Immunohistochemical detection of estrogen receptors in fish scales.

Calcium mobilization from internal stores, such as scales, induced by 17beta-estradiol during sexual maturation in salmonids is well documented. This calcium mobilization from scales is proposed to be mediated by the estrogen receptor (ER). However, the ER subtypes involved and signaling mechanisms responsible for this effect remain to be fully characterized. In the present study, we have localized ERalpha, ERbetaa and ERbetab proteins in juvenile and adult sea bream (Sparus auratus) and Mozambique tilapia (Oreochromis mossambicus) scales by immunohistochemistry with sea bream ER subtype specific antibodies. The three ERs were detected in isolated or small groups of round cells, in the basal layer of the scales of both juvenile and adult fish and the localization and signal intensity varied with the species and age of the animals. The ERs may be co-localized in cells of the scale posterior region that expressed tartrate-resistant acid phosphatase (TRAP), a marker for osteoclasts. These results suggest that the calcium mobilizing action of 17beta-estradiol on fish scales is via its direct action on ERs localized in osteoclasts.

Cloning of the glucocorticoid receptor (GR) in gilthead seabream (Sparus aurata). Differential expression of GR and immune genes in gilthead seabream after an immune challenge.

In order to determine the cortisol response after an immune challenge in the gilthead seabream (Sparus aurata), a cortisol receptor (GR) was cloned, sequenced and its expression determined after lipopolysaccharide (LPS) treatment. To clone the gilthead seabream GR (sbGR), consecutive PCR amplifications and screening of a pituitary cDNA library were performed. We obtained a clone of 4586 bp encoding a 784aa protein. Northern blot analysis from head kidney, heart and intestine revealed that the full length sbGR mRNA was approximately 6.5 Kb. A LPS treatment, used as an acute stress model, was employed to characterise the expression of sbGR and some selected genes involved in the immune response (IL-1beta, TNF-alpha, Mx protein, cathepsin D and PPAR-gamma). All genes were expressed in all tissues examined and responses were tissue and time dependent revealing differential gene expression profiles after LPS administration. Furthermore, analysis of plasma cortisol levels after LPS injection, showed an acute response to inflammatory stress with a significant increase two and six h after injection, recovering to basal levels 12 h post-stress in all LPS concentrations tested.

Ligand binding and signalling pathways of PTH receptors in sea bream (Sparus auratus) enterocytes.

Whole animal studies have indicated that Ca(2+) uptake by the gastrointestinal tract is regulated by the action of parathyroid hormone-related peptide (PTHrP) in teleost fish. We have characterised PTH receptors (PTHR) in piscine enterocytes and established, by using amino-terminal PTHrP peptides, the amino acid residues important for receptor activation and for stabilising the ligand/receptor complex. Ligand binding of (125)I-(1-35(tyr)) PTHrP to the membrane fraction of isolated sea bream enterocytes revealed the existence of a single saturable high-affinity receptor (K (D)=2.59 nM; B (max)=71 fmol/mg protein). Reverse transcription/polymerase chain reaction with specific primers for sea bream PTH1R and PTH3R confirmed the mRNA expression of only the later receptor. Fugu (1-34)PTHrP increased cAMP levels in enterocytes but had no effect on total inositol phosphate accumulation. The amino-terminal peptides (2-34)PTHrP, (3-34)PTHrP and (7-34)PTHrP bound efficiently to the receptor but were severely defective in stimulating cAMP in enterocyte cells indicating that the first six residues of piscine (1-34)PTHrP, although not important for receptor binding, are essential for activation of the adenylate cyclase/phosphokinase A (AC-PKA)-receptor-coupled intracellular signalling pathway. Therefore, PTHrP in teleosts acts on the gastrointestinal tract through PTH3R and the AC-PKA intracellular signalling pathway and might regulate Ca(2+) uptake at this site. Ligand-receptor binding and activity throughout the vertebrates appears to be allocated to the same amino acid residues of the amino-terminal domain of the PTHrP molecule.

Calcium mobilization from fish scales is mediated by parathyroid hormone related protein via the parathyroid hormone type 1 receptor.

The scales of bony fish represent a significant reservoir of calcium but little is known about their contribution, as well as of bone, to calcium balance and how calcium deposition and mobilization are regulated in calcified tissues. In the present study we report the action of parathyroid hormone-related protein (PTHrP) on calcium mobilization from sea bream (Sparus auratus) scales in an in vitro bioassay. Ligand binding studies of piscine 125I-(1-35(tyr))PTHrP to the membrane fraction of isolated sea bream scales revealed the existence of a single PTH receptor (PTHR) type. RT-PCR of fish scale cDNA using specific primers for two receptor types found in teleosts, PTH1R, and PTH3R, showed expression only of PTH1R. The signalling mechanisms mediating binding of the N-terminal amino acid region of PTHrP were investigated. A synthetic peptide (10(-8) M) based on the N-terminal 1-34 amino acid residues of Fugu rubripes PTHrP strongly stimulated cAMP synthesis and [3H]myo-inositol incorporation in sea bream scales. However, peptides (10(-8) M) with N-terminal deletions, such as (2-34), (3-34) and (7-34)PTHrP, were defective in stimulating cAMP production but stimulated [3H]myo-inositol incorporation. (1-34)PTHrP induced significant osteoclastic activity in scale tissue as indicated by its stimulation of tartrate-resistant acid phosphatase. In contrast, (7-34)PTHrP failed to stimulate the activity of this enzyme. This activity could also be abolished by the adenylyl cyclase inhibitor SQ-22536, but not by the phospholipase C inhibitor U-73122. The results of the study indicate that one mechanism through which N-terminal (1-34)PTHrP stimulates osteoclastic activity of sea bream scales, is through PTH1R and via the cAMP/AC intracellular signalling pathway. It appears, therefore, that fish scales can act as calcium stores and that (1-34)PTHrP regulates calcium mobilization from them; it remains to be established if this mechanism contributes to calcium homeostasis in vivo.

Ontogeny of osteonectin expression in embryos and larvae of sea bream (Sparus auratus).

Osteonectin (OSN) is a glycoprotein which is implicated in development, bone formation and mineralisation, tumorigenesis, angiogenesis, and wound healing. Regulation of its expression by hormones may be one of the mechanisms by which the endocrine system affects bone metabolism. As a first step to understanding OSN function in fish, the gene expression of the recently cloned cDNA for sea bream, Sparus auratus, osteonectin (sbOSN) was characterised during embryonic and larval development. sbOSN mRNA was first detected by semi-quantitative reverse transcription-polymerase chain reaction in embryos at early gastrula and its expression increased continuously until hatch, after which it decreased until 15 days post-hatch (dph), increased transiently until 24 dph and decreased thereafter. In situ hybridisation showed it had a differential tissue distribution which was age dependent. In general, sbOSN mRNA was identified in cartilaginous and calcified structures of both dermal and endochondral origin but its expression was not restricted to the skeleton. sbOSN transcripts were also detected in the skin, perichordal sheath, nerve cord, and kidney tubules.

Stimulation of cortisol release by the N terminus of teleost parathyroid hormone-related protein in interrenal cells in vitro.

The mode of action of PTHrP in the regulation of sea bream (Sparus auratus) interrenal cortisol production was studied in vitro using a dynamic superfusion system. Piscine (1-34)PTHrP (10(-6)-10(-11) M) stimulated cortisol production in a dose-dependent manner. The ED50 of (1-34)PTHrP was 2.8 times higher than that of (1-39)ACTH, and maximum increase in cortisol production in response to 10(-8) M of (1-34)PTHrP was approximately 7-fold lower than for 10(-8) M of (1-39)ACTH. In contrast to (1-34)PTHrP, piscine (10-20)PTHrP, (79-93)PTHrP, and (100-125)PTHrP (10(-9)-10(-7) M) did not stimulate cortisol production. The effect of piscine (1-34)PTHrP on cortisol production was abolished by N-terminal peptides in which the first amino acid (Ser) was absent and by simultaneous addition of inhibitors of the adenylyl cyclase-protein kinase A and phospholipase C-protein kinase C intracellular pathways but not by each separately. The PTHrP-induced signal transduction was further investigated by measurements of cAMP production and [H3]myo-inositol incorporation in an interrenal cell suspension. Piscine (1-34)PTHrP increased cAMP and total inositol phosphate accumulation, which is indicative that the mechanism of action of PTHrP in interrenal tissue involves the activation of both the adenylyl cyclase-cAMP and phospholipase C-inositol phosphate signaling pathways. These results, together with the expression of mRNA for PTHrP and for PTH receptor (PTHR) type 1 and PTHR type 3 receptors in sea bream interrenal tissue, suggest a specific paracrine or autocrine steroidogenic action of PTHrP mediated by the PTHRs.

Multiple regulatory elements control the expression of the yeast ACR1 gene.

The ACR1 gene, encoding a succinate-fumarate transporter, is required by the yeast Saccharomyces cerevisiae for ethanol utilization. Accordingly, gene expression is induced by ethanol and repressed by glucose. Here, we investigated three carbon source response elements present in its promoter region. Specific deletions as well as functional analysis of the elements in a heterologous promoter confirmed their role in transcriptional regulation. Protein binding to carbon source response elements of the ICL1 promoter was competed by all three elements to various extents by the respective ACR1 sequences. In addition, two putative stress response promoter elements present in the ACR1 promoter were investigated in deletion analyses and shown to contribute to gene expression.