In culture, lens epithelial cells undergo differentiation into clumps, which resemble lens fibres (lentoids). Lentoids express dcrystallin and undergo nuclear DNA fragmentation (detected using TUNEL). TNFa, TNFR1, and TNFR2 are all detected in lens cultures with distributions that reflect their expression patterns in sections. The addition to cultures of TNFa or agonistic antibodies to TNFR1 and TNFR2 enhances the number of TUNELpositive nuclei in lentoids, while neutralising antibodies to TNFa reduce the number of labelled nuclei. These results suggest an involvement for TNFa and its receptors in the signalling cascade that leads to the degeneration of nuclei in lentoids and developing lens fibres and these effects could be regulated by the bcl2 protein. Role of Growth Factors and Cytokines for the Specification of Neurotransmitter Phenotype of Sympathetic Neurons.U. Ernsberger, E. Reißmann, S. Heller, M. Geißen, H. Patzke, P.H. Francis-West1, P. Brickell1, H. Rohrer Max-Planck-Institut für Hirnforschung, Deutschordenstr. 46,
60528 Frankfurt/M., Germany; 1Department of Molecular Pathology,
UCL, London, UK The development of neurons involves interactions with the environment, such as neighbouring tissues or cells in the periphery, that are innervation targets. Our work focuses on approaches to identify growth factors and receptors influencing the development of chick sympathetic neurons, in particular signals involved in the specification of the adrenergic or cholinergic phenotype. We provide evidence that members of the TGFß superfamily of growth factors are signals that direct sympathetic neuron differentiation. Bone morphogenetic proteins BMP4 and 7 have the ability to enhance the formation of adrenergic sympathetic neurons both in cultures of neural crest and when ectopically expressed in the developing chick embryo. The expression of BMP4 and 7 in the vicinity of sympathetic ganglia just before adrenergic differentiation suggests that these molecules play an important role in adrenergic development in vivo. In a second differentiation step that is controlled by signals from the innervated targets a subpopulation of sympathetic neurons acquires a cholinergic transmitter phenotype, characterized by the expression of cholineacetyltransferase (ChAT) and vasoactive intestinal peptide (VIP). Members of the cytokine family of growth factors (CNTF, GPA, LIF, OSM) induce ChAT and VIP in sympathetic neurons. These effects are mediated through receptor complexes that contain specific ligand binding subunits (i.e. CNTFRa) and a common signal transduction subunit, gp130, shared by all receptors. To provide evidence for a physiological role of these cytokines in the avian embryo, the chick homologues of CNTFRa and gp130 were cloned. The function of these receptors in cholinergic sympathetic differentiation is being tested by lossoffunction approaches, both in vitro and in vivo. Cytokine Regulation of Avian Haemopoiesis.C. Siatskas, R.L. Boyd Department of Pathology and Immunology, Monash Medical School,
Commercial Road, Prahran, Victoria, 3181, Australia In the newly hatched period chickens are particularly susceptible to disease because they do not have prolonged maternal protection and their immune defence mechanisms have not yet reached maturity. The most effective first-line defence is the innate or "natural" immune system which provides immediate protection. This is the domain of bone marrow-derived myeloid cells such as granulocytes and macrophages which develop from haemopoietic precursors under the influence of cytokines. In chickens two such factors, Stem Cell Factor (SCF) and chicken myelomonocytic growth factor (cMGF) have been identified and the relevant genes cloned. In addition, we have identified two other novel factors SSL-1 and AHGF derived from embryonic spleen and bursa stromal cell lines respectively. Using an in vitro colony forming assay and multiparameter flow cytometry we have demonstrated that cMGF, SSL-1 and AHGF have multiple, similar yet distinct, effects on haemopoietic precursor cells (from the bone marrow or embryonic spleen). In a dose-dependent manner they stimulated precursor self-renewal and induced differentiation into macrophage/granulocyte-lineage cells based on colony morphology in semi-solid medium, differential staining, non-specific esterase staining, MHC class II antigen expression and phagocytosis activity. SCF was synergistic with SSL-1 in terms of cell proliferation, but had no effect on cMGF. Similarly, cMGF was not synergistic with SSL-1. There was a developmental shift in the precursor content/responsiveness of the spleen and bone marrow, which also dictated whether the cytokines acted synergistically with SCF, SCF alone only had proliferative potential in initial experiments we have further demonstrated that these factors promote macrophage development in ovo. SSL-1 supported the growth and differentiation of human granulocyte/macrophage precursors, and murine haemopoietic cells transfected with the receptor for LIF, with no such effects seen with cMGF of SCF. SSL-1 CM did not contain IFN or IL2 and no expression of mRNA for cMGF was detected as assessed by RT-PCR and northern analysis. Thus SSL-1 produces a novel haemopoietic growth factor and also LIF-like activity. Collectively, these findings provide a rational basis to minimising the incidence of disease in newly hatched chickens and for boosting the effectiveness of immunization and immune responsiveness of older birds. Localization of EGF and TGF-ß in the Quail Ovary.L. Van Nassauw, a. Schrevens, F. Harrisson, M. Callebaut RUCA, Laboratory of Human Anatomy, 171 Groenenborgerlaan,
B-2020 Antwerpen, Belgium The present study focuses on the localization of EGF, TGF-ß1 and TGF-ß2, in the ovary of the adult Japanese quail. Antibodies directed against TGF-ß1 and TGF-ß2 were kindly provided by Genzyme Corporation (Franningham, MA). EGF was detected in smooth muscle cells, in interstitial cells, in granulosa cells and in oocytes. The number of immunostained granulosa cells decreases during folliculogenesis, and increases after ovulation. In oocytes, immunoreactivity is shifted from the Balbiani complex to the zona radiata during development. TGF-ß1 was predominantly found in granulosa cells and in oocytes. In pre-ovulatory follicles, immunoreactivity was also demonstrated in the theca interna. TGF-ß2 was mainly localized to stromal interstitial cells, to the Balbiani complex of prelampbrush oocytes, and to the zona radiata of developing oocytes. These results support the hypotheses that, as in mammals, (i) EGF primarily acts on less differentiated follicles; and (ii) TGF-ß is a paracrine and autocrine modulator of granulosa and oocyte maturation. Immune Function in the Oviduct of Laying and Molting ChickensY. Yoshimura, T. Okamoto, T. Tamura Graduate School for International Development and Cooperation,
Hiroshima University, HigashiHiroshima 739, Japan Our goal is to determine the hormonal control of the immune functions of the oviduct. Immunocompetent cells were localized in all segments of the oviduct in laying and molting chickens. MHC class II positive cells were localized in the subepithelial layer of the mucosa in laying and molting birds. In laying birds, CD3+ cells were also localized in the subepithelial layer and also occasionally in the epithelium. In molting birds, they were localized in stroma but not in subepithelial layer. Although Bu1b cells were low in population number, they showed a similar pattern of localization to that of CD3+ cells. In laying birds, strong immunoreactions for IgG, but not for IgA, were also observed in the subepithelial and epithelial layers as well as stroma. In molting birds, IgG containing cells were not observed in epithelium. The population of immunocompetent cells may be greater in the magnum and vagina. These results suggest that oviducal immune function is active in the surface area of mucosa in laying hens, whereas it is reduced in molting hens. Bursectomy in ovo Effects Circulating and Organ Inhibin Levels in Growing ChicksG. Room, D. Vanmontfort, G. Verhoeven, E. Decuypere Leuven Poultry Research Group, Kard. Mercierlaan 92 (blok E),
3001 Heverlee, Belgium Recent data support the hypothesis that the fetal adrenal is the major source of circulating inhibin, while in the laying hen the ovary is far more important than the adrenals. Furthermore, in sexually immature chicks, males show higher levels of circulating inhibin than females. These data, combined with the observation of enlarged adrenal glands in hormonally bursectomized (Bx) chicks and the occurrence of a left ovotestes in male Bx chicks, led us to measure inhibin in plasma, gonads and adrenals of Bx chicks. Embryos were hormonally bursectomized on the fifth day of incubation. At days 1, 4, 8, 15, 22, 29 and 36 of age intact and Bx chicks were weighed and blood samples were taken. Adrenals and gonads were collected, weighed and homogenised. The plasma samples and supernatants were stored at -20°C until inhibin assay. Male Bx chicks showed lower levels of plasma inhibin (P<0.0001) and a lower total testes inhibin content (P<0.0003) compared to intact chicks. However no significant differences were observed in plasma inhibin concentration and ovarian inhibin content between female intact and Bx chicks. The total adrenal inhibin content as well as the adrenal inhibin concentration were significantly higher in Bx chicks than in intact chicks (P<0.0001), regardless of the sex. In conclusion, in both intact and Bx groups the testes contained the highest amount of inhibin, followed by the adrenals and the ovary respectively, although the difference in inhibin content between the latter two was less pronounced. This explains why male Bx chicks show lower plasma inhibin levels than intact males, while in females plasma inhibin levels tend to be slightly higher in Bx compared to intact chicks. GH Pulsatility Does Not Differ between Ascites Resistant and Sensitive Broiler ChickensE. Dewil, N. Buys, J. Buyse, E. Decuypere, G. Albers1, J. Veldhuis2 Leuven Poultry Research Group, Blok E, Kard. Mercierlaan 92,
B-3001 Heverlee, Belgium; 1B.V. Euribrid, Boxmeer, The
Netherlands; 2Department of Internal Medicine, University of
Virginia, Charlottesville, VA, USA Two genetic lines, both selected for a low feed conversion ratio but with a different sensitivity for the ascites syndrome, were used in this study. The ascites syndrome is characterised by a right ventricular heart failure, resulting in accumulation of fluid in the abdominal cavity, and is responsible for high mortality in meat-type chickens. The ascites sensitive line (AS) is a paternal line characterized by a fast growth and a higher deposition of muscle tissue while the ascites resistant line (AR) is a maternal line showing a lower growth and an inferior accretion of muscle tissue. At the age of 4 weeks, 3 to 6 male birds of each line were ad random selected and were cannulated in the brachial artery. Serial blood samples (0.25 ml) were taken at 10-min intervals for a 5-h period. Plasma GH concentrations were measured by a homologous RIA and the different characteristic parameters of a GH secretion were determined by multiple-parameter deconvolution analysis. Despite the faster growth and hence higher body weight of the AS chickens (AS: 1085.2±58.5 g; AR: 717.0±27.1 g), the deconvolution analysis showed no difference in the number of secretory bursts/24 hours, the t1/2 of GH or the interval between two peaks. Although the secretory burst half-duration and the mean GH burst mass are higher in the AS birds, this difference is not significant. Notwithstanding the difference in growth between both lines there was not a clear association between the growth rate and the GH pulsatility. However, the IGF-I concentration was significantly (P<0.05) higher in AS birds (16.1 ± 2.5 ng/ml) compared to AR chickens (12.8 ± 1.9 ng/ml), which may be causative for the higher protein accretion in the AS line. Endocrine-Immune Interactions in White Crown SparrowsN. Hillgarth, J.C. Wingfield Department of Zoology, Box 351800, University of Washington,
Seattle, WA 98195-1800, USA The immunosuppressive effects of testosterone and other sex hormones in birds are reviewed. We present data on white crown sparrows showing that male birds with high testosterone levels have a depressed immune system. We discuss these results in the context of parasitemediated sexual selection theory, and suggest that corticosteroids may also play an important role in the expression of secondary sex characters as well as regulation of the immune system. |