Time-resolved near backscatter imaging system on Laser MegaJoule.

The newly operating near-backscattering imaging (NBI) system on the Laser MegaJoule (LMJ) is briefly described with emphasis on the temporally resolved measurements and their synchronization with the LMJ laser pulse through target shots taken as part of the diagnostic commissioning campaign. The NBI measures the stimulated Brillouin and Raman scattered light around two quadruplets (one inner and one outer) of the upper LMJ hemisphere. The temporal resolution is achieved with a unique system: a specifically designed wide-open optical lens images 40 points of a diffuser onto an array of optical fibers with the scattered light recorded on a multiplexed photodiode array.

Animal board invited review: OneARK: Strengthening the links between animal production science and animal ecology.

Wild and farmed animals are key elements of natural and managed ecosystems that deliver functions such as pollination, pest control and nutrient cycling within the broader roles they play in contributing to biodiversity and to every category of ecosystem services. They are subjected to global changes with a profound impact on the natural range and viability of animal species, the emergence and spatial distribution of pathogens, land use, ecosystem services and farming sustainability. We urgently need to improve our understanding of how animal populations can respond adaptively and therefore sustainably to these new selective pressures. In this context, we explored the common points between animal production science and animal ecology to identify promising avenues of synergy between communities through the transfer of concepts and/or methodologies, focusing on seven concepts that link both disciplines. Animal adaptability, animal diversity (both within and between species), selection, animal management, animal monitoring, agroecology and viability risks were identified as key concepts that should serve the cross-fertilization of both fields to improve ecosystem resilience and farming sustainability. The need for breaking down interdisciplinary barriers is illustrated by two representative examples: i) the circulation and reassortment of pathogens between wild and domestic animals and ii) the role of animals in nutrient cycles, i.e. recycling nitrogen, phosphorus and carbon through, for example, contribution to soil fertility and carbon sequestration. Our synthesis identifies the need for knowledge integration techniques supported by programmes and policy tools that reverse the fragmentation of animal research toward a unification into a single Animal Research Kinship, OneARK, which sets new objectives for future science policy. At the interface of animal ecology and animal production science, our article promotes an effective application of the agroecology concept to animals and the use of functional diversity to increase resilience in both wild and farmed systems. It also promotes the use of novel monitoring technologies to quantify animal welfare and factors affecting fitness. These measures are needed to evaluate viability risk, predict and potentially increase animal adaptability and improve the management of wild and farmed systems, thereby responding to an increasing demand of society for the development of a sustainable management of systems.

Physiological and behavioral flexibility to an acute CO2 challenge, within and between genotypes in rainbow trout.

Adaptive capacities, governing the ability of animals to cope with an environmental stressor, have been demonstrated to be strongly dependent upon genetic factors. Two isogenic lines of rainbow trout, previously described for their sensitivity and resilience to an acute confinement challenge, were used in the present study to investigate whether adaptive capacities remain consistent when fish are exposed to a different type of challenge. For this purpose, the effects of a 4-hour hypercapnia (CO2 increase) challenge at concentrations relevant in aquaculture conditions are described for the two isogenic lines. Oxygen consumption, cortisol release, group dispersion and group swimming activity were measured before, during and after the challenge. Sensitivity and resilience for each measure were extracted from temporal responses and analyzed using multivariate statistics. The two fish lines displayed significant differences in their cortisol response, translating differences in the stress axis sensitivity to the stressor. On the contrary, both lines showed, for other measures, similar temporal patterns across the study. Notable within line variability in the stress response was observed, despite identical genome between fish. The results are discussed in the context of animal robustness.

Adaptive capacities from survival to stress responses of two isogenic lines of rainbow trout fed a plant-based diet.

The composition of feed for farmed salmonids has strongly evolved during the last decades due to the substitution of fishery-derived fish oil and fishmeal by ingredients of plant origin. Little information is available regarding the effects of this transition on adaptive capacities in fish. Two rainbow trout isogenic lines, known for their divergent ability to grow on a plant-based diet (PBD), were fed for seven months from first feeding either a fully PBD or a control marine-resources diet and were compared for their growing and survival capacities over time and their behavioral and stress responses at similar sizes but different ages. Although fish displayed similar appetitive behaviour, the two lines were highly affected by the PBD translated in decreased growth and apathetic behaviour, but also stronger stress responses displayed by stronger cortisol increases and more stress-related behaviour when isolated. The two lines were found to be similarly sensitive to a PBD for the assessed stress-related parameters, but one line displayed a lower survival during the early rearing period. Overall, these results suggest that a PBD supplied to fish from the alevin stage has strong effects on physiological and behavioural parameters, with possible impairment of fish welfare, but also genome-dependent survival.

Quantitative trait loci for magnitude of the plasma cortisol response to confinement in rainbow trout.

Better understanding of the mechanisms underlying interindividual variation in stress responses and their links with production traits is a key issue for sustainable animal breeding. In this study, we searched for quantitative trait loci (QTL) controlling the magnitude of the plasma cortisol stress response and compared them to body size traits in five F2 full-sib families issued from two rainbow trout lines divergently selected for high or low post-confinement plasma cortisol level. Approximately 1000 F2 individuals were individually tagged and exposed to two successive acute confinement challenges (1 month interval). Post-stress plasma cortisol concentrations were determined for each fish. A medium density genome scan was carried out (268 markers, overall marker spacing less than 10 cM). QTL detection was performed using qtlmap software, based on an interval mapping method (http://www.inra.fr/qtlmap). Overall, QTL of medium individual effects on cortisol responsiveness (<10% of phenotypic variance) were detected on 18 chromosomes, strongly supporting the hypothesis that control of the trait is polygenic. Although a core array of QTL controlled cortisol concentrations at both challenges, several QTL seemed challenge specific, suggesting that responses to the first and to a subsequent exposure to the confinement stressor are distinct traits sharing only part of their genetic control. Chromosomal location of the steroidogenic acute regulatory protein (STAR) makes it a good potential candidate gene for one of the QTL. Finally, comparison of body size traits QTL (weight, length and body conformation) with cortisol-associated QTL did not support evidence for negative genetic relationships between the two types of traits.

Identifying and monitoring pain in farm animals: a review.

One important objective for animal welfare is to maintain animals free from pain, injury or disease. Therefore, detecting and evaluating the intensity of animal pain is crucial. As animals cannot directly communicate their feelings, it is necessary to identify sensitive and specific indicators that can be easily used. The aim of the present paper is to review relevant indicators to assess pain in several farm species. The term pain is used for mammals, birds and fish, even though the abilities of the various species to experience the emotional component of pain may be different. Numerous behavioural changes are associated with pain and many of them could be used on farms to assess the degree of pain being experienced by an animal. Pain, as a stressor, is associated with variations in the hypothalamic-pituitary-adrenal axis as well as in the sympathetic and immune systems that can be used to identify the presence of pain rapidly after it started. However, most of these measures need sophisticated equipment for their assessment. Therefore, they are mainly adapted to experimental situations. Injuries and other lesional indicators give information on the sources of pain and are convenient to use in all types of situations. Histopathological analyses can identify sources of pain in experimental studies. When pronounced and/or long lasting, the pain-induced behavioural and physiological changes can decrease production performance. Some indicators are very specific and sensitive to pain, whereas others are more generally related to stressful situations. The latter can be used to indicate that animals are suffering from something, which may be pain. Overall, this literature review shows that several indicators exist to assess pain in mammals, a few in birds and very few in fish. Even if in some cases, a single indicator, usually a behavioural indicator, may be sufficient to detect pain, combining various types of indicators increases sensitivity and specificity of pain assessment. Research is needed to build and validate new indicators and to develop systems of pain assessment adapted to each type of situation and each species.

Fish welfare and genomics.

There is a considerable public and scientific debate concerning welfare of fish in aquaculture. In this review, we will consider fish welfare as an integration of physiological, behavioral, and cognitive/emotional responses, all of which are essentially adaptative responses to stressful situations. An overview of fish welfare in this context suggests that understanding will rely on knowledge of all components of allostatic responses to stress and environmental perturbations. The development of genomic technologies provides new approaches to this task, exemplified by how genome-wide analysis of genetic structures and corresponding expression patterns can lead to the discovery of new aspects of adaptative responses. We will illustrate how the genomic approach may give rise to new biomarkers for fish welfare and also increase our understanding of the interaction between physiological, behavioral, and emotional responses. In a first part, we present data on expression of candidate genes selected a priori. This is a common avenue to develop molecular biomarkers capable of diagnosing a stress condition at its earliest onset, in order to allow quick corrective intervention in an aquaculture setting. However, most of these studies address isolated physiological functions and stress responses that may not be truly indicative of animal welfare, and there is only rudimentary understanding of genes related to possible cognitive and emotional responses in fish. We also present an overview on transcriptomic analysis related to the effect of aquaculture stressors, environmental changes (temperature, salinity, hypoxia), or concerning specific behavioral patterns. These studies illustrate the potential of genomic approaches to characterize the complexity of the molecular mechanisms which underlies not only physiological but also behavioral responses in relation to fish welfare. Thirdly, we address proteomic studies on biological responses to stressors such as salinity change and hypoxia. We will also consider proteomic studies developed in mammals in relation to anxiety and depressive status which may lead to new potential candidates in fish. Finally, in the conclusion, we will suggest new developments to facilitate an integrated view of fish welfare. This includes use of laser microdissection in the transcriptomic/proteomic studies, development of meta-analysis methods for extracting information from genomic data sets, and implementation of technological advances for high-throughput proteomic studies. Development of these new approaches should be as productive for our understanding of the biological processes underlying fish welfare as it has been for the progress of pathophysiological research.

Effect of seawater transfer on CYP1A gene expression in rainbow trout gills.

During the transfer of rainbow trout from freshwater to seawater, the gills have to switch from an ion-absorption epithelium to an ion-secretion epithelium in order to maintain equilibrium of their hydromineral balance. After a change to ambient salinity, several gill modifications have already been demonstrated, including ion transporters. In order to identify new branchial mechanisms implicated in seawater acclimation, we carried out an extensive analysis of gene expression in gills using microarray technology. This strategy allowed us to show that CYP1A gene expression was up-regulated in the gills after salinity transfer. This increase was confirmed by real-time reverse transcription PCR. Furthermore, measurements of CYP1A enzyme activity (EROD) showed a significant increase after transfer to seawater. Immunohistochemistry analysis in the gills revealed that cells with a higher expression of CYP1A protein were principally pillar cells and those in the primary lamellae not in contact with the external medium. The results of this study suggest for the first time that CYP1A may be implicated in the seawater acclimation of the gills of rainbow trout.

Trout gill cells in primary culture on solid and permeable supports.

Trout gill cells in primary culture on solid and permeable supports were compared. Cultures were carried out by directly seeding cells on each support after gill dissociation. Most of the cell types present in culture were similar, regardless of culture support (pavement cells, mucous cells (3-4%), but no mitochondria-rich cells). However, insertion of mucous cells in cultured epithelium on permeable support presented a morphology more similar to gills in situ. Gene expression of ion transporters and hormonal receptors indicated similar mRNA levels in both systems. Cortisol inhibited cell proliferation on both supports and maintained or increased the total cell number on solid and permeable membranes, respectively. This inhibition of mitosis associated with an increase or maintenance of total gill cells suggests that cortisol reduced cell degeneration. In the presence of cortisol, transepithelial resistance of cultured gill cells on permeable membranes was increased and maintained for a longer time in culture. In conclusion, gill cells in primary culture on permeable support present: (i) a morphology more similar to epithelium in situ; and (ii) specific responses to cortisol treatment. New findings and differences with previous studies on primary cultures of trout gill cells on permeable membrane are discussed.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.