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P Prunet

Publications and source records attributed to P Prunet.

At least 19 recordsLinked to original sources
PubMed2006

Multiple corticosteroid receptors in fish: from old ideas to new concepts.

The effect of corticosteroid hormones in fish are mediated through intracellular receptors that act as ligand-binding transcription factors. Many studies have been devoted to cortisol binding using radiolabeled ligand in fish and allowed characterization of a single class of high affinity binding sites in various tissues. Molecular characterization of cortisol receptors has only been initiated recently by cloning the different receptor forms: Following a isolation of a first glucocorticoid receptor (GR), a mineralocorticoid receptor (MR) was described and the presence of various GR isoforms was recently reported. Sequence comparison and phylogenetic analysis of these sequences confirm that fish possess both GR and MR and that GR gene is duplicated. The importance of these various corticosteroid receptor forms is also illustrated by analysis of their transcriptional activity. When tested in human cell lines, these receptors showed functionally distinct actions on GR-sensitive promotors, thus suggesting a more complicated corticosteroid signaling system than initially anticipated from binding studies. These results also suggest that, whereas cortisol is certainly the physiological ligand for GR, this may not be the case for MR which showed high sensitivity for deoxycorticosterone (DOC) and aldosterone. As this last hormone is probably absent in fish, these results raise the question as to whether DOC could be a physiological ligand for MR in fish. Information on DOC effect in fish is very scarce and clarification of the differential osmoregulatory roles of cortisol and DOC in fish needs ellucidation. This will require analysis of all actors of the corticosteroid signaling system at pre-receptor, receptor, and post-receptor levels.

Amino Acid Sequence↗
PubMed2004

11-deoxycorticosterone is a potent agonist of the rainbow trout (Oncorhynchus mykiss) mineralocorticoid receptor.

The teleost fish are thought to lack the mineralocorticoid hormone aldosterone but possess mineralocorticoid receptor (MR) homologs. Here we describe the characterization of two rainbow trout (Oncorhynchus mykiss) MRs, called rtMRa and rtMRb. The open reading frame of rtMRa cDNA encoded a protein of 1041 amino acids. The rtMRb predicted protein sequence is similar, differing in only 10 amino acids in the nonconserved A/B domain and lacking a three-amino acid insertion between the two zinc fingers of the C domain. Expression of rtMR mRNA (sum of both forms), measured in juvenile trout by real-time RT-PCR, shows that the transcripts are ubiquitous. Expression was significantly higher in brain than the other tissues studied (eye, trunk kidney, head kidney, gut, gills, liver, spleen, ovary, heart, white muscle, skin). Hormonal stimulation of receptor transactivation activity was studied in COS-7 cells transiently cotransfected with receptor cDNA and a mouse mammary tumor virus-luciferase reporter. The mineralocorticoids 11-deoxycorticosterone and aldosterone were more potent enhancers of rtMRa transcriptional activity (EC50 = 1.6 +/- 0.5 x 10(-10) and 1.1 +/- 0.4 x 10(-10) M, respectively) than the glucocorticoids cortisol and 11-deoxycortisol (EC50 = 1.1 +/- 0.3 x 10(-9) and 3.7 +/- 1.9 x 10(-9) M, respectively). A similar response was observed in transactivation assays with rtMRb. These results are discussed in the view of reported circulating levels of corticosteroids in trout.

Aldosterone↗
PubMed2003

Evidence for two distinct functional glucocorticoid receptors in teleost fish.

Using RT-PCR with degenerated primers followed by screening of a rainbow trout (Oncorhynchus mykiss) intestinal cDNA library, we have isolated from the rainbow trout a new corticosteroid receptor which shows high sequence homology with other glucocorticoid receptors (GRs), but is clearly different from the previous trout GR (named rtGR1). Phylogenetic analysis of these two sequences and other GRs known in mammals, amphibians and fishes indicate that the GR duplication is probably common to most teleost fish. The open reading frame of this new trout GR (named rtGR2) encodes a protein of 669 amino acids and in vitro translation produces a protein of 80 kDa that appears clearly different from rtGR1 protein (88 kDa). Using rtGR2 cDNA as a probe, a 7.3 kb transcript was observed in various tIssues suggesting that this gene would lead to expression of a steroid receptor. In vitro studies were used to further characterize this new corticosteroid receptor. Binding studies with recombinant rtGR1 and rtGR2 proteins show that the two receptors have a similar affinity for dexamethasone (GR1 K(d)=5.05+/-0.45 nM; GR2 K(d)=3.04+/-0.79 nM). Co-transfection of an rtGR1 or rtGR2 expression vector into CHO-K1 or COS-7 cells, along with a reporter plasmid containing multiple consensus glucocorticoid response elements, shows that both clones are able to induce transcriptional activity in the presence of cortisol and dexamethasone. Moreover, at 10(-)(6 )M 11-deoxycortisol and corticosterone partially induced rtGR2 transactivation activity but were without effect on rtGR1. The other major teleost reproductive hormones, as well as a number of their precursors or breakdown products of these and corticosteroid hormones, were without major effects on either receptor. Interestingly, rtGR2 transactivational activity was induced at far lower concentrations of dexamethasone or cortisol (cortisol EC(50)=0.72+/-0.87 nM) compared with rtGR1 (cortisol EC(50)=46+/-12 nM). Similarly, even though RU486 inhibited transactivation activity in both rtGR1 and rtGR2, rtGR1 was more sensitive to this GR antagonist. Altogether, these results indicate that these two GR sequences encode for two functionally distinct GRs acting as ligand-inducible transcription factors in rainbow trout.

Amino Acid Sequence↗
PubMed2001

Evidence of rainbow trout prolactin interaction with its receptor through unstable homodimerisation.

This study aims to characterise Prolactin receptor (PRLR) in rainbow trout for which no information is available despite the availability of Salmonid PRL preparations. By screening a freshwater rainbow trout intestine cDNA library with a probe corresponding to the extracellular domain (ECD) of tilapia PRLR, we have cloned a 2.5 kb insert coding for the PRLR. The mature protein of 614 amino acid residues is similar to PRLR isolated in tilapia and also the long form of mammalian PRLR. Analysis of PRLR gene expression in osmoregulatory organs revealed the presence of a unique transcript, thus confirming the involvement of this hormone in the control of osmoregulation in this fish species. By using surface plasmon resonance (SPR) technology, kinetic measurement of interaction between trout PRL and its receptor ECD was studied. This approach allowed us to demonstrate the formation of a transient, unstable homodimeric complex. This unstability could explain the inability to perform binding experiments using homologous PRL. In contrast, heterologous lactogenic ligands were able to interact through a more stable complex. Whether these characteristics of PRL-receptor interaction in rainbow trout are different to what occurs in tilapia where a homologous radioreceptor assay was developed would require further studies.

Animals↗
PubMed2001

Transfer of tilapia (Oreochromis niloticus) to a hyperosmotic environment is associated with sustained expression of prolactin receptor in intestine, gill, and kidney.

Expression of the tilapia prolactin receptor (tiPRL-R) has been characterized in the intestine of Oreochromis niloticus and the levels of both tiPRL-R transcripts and tiPRL binding sites have been further analyzed in this organ, as well as in gill and kidney, during adaptation of tilapia to a hyperosmotic environment. A single high-affinity binding site for tilapia PRL-I (tiPRL-I) was determined in full-length intestine by Scatchard analysis. A heterogeneous distribution of tiPRL-R was detected in this organ, with the posterior part always displaying a higher expression of both tiPRL-R transcript and tiPRL binding sites than the anterior and medial parts. Transfer of tilapia to brackish water (BW) led to an apparent increase in the specific binding of tiPRLs in intestine and gill even for long-term-adapted fish, whereas the high level of kidney tiPRL binding sites measured in control fish reared in fresh water was still detected in BW-adapted tilapia. There was no overall significant modification of tiPRL-R transcript levels in any organ during short-term or long-term adaptation, although a limited decrease occurred in the gill of BW-adapted fish, as shown earlier. Therefore, in O. niloticus adapted to BW, high and sustained levels of tiPRL-R were observed in the three major osmoregulatory organs, gill, kidney, and intestine.

Adaptation, Physiological↗
PubMed2001

In vitro effect of various xenobiotics on trout gill cell volume regulation after hypotonic shock.

Their functions and localisation can expose gill cells to volume changes. To maintain their vital functions, these gill cells must regulate their own volume after cellular swelling or shrinkage. Recently, we showed that rainbow trout pavement gill cells in primary culture have the capacity to regulate their own volume after cellular swelling induced by hypotonic shock. This so-called regulatory volume decrease (RVD) is associated with intracellular calcium increase, which occurs as a transient peak followed by a plateau when maintained a hypotonic condition. Return to an isotonic medium restores baseline [Ca2+]i level. In this study, the effect of different xenobiotics on cellular swelling induced RVD and its calcium signal was investigated in trout pavement gill cells in primary culture. These cells were exposed to different pollutants after confluent epithelium was obtained. After 36 h in xenobiotics exposure in vitro, cellular volume and intracellular calcium concentration were measured. Nonylphenol poly- and di-ethoxylate were lethal at concentrations of 10 and 100 microM, respectively. With 10 microM of the diethoxylate form, cells did not die but, unlike non-treated cells, burst during hypotonic shock (2/3rd strength Ringer solution). With 1 microM nonylphenol polyethoxylate (NPnEO), RVD and [Ca2+]i were reduced. Copper (10 and 100 microM) had no significant effect on gill cell volume regulation. However, the heavy metal modified calcium response to hypotonic shock by inhibiting return to baseline level under isotonic conditions. 10 microM prochloraz and 2,4-dichloroaniline had no effect on cell morphology, volume and [Ca2+]i concentration. With 100 microM, however, prochloraz was lethal and dichloroaniline increased baseline [Ca2+]i. These results indicate that the effects observed on gill cells are consistent with the known toxic properties of the molecules tested, thus confirming the validity of primary culture to investigate the toxic effects of xenobiotics on fish gill epithelium.

Aniline Compounds↗
PubMed2001

Biological functions of trout pavement-like gill cells in primary culture on solid support: pH(i) regulation, cell volume regulation and xenobiotic biotransformation.

This review presents results obtained on rainbow trout gill cells in primary culture on solid support. Ultrastructural analysis showed that cultured gill cells displayed features of pavement cells in situ. Several biological functions have been investigated on these cultured cells. First, it was shown that their intracellular pH at rest and after acidosis is regulated by a Na+/H+ exchanger. Second, gill cells in primary culture can regulate their volume after a cell swelling. Intracellular calcium appears to be involved in this regulation. The effects of different xenobiotics on the capacity of gill cells to regulate their volume are presented. Third, cultured pavement cells contain biotransformation enzymes to metabolize xenobiotics. All these results demonstrate that gill cells in primary culture on solid support represent a promising in vitro model for the study of pavement cells physiology. In conclusion, applications of this culture are discussed and compared with the permeable filter method, together with the limitations and prospects of this in vitro model on solid support.

Animals↗
PubMed2001

Molecular cloning and characterization of thyroid hormone receptors in teleost fish.

Thyroid hormones are pleiotropic factors important for many developmental and physiological functions in vertebrates. Their effects are mediated by two specific receptors (TRalpha and TRbeta) which are members of the nuclear hormone receptor superfamily. To clarify the function of these receptors, our laboratory has started a comparative study of their role in teleost fish. This type of approach has been hampered by the isolation of specific clones for each fish species studied. In this report, we describe an efficient reverse transcription/PCR procedure that allows the isolation of large fragments corresponding to TRalpha and TRbeta of a wide range of teleost fish. Phylogenetic analysis of these receptors revealed a placement consistent with their origin, sequences from teleost fish being clearly monophyletic for both TRalpha and TRbeta. Interestingly, this approach allowed us to isolate (from tilapia and salmon) several new TRalpha or TRbeta isoforms resulting from alternative splicing. These isoforms correspond to expressed transcripts and thus may have an important physiological function. In addition, we isolated a cDNA encoding TRbeta in the Atlantic salmon (Salmo salar) encoding a functional thyroid hormone receptor which binds specific thyroid hormone response elements and regulates transcription in response to thyroid hormones.

Alternative Splicing↗
PubMed2000

Chronology of the appearance of beta, A, and alpha mitochondria-rich cells in the gill epithelium during ontogenesis of the brown trout (Salmo trutta).

Three types of mitochondria-rich (MR) cells, the alpha, beta, and accessory cells, are observed in the gill epithelium of juvenile and adult freshwater teleosts. In addition to numerous mitochondria, their cytoplasm contains a network of membranous tubules, the tubular system, connected to the laterobasal plasma membrane. Because they are believed to play a role in ionic regulation, it is of interest to examine the order of appearance and the ultrastructural characteristics of such cells during the embryogenesis and larval life of the brown trout. Gills of embryos and fry maintained in freshwater were thus removed at different stages and prepared for transmission and scanning electron microscopic examination. One week before hatching, cells resembling the beta cells of juvenile and adult teleosts appeared first among the epithelial cells located at the base of the filaments in the gills of the brown trout larva. In addition to their tubular system, they contained numerous and large apical structures seemingly originating from the Golgi apparatus. At approximately hatching time, small pear-shaped cells were seen to be closely apposed to the lateral side of the beta cells; they were usually devoid of apical structures and were considered to be accessory cells. After yolk sac resorption, additional cells, the alpha cells, were present along the lamellae. In contrast to the beta cells, they only exhibited poorly developed apical structures. The possible role of these three types of MR cells in osmoregulation during fish development is discussed.

Animals↗
PubMed2000

Cloning and expression analysis of an inducible HSP70 gene from tilapia fish.

We isolated and characterized the tilapia (Oreochromis mossambicus) HSP70 gene, highly homologous to other HSP70 genes. A dramatic increase of tilapia HSP70 mRNA levels was observed after heat shock of whole animals in all organs tested. Reporter constructs were tested for transient expression in carp cells and in microinjected zebrafish embryos. The entire isolated regulatory region (-851/+157) was able to mediate heat shock inducible expression of the reporter gene, with no preference for a particular tissue. Our studies represent the first transcriptional analysis of a HSP70 promoter from fish, revealing a powerful tool to direct controlled, tissue-independent gene expression in fish.

Amino Acid Sequence↗
PubMed2000

Xenobiotic and steroid biotransformation activities in rainbow trout gill epithelial cells in culture.

The biotransformation of xenobiotics and steroids was investigated in cultured respiratory epithelial cells from rainbow trout (Oncorhynchus mykiss) gills. As a first approach, ethoxyresorufin-O-deethylase (EROD), chosen as a marker of CYP1A activity, was measured in monolayers of adherent cells. The induction of this enzyme was studied in cells exposed to beta-naphthoflavone (BNF) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in concentrations ranging from 10(-6) to 10(-12) M. After 24 h, TCDD showed a maximal induction at a concentration of 10(-9) M while BNF showed a maximal induction at a concentration of 10(-7) M. Concurrently, a variety of substrates involved in cytochrome P450-dependent metabolism as well as phase II reactions, namely ethoxycoumarin, aniline and testosterone were incubated with cultured gill cells for 2 or 8 h and with freshly isolated hepatocytes for comparison. Our results revealed a significant cytochrome P450-dependent activity in gill cells with ethoxycoumarin and aniline, but no hydroxylation was observed with testosterone as substrate. No trace of sulfate conjugate was detected. With 2.5 µM aniline as substrate, 2-hydroxyaniline accounted for 32.1% of the radioactivity after 2 h incubation whereas acetanilide amounted to 6.4%. Significant differences were found between gill cells and isolated hepatocytes in the capacity of these systems to conduct oxidative and conjugating metabolic pathways. Qualitatively, the main difference was observed for testosterone which is hydroxylated in position 6beta and 16beta and conjugated to glucuronic acid in liver cells, whereas reductive biotransformation giving rise to dihydrotestosterone and androstanediol and traces of androstenedione were observed in gill cells. Quantitatively, the biotransformation activity in gill epithelial cells, expressed as pmol/h per mg protein, was between 1.5 and 14% of the activity level observed in isolated hepatocytes, depending on the substrate.

Journal Article↗
PubMed2000

Effect of egg deprivation on sex steroids, gonadotropin, prolactin, and growth hormone profiles during the reproductive cycle of the mouthbrooding cichlid fish Oreochromis niloticus.

Various hormones were analyzed during the course of a reproductive cycle in the cichlid fish Oreochromis niloticus: plasma levels of the gonadal steroids 17beta-estradiol (E2), testosterone (T), 17, 20beta-OH progesterone (17,20beta-P), gonadotropin (taGtH), and plasma and pituitary concentrations of prolactin (tiPRL(I) and tiPRL(II)) and growth hormone (tiGH). Two categories of fish were sampled and sacrificed on days 1 and 3 postspawning and at 3-day intervals thereafter: typical incubating females (INC), and nonincubating females (NI), deprived of their eggs just after spawning. Such deprivation is known to suppress maternal behavior and to accelerate ovarian development and especially vitellogenesis, thus shortening the mean interspawning interval. In both groups, variations of the plasma concentrations of E2 and T appeared to depend on ovarian stages, and differences between groups appeared to reflect underlying differences in the kinetics of ovarian development. The observation of noticeable levels of 17,20beta-P in plasma before spawning, when high values of taGtH could also be detected in NI females, suggests the implication of this progestin in the control of final maturation events, as in some other teleosts. Moreover, 17,20beta-P, which was still detected a few days after spawning, but at low concentrations and only in the plasma of INC females, might play a role at the beginning of the reproductive cycle in incubating females (maternal behavior and/or slowing down of ovarian growth). The pituitary and plasma profiles of both tiPRLs isoforms appeared to depend mainly on the kinetics of ovarian development in each group of fish, suggesting a role during the beginning of vitellogenesis. However, the variance of plasma tiPRL(II), which was significantly enhanced during maternal behavior in INC females, also suggests an implication of this hormone in the control of that behavior. Concerning tiGH, comparison of the plasma profiles in INC and NI fish also suggest an influence on the control of maternal behavior, but a main effect of starvation of INC during mouthbrooding cannot be excluded.

Animals↗
PubMed2000

Recombinant prolactin receptor extracellular domain of rainbow trout (Oncorhynchus mykiss): subcloning, preparation, and characterization.

The cDNA of the extracellular domain of rainbow trout (Oncorhynchus mykiss) prolactin receptor (trPRLR-ECD) was cloned in the prokaryotic expression vector pMON to enable its expression in Escherichia coli after induction with nalidixic acid. The bacterially expressed trPRLR-ECD protein, contained within the refractile body pellet, was solubilized in 4.5 M urea, refolded, and purified on a Q-Sepharose column, pH 8, by stepwise elution with NaCl. The bioactive monomeric 26-kDa fraction was eluted in 0.2 M NaCl, yielding 20 mg/2.5 L of induced culture. The purified protein was over 98% homogeneous, as shown by SDS-PAGE in the presence or absence of reducing agent and by chromatography on a Superdex column. Binding experiments using [125I]ovine placental lactogen (oPL) as a ligand revealed that human growth hormone (hGH), oPL, and ovine prolactin (oPRL) were the most effective competitors, with respective IC50 values of 1.32, 2.27, and 2.70 nM. Chicken (ch) PRL did not compete at all, and homologous trPRL was much less effective, with a corresponding IC50 value of 1826 nM. Gel-filtration was used to determine the stoichiometry of trPRLR-ECD's interaction with oPL, hGH, and oPRL. Only oPL yielded a 2:1 complex, whereas hGH and oPRL formed only 1:1 complexes, with excess trPRLR-ECD being seen at the initial 2:1 trPRLR-ECD:hGH or trPRLR-ECD:oPRL ratios. No studies were performed with chPRL because of its inability to compete with [125I]oPL or with trPRL because of its low affinity toward trPRLR-ECD. The present results agree with previous findings indicating, as in mammals, that homologous PRL interacts transiently with its receptor and suggest that transient homologous PRL-induced homodimerization of the receptor is sufficient to initiate a biological signal, despite the fact that, in classical binding experiments, only low specific binding can be detected.

Animals↗
PubMed2000

Synthesis of gill Na(+)-K(+)-ATPase in Atlantic salmon smolts: differences in alpha-mRNA and alpha-protein levels.

Several parameters were analyzed to determine the mechanisms responsible for the enhancement of the gill Na(+)-K(+)-ATPase activity of Atlantic salmon smolts. A major alpha-subunit transcript of 3.7 kb was revealed by Northern blot in both parr and smolt gills when hybridized with two distinct cDNA probes. The alpha-mRNA abundance demonstrated an increase to maximal levels in smolts at an early stage of the parr-smolt transformation. This was followed by a gradual rise in alpha-protein levels, revealed by Western blots with specific antibodies and by an increase in gill Na(+)-K(+)-ATPase hydrolytic activity, both only reaching maximum levels a month later, at the peak of the transformation process. Parr fish experienced a decrease in alpha-mRNA abundance and had basal levels of alpha-protein and enzyme activity. Measurement of the binding of [(3)H]ouabain to Na(+)-K(+)-ATPase was characterized in smolts and parr gill membranes showing more than a twofold elevation in smolts and was of high affinity in both groups (dissociation constant = 20-23 nM). Modulation of the enzyme due to increased salinity was also observed in seawater-transferred smolts, as demonstrated by an increase in alpha-mRNA levels after 24 h with a rise in Na(+)-K(+)-ATPase activity occurring only after 11 days. No qualitative change in alpha-expression was revealed at either the mRNA or protein level. Immunological identification of the alpha-protein was performed with polyclonal antibodies directed against the rat alpha-specific isoforms, revealing that parr, freshwater, and seawater smolts have an alpha(3)-like isoform. This study shows that the increase in Na(+)-K(+)-ATPase activity in smolt gills depends first on an increase in the alpha-mRNA expression and is followed by a slower rise in alpha-protein abundance that eventually leads to a higher synthesis of Na(+)-K(+) pumps.

Aging↗
PubMed2000

Expression of the prolactin receptor (tiPRL-R) gene in tilapia Oreochromis niloticus: tissue distribution and cellular localization in osmoregulatory organs.

The expression of the prolactin receptor (PRL-R) gene has been investigated in various tissues of tilapia (Oreochromis niloticus) reared in fresh or brackish water. Using a cDNA probe spanning the extracellular domain of the tilapia PRL-R and Northern blot analysis, the presence of tilapia PRL-R mRNA has been confirmed in the osmoregulatory organs and has been detected in other tissues, including the skin, the brain, the reproductive organs, and the two major hematopoietic organs (spleen and head kidney), as well as circulating lymphocytes. These findings suggest a conservation of the physiological processes regulated by prolactin throughout the vertebrates, including immunity and central nervous activity. A non-radioactive in situ hybridization procedure has allowed us to detect the expression of the tilapia PRL-R in the branchial chloride cells and the intestinal mucosal layer of fresh water animals, confirming the direct control exerted by prolactin on the water and ionic exchanges in tilapia. In all the tissues examined one unique PRL-R transcript has been detected with a similar size (3.2 kb) whatever the salinity conditions. Thus, the transcriptional expression of the tilapia PRL-R strongly differs from the complex RNA pattern reported for the higher vertebrates PRL-R and provides an additional argument for the existence of a single PRL-R for both prolactin isoforms in this fish species.

Animals↗
PubMed2000

Transgenic rainbow trout expressed sGnRH-antisense RNA under the control of sGnRH promoter of Atlantic salmon.

A recombinant vector containing antisense DNA complementary to Atlantic salmon (Salmo salar) sGnRH cDNA driven by specific promoter Pab derived from a corresponding sGnRH gene was introduced into rainbow trout (Oncorhynchus mykiss) eggs. This resulted in transgenic animals that had integrated one copy of the transgene into their genome and transmitted it through the germline. Antisense-sGnRH mRNA (AS) was expressed mainly in the brain of transgenic AS(+) fish. Levels of sGnRH endogenous mRNA in the brain were lower in 11-month-old AS(+) fish compared with nontransgenic AS(-) individuals from the same F2 progeny. sGnRH levels significantly decreased in the pituitary of transgenic males and females around the maturation period and in the brain of AS(+) immature females compared with controls. No reliable statistical difference was found in the levels of FSH and LH between AS(+) and AS(-) groups either in immature or mature fish. The majority of transgenic fish reached maturity at the same time as did nontransgenic individuals, although the maturation of AS(+) animals seemed to be more asynchronous. For the first time, the influence of antisense messengers on endogenous mRNA in transgenic fish and the corresponding protein is described.

Animals↗
PubMed2000

Molecular characterization of the prolactin receptor in two fish species, tilapia Oreochromis niloticus and rainbow trout, Oncorhynchus mykiss: a comparative approach.

We present recent information on the molecular characterization of the prolactin receptor (PRL-R) in two teleost species, tilapia (Oreochromis niloticus) and rainbow trout (Oncorhynchus mykiss), in the perspective of improved understanding of the physiological differences in the control of osmoregulatory function between these two fish species. Although our interest will mainly focus on osmoregulatory organs, we will also discuss evidence of the presence of PRL-R in other tissues such as gonads and hematopoietic organs. The first fish PRL-R was characterized in tilapia. This receptor is similar to that of the long form of mammalian PRL-R, but the most conserved region (extracellular domain) has only 53% identity with mammalian PRL-R. A rainbow trout PRL-R cDNA has been also isolated and appeared very similar in structure to tilapia PRL-R. Expression of the PRL-R gene was studied by Northern blotting for various tissues from tilapia and trout, and a unique transcript size of 3.2-3.4 kb was observed in all tissues studied (including male and female gonads, skin, brain, spleen, head, kidney, and circulating lymphocytes). Osmoregulatory organs (gills, kidney, intestine) were the richest tissues. Using in situ hybridization, PRL-R transcripts were localized in gill chloride cells, both in trout and tilapia. Analysis of PRL-R transcript levels in gills, kidney, and intestine indicated the maintenance of a high level of expression during adaptation to a hyperosmotic environment. These results support PRL being a pleiotropic hormone in fish and suggest the presence of a unique PRL-R form in tilapia and in trout. Finally, characterization of hormone receptor binding has been carried out in both species using a radioreceptor assay (in tilapia) or surface plasmon resonance (SPR) technology (in trout). These studies indicated the presence of a stable hormone-receptor complex in tilapia, while PRL binds to its receptor through an unstable homodimeric complex in trout. Thus, the characteristics of PRL binding on its receptor appear to be significantly different in tilapia and trout. Whether such differences may lead to different signal transduction mechanisms and osmoregulatory actions of PRL in these two euryhaline species merits further investigation.

Amino Acid Sequence↗