Armin Sturm

Corticosteroid receptors involved in stress regulation in common carp, Cyprinus carpio

In higher vertebrates, mineralo- (aldosterone) and glucocorticoids (cortisol/corticosterone) exert their multiple actions via specific transcription factors, glucocorticoid (GR) and mineralocorticoid (MR) receptors. Teleostean fishes lack aldosterone and mineral regulatory processes seem under dominant control by cortisol. Despite the absence of the classical mineralocorticoid aldosterone, teleostean fishes do have an MR with cortisol and possibly 11-deoxycorticosterone (DOC) (as alternative for aldosterone) as predominant ligands. We studied corticoid receptors in common carp (Cyprinus carpio L). Through homology cloning and bioinformatic analysis, we found duplicated GR genes and a single MR gene. The GR genes likely result from a major genomic duplication event in the teleostean lineage; we propose that the gene for a second MR was lost. Transactivation studies show that the carp GRs and MR have comparable affinity for cortisol; the MR has significantly higher sensitivity to DOC, and this favours a role for DOC as MR ligand in fish physiology. mRNA of the GRs and the MR is expressed in forebrain (in pallial areas homologous to mammalian hippocampus), corticotrophin-releasing hormone (CRH) cells in the pre-optic nucleus (NPO) and pituitary pars distalis ACTH cells, three key neural/endocrine components of the stress axis. After exposure to prolonged and strong (not to mild acute) stressors, mRNA levels of both GRs and MR become down-regulated in the brain, but not in the NPO CRH cells or pituitary ACTH cells. Our data predicts a function in stress physiology for all CRs and suggest telencephalon as a first line cortisol target in stress.

The ABC transporter gene family of Daphnia pulex

BackgroundThe large gene superfamily of ABC (ATP-binding cassette) transporters encodes membrane proteins involved in trafficking processes across biological membranes and further essential cell biological functions. ABC transporters are evolutionary ancient and involved in the biochemical defence against toxicants. We report here a genome-wide survey of ABC proteins of Daphnia pulex, providing for the first time information on ABC proteins in crustacea, a primarily aquatic arthropod subphylum of high ecological and economical importance.ResultsWe identified 64 ABC proteins in the Daphnia genome, which possesses members of all current ABC subfamilies A to H. To unravel phylogenetic relationships, ABC proteins of Daphnia were compared to those from yeast, worm, fruit fly and human. A high conservation of Daphnia of ABC transporters was observed for proteins involved in fundamental cellular processes, including the mitochondrial half transporters of the ABCB subfamily, which function in iron metabolism and transport of Fe/S protein precursors, and the members of subfamilies ABCD, ABCE and ABCF, which have roles in very long chain fatty acid transport, initiation of gene transcription and protein translation, respectively. A number of Daphnia proteins showed one-to-one orthologous relationships to Drosophila ABC proteins including the sulfonyl urea receptor (SUR), the ecdysone transporter ET23, and the eye pigment precursor transporter scarlet. As the fruit fly, Daphnia lacked homologues to the TAP protein, which plays a role in antigene processing, and the cystic fibrosis transmembrane conductance regulator (CFTR), which functions as a chloride channel. Daphnia showed two proteins homologous to MDR (multidrug resistance) P-glycoproteins (ABCB subfamily) and six proteins homologous to MRPs (multidrug resistance-associated proteins) (ABCC subfamily). However, lineage specific gene duplications in the ABCB and ABCC subfamilies complicated the inference of function. A particularly high number of gene duplications were observed in the ABCG and ABCH subfamilies, which have 23 and 15 members, respectively.ConclusionThe in silico characterisation of ABC transporters in the Daphnia pulex genome revealed that the complement of ABC transporters is as complex in crustaceans as that other metazoans. Not surprisingly, among currently available genomes, Daphnia ABC transporters most closely resemble those of the fruit fly, another arthropod.

Stress and innate immunity in carp: corticosteroid receptors and pro-inflammatory cytokines.

The stress hormone cortisol is deeply involved in immune regulation in all vertebrates. Common carp (Cyprinus carpio L.) express four corticoid receptors that may modulate immune responses: three glucocorticoid receptors (GR); GR1, with two splice variants (GR1a and GR1b), GR2 and a single mineralocorticoid receptor (MR). All receptors are expressed as of 4 days post-fertilization and may thus play a critical role in development and functioning of the adult immune system. Immune tissues and cells predominantly express mRNA for GRs compared to mRNA for the MR. Three-dimensional protein structure modeling predicts, and transfection assays confirm that alternative splicing of GR1 does not influence the capacity to induce transcription of effector genes. When tested for cortisol activation, GR2 is the most sensitive corticoid receptor in carp, followed by the MR and GR1a and GR1b. Lipopolysacharide (LPS) treatment of head kidney phagocytes quickly induces GR1 expression and inhibits GR2 expression. Cortisol treatment in vivo enhances GR1a and MR mRNA expression, but only mildly, and cortisol treatment in vitro does not affect receptor expression of phagocytes. Cortisol has no direct effect on the LPS-induced receptor profile. Therefore, an immune rather than a stress stimulus regulates GR expression. Cortisol administered at stress levels to phagocytes in vitro significantly inhibits LPS-induced expression of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin-12 (IL-12) (subunit p35) and of inducible nitric oxide synthase (iNOS) expression. A physiologically differential function for GR1 and GR2 in the immune response of fish to infection is indicated.

Plasma 11-deoxycorticosterone (DOC) and mineralocorticoid receptor testicular expression during rainbow trout Oncorhynchus mykiss spermiation: implication with 17alpha, 20beta-dihydroxyprogesterone on the milt fluidity?

BackgroundIn rainbow trout (Oncorhynchus mykiss), the endocrine control of spermiation is not fully understood. Besides 11ketotestosterone (11KT) and 17alpha, 20beta-dihydroxyprogesterone (MIS), the potential physiological ligand of the mineralocorticoid receptor (MR) 11-deoxycorticosterone (DOC), is a credible candidate in O. mykiss spermiation regulation as spermiation is accompanied with changes in aqueous and ionic flows.MethodsIn this study, we investigated potential roles of DOC during spermiation 1) by describing changes in blood plasma DOC level, MR mRNA abundance during the reproductive cycle and MR localization in the reproductive tract 2) by investigating and comparing the effects of DOC (10 mg/kg) and MIS (5 mg/kg) supplementations on sperm parameters 3) by measuring the in vitro effect of DOC on testis MIS production.ResultsThe plasma concentration of DOC increased rapidly at the end of the reproductive cycle to reach levels that were 10–50 fold higher in mature males than in immature fish. MR mRNA relative abundance was lower in maturing testes when compared to immature testes, but increased rapidly during the spermiation period, immediately after the plasma rise in DOC. At this stage, immunohistochemistry localized MR protein to cells situated at the periphery of the seminiferous tubules and in the efferent ducts. Neither DOC nor MIS had significant effects on the mean sperm volume, although MIS treatment significantly increased the percentage of males producing milt. However, a significant reduction in the spermatocrit was observed when DOC and MIS were administrated together. Finally, we detected an inhibitory effect of DOC on testis MIS production in vitro.ConclusionThese results are in agreement with potential roles of DOC and MR during spermiation and support the hypothesis that DOC and MIS mechanisms of action are linked during this reproductive stage, maybe controlling milt fluidity. They also confirm that in O. mykiss MIS is involved in spermiation induction.

Molecular cloning and characterisation of a novel P-glycoprotein in the salmon louse Lepeophtheirus salmonis

The salmon louse, Lepeophtheirus salmonis, is a crustacean ectoparasite of salmonid fish. At present, sea louse control on salmon farms relies heavily upon chemical treatments. Drug efflux transport, mediated by ABC transporters such as P-glycoprotein (Pgp), represents a major mechanism for drug resistance in parasites. We report here the molecular cloning of a new Pgp from the salmon louse, called SL-PGY1. A partial Pgp sequence was obtained by searching sea louse ESTs, and extended by rapid amplification of cDNA ends (RACE). The open reading frame of SL-PGY1 encodes a protein of 1438 amino acids that possesses typical structural traits of P-glycoproteins, and shows a high degree of sequence homology to invertebrate and vertebrate P-glycoproteins. In the absence of drug exposure, SL-PGY1 mRNA expression levels did not differ between a drug-susceptible strain of L. salmonis and a strain showing a ~7-fold decrease in sensitivity against emamectin benzoate, the active component of the in-feed sea louse treatment SLICE (Merck Animal Health). Aqueous exposure of the hyposensitive salmon louse strain to emamectin benzoate (24h, 410 μg/L) provoked a 2.9-fold upregulation of SL-PGY1. Adult male lice of both strains showed a greater abundance of SL-PGY1 mRNA than adult females.

Implication of the mineralocorticoid axis in rainbow trout osmoregulation during salinity acclimation

Cortisol and glucocorticoid receptors (GRs) play an important role in fish osmoregulation, whereas the involvement of the mineralocorticoid receptor (MR) and its putative ligand 11-deoxycorticosterone (DOC) is poorly investigated. In this study, we assessed the implication of DOC and MR in rainbow trout (Oncorhynchus mykiss) osmoregulation during hypo- and hypersaline acclimation in parallel with the cortisol-GR system. A RIA for DOC was developed to measure plasma DOC levels, and a MR-specific antibody was developed to localize MR protein in the gill, intestine, and kidney. This is the first study to report DOC plasma levels during salinity change and MR localization in fish osmoregulatory tissue. Corticosteroid receptor mRNA abundance was investigated in osmoregulatory tissue during salinity acclimation, and the effect of cortisol and DOC on ionic transporters gene expression was assayed using an in vitro gill incubation method. Differential tissue-, salinity-, and time-dependent changes in MR mRNA levels during both hyper- and hyposaline acclimations and the ubiquitous localization of MR in osmoregulatory tissue suggest a role for the MR in osmoregulation. Presumably, DOC does not act as ligand for MR in osmoregulation because there were no changes in plasma DOC levels during either freshwater-seawater (FW-SW) or SW-FW acclimation or any effect of DOC on gill ionic transporter mRNA levels in the gill. Taken together, these results suggest a role for MR, but not for DOC, in osmoregulation and confirm the importance of cortisol as a major endocrine regulator of trout osmoregulation.

Identification of a sex-linked SNP marker in the salmon louse (Lepeophtheirus salmonis) using RAD sequencing.

The salmon louse (Lepeophtheirus salmonis (Krøyer, 1837)) is a parasitic copepod that can, if untreated, cause considerable damage to Atlantic salmon (Salmo salar Linnaeus, 1758) and incurs significant costs to the Atlantic salmon mariculture industry. Salmon lice are gonochoristic and normally show sex ratios close to 1:1. While this observation suggests that sex determination in salmon lice is genetic, with only minor environmental influences, the mechanism of sex determination in the salmon louse is unknown. This paper describes the identification of a sex-linked Single Nucleotide Polymorphism (SNP) marker, providing the first evidence for a genetic mechanism of sex determination in the salmon louse. Restriction site-associated DNA sequencing (RAD-seq) was used to isolate SNP markers in a laboratory-maintained salmon louse strain. A total of 85 million raw Illumina 100 base paired-end reads produced 281,838 unique RAD-tags across 24 unrelated individuals. RAD marker Lsa101901 showed complete association with phenotypic sex for all individuals analysed, being heterozygous in females and homozygous in males. Using an allele-specific PCR assay for genotyping, this SNP association pattern was further confirmed for three unrelated salmon louse strains, displaying complete association with phenotypic sex in a total of 96 genotyped individuals. The marker Lsa101901 was located in the coding region of the prohibitin-2 gene, which showed a sex-dependent differential expression, with mRNA levels determined by RT-qPCR about 1.8-fold higher in adult female than adult male salmon lice. This study’s observations of a novel sex-linked SNP marker are consistent with sex determination in the salmon louse being genetic and following a female heterozygous system. Marker Lsa101901 provides a tool to determine the genetic sex of salmon lice, and could be useful in the development of control strategies.

Chapter 18 P-glycoproteins and xenobiotic efflux transport in fish

Publisher Summary This chapter reviews the more recent data and focuses on P-glycoproteins (P-gp) in fish and general biochemistry of P-gp. Biological membranes are barriers for the uptake, distribution, and elimination of xenobiotics in organisms. According to a traditional view in toxicology, the bioaccumulation of organic chemicals occurs mainly passively, driven by the compounds hydrophobicity. Once bioaccumulated, the xenobiotic may undergo phase I and II biotransformation metabolisms, the products of which in general are less toxic than the mother compound. Hydrophobicity and biotransformation rate have traditionally been considered the main factors determining whether a compound is adequately detoxified. The chapter explains general aspects of P-glycoprotein and P-glycoproteins in aquatic animals. General aspect of P-glycoprotein is explained with the help of ATP binding cassette (ABC) transporters, functional and structural characteristics of P-glycoprotein, and physiological function of P-glycoprotein. The functional ABC transporter comprises multiple functional units organized in a characteristic fashion. The most astonishing characteristic of P-gp is that it transports structurally and functionally very diverse substrates.

Tributyltin is a potent inhibitor of piscine peroxisome proliferator-activated receptor α and β

Increasing evidence suggests that common environmental contaminants can act as endocrine disrupters in fish. However, current data are biased towards environmental estrogens, highlighting the need to elucidate potential pollutant impact on other endocrine axes. Here, we report a high-throughput assay to identify chemicals interacting with piscine peroxisome proliferator-activated receptors (PPARs). Our transactivation assay employs a fish cell line and uses recombinant proteins combining the yeast Gal4 DNA-binding domain with the ligand-binding domain of PPARs from plaice (Pleuronectes platessa). Compared to assays with full-length PPARs, this approach circumvents interaction of chemicals binding to retinoid X receptors, which form heterodimers with PPAR and many other nuclear receptors. Plaice PPARα and PPARβ are activated by fibrate drugs and by phthalate mono-esters at concentrations similar to those activating the homologous mammalian receptors. In line with their assumed role as central transcriptional regulators of energy homeostasis, a number of fatty acids activate plaice PPARα and PPARβ. In contrast, tributyl tin oxide (TBTO) is a potent antagonist of PPARα and PPARβ, showing activity at environmentally relevant concentrations of TBTO (1-50 nM). Given the ubiquitous and persistent nature of TBTO, the possibility that chronic environmental effects are occurring via disruption of PPAR signalling in fish should be further investigated.

Molecular determinants of hormone sensitivity in rainbow trout glucocorticoid receptors 1 and 2.

Many teleost fish possess two glucocorticoid receptors (GR). In the rainbow trout rtGR1 and rtGR2 differ in their affinities to dexamethasone and EC50 values for glucocorticoids in transactivation assays, with rtGR2 being more sensitive. The objective of this study was to identify the molecular traits underlying the sensitivity difference. Domain-swap mutants between rtGR1 and rtGR2 showed that sensitivity was mainly determined by the hormone binding domain (E-domain). Chimeras exchanging three E-domain subregions indicated that all subregions influenced sensitivity, with the most C-terminal region that included AF2 having the greatest (12.6-fold) effects on cortisol transactivation EC50. The C-terminal extremity (CTE) in rtGR1 departs from a consensus preserved in other GRs. Introducing the consensus CTE into rtGR1 provoked a 4.2-fold decrease in transactivation EC50, suggesting CTE is one of several determinants of rtGR1s hyposensitivity. GRs with similar unusual CTEs exist in other salmonids, suggesting hyposensitive GR have evolved in this highly successful teleost lineage.