Browsing by Author "Fuentes, J."
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- Adaptation to different salinities exposes functional specialization in the intestine of the sea bream (Sparus aurata L.)Publication . Gregorio, Silvia; Carvalho, Edison Samir Mascarelhas; Encarnação, Sandra; Wilson, J.; Power, Deborah; Canario, Adelino V. M.; Fuentes, J.The processing of intestinal fluid, in addition to a high drinking rate, is essential for osmoregulation in marine fish. This study analyzed the long-term response of the sea bream (Sparus aurata L.) to relevant changes of external salinity (12, 35 and 55p.p.t.), focusing on the anterior intestine and in the less-often studied rectum. Intestinal water absorption, epithelial HCO3– secretion and gene expression of the main molecular mechanisms (SLC26a6, SLC26a3, SLC4a4, atp6v1b, CFTR, NKCC1 and NKCC2) involved in Cl– and HCO3– movements were examined. The anion transporters SLC26a6 and SLC26a3 are expressed severalfold higher in the anterior intestine, while the expression of Atp6v1b (V-type H+-ATPase β-subunit) is severalfold higher in the rectum. Prolonged exposure to altered external salinity was without effect on water absorption but was associated with concomitant changes in intestinal fluid content, epithelial HCO3– secretion and salinity-dependent expression of SLC26a6, SLC26a3 and SLC4a4 in the anterior intestine. However, the most striking response to external salinity was obtained in the rectum, where a 4- to 5-fold increase in water absorption was paralleled by a 2- to 3-fold increase in HCO3– secretion in response to a salinity of 55p.p.t. In addition, the rectum of high salinity-acclimated fish shows a sustained (and enhanced) secretory current (Isc), identified in vitro in Ussing chambers and confirmed by the higher expression of CFTR and NKCC1 and by immunohistochemical protein localization. Taken together, the present results suggest a functional anterior–posterior specialization with regard to intestinal fluid processing and subsequently to salinity adaptation of the sea bream. The rectum becomes more active at higher salinities and functions as the final controller of intestinal function in osmoregulation.
- AVT is involved in the regulation of ion transport in the intestine of the sea bream (Sparus aurata)Publication . Martos-Sitcha, J. A.; Gregorio, Silvia; Carvalho, Edison Samir Mascarelhas; Canario, Adelino V. M.; Power, Deborah; Mancera, J. M.; Martínez-Rodriguez, G.; Fuentes, J.The intestine of marine fish plays a crucial role in ion homeostasis by selective processing of ingested fluid. Although arginine vasotocin (AVT) is suggested to play a role in ion regulation in fish, its action in the intestine has not been demonstrated. Thus, the present study investigated in vitro the putative role of AVT in intestinal ion transport in the sea bream (Sparus aurata). A cDNA encoding part of an AVT receptor was isolated and phylogenetic analysis revealed it clustered with the V1a2-type receptor clade. V1a2 transcripts were expressed throughout the gastrointestinal tract, from esophagus to rectum, and were most abundant in the rectum regardless of long-term exposure to external salinities of 12, 35 or 55 p.p.t. Basolateral addition of AVT (10 6 M) to the anterior intestine and rectum of sea bream adapted to 12, 35 or 55 p.p.t. mounted in Ussing chambers produced rapid salinity and region dependent responses in short circuit current (Isc), always in the absorptive direction. In addition, AVT stimulation of absorptive Isc conformed to a dose–response curve, with significant effects achieved at 10 8 M, which corresponds to physiological values of plasma AVT for this species. The effect of AVT on intestinal Isc was insensitive to the CFTR selective inhibitor NPPB (200 lM) applied apically, but was completely abolished in the presence of apical bumetanide (200 lM). We propose a role for AVT in the regulation of ion absorption in the intestine of the sea bream mediated by an absorptive bumetanide-sensitive mechanism, likely NKCC2.
- Branchial osmoregulatory response to salinity in the gilthead sea bream,Sparus auratusPublication . Laiz-Carrión, R.; Guerreiro, P. M.; Fuentes, J.; Canario, Adelino V. M.; Martín Del Río, María P.; Mancera, J. M.The branchial osmoregulatory response of gilthead sea bream (Sparus auratus L.) to short-term (2–192 hr) and long-term (2 weeks) exposure to different environmental salinities (5%, 15%, 25%, 38% and 60%) was investigated. A ‘‘U-shaped’’ relationship was observed between environmental salinity and gill Naþ,Kþ-ATPase activity in both long- and short-term exposure to altered salinity, with the increase in activity occurring between 24 and 96 hr after the onset of exposure. Plasma osmolality and plasma ions (sodium, chloride, calcium and potassium) showed a tendency to increase in parallel with salinity. These variables only differed significantly (Po0.05) in fish adapted to 60% salinity with respect to fish adapted to full-strength sea-water (SW). Plasma glucose remained unchanged whereas plasma lactate was elevated at 5% and 60%. Muscle water content (MWC) was significantly lower in fish adapted to 60%. Chloride cells (CC) were only present on the surface of the gill filaments and absent from the secondary lamellae. CC distribution was not altered by external salinity. However, the number and size of CC were significantly increased at salinity extremes (5% and 60%), whereas fish exposed to intermediate salinities (15% and 25%) had fewer and smaller cells. Furthermore, the CC of fish exposed to diluted SW became rounder whereas they were more elongated in fish in full-strength and hypersaline SW. This is consistent with previous reports indicating the existence of two CC types in euryhaline fish. At likely environmental salinities, gilthead sea bream show minor changes in plasma variables and the effective regulation of gill Naþ,Kþ-ATPase. However, at very low salinities both haemodilution and up-regulation of gill Naþ,Kþ-ATPase predict a poor adaptation most likely related to deficiency or absence of specific components of the CC important for ion uptake.
- Ca2+-Calmodulin regulation of testicular androgen production in Mozambique tilapia (Oreochromis mossambicus)Publication . Martins, Rute S. T.; Fuentes, J.; Almeida, O. G.; Power, Deborah; Canario, Adelino V. M.The Ca2+-Calmodulin (CaM) signaling pathway has previously been shown to be involved in the regulation of teleost fish ovarian steroidogenesis. However, a putative role of CaM in testicular steroidogenesis and potential targets has not been examined. To examine whether basal steroidogenesis is modulated by Ca2+ and CaM levels in the testis of Mozambique tilapia (Oreochromis mossambicus) we have incubated testicular fragments in vitro under different conditions and analyzed steroid output. Calcium-free medium with or without EGTA did not affect testicular basal 11-ketotestosterone (11-KT) and testosterone (T) secretion. However, addition of 80 lM the CaM inhibitor W7 significantly reduced basal 11-KT, T and androstenedione secretion. Interestingly, the decreased androgen production by 80 lM of W7 was accompanied by increased 11-desoxicortisol output and by the activation of cortisol synthesis in the testis, the latter undetected in untreated tissues. However, production of 17,20a-dihydroxy-4-pregnen- 3-one was unaltered by W7. This suggests that C17,20 desmolase, 21-hydroxylase and possibly 11bhydroxysteroid dehydrogenase are targets for CaM. In addition, androgen production was also found to be regulated by the level of cAMP since incubations with forskolin (FK) significantly increased 11-KT and T output. A cross-talk between the cAMP and Ca2+-CaM signaling pathways was detected since W7 administration also decreased FK stimulated androgen production. Altogether, these data show that both basal and cAMP stimulated androgen levels were modulated by intracellular Ca2+-dependent CaM and that possibly Ca2+-CaM determines the shift in steroidogenesis from C21 steroids to androgens.
- Calcium balance in sea bream (Sparus aurata): the effect of oestradiol-17 betaPublication . Guerreiro, P. M.; Fuentes, J.; Canario, Adelino V. M.; Power, DeborahIn all teleost fishes vitellogenesis is triggered and maintained by oestradiol-17 (E2) and is accompanied by an increase of blood plasma calcium and phosphate. The action of this hormone on calcium metabolism was investigated by treating fast-growing immature juvenile sea bream (Sparus aurata) with coconut butter implants alone (control) or implants containing 10 μg/g E2. Treatment with E2 induced the production of circulating vitellogenin, a 2·5-fold increase in plasma ionic Ca2+ and a 10-fold increase in plasma total calcium, largely bound to protein. In contrast to freshwater species, which obtain most of their calcium from the environment directly through the gills, the intestinal component of calcium uptake of the salt water-living sea bream represented up to 60–70% of the total uptake. The whole body calcium uptake, expressed as the sum of calcium obtained via intestinal and extra-intestinal (likely branchial) routes increased significantly in response to E2. Combined influx and unchanged efflux rates resulted in a significant 31% increase in net calcium uptake. There was no evidence for an effect of E2 on the calcium and phosphate content of the scales or the tartrate-resistant acid phosphatase activity (an index for bone/scale osteoclast activity). While most freshwater fish appear to rely on internal stores of calcium, i.e. bone and/or scales to increase calcium availability, the marine sea bream accommodates calcium-transporting mechanisms to obtain calcium from the environment and preserve internal stores. These observations suggest that a fundamental difference may exist in the E2-dependent calcium regulation between freshwater and marine teleosts.
- Cloning of the cDNA for sea bream (Sparus aurata) parathyroid hormone-related proteinPublication . Flanagan, J. A.; Power, Deborah; Bendell, L. A.; Guerreiro, P. M.; Fuentes, J.; Clark, M. S.; Canario, Adelino V. M.; Danks, J. A.; Brown, B. L.; Ingleton, P. M.This paper reports cloning of the cDNA for sea bream (Sparus aurata) parathyroid hormone-related protein (PTHrP). The gene codes for a 125-amino acid mature protein with a 35-residue prepeptide. The total gene sequence is 1.8 kb with approximately 75% noncoding. The N-terminus of the protein resembles mammalian and chicken PTHrP peptides with 12 of the first 21 amino acids identical and for which there is homology with mammalian parathyroid hormone. Toward the C-terminus, the nuclear transporter region between residues 79 and 93 in sea bream is 73% homologous to tetrapod PTHrP, and the RNA binding domain, 96–117, is 50% homologous, moreover starting with the conserved lysine and terminating with the lysine/arginine sequence. Sea bream PTHrP differs significantly from mammalian and chicken PTHrP, having a novel 16-amino acid segment between residues 38 and 54 and completely lacking the terminal domain associated in mammals with inhibition of bone matrix lysis. RT-PCR and in situ hybridization of sea bream tissues show that the gene is expressed widely and the results confirm observations of a PTHrP-like factor in sea bream detected with antisera to human PTHrP.
- Cortisol and parathyroid hormone-related peptide are reciprocally modulated by negative feedbackPublication . Guerreiro, P. M.; Rotllant, J.; Fuentes, J.; Power, Deborah; Canario, Adelino V. M.In previous in vitro studies, we have shown that the N-terminal region of parathyroid hormone-related protein (PTHrP) can stimulate cortisol production in sea bream, Sparus auratus, interrenal tissue, possibly through a paracrine action. In the current study, the systemic interaction between cortisol and PTHrP was studied in vivo. Sustained elevated blood cortisol levels, induced either by cortisol injection or conWnement stress, suppressed circulating PTHrP 6 and 24-fold,respectively, by comparison to control Wsh.reduced cortisol levels, prevented the decrease of plasma PTHrP observed in conWned Wsh and raised plasma PTHrPrespectively, by comparison to control fish.
- DAX1 regulatory networks unveil conserved and potentially new functionsPublication . Martins, Rute S. T.; Power, Deborah; Fuentes, J.; Deloffre, Laurence A. M.; Canario, Adelino V. M.DAX1 is an orphan nuclear receptorwith actions in mammalian sex determination, regulation of steroidogenesis, embryonic development and neural differentiation. Conserved patterns of DAX1 gene expression frommammals to fish have been taken to suggest conserved function. In the present study, the European sea bass, Dicentrarchus labrax, DAX1 promoter was isolated and its conserved features compared to other fish and mammalian DAX1 promoters in order to derive common regulators and functional gene networks. Fish andmammalian DAX1 promoters share common sets of transcription factor frameworkswhichwere also present in the promoter region of another 127 genes. Pathway analysis clustered these into candidate gene networks associated with the fish and mammalian DAX1. The networks identified are concordantwith described functions for DAX1 in embryogenesis, regulation of transcription, endocrine development and steroid production. Novel candidate gene network partners were also identified, which implicate DAX1 in ion homeostasis and transport, lipid transport and skeletal development. Experimental evidence is provided supporting roles for DAX1 in steroid signalling and osmoregulation in fish. These results highlight the usefulness of the in silico comparative approach to analyse gene regulation for hypothesis generation. Conserved promoter architecture can be used also to predict potentially newgene functions. The approach reported can be applied to genes from model and non-model species.
- Determination of tissue and plasma concentrations of PTHrP in fish: development and validation of a radioimmunoassay using a teleost 1–34 N-terminal peptidePublication . Rotllant, J.; Worthington, G. P.; Fuentes, J.; Guerreiro, P. M.; Teitsma, C. A.; Ingleton, P. M.; Balment, R.; Canario, Adelino V. M.; Power, DeborahA specific and sensitive radioimmunoassay (RIA) for the N-terminus of sea bream (Sparus auratus) and flounder (Platichthys flesus) parathyroid hormone-related protein (PTHrP) was developed. A (1–34) amino-terminal sequence of flounder PTHrP was synthesized commercially and used as the antigen to generate specific antiserum. The same sequence with an added tyrosine (1– 35Tyr) was used for iodination. Human (1–34) parathyroid hormone (PTH), human (1–34) PTHrP, and rat (1–34) PTHrP did not cross-react with the antiserum or displace the teleost peptide. Measurement of PTHrP in fish plasma was only possible after denaturing by heat treatment due to endogenous plasma binding activity. The minimum detectable concentration of (1–34) PTHrP in the assay was 2.5 pg/tube. The level of immunoreactive (1–34) PTHrP in plasma was 5.2 0.44 ng/ml (mean SEM, n ¼ 20) for flounder and 2.5 0.29 ng/ml (n ¼ 64) for sea bream. Dilution curves of denatured fish plasma were parallel to the assay standard curve, indicating that the activity in the samples was indistinguishable immunologically from (1–34) PTHrP. Immunoreactivity was present, in order of abundance, in extracts of pituitary, oesophagus, kidney, head kidney, gills, intestine, skin, muscle, and liver. The pituitary gland and oesophagus contained the most abundant levels of PTHrP, 37.7 6.1 ng/g wet tissue and 2.3 0.7 ng/g wet tissue, respectively. The results suggest that in fish PTHrP may act in a paracrine and/or autocrine manner but may also be a classical hormone with the pituitary gland as a potential major source of the protein.
- Dietary aflatoxin B1 (AFB1) reduces growth performance, impacting growth axis, metabolism, and tissue integrity in juvenile gilthead sea bream (Sparus aurata)Publication . Barany, A.; Guilloto, M.; Cosano, J.; de Boevre, M.; Oliva, M.; de Saeger, S.; Fuentes, J.; Martinez-Rodriguez, G.; Mancera, J. M.Mycotoxins are an increasing threat to all the related commodities from agriculture. Its occurrence is expected to increase due to climate change. Here, we examined the impacts of dietary toxicity of aflatoxin B1 (AFB1) in gilthead sea bream (Sparus aurata) at levels of 1 or 2 mg AFB1 kg(-1)- fish feed. Inclusion of AFB1 in the diet resulted in 80% inhibition of the total weight gain during the 85-day trial. Carbohydrate and lipid energetic metabolites, both in plasma and liver, were depleted. Moreover, the histopathological analysis revealed several tissue anomalies in the liver, kidney, and spleen. Furthermore, the relative expression of gene transcripts for growth regulation was affected by AFB1. Adenohypophyseal gh and hepatic igf1 were inversely correlated due to AFB1 effects. Relative expression levels of gene transcripts as stress indicators were increased at AFB1 highest doses, such as hypothalamic trh, crh, and crhbp, as well as star in head kidney. Interestingly circulating levels of cortisol were unaffected. Overall, our results showed that aquafeeds with AFB1 impaired growth, alter metabolism, tissue integrity, and transcriptomic responses. However, we did find AFB1 residue neither in the liver nor muscle.
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