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.
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O Sandra, P Le Rouzic, C Cauty, M Edery, and P Prunet
B Auperin, F Rentier-Delrue, J A Martial, and P Prunet
In tilapia, there are two forms of prolactin (PRL) whose effects on sodium and chloride movements differ and depend on the living environment of the fish. To see whether different receptors or the same receptor mediates these different effects, we have characterized the specific binding of both forms of tilapia (ti)PRL in two osmoregulatory organs, the gill and kidney. Two recombinant tiPRLs were used for this analysis. The recombinant hormones had the same properties as the native hormones in a tilapia gill radioreceptor assay. Specific binding to gill and kidney membranes was increased by optimizing the quality of the tissue preparations (physiological state of fish, membrane preparation) and the incubation conditions (pH, salt concentrations, temperature, time). Under these optimized conditions, we detected only one class of high affinity PRL receptor in gill and kidney. Its binding affinity was higher for tiPRLI than for tiPRLII in both gill and kidney (for tiPRLI the respective affinity values were 2·9 and 2·3 × 1010 per m, for tiPRLII they were 1·9 and 0·5 × 1010 per m). In competition studies, tiPRLI was more potent, followed by tiPRLII and ovine (o)PRL. tiGH and oGH did not significantly displace either tiPRL. The receptor we have characterized thus recognizes quite specifically both tiPRLs.
B Auperin, F Rentier-Delrue, J A Martial, and P Prunet
Two forms of prolactin (tiPRLI and tiPRLII), with only 69% sequence identity, have been previously described in the cichlid fish tilapia (Oreochromis species). In the present study we have attempted to investigate the biological activity of these two prolactin forms during adaptation to a hyperosmotic environment. For this purpose, we have developed two highly sensitive (sensitivity: 0·05 ng/ml) and specific (cross-reactivity <0·04%) radioimmunoassays for tiPRLI and tiPRLII, using recombinant hormones.
When fish were directly transferred from fresh to brackish water, the measured levels of plasma tiPRLI and tiPRLII dropped abruptly until 12 h after transfer. Thereafter, plasma tiPRLII remained stable (around 0·5 ng/ml) until the end of the experiment, whereas plasma tiPRLI continued to decrease to undetectable levels. These different patterns of change are reflected in the calculated ratio of plasma tiPRLII to tiPRLI, which increased from 2–3 in fresh water-adapted fish to over 10 in fish which had spent 3 days or more in brackish water. The pituitary contents of tiPRLI and tiPRLII varied in a qualitatively similar fashion after transfer to brackish water. The tiPRLI content dropped continuously after 12 h, reaching one-twelfth of its initial level after 2 weeks. The pituitary tiPRLII content, on the other hand, did not decrease significantly until day 7, and after a 2-week exposure to brackish water it had only decreased by 50%.
When injected into tilapia adapted to brackish water, both ovine prolactin and recombinant tiPRLI induced a clear dose-dependent ion-retaining effect. In contrast, the effect induced by tiPRLII treatment was markedly smaller and not dose-dependent.
Northern blot analysis of tiPRL mRNAs using either a tiPRLI or a tiPRLII cDNA probe indicated the presence of two mRNAs differing in size: a 1·7 kb mRNA coding for tiPRLI and a 1·3 kb mRNA coding for tiPRLII. After transfer to brackish water, levels of the two mRNAs decreased similarly.
The present study indicates that, in O. niloticus, the two forms of prolactin have different osmoregulatory roles during adaptation to brackish water. Accordingly, their syntheses are differentially regulated after transfer to a hyperosmotic environment, presumably at a post-transcriptional level.
P. Le Goff, C. Weil, Y. Valotaire, J. F. Gonnard, and P. Prunet
To study the control of prolactin secretion in fish, an in-vitro technique using a monolayer cell culture system of rainbow trout pituitary glands was developed. Such secretion was characterized by measurement of both prolactin release and prolactin mRNA content using a trout prolactin cDNA as a probe. This cell culture technique, already used to study the regulation of gonadotrophin secretion in rainbow trout, was further validated by measuring total DNA and protein content. Both parameters appeared to be stable after 2 days of culture. Studying the effect of somatostatin (SRIF) on prolactin cells indicated that a maximal inhibitory effect (62%) was observed after 24 h of treatment. Significant inhibition of prolactin release was obtained for SRIF doses ranging from 50 nm to 1 μm. However, in the same experiment, SRIF was much more potent as an inhibitor of growth hormone release. Short-term (<12h) incubation with SRIF did not induce a significant change in prolactin release, whereas growth hormone release was reduced at as early as 1 h after SRIF exposure. SRIF did not have a significant effect on total prolactin content or prolactin mRNA levels, suggesting the absence of an effect on prolactin synthesis. No increase in the magnitude of the inhibitory effect of SRIF was observed when using pituitary cells from immature, mature male or mature female trout. When comparing effects on primary cultures containing cells from the whole pituitary with a prolactin cell-enriched population, SRIF appeared to have the same inhibitory effect on prolactin release, supporting a direct action of SRIF on prolactin cells. These results provide further support for SRIF being a prolactin-inhibiting factor in rainbow trout and acting as a modulator of a dominant stimulatory control of prolactin release.
S Uzbekova, J Chyb, F Ferriere, T Bailhache, P Prunet, P Alestrom, and B Breton
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.
O Marchand, R Safi, H Escriva, E Van Rompaey, P Prunet, and V Laudet
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.
NR Bury, A Sturm, P Le Rouzic, C Lethimonier, B Ducouret, Y Guiguen, M Robinson-Rechavi, V Laudet, ME Rafestin-Oblin, and P Prunet
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.