Ovine Prolactin Research Articles

Relative localization of the prolactin receptor binding sites for lactogenic hormones.

A monoclonal antibody termed MAb R7B4, directed to an epitope present in prolactin receptors (PRLRs), was used as a tool to map the receptor binding sites for human growth hormone (hGH), ovine prolactin (oPRL) and human placental lactogen (hPL). Although the three hormones completely inhibited the binding of each other to Nb2 cells or rat liver receptors, MAb R7B4 behaviour was different depending on the hormone tested and the receptor source. According to the MAb effects, PRLR from Nb2 cells would locate both hGH and oPRL close to R7B4 epitope, whereas hPL would bind far from the MAb binding site. On the other hand, PRLR from rat liver should bind hGH close to the R7B4 epitope but oPRL and hPL would be recognized by a separate region of the same receptor. Thus, results presented in this paper suggest that PRLR binding sites for hGH, oPRL and hPL do not exactly overlap in spite of full competition between ligands.

Prolactin actions in the sheep testis: a test of the priming hypothesis.

This study investigated whether prolactin (PRL) plays a priming role in the testis during the nonmating season and thereby facilitates gonadal reactivation. Sexually inactive Soay rams under long days were treated as follows: 1) group C (control) received vehicle, 2) group B received bromocriptine to suppress PRL secretion, 3) group B + PRL received bromocriptine + ovine PRL to reinstate physiological levels of PRL (n = 5/group). Treatments were for 10 wk. The photoperiod was then switched to short days to reactivate the reproductive axis. Testis diameter and sex skin coloration were recorded, and routine blood samples were collected to measure concentrations of FSH, inhibin A, and testosterone (T). At the end of the treatments, blood samples were collected every 10 min for 10 h to monitor LH pulses and the T-response to exogenous LH, and a testis biopsy was collected to assess spermatogenic activity (bromodeoxyuridine [BrDU] method) and expression of PRL receptor (reverse transcription-polymerase chain reaction and immunocytochemistry). There were no significant differences between groups in spermatogenesis (BrDU index) or steroidogenesis (T-response), and no difference in the time taken to achieve full testicular redevelopment under short days. Testis diameter and inhibin A were marginally increased in group B + PRL. Overall, this thorough experiment provides minimal support for the priming hypothesis.

Nuclear translocation of STAT5 and increased expression of Fos related antigens (FRAs) in hypothalamic dopaminergic neurons after prolactin administration

Ample evidence indicates feedback relationships between pituitary prolactin and hypothalamic dopaminergic neurons. Since the presence of prolactin receptors was earlier demonstrated in hypothalamic dopaminergic neurons, our working hypothesis was that prolactin induced activation of prolactin receptor coupled signaling leads to increased neuronal activity in these neurons. The aim of this study was to correlate prolactin receptor mediated signaling and prolactin induced activation in hypothalamic dopaminergic neurons. We used nuclear translocation of STAT5 as a marker of prolactin receptor induced signaling and expression of Fos related antigens (FRAs) as an indicator of neuronal activation. We performed double label immunocytochemical studies to determine the time course of the presence of FRAs and STAT5 in the nuclei of hypothalamic dopaminergic neurons after ovine prolactin treatment. Exogenous ovine prolactin treatment of ovariectomized rats resulted in an increase in serum ovine prolactin levels and a decrease in endogenous serum prolactin levels, indicating that ovine prolactin activated mechanisms inhibited pituitary prolactin secretion. Indeed, ovine prolactin activated the prolactin receptors in most subpopulations of hypothalamic dopaminergic neurons, resulting in nuclear translocation of STAT5. Also, increased neuronal activity, indicated by expression of FRAs, was observed in the same neuron populations after ovine prolactin treatment. These results suggest that signal transduction mechanisms coupled to prolactin receptors in hypothalamic dopaminergic neurons resemble those observed in other tissues; and nuclear translocation of STAT5 can be used as a marker of prolactin receptor activation in hypothalamic dopaminergic neurons.

Hormonal Control of Osmoregulation in the Channel Catfish Ictalurus punctatus

Prolactin (PRL) is an important hormone for freshwater adaptation in many teleost species. In some euryhaline fishes, growth hormone (GH) and cortisol are involved in seawater adaptation by stimulating ion extrusion. When channel catfish (Ictalurus punctatus) were transferred from fresh water to dilute seawater (300–400 mOsm), their plasma osmolality was always higher than the environmental salinity. In correlation with the increase in plasma osmolality, significant increases in plasma cortisol were observed. However, no effect of ovine GH or cortisol was seen in plasma osmolality or gill Na, K-ATPase activity when the hormones were given during the course of acclimation to dilute seawater. When catfish in fresh water were hypophysectomized, plasma osmolality was significantly decreased by 24 h, reaching a minimum level after 2 days. When they were transferred to dilute seawater, the plasma osmolality of the sham-operated fish was consistently higher than that of environmental water, whereas the osmolality of the hypophysectomized fish was equivalent to the environmental salinity. Ovine PRL restored the plasma osmolality of the hypophysectomized fish in fresh water to the level of sham-operated fish. Cortisol was also effective, but the effect was less pronounced than the effect of PRL. Injection of PRL in combination with cortisol resulted in a marked additive increase in plasma osmolality to a level even above that of the sham-operated fish. Ovine GH was without effect. These treatments in hypophysectomized fish transferred to dilute seawater produced essentially the same results as those in fish in fresh water. Plasma osmolality was also increased after PRL treatment of the intact fish in fresh water. There was a synergistic effect between PRL and cortisol in hypophysectomized fish in dilute seawater as well as in intact fish in fresh water. PRL did not stimulate cortisol secretion either in hypophysectomized fish or in intact fish. In the stenohaline catfish, both PRL and cortisol seem to be involved importantly in ion uptake from the environment not only in fresh water but also in brackish water.

Anxiolytic and Anti-Stress Effects of Brain Prolactin: Improved Efficacy of Antisense Targeting of the Prolactin Receptor by Molecular Modeling

We provide the first evidence that prolactin is a neuromodulator of behavioral and neuroendocrine stress coping in the rat. In virgin female and male rats, intracerebral infusion of ovine prolactin (oPRL) into the lateral cerebral ventricle (intracerebroventricular) exerted an anxiolytic effect on the elevated plus-maze in a dose-dependent manner (0.1 and 1.0 microg/5 microl; p < 0.01). In contrast, downregulation of the expression of the long form of brain prolactin receptors by chronic intracerebroventricular infusion of an antisense oligodeoxynucleotide (ODN) (osmotic minipump, 0.5 microg. 0.5 microl(-1). hr(-1); 5 d) increased anxiety-related behavior on the plus-maze compared with mixed bases-treated and vehicle-treated rats (p < 0.01), again demonstrating an anxiolytic effect of PRL acting at brain level. Furthermore, in jugular vein-catheterized female rats, the stress-induced increase of corticotropin secretion was decreased after chronic intracerebroventricular infusion of oPRL (osmotic minipump, 1.0 microg. 0.5 microl(-1). hr(-1); p < 0.05) and, in contrast, was further elevated by antisense targeting of the brain prolactin receptors (p < 0.01). This provides evidence for a receptor-mediated attenuation of the responsiveness of the hypothalamo-pituitary-adrenal (HPA) axis by prolactin. The antisense ODN sequence was selected on the basis of secondary structure molecular modeling of the target mRNA to improve antisense ODN-mRNA hybridization. Receptor autoradiography confirmed the expected improvement in the efficacy of downregulation of prolactin receptor expression [empirically designed antisense, 30%; p > 0.05, not significant; adjustment of target position after mRNA modeling, 72%; p < 0.05). Taken together, prolactin acting at brain level has to be considered as a novel regulator of both emotionality and HPA axis reactivity.

Orexins/hypocretins: waking up the scientific world.

Orexins/hypocretins: waking up the scientific world.

In vivo and in vitro effects of prolactin and growth hormone on lipid metabolism in a teleost, Anabas testudineus (Bloch)

Prolactin (PRL) has an important role in the regulation of water and electrolyte homeostasis in teleosts. The present study was designed to evaluate the role of PRL and GH on malic enzyme (ME), glucose-6-phosphate dehydrogenase (G6PDH) and isocitrate dehydrogenase (ICDH) in Anabas testudineus. Ovine prolactin significantly inhibited ME, G6PDH and ICDH activities when administered in vivo compared to vehicle treated controls. In vivo administration of PRL reversed the action of bromocryptine on enzyme activities. Ovine growth hormone in vivo also modified the effect of bromocryptine but not to the level of prolactin. Combined action of PRL+GH in vivo was most effective in keeping the enzyme activities at normal level after bromocryptine treatment. Prolactin in vitro also reversed the action of bromocryptine on enzyme activities, while GH in vitro failed to do so. Hence, prolactin seems to have an inhibitory effect on lipid metabolism in this teleost. Combined action of PRL+GH is more prominent in in vivo conditions at low PRL levels. Dopaminergic pathways may be involved in the control of prolactin and to some extent on growth hormone secretion.

Growth hormone induces tyrosyl phosphorylation of the transcription factors Stat5a and Stat5b in CMT-U335 canine mammary tumor cells

It has now been well documented that the normal and tumorous canine mammary glands can be extra-pituitary sites of substantial growth hormone (GH) synthesis. Until now, attempts to reproduce the GH synthesis in-vitro using canine mammary explants or mammary tumor cells have not been successful. Therefore, the response of CMT-U335 canine mammary tumor cells to administered porcine GH (pGH) was investigated as an in-vitro model to study the possible effects of GH synthesis on this site. CMT-U335 cells spontaneously express the growth hormone receptor (GHR) as well as the prolactin receptor (PRLR). Twenty five minutes after administration, GH induced, in a dose-dependent manner, phosphorylation of the transcription factors Stat5a and Stat5b. Clear phosphorylation was induced by 10 −7 M and 10 −8 M pGH, with virtually no phosphorylation at 10 −9 M pGH. A similar dose-dependent phosphorylation of Stat5a by ovine prolactin was found in these cells. Although at high concentrations binding of pGH to the canine PRLR can occur (albeit with a low pKa), the similar dose-dependent effect of oPRL on Stat5a phosphorylation indicated that pGH signaled through the GHR. Remarkably, pGH induced a moderately decreased proliferation of CMT-U335 tumor cells, which may indicate that GH induces differentiation in these tumor cells. The GH-induced activation of Stat5a and Stat5b in these cells, as part of the JAK/Stat signal transduction pathway, is consistent with mammary GH playing a role in autocrine and/or paracrine stimulation of (tumorous) mammary cells.

Calmodulin and a cyclic nucleotide-dependent protein kinase facilitate the prolactin-induced increase in tyrosine hydroxylase activity in tuberoinfundibular dopaminergic neurons.

Many aspects of tuberoinfundibular dopaminergic neuronal function are increased by elevated prolactin (PRL) levels, including the activity of tyrosine hydroxylase, the rate-limiting enzyme in the biosynthesis of dopamine. This study evaluated the roles of calmodulin, cyclic nucleotide-dependent protein kinase, and calcium/calmodulin-dependent protein kinase II in the PRL-induced increase in tyrosine hydroxylase activity. Ovariectomized rats were treated with haloperidol or ovine PRL (oPRL) for 20-30 h before the experiment, respectively. Treatment with haloperidol increased circulating PRL levels 8-fold and tyrosine hydroxylase activity in the stalk-median eminence 1.8-fold. Treatment with oPRL increased tyrosine hydroxylase activity 1.9-fold. W-7, a calmodulin antagonist, reversed both the haloperidol- and oPRL-induced increase in tyrosine hydroxylase activity to control levels. H-8, a cyclic nucleotide-dependent protein kinase inhibitor, also reversed the haloperidol induced increase in tyrosine hydroxylase activity. KN62, a selective calcium/calmodulin-dependent protein kinase II inhibitor, attenuated the haloperidol-induced increase in tyrosine hydroxylase activity, but KNO4, a structurally related control compound, had no effect. By contrast, the oPRL- and haloperidol-induced increases in tyrosine hydroxylase activity were not altered by KN93, a selective calcium/calmodulin-dependent protein kinase II inhibitor. These data indicate that calmodulin and a cyclic nucleotide-dependent protein kinase contribute to the PRL-induced increase in tyrosine hydroxylase activity, but the role of calcium/calmodulin-dependent protein kinase II is still unclear.

Cytokine-like effects of prolactin in human mononuclear and polymorphonuclear leukocytes

Some biochemical events following the binding of prolactin (PRL) to its receptor in normal human leukocytes were investigated. PRL enhanced JAK2 phosphorylation in peripheral blood mononuclear cells (PBMC) but not in granulocytes. PRL also induced phosphorylation of Stat-5 in PBMC and Stat-1 in granulocytes. Subsequent binding of Stat-5- and of Stat-1-like molecules to a GAS responsive element from the β-casein promoter was detected by EMSA. p38 MAPK (but not p42/p44 MAPK) was activated by PRL in both leukocyte populations. PRL induced iNOS and CIS mRNA expression in granulocytes. Increased expression of IRF-1 and SOCS-2 was observed in granulocytes and of SOCS-3 and iNOS in PBMC. Similar effects were obtained with ovine and human PRL. Antiserum to PRL reduced iNOS and IRF-1 expression induced by PRL in granulocytes and reduced iNOS expression in PBMC. Also, pretreatment of granulocytes with a p38 MAPK inhibitor (SB 203580) prevented in part PRL-induced iNOS and IRF-1 expression. In PBMC, the p38 inhibitor decreased PRL-induced iNOS gene expression. These results indicate that PRL-induced gene regulation in leukocytes requires the activation of at least two different pathways: the Stat and the MAP kinase pathways. Moreover, although PRL activates Stat in both leukocyte types, signal transduction is different in granulocytes and in PBMC. Most importantly, PRL modulates the expression of genes crucial to leukocyte function. The present findings reinforce the concept that PRL has “cytokine-like” activity in human leukocytes.

Differential regulation of hepatic bile salt and organic anion transporters in pregnant and postpartum rats and the role of prolactin

We characterized expression and activity of the bile salt transporters Na +/taurocholate (TC) cotransporting polypeptide (Ntcp), and bile salt export pump (Bsep), and the expression of organic anion transporting polypeptides 1 and 2 (Oatp1 and 2) and multidrug resistance associated protein-2 (Mrp2) in pregnancy and throughout lactation in rats. The V max for Na +/TC cotransport in basolateral liver plasma membrane was increased 1.7-fold in 2 days postpartum relative to control and pregnant rats. This correlated well with an increase in Ntcp messenger RNA (mRNA) and a 2-fold increase in Ntcp protein. Ntcp mRNA remained significantly elevated until 14 days postpartum but had begun to decline by 21 days postpartum. The maximal secretory rate (nmol/min/g liver) for TC in the single pass isolated perfused liver was also increased by 10%, 31%, and 24% at 2, 14, and 21 days postpartum and correlated with increased expression of Ntcp and Bsep mRNA and protein. Infusion of ovine prolactin (oPRL) to ovariectomized rats increased expression of both Ntcp and Bsep mRNA and protein. These data indicate a coordinate increased expression of bile salt transporters postpartum and by PRL. Mrp2 mRNA was stable in pregnancy and postpartum, whereas Mrp2 protein expression decreased significantly in pregnancy, but returned to control levels postpartum. Organic anion transporting polypeptide 2 (Oatp2) mRNA was decreased in pregnancy and increased postpartum, but changes in Oatp2 protein were not significant. Oatp1 mRNA and protein were unchanged in pregnancy and postpartum. (H EPATOLOGY 2001;33:140-147.)

The Role of Prolactin in the Regulation of Clutch Size and Onset of Incubation Behavior in the American Kestrel

In most bird species, the timing of incubation onset may influence the degree of hatching asynchrony, which, together with variation in clutch size, affects reproductive success. In some domesticated species that usually show no hatching asynchrony, plasma prolactin concentrations in females rise with the onset of incubation and the end of laying, and this rise enhances incubation behavior and may terminate laying. To investigate whether a rise in prolactin during laying is involved in the regulation of clutch size and incubation onset in a species with hatching asynchrony, we measured plasma concentrations of immunoreactive prolactin (ir-prolactin) in laying American kestrels, Falco sparverius, and quantified clutch size and incubation behavior. In a separate study, we administered one of three concentrations of ovine prolactin (o-prolactin) via osmotic pumps implanted in females when egg 2 of a clutch was laid. ir-Prolactin concentrations during laying were higher in small than in large clutches and increased in parallel with the development of incubation behavior. o-Prolactin treatment enhanced incubation behavior, but did not affect clutch size, possibly because the manipulation was performed after clutch size had already been determined. Consistent with studies on domesticated species that show synchronous hatching, our results indicate that rising prolactin during laying enhances the expression of incubation behavior in a species that shows hatching asynchrony. Further studies are necessary to determine whether the relationship between prolactin and clutch size in the American kestrel is one of causation or of mere association.

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±2.06×10 −11 M) and human prolactin ( K a=3.1±0.12 nM −1 and B max=2.87±0.66×10 −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.

Prolactin signaling in erythrophores and xanthophores of teleost fish.

Prolactin directly affects erythrophores and xanthophores of teleost fish, resulting in pigment dispersion. In the present study, signal transduction elicited by prolactin was examined using split-tail fin preparations of the rose bitterling and Nile tilapia, and cultured erythrophores and xanthophores from the paradise goby and rose bitterling. When antibodies to the prolactin receptor were added to an ovine prolactin (oPRL) solution, pigment dispersion within cultured cells was significantly inhibited, suggesting the existence of a prolactin receptor in the cell membrane. In mammals and birds, prolactin receptors belong to a cytokine receptor superfamily and signal through a tyrosine kinase-mediated pathway. Therefore, we examined the effects of three kinds of protein tyrosine kinase inhibitors on pigment dispersion elicited by oPRL. None of those inhibitors depressed the response. On the other hand, lithium ions (an inhibitor of adenylate cyclase) and H-88 and H-89 (inhibitors of protein kinase A) decreased the levels of oPRL-induced pigment dispersion in a dose-dependent manner. In cultured cells treated with cholera toxin for 3 hrs, the effect of oPRL was irreversible, indicating the possible involvement of Gs protein in the prolactin action. From these results, we conclude that cAMP may be a second messenger in the dispersion of pigment induced by prolactin and that a novel protein receptor coupled with a Gs protein may be present in the membrane of erythrophores and xanthophores of teleost fish.

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.

Effects of a 3beta-hydroxysteroid dehydrogenase inhibitor on monocyte-macrophage infiltration into rat corpus luteum and on apoptosis: relationship to the luteolytic action of prolactin

The administration of prolactin to hypophysectomized rats results in regression of the corpora lutea, accompanied by immune-inflammatory events such as infiltration of monocytes and macrophages. Recent reports indicate an autocrine role for progesterone during the lifespan of the corpus luteum. In the present study, an inhibitor of 3beta-hydroxysteroid dehydrogenase, Trilostane, was used to investigate the hypothesis that a decrease in luteal tissue steroids precipitates the cascade of immune-inflammatory events leading to luteal regression in prolactin-treated hypophysectomized rats. Immature rats were induced to ovulate by administering eCG-hCG, and hypophysectomized on the day after ovulation (at 32 days of age). Rats were injected s.c. 9-11 days after hypophysectomy with (a) Trilostane (80 mg kg(-1) day(-1)), (b) ovine prolactin (500 mg day(-1)), (c) Trilostane plus prolactin, or (d) vehicle. Plasma and luteal tissue progesterone and 20alpha-dihydroprogesterone ('progestin') were quantified; luteal tissue monocytes-macrophages and apoptotic nuclei were counted, and luteal wet mass was determined. Rats treated with prolactin alone showed the expected markers of luteal regression: decreased plasma progestin, increased numbers of monocytes-macrophages and apoptotic nuclei in luteal tissue, and decreased luteal wet mass; however, progestin concentration in luteal tissue was unchanged. Treatment with Trilostane reduced plasma and luteal tissue progestin, but did not result in an infiltration of monocytes-macrophages or increased numbers of apoptotic nuclei in the corpora lutea, or any change in luteal wet mass. Trilostane in combination with prolactin reduced plasma and luteal tissue progestin and produced the expected markers of regression, with the exception of luteal tissue mass, which remained unchanged. In conclusion, inhibition of steroidogenesis does not initiate luteal regression or augment prolactin-induced luteal regression in hypophysectomized rats. Prolactin-induced infiltration of monocytes-macrophages is not accompanied by a decrease in luteal tissue progestin, at least in the early stages of luteal regression.

Ovine placental lactogen and ovine prolactin: partial proteolysis and conformational stability

The high-resolution structure of ovine placental lactogen (oPL) and ovine prolactin (oPRL), not yet established in detail, was probed by limited proteolysis with the Glu-specific protease from Staphylococcus aureus V8. While in hGH there were no cleavage sites inside of any of the four α–helices, the analysis of the fragments obtained after partial proteolysis of oPL showed a site of cleavage at the putative third helix, suggesting that this helix is partially unwound at this point. The partial proteolysis of the rest of the molecule was compatible with a similar folding pattern for oPL, hGH and pGH, on the basis of the crystal structure of these last hormones. In the case of oPRL, proteolytic cleavage occurred at Glu residues which would be located at the end of the first helix and the beginning of the second in the hGH folding model, suggesting that these helices are shorter in oPRL than in hGH. In order to gain further insight on the folding of these molecules, circular dichroism and intrinsic fluorescence measurements were used to examine the effect of denaturing conditions on oPL and oPRL. After exposure to 6 M guanidine the unfolding of both proteins was completely reversed upon elimination of the denaturing agent. In contrast, exposure to pH 3.0 caused an irreversible decrease in the α-helical content in both hormones, most striking for oPL, indicating that this hormone is less stable than oPRL or hGH.

Prolactin is not a juvenile hormone in Xenopus laevis metamorphosis.

Prolactin (PRL) is widely considered to be the juvenile hormone of anuran tadpoles and to counteract the effects of thyroid hormone (TH), the hormone that controls amphibian metamorphosis. This putative function was concluded mainly from experiments in which mammalian PRL was injected into tadpoles or added to cultured tadpole tissues. In this study, we show that overexpression of ovine or Xenopus laevis PRL in transgenic X. laevis does not prolong tadpole life, establishing that PRL does not play a role in the life cycle of amphibians that is equivalent to that of juvenile hormone in insect metamorphosis. However, overexpression of PRL produces tailed frogs by reversing specifically some but not all of the programs of tail resorption and stimulating growth of fibroblasts in the tail. Whereas TH induces muscle resorption in tails of these transgenics, the tail fibroblasts continue to proliferate resulting in a fibrotic tail that is resistant to TH.

Effect of a prolactin pharmacological stimulant (sulpiride) and suppressant (bromocriptine) on heat shock protein 70 expression in silver sea bream, Sparus sarba

Heat shock protein 70 (HSP70) expression was assessed in hepatic tissue of silver sea bream, Sparus sarba. Using a combination of reverse transcription and PCR we have cloned a 794 base pair HSP70 cDNA fragment. This clone hybridized to a 2.3kb HSP70 mRNA transcript which was elevated 2.5 fold after acute heat shock exposure. Cold shock however, had no effect on HSP70 expression. In order to study the effect of prolactin on silver sea bream hepatic HSP70, groups of fish received either ovine prolactin (oPRL), sulpiride or bromocriptine, daily, over a seven day period. Treatment with oPRL and sulpiride resulted in a reduction in the relative abundance of hepatic HSP70 mRNA by 48 and 52%, respectively. Also hepatic HSP70 levels were reduced by 69 and 46% (as determined by immunoblotting) after oPRL and sulpiride administration, respectively. The administration of bromocriptine significantly increased both HSP70 mRNA and protein levels by 197 and 240%, respectively. The data from this study provides strong support for the importance of prolactin in regulating HSP70 expression in silver sea bream.

Molecular cloning and expression studies of a prolactin receptor in goldfish ( Carassius auratus)

A full-length cDNA clone, of a size of 4.6 kb, for the goldfish prolactin receptor has been isolated. This cDNA clone encodes a protein of 600 amino acids homologous to prolactin receptors of other species. A Kyte-Doolittle hydropathy analysis of the receptor indicates that the translated protein consists of a signal peptide of 22 amino acids, an extracellular domain of 228 amino acids, a single transmembrane domain of 24 amino acids, and an intracellular domain of 346 amino acids. Several characteristic landmarks of prolactin receptor could be identified in this clone. These include the four conserved cysteine residues and the WS motif within the extracellular domain, and the box 1 and box 2 regions of the intracellular domain. Among all the prolactin receptor sequences known to date, this clone bears the closest resemblance to the tilapia prolactin receptor, although homology between these two fish prolactin receptors is rather low. There are only 57.4% of nucleotide and 48.3% of amino acid sequence identities between these two fish receptors. This receptor cDNA was transfected into CHO-K1 cells for functional analysis. RT-PCR analysis with a pair of gene specific primers indicate that the receptor was transcribed in the transfected cells. Using a cell proliferation assay based on the reduction of the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, the receptor transfected CHO-K1 cells can be stimulated to proliferate upon the addition of ovine prolactin in the culture medium. The tissue distribution of the prolactin receptor in goldfish was studied by RT-PCR/Southern analysis and by Northern analysis. The results indicated that the receptor is expressed mostly in the kidney, the gill and the intestine of goldfish, corroborating with the osmoregulatory role of prolactin in fish. In addition, an appreciable level of the receptor is also found in the brain and gonads of goldfish. Northern analysis showed that there are two transcript sizes, a major 4.6 kb and a minor 3.5 kb mRNAs, in the kidney, gill and intestine.