Acute toxic effects of 8-epidiosbulbin E, a 19-norclerodane diterpene from yam Dioscorea antaly, on medaka Oryzias latipes embryos.

The fractionation of an aqueous extract of yam Dioscorea antaly from Madagascar led to the isolation of terpenoids and flavonoids. Compounds were identified on the basis of modern mass spectrometry and two-dimensional nuclear magnetic resonance (2D-NMR). Toxicological effects of the most abundant isolated compound, 8-epidiosbulbin E were studied on medaka Oryzias latipes embryo-larval development. The lethal concentration (killing 50%; LC(50) ) to embryos treated 24 h before hatching and for 3 days after hatching was estimated to be 0·56 mg ml(-1) (P< 0·05). No mortality was observed with O. latipes larvae exposed after hatching until day 4. Anatomo-pathological studies of embryos exposed to 0·56 mg ml(-1) showed development anomalies of the central nervous system, liver, muscle and intestine. The present data thus extend the model of O. latipes embryos as a useful animal model to analyse the effects of food toxins.

Proteomic and phosphoproteomic analysis of cellular responses in medaka fish (Oryzias latipes) following oral gavage with microcystin-LR.

Chronic and subchronic toxicity resulting from exposure to microcystins (MCs) receives increasing attention due to the risk of bioaccumulation of these toxins by aquatic animals, including fish. The mechanisms of action of MCs that target the liver, involve modifications of protein phosphorylation resulting from phosphatases 1 and 2A inhibition. Therefore, studying phosphoprotein modifications by using a specific phosphoprotein stain Pro-Q Diamond in fish liver contaminated with MC-leucine-arginine (MC-LR), the most toxic MC, should help dissecting disturbed signaling and metabolic networks. We have recently used this technology to identify several proteins that are modulated either in expression or phosphorylation in the liver of medaka following short-term exposure to MC-LR by balneation. In the present study, we have decided to use an alternative way of introducing the toxin into fish; that is by gavage (force-feeding). This was first achieved using tritiated MC-LR and allowed us to quantify the quantity of toxin incorporated into fish and to demonstrate that the toxin is mainly accumulated in liver. Afterwards a proteomics study limited to liver cytosolic proteins of contaminated animals showed that several proteins were up or down regulated either in quantity or in phosphorylation or both. Some of them had been previously detected as modified in balneation experiments but new molecules were identified as involved in signal transduction pathways activated by the toxin. In addition, in the conditions used (5 microg toxin/g body weight) anatomopathological studies supported a process of apoptonecrosis established after 24h, which was suggested to proceed by the evolution of some of the proteins after 2h contamination.

Cytokine-inducible SH2-containing protein suppresses PRL signaling by binding the PRL receptor.

Inhibition of PRL hormone signaling by suppressor of cytokine signaling (SOCS)/cytokine-inducible SH2-containing protein (CIS) was investigated in transfected HEK 293 cells. We used the physiologically relevant wild-type beta-casein promoter as a target gene for PRL action. We demonstrate that CIS produces a 70% inhibition of PRL signaling by a mechanism distinct from, and downstream of, the effect of SOCS-1 on JAK2. This inhibition involves association with the PRL receptor (PRLR), resulting in the inhibition of signal transducer and activator of transcription 5 (STAT5) activation. Further, we show that SOCS-3 coimmunoprecipitates with the PRLR. These data suggest that SOCS-3 involves a second pathway for the inhibition of PRL signaling other than JAK2 inhibition. Additional results indicate that SOCS-2 can play a more important potentiator role on PRL signaling, resulting in a restoration of 50% of transcriptional inhibition induced by SOCS-3 and a restoration of 100% of transcriptional inhibition induced by CIS. SOCS-2 was able to block the inhibitory effect of SOCS-1. These results indicate that SOCS-2 seems to be an antagonist of the other SOCS. SOCS-1 binds JAK2 and inhibits its phosphorylation; SOCS-3 does not bind JAK2 but binds the PRLR that may mediate its inhibition of JAK2; and finally, CIS binds the PRLR but inhibits signal transducer and activator of transcription 5 rather than JAK2.

Cloning, characterization, and tissue distribution of prolactin receptor in the sea bream (Sparus aurata).

The prolactin receptor (PRLR) was cloned and its tissue distribution characterized in adults of the protandrous hermaphrodite marine teleost, the sea bream (Sparus aurata). An homologous cDNA probe for sea bream PRLR (sbPRLR) was obtained by RT-PCR using gill mRNA. This probe was used to screen intestine and kidney cDNA libraries from which two overlapping clones (1100 and 2425 bp, respectively) were obtained. These clones had 100% sequence identity in the overlapping region (893 bp) and were used to deduce the complete amino acid sequence of sbPRLR. The receptor spans 2640 bp and encodes a protein of 537 amino acids. Features characteristic of PRLR, two pairs of cysteines, WS box, hydrophobic transmembrane domain, box 1, and box 2, were identified and showed a high degree of sequence identity to PRLRs from other vertebrate species. SbPRLR is 29 and 32% identical to tilapia (Oreochromis niloticus) and goldfish (Carassius auratus) PRLRs, respectively. In the sea bream two PRLR transcripts of 2.8 and 3.2 kb were detected in the intestine, kidney, and gills and a single transcript of 2.8 kb was detected in skin and pituitary by Northern blot. Spermiating gonads (more than 95% male tissue; gonado-somatic index of 0.6) contained, in addition to the 2.8-kb transcript, three more transcripts of 1.9, 1.3, and 1.1 kb. RT-PCR, which is a far more sensitive method than Northern blot, detected PRLR mRNA in gills, intestine, brain, pituitary, kidney, liver, gonads, spleen, head-kidney, heart, muscle, and bone. Immunohistochemistry using specific polyclonal antibodies raised against an oligopeptide from the extracellular domain of sbPRLR detected PRLR in several epithelial tissues of juvenile sea bream, including the anterior gut, renal tubule, choroid membrane of the third ventricle, saccus vasculosus, branchial chloride cells, and branchial cartilage.

Potentiation of growth hormone-induced liver suppressors of cytokine signaling messenger ribonucleic acid by cytokines.

Endotoxin and proinflammatory cytokines such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNFalpha) induce a state of GH resistance. A new family of suppressors of cytokine signaling (SOCS), induced by cytokines activating the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway, has been recently identified as a negative feedback loop of intracellular signaling. Overexpression of some SOCS (SOCS-3, CIS, and SOCS-2) has been reported to inhibit the JAK-STAT pathway stimulated by GH. To assess the possible role of these three SOCS proteins in the GH resistance induced by endotoxin and cytokines, we investigated the regulation of their gene expression by endotoxin and GH in rat liver and by proinflammatory cytokines and GH in primary culture hepatocytes. Both GH and lipopolysaccharide induced the three SOCS messenger RNAs (mRNAs) in vivo. In vitro, GH also increased the liver mRNAs encoding SOCS-2, SOCS-3, and CIS. Although IL-1/beta and TNFalpha alone induced only weakly the expression of SOCS-3 and CIS, these cytokines strongly potentiated the induction of these two SOCS by GH. In contrast, IL-6 alone markedly induced SOCS-3 mRNA, but did not potentiate the GH action on SOCS-3 and CIS mRNAs. The GH induction of SOCS-2 was not potentiated by any of these cytokines. Considering the ability of these SOCS to inhibit the JAK-STAT pathway induced by GH, these results suggest that the overexpression of SOCS-3 and CIS mRNAs induced by IL-1beta and TNFalpha or by endotoxin in vivo may play a role in the GH resistance induced by sepsis.

Sequence and functional characterisation of the marmoset monkey (Callithrix jacchus) prolactin receptor: comparative homology with the human long-form prolactin receptor.

This study demonstrates the cloning and in-vitro characterisation of the marmoset monkey (Callithrix jacchus) prolactin receptor cDNA. The marmoset prolactin receptor cDNA was generated by reverse transcription-polymerase chain reaction using adrenal RNA and primers designed from prolactin receptor conserved regions. Sequence analysis predicts a mature protein of 598 amino acids exclusive of the 24 amino acid signal peptide. The marmoset prolactin receptor cDNA shares 93 and 61% base pair, and 89 and 61% amino acid sequence homologies with the long form human and rat prolactin receptor cDNA, respectively. The marmoset prolactin receptor cDNA sequence retains all the receptor sequences that have been shown previously to be essential for ligand binding, structural integrity and signal transduction. Transfection of human 293 fibroblast cells with the marmoset prolactin receptor cDNA (three independent experiments) confirmed the expression of a receptor that has high binding affinity to human growth hormone (K(a)=3.6+/-0.07 nM(-1) and B(max)=7.55+/-2.06x10(-11) M) and human prolactin (K(a)=3.1+/-0.12 nM(-1) and B(max)=2.87+/-0.66x10(-11) M). Functionality of the receptor was assessed by co-transfection of 293 fibroblast cells with marmoset prolactin receptor cDNA and the Jak2 cDNA, or marmoset prolactin receptor and a Stat5 responsive element linked to the luciferase coding sequence. Incubation of the cells with 18 nM ovine prolactin resulted in rapid phosphorylation of Jak2 as ascertained by Western blotting. In addition, the marmoset prolactin receptor cDNA led to 9.06+/-0.47-fold induction of luciferase gene activity. This was comparable with the induction observed following transfection with the human prolactin receptor cDNA (8.55+/-0. 5-fold). In-vivo prolactin receptor expression in the marmoset monkey was assessed by ribonuclease protection assay and detected in a number of tissues including female reproductive organs. These data confirm the cloning and functionality of the marmoset prolactin receptor cDNA. The marmoset prolactin receptor shares a high sequence homology with the long-form human prolactin receptor, and both receptors bind hormones with comparable affinity and confer a similar intracellular response. The marmoset monkey may provide a useful tool to investigate the role of prolactin in primate reproduction.

Cross-talk between signal transducer and activator of transcription (Stat5) and thyroid hormone receptor-beta 1 (TRbeta1) signaling pathways.

PRL and T3 are involved in antagonistic regulations during various developmental processes in vertebrate species. We have studied cross-talk between transcription factors activated by these signaling pathways, i.e. signal transducer and activator of transcription 5 (Stat5) and thyroid hormone receptor beta1 (TRbeta1). Liganded TRbeta1 in the presence of its heterodimeric partner, retinoid X receptor gamma (RXRgamma), inhibited the PRL-induced Stat5a- and Stat5b-dependent reporter gene expression by up to 60%. This T3-inhibitory effect studied on Stat5 activity was partly reversed by overexpression of a TRbeta1 dominant negative variant mutated within its nuclear localization signal (TR2A). We next showed that TRbeta1 and TR2A in the presence of RXRgamma increased and decreased, respectively, Stat5 localization into the nucleus regardless of hormonal stimulation. Thus, our data suggest that TRbeta1 can be associated with Stat5 in the cytoplasm and may be involved in Stat5 nuclear translocation. In PRL-treated cells overexpressing TRbeta1/RXRgamma, both Stat5 and TRbeta1 were coimmunoprecipitated, indicating physical association of the two transcription factors. In these cells, addition of T3 with ovine (o)PRL decreased the amounts of total and tyrosine-phosphorylated Stat5 in the cytoplasm compared with oPRL-treated cells. In the nucleus, no clear difference was observed on Stat5 DNA-binding after treatment with PRL and T3 vs. PRL alone in TRbeta1/RXRgamma transfected cells. However, antibodies directed against TRbeta1 lowered Stat5-DNA binding and addition of the deacetylase inhibitor trichostatin A (TSA) relieved T3 inhibition on Stat5 transcriptional activity. Thus, we postulated that the negative cross-talk between TR and Stat5 on target genes could involve histone deacetylase recruitment by liganded TRbeta1.

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.

From the molecular biology of prolactin and its receptor to the lessons learned from knockout mice models.

Prolactin (PRL), a polypeptide hormone secreted mainly by the pituitary and, to a lesser extent, by peripheral tissues, affects more physiological processes than all other pituitary hormones combined since it is involved in > 300 separate functions in vertebrates. Its main actions are related to lactation and reproduction. The initial step of PRL action is the binding to a specific membrane receptor, the PRLR, which belongs to the class 1 cytokine receptor superfamily. PRL-binding sites have been identified in a number of tissues and cell types in adult animals. Signal transduction by this receptor is mediated, at least in part, by two families of signaling molecules: Janus tyrosine kinases and signal transducers and activators of transcription (STATs). Disruption of the PRLR gene has provided a new mouse model with which to identify actions directly associated with PRL or any other PRLR ligands, such as placental lactogens. To date, several different phenotypes have been analyzed and are briefly described in this review. Coupled with the SAGE technique, this PRLR knockout model is being used to qualitatively and quantitatively evaluate the expression pattern of hepatic genes in two physiological situations: transcriptomes corresponding to livers from both wild type and PRLR KO mice are being compared, and following statistical analyses, candidate genes presenting a differential profile will be further characterized. Such a new approach will undoubtedly open future avenues of research for PRL targets. To date, no pathology linked to any mutation in the genes encoding PRL or its receptor have been identified. The development of genetic models provides new opportunities to understand how PRL can participate to the development of pathologies throughout life, as for example the initiation and progression of breast cancer.

A beta-turn endocytic code is required for optimal internalization of the growth hormone receptor but not for alpha-adaptin association.

Intracellular trafficking of GH and its receptor, more particularly the chicken GH receptor (cGHR), was examined in COS-7 cells using biochemical and structural studies. Internalization of radioactive GH by the cGHR is reduced as compared with the rat GHR. On the contrary, activation of gene transcription through Janus kinase-2 was similar for both species. Secondary structures of the cytoplasmic domain of chicken and rat GHR were compared, since beta-turns were reported as internalization signals. The substitution of Pro335-Asp335, present in mammalian GH receptors, with Thr307-Gln308 in the cGHR leads to the loss of a beta-turn within a conserved cytoplasmic region. Mutational analysis indicated that the lower rate of internalization of cGHR, as compared with mammalian GHR, was due to this motif. Our data further show that alpha-adaptin, a subunit of adaptor protein AP-2, associates with the GHR upon hormone stimulation. The clathrin-coated pit pathway therefore seems to be involved in the endocytosis of cGHR, as AP-2 is known to intervene in the recruitment of receptors to these pits. Interaction with alpha-adaptin may occur through a common epitope of the chicken and mammalian GHR, since receptors from both species bind similar amounts of alpha-adaptin; alternatively, two different epitopes with similar affinity may be involved. Therefore, not alpha-adaptin but an uncharacterized factor, presumably interacting with the identified beta-turn endocytic code, is responsible for the difference in internalization kinetics. Finally, the present study illustrates that functional amino acid motifs of receptors can be derived from comparative studies.

Inhibition and restoration of prolactin signal transduction by suppressors of cytokine signaling.

Prolactin (PRL) has been shown to activate the cytoplasmic tyrosine kinase Janus kinase 2 (Jak2) and the subsequent recruitment of various signaling molecules including members of the signal transducer and activator of transcription family of transcription factors. Recently, an expanding family of cytokine-inducible inhibitors of signaling has been identified that initially included four members: suppressor of cytokine signaling (SOCS)-1, SOCS-2, SOCS-3, and cytokine-inducible src homology domain 2 (SH-2) proteins. The present study analyzes the role of these members in PRL signaling. Constitutive expression of SOCS-1 and SOCS-3 suppressed PRL-induced signal transducer and activator of transcription 5-dependent gene transcription, and Jak2 tyrosine kinase activity was greatly reduced in the presence of SOCS-1 or SOCS-3. SOCS-1 was shown to associate with Jak2, whereas SOCS-2 was associated with the prolactin receptor. Co-transfection studies were conducted to further analyze the interactions of SOCS proteins. SOCS-2 was shown to suppress the inhibitory effect of SOCS-1 by restoring Jak2 kinase activity but did not affect the inhibitory effect of SOCS-3 on PRL signaling. Northern blot analysis revealed that SOCS-3 and SOCS-1 genes were transiently expressed in response to PRL, both in vivo and in vitro, whereas the expression of SOCS-2 and CIS genes was still elevated 24 h after hormonal stimulation. We thus propose that the early expressed SOCS genes (SOCS-1 and SOCS-3) switch off PRL signaling and that the later expressed SOCS-2 gene can restore the sensitivity of cells to PRL, partly by suppressing the SOCS-1 inhibitory effect.

Endothelial cell chemotaxic activity expressed in rat placenta is not associated with prolactin-like proteins B and C.

Conditioned medium from gestation day 18 rat placental cultures showed potent stimulation of the directional migration of human retinal endothelial cells. To examine the role of major secreted placental proteins in this chemotaxic activity, prolactin-like proteins (PLPs)-B and C were purified from rat placenta using immuno-affinity chromatography. In contrast to conditioned medium, native PLP-B and PLP-C preparations failed to show any significant stimulation of endothelial cell migration. This study further examined the ability of PLP-B to bind to rat receptors for growth hormone (GH-R) and prolactin (PRL-R). In competitive binding assays with [125I]-hGH, neither native nor recombinant PLP-B preparations showed significant high affinity binding to the transfected rat GH-R or PRL-R. In summary, neither PLP-B nor PLP-C exhibit the potent chemotaxis stimulatory activity of placental conditioned media, nor does PLP-B show evidence of ability to act via rat GH or PRL receptors.

Dual effects of suppressor of cytokine signaling (SOCS-2) on growth hormone signal transduction.

A family of suppressors of cytokine signaling (SOCS) has recently been identified of which two members have been shown to block growth hormone (GH) signaling. Dose-response experiments were conducted in 293 cells and SOCS-1 and SOCS-3 were shown to inhibit the transcriptional activation of a GH-responsive element and suppressed Jak2 tyrosine kinase activity. SOCS-2 had two opposite effects: at low concentrations it inhibited GH-induced STAT5-dependent gene transcription, but restoration of GH signaling was observed at higher concentrations. In cotransfection studies, SOCS-2 was able to block the inhibitory effect of SOCS-1 but not that of SOCS-3 on GH signaling. These findings suggest that a major function for SOCS-2 is to restore the sensitivity to GH by overcoming the initial inhibitory effects of other endogenous SOCS molecules.

Generation of chicken growth hormone-binding proteins by proteolysis.

A soluble protein that specifically bound growth hormone (GH) was characterized in culture medium of a COS-7 cell line transfected with the cDNA of the full-length chicken GH receptor (cGHR). Incubation of culture medium with 125I-labeled human GH resulted in the formation of a single specific complex with high affinity (KD = 0.36 nM) and apparent molecular weight of 75 kDa. The production of large quantities of GH-binding protein (GHBP) amounting to, per hour, 23% of the cell's GHR, points to the importance of partial proteolysis for GHR turnover. Considerable amounts of GHBP were also detected in a cytosolic fraction. These results strongly suggest that in chicken, as in rabbit and monkey, the GHBP is generated, at least partially, by proteolytic cleavage of the membrane-anchored GHR.

Activation of gene transcription by tilapia prolactin variants tiPRL188 and tiPRL177.

In the tilapia species Oreochromis niloticus, the pituitary releases two forms of prolactins (tiPRL188 and tiPRL177). The binding parameters and the activation of tiPRL-induced JAK2/Stat5 signalling pathway were analysed using a mammalian cell line transiently transfected with the tiPRL receptor (tiPRLR). Our data indicate that the tiPRLR is able to mediate transcriptional activation of the PRL responsive element. At nanomolar concentrations, tiPRL188 activates gene transcription whereas at micromolar concentrations it inhibits luciferase transcription from the lactogenic responsive element. This is consistent with a model of receptor dimerisation. In contrast, the activation by tiPRL177 was only reached at high (microM) concentrations. The transcriptional activities induced by tiPRL177 and tiPRL188 are discussed in the context of the physiology of these hormones.

Alternative splicing of the prolactin receptor gene generates a 1.7 kb RNA transcript that is linked to prolactin function in the red deer testis.

A cDNA encoding a putative non-membrane bound prolactin receptor was amplified by RT-PCR from red deer (Cervus elaphus) testis. Sequence analysis suggests that the testicular cDNA is generated by alternative splicing resulting in the deletion of exons 7 and 8, which code for: (a) the final 53 aa of the extracellular domain of the receptor including the fifth conserved cysteine residue and the WS x WS motif, (b) the entire transmembrane domain, (c) the first three cytoplasmic amino acid residues, and (d) two nucleotides of the fourth cytoplasmic amino acid codon. The resultant RNA would encode a putative protein of 174 aa due to a single bp frame shift and a premature stop codon. Northern blot analysis confirmed that the PCR-amplified cDNA is encoded by a specific 1.7 kb RNA transcript whereas the membrane bound receptor is encoded by transcripts of 3.5 and 2.5 kb. HPLC studies using media from 293 cells transfected with the 1.7 kb cDNA failed to detect any specific binding for prolactin. These data suggest that: (a) the deletion in the 1.7 kb transcript alters the structure of the prolactin binding domain in the putative protein encoded by the 1.7 kb transcript, and (b) alternative splicing of the prolactin receptor gene toward the 1.7 kb transcript is a means of down-regulating the expression of the full length prolactin receptor and hence may modify the role of prolactin in the testis of seasonally breeding mammals such as red deer. The sequence reported in this paper has been deposited in the Genbank/EMBL data base with accession number Y14753.

Identification and modulation of a growth hormone-binding protein in rainbow trout (Oncorhynchus mykiss) plasma during seawater adaptation.

A soluble protein that specifically bound 125I-human growth hormone (hGH) was identified in rainbow trout plasma, using HPLC-gel filtration. The binding affinity of the protein for hGH was 1.2 x 10(9)M-1. 125I-rainbow trout GH (tGH) was also able to bind to the protein albeit with a lower affinity (6.6 x 10(7)M-1) than hGH. Crosslinking experiments using 125I-hGH revealed two specific bands of 150 and 130 kDa. The complex 125I-hGH-BP could be precipitated by a monoclonal anti-GH receptor antibody, suggesting a close relationship between the plasma GH-BP and the GH receptor. A fourfold increase in the hGH binding to the GH-BP was shown 48 h after transfer of the fishes from freshwater to seawater. The increase in binding was related to a high binding capacity without significant changes in binding affinity. These results suggest a potential role of this related GH-BP as an index of GH effects during seawater adaptation in salmonids.

Prolactin: a hormone at the crossroads of neuroimmunoendocrinology.

Prolactin (PRL), secreted by the pituitary, decidua, and lymphoid cells, has been shown to have a regulatory role in reproduction, immune function, and cell growth in mammals. The effects of PRL are mediated by a membrane-bound receptor that is a member of the superfamily of cytokine receptors. Formation of a trimer, consisting of one molecule of ligand and two molecules of receptor, appears to be a necessary prerequisite for biological activity. The function of these receptors is mediated, at least in part, by two families of signaling molecules: Janus tyrosine kinases (JAKs) and signal transducers and activators of transcription (STATs). To study these receptors, we have used two approaches: mutational analysis of their cytoplasmic domains coupled with functional tests and inactivation (knockout) of the receptor gene by homologous recombination in mice. We have produced mice by gene targeting in embryonic stem cells carrying a germline null mutation of the prolactin receptor gene. Heterozygous (+/-) females show almost complete failure to lactate, following their first, but not subsequent pregnancies. Homozygous (-/-) females are infertile as a result of multiple reproductive abnormalities, including ovulation of premiotic oocytes, reduced fertilization of oocytes, reduced preimplantation oocyte development, lack of embryo implantation, and the absence of pseudopregnancy. Half of the homozygous males are infertile or show reduced fertility. In view of the wide-spread distribution of PRL receptors, other phenotypes including those on the immune system, are currently being evaluated in -/- animals. This study establishes the prolactin receptor as a key regulator of mammalian reproduction and provides the first total ablation model to further study the role of the prolactin receptor and its ligands.

Prolactin (PRL) and its receptor: actions, signal transduction pathways and phenotypes observed in PRL receptor knockout mice.

PRL is an anterior pituitary hormone that, along with GH and PLs, forms a family of hormones that probably resulted from the duplication of an ancestral gene. The PRLR is also a member of a larger family, known as the cytokine class-1 receptor superfamily, which currently has more than 20 different members. PRLRs or binding sites are widely distributed throughout the body. In fact, it is difficult to find a tissue that does not express any PRLR mRNA or protein. In agreement with this wide distribution of receptors is the fact that now more than 300 separate actions of PRL have been reported in various vertebrates, including effects on water and salt balance, growth and development, endocrinology and metabolism, brain and behavior, reproduction, and immune regulation and protection. Clearly, a large proportion of these actions are directly or indirectly associated with the process of reproduction, including many behavioral effects. PRL is also becoming well known as an important regulator of immune function. A number of disease states, including the growth of different forms of cancer as well as various autoimmune diseases, appear to be related to an overproduction of PRL, which may act in an endocrine, autocrine, or paracrine manner, or via an increased sensitivity to the hormone. The first step in the mechanism of action of PRL is the binding to a cell surface receptor. The ligand binds in a two-step process in which site 1 on PRL binds to one receptor molecule, after which a second receptor molecule binds to site 2 on the hormone, forming a homodimer consisting of one molecule of PRL and two molecules of receptor. The PRLR contains no intrinsic tyrosine kinase cytoplasmic domain but associates with a cytoplasmic tyrosine kinase, JAK2. Dimerization of the receptor induces tyrosine phosphorylation and activation of the JAK kinase followed by phosphorylation of the receptor. Other receptor-associated kinases of the Src family have also been shown to be activated by PRL. One major pathway of signaling involves phosphorylation of cytoplasmic State proteins, which themselves dimerize and translocate to nucleus and bind to specific promoter elements on PRL-responsive genes. In addition, the Ras/Raf/MAP kinase pathway is also activated by PRL and may be involved in the proliferative effects of the hormone. Finally, a number of other potential mediators have been identified, including IRS-1, PI-3 kinase, SHP-2, PLC gamma, PKC, and intracellular Ca2+. The technique of gene targeting in mice has been used to develop the first experimental model in which the effect of the complete absence of any lactogen or PRL-mediated effects can be studied. Heterozygous (+/-) females show almost complete failure to lactate after the first, but not subsequent, pregnancies. Homozygous (-/-) females are infertile due to multiple reproductive abnormalities, including ovulation of premeiotic oocytes, reduced fertilization of oocytes, reduced preimplantation oocyte development, lack of embryo implantation, and the absence of pseudopregnancy. Twenty per cent of the homozygous males showed delayed fertility. Other phenotypes, including effects on the immune system and bone, are currently being examined. It is clear that there are multiple actions associated with PRL. It will be important to correlate known effects with local production of PRL to differentiate classic endocrine from autocrine/paracrine effects. The fact that extrapituitary PRL can, under some circumstances, compensate for pituitary PRL raises the interesting possibility that there may be effects of PRL other than those originally observed in hypophysectomized rats. The PRLR knockout mouse model should be an interesting system by which to look for effects activated only by PRL or other lactogenic hormones. On the other hand, many of the effects reported in this review may be shared with other hormones, cytokines, or growth factors and thus will be more difficult to study. (ABSTRACT TRUNCATED)

Is prolactin a gonadotrophic hormone in red deer (Cervus elaphus)? Pattern of expression of the prolactin receptor gene in the testis and epididymis.

This study investigated the pattern and site of expression of the prolactin receptor gene in the testis and epididymis of red deer collected during the breeding season (n=3). Ribonuclease protection assays using 50 microg total RNA and a 300 bp [32P]-labelled antisense cRNA probe, generated from the extracellular domain of the red deer prolactin receptor, confirmed the expression of the receptor in both the testis and epididymis; a higher level of prolactin receptor mRNA was detected in the epididymis compared with the testis (170.4+/-1.5 x 10(3) and 26.3+/-2.7 x 10(3) arbitrary units respectively; P<0.05). In situ hybridisation using 300 bp [33P]-labelled sense and antisense cRNA probes generated from the extracellular domain of the receptor localised the expression sites to the seminiferous tubules and interstitial compartments of the testis and the epithelial layer of the epididymal duct. Quantification of grain numbers demonstrated a higher level of expression of the receptor in the epididymis compared with the interstitial and seminiferous tubule compartments of the testis (18.1+/-4.4 x 10(2), 10.1+/-2.0 x 10(2) and 8.3+/-0.8 x 10(2) grains/microm2 respectively; P<0.05). However, no differences were detected in the level of expression of the receptor between the interstitial and seminiferous tubule compartments of the testis. Immunocytochemistry using an anti-prolactin receptor antibody, raised against a peptide sequence from the extracellular domain of the rat prolactin receptor, localised expression of the receptor gene to the Leydig cells, pachytene spermatocytes, round spermatids and elongating spermatids. In the epididymis, the receptor was localised to the epithelial layer within the epididymal ducts. Expression of the prolactin receptor gene in the red deer testis and epididymis suggests a role for the hormone in steroidogenesis and spermatogenesis.