Objetivo
Parathyroid hormone related protein is apparently a multifunctional protein produced by both normal and malignant tissues and during embryonic and fetal development in mammals and birds. Immunoreactive PTHrP was detected in plasma and tissues of larval and adult sea bream, Sparus aurata, and in other fish. These observations suggest that PTHrP has important physiological functions in fish that require investigation. The objectives of the "PTHrP in sea bream development and physiology (project FAIR-CT96-1742)" are the following:
i) The identification of the physiological roles of PTHrP in adult and juvenile sea bream. The effect of PTHrP on calcium metabolism (influx and eflux) either in relation to recombinant PTHrP administration or in the absence of biologically active PTHrP, as a result of passive immunisation with anti-PTHrP sera will be studied. The contribution of prolactin (PRL) to calcium metabolism will be assessed by passive immunisation with anti-PRL sera. The effect of PTHrP on growth will be studied by hypophysectomy and replacement therapy. In vitro culture system will be used to study the effects of calcium regulating factors in Sparus cells and tissue
ii) The determination of the functions of PTHrP in normal and dystrophic embryonic and larval development. The effects of PTHrP on Sparus embryonic and larval development will be studied by culturing eggs and larvae at defined stages in the presence or absence of PTHrP, or by applying PTHrP to specific tissues. The effect of PTHrP will be monitored using macroscopic, histological and histochemical methods. Particular attention will be paid to critical stages of metamorphosis such as mouth opening, functionality of heart, gut differentiation and swim bladder, nervous system and muscle formation. The interaction of environmental factors, such as salinity, calcium and temperature fluctuations with PTHrP expression and occurrence of axial dysplasias will also be studied.
iii) Cloning the gene for PTHrP receptor(s) in sea bream. cDNA libraries will be prepared from Sparus tissues and used to clone the gene for the PTH/PTHrP receptor; its tissue distribution and level of expression in different physiological/developmental conditions will be determined.
The results obtained so far indicate that Sparus accumulates calcium for growth and development and that the gills play a significant role in the calcium balance. In particular very young fish seem to take up calcium from the water at a very high rate. No effect of hPTHrP injections on any form of calcium uptake from the water or on plasma calcium levels has yet been found.
For other aspects of the physiology of PTHrP an antiserum which reacts specifically with PTHrP was required and this has been developed in rabbits using a short pentapeptide region of the molecule which is common to tetrapod PTHrP already analysed. The specificity of the antiserum has been checked by immunohistochemistry of Sparus tissues.
Examination of tissues from normal and dystrophic juvenile Sparus has shown that the distribution and abundance of immunoreactive PTHrP (irPTHrP) protein and its gene expression differ in the two conditions. The protein is present in brain and spinal cord, pituitary, skin, muscle, gills, kidney, motor neurones and dorsal root ganglia and remnant notochord cells. However, the dystrophic spinal columns in addition to these sites of irPTHrP also retain chondrocytes in the areas of cartilage overgrowth in the spinal column.
Primary cell cultures from sea bream bone tissues can now be easily prepared. These cells are therefore a first hand material that will unable us to look for specific gene expression and regulation.
A clear description of the chronology of development of the diverse bone structures, head, vertebral column, and fins has been generated in sea bream.
PRC probes for myosin, alpha-actin and gamma-actin are now available and can be used to study sea bream development and the effect of PTHrP on development. cDNA libraries including a kidney library developed in this project is now available to isolate PTHrP and its receptor.
DISCUSSION
Since the project is still at an early stage it is only possible to speculate on the roles of PTHrP. So far we have not been able to show effects of hPTHrP injections on any form of calcium uptake from the water or on plasma calcium levels. In the coming year other doses and administration regimes will be evaluated once more for their effect on branchial calcium handling. Also conditions will be set in which calcium uptake is stimulated (enhanced growth, salinity variations).
The effect of PTHrP on bone cell lines can now be studied before in vivo experiments are to be done in order to save costly hormone.
After some preliminary experiments the technique of hypophysectomy will be developed for the sea bream and hormone replacement therapy experiments will be carried using several different hormones.
The effect of PTHrP on early development will be studied and the distribution of muscle proteins will be studied with oligonucleotide probes.
Finally it is expected to have isolated and characterised the sea bream PTHrP receptor.
ACKNOWLEDGMENTS
The study has been carried out with financial support from the Commission of the European Communities, Agriculture and Fisheries (FAIR) specific RTD programme, CT96-1742 "PTHrP in sea bream development and physiology". It does not necessarily reflect its views and in no way anticipates the Commission's future policy in this area"
REFERENCES
Danks, J. A., A. J. Devlin, et al. (1993). "Parathyroid hormone-related protein is a factor in normal fish pituitary." Gen Comp Endocrinol 92(2):201-12.
Devlin, A. J., J. A. Danks, et al. (1996). "Immunochemical detection of parathyroid hormone-related protein in the saccus vasculosus of a teleost fish." Gen Comp Endocrinol 101(1): 83-90.
Ingleton, P. M., N. Hazon, et al. (1995). "Immunodetection of parathyroid hormone-related protein in plasma and tissues of an elasmobranch (Scyliorhinus canicula)." Gen Comp Endocrinol 98(2): 211-8.
Karaplis, A. C., A. Luz, et al. (1994). "Lethal skeletal dysplasia from targeted disruption of the parathyroid hormone-related peptide gene." Genes Dev 8(3): 277-89.
Kong, X. F., E. Schipani, et al. (1994). "The rat, mouse, and human genes encoding the receptor for parathyroid hormone and parathyroid hormone-related peptide are highly homologous." Biochem Biophvs Res Commun 201(2): 1058.
INTRODUCTION
Parathyroid hormone-related protein (PTHrP) was identified as a factor in fish (Sparus aurata) pituitary and plasma using antisera to human PTHrP (Danks, Devlin et al. 1993). Since then we have shown that immunological PTHrP is present in tissues and plasma of elasmobranch (Ingleton, Hazon et al. 1995), in the sacus vasculosus of Sparus (Devlin, Danks et al. 1996) and in the urophysis and corpuscles of Stannius of the euryhaline teleost Plachthys flesus. These observations suggest that PTHrP has important physiological functions in fish that require investigation. The cross-reactivity between antibodies to human PTHrP and fish tissues indicate that there has been evolutionary conservation of the molecular structure and composition so predicting that the functions of PTHrP are fundamental and vital. Thus functions of PTHrP in mammals are likely to have originated from those established in fish and hence form the basis for areas of investigation in fish species. Furthermore the vital importance of PTHrP is reinforced by studies in mice showing that offspring in which the gene has been deleted fail to survive the perinatal period (Karaplis, Luz et al. 1994).
Because fish do not have a parathyroid gland it is possible that PTHrP acts as a hypercalcaemic agent and its location in the kidney tubules, corpuscles of Stannius and saccus vasculosus of some teleosts and rectal gland elasmobranch suggest that it may be involved in control of ion balance. However, unlike mammals and birds, fish appear to have high circulating concentrations of PTHrP, which may be derived from the pituitary (Danks, Devlin et al. 1993), suggesting that, in this group of vertebrates, it acts as a classical hormone in adults in addition to local tissue interactions. It is more likely that PTHrP functions as a paracrine or autocrine factor in the embryonic and larval development of fish, as it does in chickens and mammals.
PTHrP is a candidate factor involved in both normal and abnormal development of sea bream embryos and larvae. The production of PTHrP may depend both upon intrinsic and environmental factors at the early stages of development of sea bream and it is important to determine both PTHrP gene expression during development and to investigate the environmental factors which may be involved in affecting both this expression and the induction of axial malformations. It seems likely that PTHrP is a factor involved in the embryonic development of sea bream tissues and particularly affecting the integrated differentiation of the vertebral column, spinal cord and segmental muscles. PTHrP also has potential involvement in development and function of the swim bladder, which also fails to grow properly in dystrophic.
In order to determine the actions of PTHrP in Sparus it is necessary to demonstrate which tissues are susceptible to PTHrP action through specific receptors. The gene for the common PTH/PTHrP receptor in mammals, the only receptor so far demonstrated for PTHrP, is highly conserved between tissues and species examined and prescribes a G-protein-linked seventrans membrane domain construct (Kong, Schipani et al. 1994). Such receptor forms appear to be conserved across vertebrate groups.
The objectives of this project are the identification of the physiological roles of PTHrP in adult and juvenile sea bream, the determination of the functions of PTHrP in normal and dystrophic embryonic and larval development and the cloning the gene for PTHrP receptor(s) in sea bream.
Ámbito científico
- medical and health sciencesbasic medicineimmunologyimmunisation
- natural scienceschemical sciencesinorganic chemistryalkaline earth metals
- natural sciencesbiological sciencesbiochemistrybiomoleculesproteins
- medical and health sciencesclinical medicineobstetricsfetal medicine
- medical and health sciencesbasic medicinephysiology
Tema(s)
Convocatoria de propuestas
Data not availableRégimen de financiación
CSC - Cost-sharing contractsCoordinador
8000 FARO
Portugal