Anti-ovine Prolactin Research Articles

The Rabbit Mammary Gland Prolactin Receptor Is Tyrosine-phosphorylated in Response to Prolactin in Vivo and in Vitro

We report the first in vivo study demonstrating tyrosine phosphorylation of mammary gland proteins including the prolactin receptor, in response to the injection of prolactin. Immunoblotting of mammary gland membrane extracts revealed that subunits of 200, 130, 115, 100, 90, 70, and 45 kDa display increased tyrosine phosphorylation within 5 min of prolactin administration. The 100-kDa component was identified as the full-length prolactin receptor by a variety of means including immunoprecipitation and immunoblotting with monoclonal (U5, 917, 110, and 82) and polyclonal (46) antibodies to the prolactin receptor. Maximal receptor phosphorylation was seen within 1 min of hormone injection, and to obtain a strong response it was necessary to deprive rabbits of their endogenous prolactin for 36 h. Rapid tyrosine phosphorylation of the full-length receptor was verified by its demonstration in Chinese hamster ovary cells stably transfected with rabbit prolactin receptor cDNA. Both in vivo and in vitro, the phosphorylation signal was transient, being markedly reduced within 10 min of exposure to prolactin. Tyrosine-phosphorylated receptor was shown to be associated with JAK 2 by immunoblotting of receptor immunoprecipitated from transfected Chinese hamster ovary cells with polyclonal 46. A 48-kDa ATP-binding protein was also shown to be associated with the mammary gland receptor by U5 or polyclonal 46 immunoprecipitation of receptor complexes following covalent labeling with [alpha-32P]azido-ATP. Our demonstration of prolactin receptor tyrosine phosphorylation raises the possibility of signaling pathways regulated by receptor/SH2 protein interaction, which would facilitate prolactin specific responses. The fact that a period of hormone deprivation is needed for significant hormone triggered receptor phosphorylation indicates that the mammary gland receptor exists in a largely desensitized state in vivo, analogous to the related growth hormone receptor.

Presence of immunoreactive growth hormone and prolactin in the ovine pineal gland

The use of antisera raised against bovine growth hormone (GH) and ovine prolactin (PRL) enabled the detection of related immunoreactive (ir) sequences of proteins in ovine pineal tissue. The isolation of PRL-like ir-material was accomplished using a 0.25 M ammonium sulphate (pH 5.5) extraction followed by ethanol precipitation, whereas the resulting 2.0 M ammonium sulphate (pH 7.0) precipitate contained a GH-like immunoreactivity. Gel chromatography of the GH-like immunoreactivity (Sephadex G-100) indicated the presence of several GH-like fragments ranging in the M(r) range of 7,000 to 55,000. Analyses of the PRL-like ir-material found in pineal tissue on HPLC using a TSK 545-DEAE column led to the resolution into a single peak of immunoreactivity. A single peak of activity was also observed following chromatofocusing and hydrophobic interaction chromatography of the ir-peak from the TSK 545-DEAE column. The PRL-like ir-material inhibited the binding of [125I]ovine PRL-S14 to anti-ovine PRL antibodies without showing an affinity for binding to anti-rat PRL or anti-bovine GH antibodies. Scatchard analysis of the binding of pineal PRL-like ir-material and pituitary ovine PRL-S14 to liver membranes from day-20 pregnant rats revealed similar affinity constants (Ka of 4.7 +/- 0.2 x 10(9) M-1). In addition, the replication of Nb 2 Node rat lymphoma cells was stimulated by pineal PRL-like ir-material, an effect known to be specific for lactogenic hormones. The pineal PRL-like immunoreactivity appeared on sodium dodecyl sulfate polyacrylamide gels as a single major band of M(r) 24,000. The functional status of PRL- and GH-like ir-material in the ovine pineal remains to be determined, but evidence is presented that the overall protein synthesis rate of the rat pineal responded to circulating concentrations of PRL.

An enzyme-linked immunosorbent assay (ELISA) for measuring prolactin levels in ovine and cervine plasma

Abstract An indirect enzyme-linked immunosorbent assay (ELISA) for ovine plasma prolactin that can be completed in less than 8 h is described and validated for ovine and cervine plasma. Ovine prolactin is covalently bound to bovine thyroglobulin and passively adsorbed in guanidine hydrochloride to a standard 96-well microtitre plate. Addition of standards, samples, and rabbit anti-ovine prolactin is specific for this assay, but subsequent steps which include addition of peroxidase-labelled goat-antirabbit IgG, washing, addition of o-phenylenediamine substrate with colour development, and reading of plates at 492 nm using an automatic ELISA processor are common to most ELISAs currently being performed in this laboratory. Assay sensitivity is less than 2.5 ng/ml, and intra- and inter-assay coefficients of variation are less than 9 and 16 % respectively. All steps except for sample addition are performed on a Behring Elisa Processor M automatic machine. This ELISA is quick, simple, and cost-effective compare...

Immunocytochemical study of cell type distribution in the pituitary of Barbus barbus (Teleostei, Cyprinidae)

Antisera to mammalian pituitary and placental hormones have been used to identify and localize the different cell types in the pituitary of the barbel ( Barbus barbus, L.). The immunocytochemical labeling employed the immunoperoxidase technique or the immunogold silver staining procedure. Corticotrophic and prolactin cells, visualized using antisera to human adrenocorticotropic hormone and ovine prolactin (PRL), respectively, occur in the rostral pars distalis (RPD). Antisera against mammalian gonadotropins [ovine folliclestimulating hormone (FSH); bovine luteinizing hormone] or porcine growth hormone selectively cross-react with two different cell populations occupying the major part of the proximal pars distalis (PPD). Thyrotropic cells, stained by an antiserum to whole human thyroidstimulating hormone preabsorbed with porcine FSH, are scattered throughout the PPD and found amongst growth hormone and gonadotrophic cells. The majority of pars intermedia cells are stained with anti-melanophore stimulating hormone whereas the scattered PAS positive cells are revealed by both anti-ovine PRL and anti-bovine placental lactogen (or chorionic somatomammotropin). The latter antiserum also cross-reacts with the PRL cells of the RPD. Our results indicate that the distribution of the different cell types in Barbus barbus is similar to that described in other families of teleosts. This report is also the first demonstration of antigenic similarity between mammalian placental lactogen and fish prolactin.

The effect of time of day and photoperiod on prolactin content and electrophoretic mobility in the pituitary of the goldfish, Carassius auratus L.

Prolactin was identified in goldfish pituitaries by its location within the pituitary, the absence of carbohydrate, its cross reactivity with antiovine prolactin, and its molecular weight. The optimal conditions for the separation of goldfish pituitary prolactin by disc electrophoresis were determined. On a long photoperiod (10L:14D) prolactin showed two peaks of concentration: a lesser one early in the dark phase, followed by a major one at the end of the dark phase. On a short photoperiod (10L:14D) a single peak occurred early in the dark phase. The electrophoretic mobility of prolactin varied diurnally and with change in photoperiod. Mobility was greatest when pituitary content of prolactin was least and vice versa. Evidence is presented that the change in mobility reflects changes in the prolactin molecule and is not an artifact induced by varying protein concentration or other variables in the electrophoretic technique.

Isolation and identification of a cDNA clone of rat placental lactogen II.

The developing rat placenta expresses two placental lactogens at different stages of pregnancy: rat placental lactogen I from Days 11 to 13 of pregnancy and rat placental lactogen II (rPLII) from Day 12 to term. In this paper, we describe cDNA clones for rPLII, which have been isolated from a Day 18 rat placental cDNA library. The rPLII clones hybrid-select a mRNA which translates in vitro to a protein of 25,000 daltons. This protein is processed by dog pancreatic microsomes to a 22,000-dalton form, identical in size to rPLII isolated from pregnant rat serum. Both forms are precipitated by an anti-rPLII antiserum and an anti-ovine prolactin antiserum. The mRNA for rPLII is first expressed in Day 12 placenta and reaches a maximum at about Day 18 of pregnancy, in parallel with the appearance of the hormone in serum. Sequencing of the cDNA shows that, unlike human placental lactogen which is 85% homologous to human growth hormone at the amino acid level, rPLII is much more closely related to the prolactins. Thus, rPLII is 52% homologous to rat prolactin at the amino acid level, but only 34% related to rat growth hormone. This is the second placental lactogen to be fully characterized, and in the rat this hormone appears to have evolved by a route quite different from that which produced placental lactogen in humans.

Autoantibodies in sera from patients with multiple sclerosis directed against antigenic determinants in pituitary growth hormone-producing cells and in structures containing vasopressin/oxytocin

We have reported previously that autoantibodies in sera from patients with multiple sclerosis (MS) were reactive with rat brain, including pituitary, and with swine pituitary in areas thought to contain peptides of the somatotropin family and/or vasopressin/oxytocin. We have now tested the same patient sera for their specificity to antigenic determinants which are common to animal and human peptides. Localization of the binding sites of the MS sera was demonstrated in rat pituitaries and brains using the double immunofluorescence staining method, employing anti-bovine somatotropin (STH), anti-ovine prolactin (PRL), anti-neurophysin I and II, anti-somatostatin, and anti-vasopressin as reference antibodies. In the pituitary, the positive MS sera reacted specifically with cells which were also reactive with anti-bSTH. In the brain, positivity of MS sera was mainly localized in structures reactive with anti-neurophysin I and II and anti-vasopressin. Absorption experiments, immunocytochemical model assays, and radioimmunoassays, however, did not show specific binding of the MS sera to any of the above-mentioned peptides. Therefore, while these data present additional evidence on the localization of the immunocytochemical reaction sites of the MS autoantibodies, they do not enable us to identify the specificity of these antibodies.

Identification by immunofluorescence of prolactin- and somatotrophin-producing cells in the pituitary gland of the tree frog Hyla arborea

The indirect immunofluorescence procedure was used to localize prolactin (PRL)- and somatotrophin (STH)- producing cells in the pituitary distal lobe from Hyla arborea adult specimens. The following mammalian antisera were employed: rabbit anti-ovine PRL, antibovine PRL, anti-human PRL, anti-rat PRL, anti-ovine STH, anti-bovine STH, anti-human STH; monkey anti-rat STH. Immunocytochemical staining was suppressed by solid phase absorption of both anti-PRL and anti-STH with the specific antigen. Absorption of anti-PRL with STH and of anti-STH with PRL did not appreciably affect immunocytochemical staining. Treatment with the two antisera revealed two different reactive cell types, both acidophils. Using PRL antisera a strong fluorescence was found in the large acidophils located chiefly in the rostro-central and ventral areas of the distal lobe sagittal sections. A somewhat weaker fluorescence was observed using STH antisera in the fewer, small acid-ophils mostly concentrated in the dorso-caudal region and only sparsely scattered in the other areas of the pars distalis. Strikingly, the overall pattern of localization shown by the two cell types is similar to their already known distribution in the pituitary distal lobe of some other species of urodele and anuran amphibians.

Seasonal variations in mink ( Mustela vison) plasma prolactin measured by heterologous radioimmunoassay

Monthly prolactin levels were measured in mink by a heterologous radioimmunoassay using porcine prolactin SP162C as the labeled hormone and an antiovine prolactin guinea-pig serum. The yearly variation of plasma prolactin concentration closely followed the day-length ratio throughout the year in males as in females.

Microelectrophoretic identification of pituitary prolactin in Carassius auratus

A microelectrophoretic polyacrylamide slab gel system has been adapted to analyze prolactin levels in individual goldfish pituitaries. The putative prolactin (Rf = 0.40) was identified within the gels by use of what is known about hypothalamic, dopaminergic control of secretion of this hormone, the effect of osmotic pressure on prolactin secretion, and the effect of administration of ergot drugs in fish. The putative goldfish prolactin was also shown to cross-react with anti-ovine and anti-pollack prolactin in two systems.

Radioimmunoassay for porcine prolactin: plasma levels during lactation, suckling and weaning and after TRH administration.

A sensitive heterologous radioimmunoassay for porcine prolactin (pPRL) has been developed. Anti-ovine prolactin antibody was raised in rabbits which allowed a final dilution of 1:500 000. The separation of free and antibody bound [125I]pPRL is based on the double antibody solid phase system. The assay is specific for pPRL. There is no cross-reaction with pLH, pFSH and pTSH; little cross-reaction (1.35%) was found with pGH. The smallest detectable amount was 0.08 ng per tube. During lactation high plasma levels are found with great fluctuations. After weaning the plasma PRL levels fall to basal within a few hours. After thyrotrophin-releasing hormone (TRH) administration plasma concentrations increase within a few minutes.

Histological, immuno- and enzyme-histochemical investigations on the adenohypophysis of the urodeles, Mertensiella caucasica and Triturus cristatus and the caecilian, Chthonerpeton indistinctum

The pituitary glands of two urodelan species (Mertensiella caucasica, Triturus cristatus) and one one caecilian species (Chthonerpeton indistinctum) were examined with histological (Alcian blue, Brookes' trichrome stain), enzyme histochemical (acid phosphatase, alpha-naphthylacetate-esterase, acetylcholinesterase) and immunofluorescence techniques (anti-carp GTH, anti-ovine prolactin, anti-synthetic alpha-MSH). In the pituitary gland of Mertensiella and Triturus six chromophilic cell types could be distinguished. A strong fluorescence was observed in the MSH-, GTH- and TSH-cells. In the pituitary gland of Chthonerpeton only five chromophilic cell types could be distinguished: in the rostral part of the pituitary gland the B3-cell; in the basal region of the central area the B2-cell; dorsocaudally the B1-cell. The acidophilic cells were found in the central and caudal part of the pars distalis. The basophils of the pars intermedia could be observed in the dorsocaudal part of the pituitary gland surrounding the neurohypophysis. All acidophilic cells showed a strong immunofluorescence with anti-ovine prolactin (LTH).

Modifications hypophysaires et thyroïdiennes chez le pleurodèle en milieu salin

In the pituitary gland of Pleurodeles kept in a saline solution (NaCl, 9 g/liter) for 15 days, ventral orangeophilic cells appear less active, dorsal erythrosinophilic cells enlarge, and the intermediate lobe seems less active than in freshwater animals. Caryometric data corroborate these findings. Published results on osmoregulation generally indicate that prolactin (PRL) secretion is reduced in a saline environment, an observation which may be correlated with the hypoactivity detected in the orangeophilic cells simultaneously labelled with an anti-ovine prolactin antibody. The localization of growth hormone (STH) and melanocorticotropic hormone secretion is in accordance with previous cytoimmunological data. The histological structure of the thyroid gland suggests an increased activity in saline solution which may facilitate osmotic adaptation.

Immunohistochemical localization and identification of the prolactin and growth hormone producing cells in the pituitary gland of the toad Bufo bufo bufo (L)

The two acidophilic cell types in the pars distalis of the pituitary gland of Bufo bufo were tinctorially differentiated and immunohistochemically identified with rabbit antiserum to ovine prolactin and to bovine growth hormone. By means of immunoglobulin-enzyme bridge and indirect immunofluorescence technique, the antio vine prolactin was localized in only the carminophils and antibovine growth hormone was localized in only the orangeophils.

Immunocytochemical localization of prolactin in the pars distalis of the salamander Necturus maculosus

The prolactin cells in the anterior pituitary of Necutrus maculosus have been localized with anti-ovine prolactin using the peroxidase-labeled antibody technique. Two histological stains, carmoisine-L, orange G and hematolylin and eosin, were used to corroborate the immunocytochemical results. In low magnification micrographs, the peroxidase positive area corresponds to comparable acidophilic and carmoisine-L staining areas of other sections from the same part of the gland. Carmoisine-L stains the heavily granulated prolactin cells bright orange. These cells correspond in morphology and arrangement to those containing the dark brown precipitate which indicates the site where antigen bound peroxidase has reacted with its substrate. When tissue was stained with nonimmune serum or with immune serum adsorbed with prolactin instead of the first antibody, the results were negative.

Effect of antiserum to rat prolactin on milk yield and food intake in the rat.

Department of Applied Pharmacology, School of Pharmacy, Hebrew University, Jerusalem, Israel (Received 5th December 1974) Anti-ovine prolactin injected into the duct of a rabbit mammary gland inhibits the local lactogenic response of prolactin in the same duct (Saji & Crighton, 1968), and inhibits the proliferative effect of concurrently-injected prolactin in the pigeon crop-sac assay (Hayashida, 1962). Anti-ovine prolactin also decreases the weights of the prostate and seminal vesicle of male rabbits (Asano, Kanzaki, Sekiguchi & Tasaka, 1971). Anti-mouse prolactin has been reported to suppress the growth of mammary tumours in mice (Sinha, Lewis & Vanderlaan, 1974) and to inhibit the biological activity of mouse prolactin as measured by the pigeon crop-sac test (Kosugiyama, Mori & Nagasawa, 1970). Anti-rat prolactin injected into lactating rats lowered the progesterone secretion rate (Yoshinaga, 1973), while anti-human placental lactogen injected to pregnant rats during Days 1 to 5 of gestation reduced the weight of the

Immunohistochemical Localization of Growth Hormone and Prolactin in the Pituitary Gland ofAcipenser güldenstaedtiBrandt (Chondrostei)

Abstract Prolactin (LTH) and growth hormone (GH) containing cells in A. güldenstaedti have been localized by means of anti-ovine prolactin and anti-bovine growth hormone respectively, coupled indirectly to peroxidase, and localized histochemically with hydrogen peroxide as substrate and 3,3′-diaminobenzidine as capturing agent. The distribution of the anti-prolactin positive cells has been demonstrated and correlated histologically with the acidophilic cells both in the rostral and proximal pars distalis. This cell type is elongated and arranged in follicles in the rostral pars distalis; in the proximal pars distalis they are smaller and oval, without any special orientation. Neither of the other cell types which are scattered among these acidophils contain prolactin. The anti-bovine growth hormone positive cells are evenly distributed in the proximal pars distalis above the hypophysial cleft, and some are also found in the pars intermedia. The anti-GH positive cells have been correlated histologically with the amphiphilic cells in the proximal pars distalis. These cells are arranged in cell cords in close contact with the secondary capillary plexus, near its origin from the primary capillary plexus covering the median eminence.

A New Modification of the Radioimmunoassay for Human Prolactin

Jqcobs等の方法によるとヒトプロラクチンのRadioimmunoassay法を更に改良, 簡便化した.標準品として, Jacobs等が患者血清を使用しているのに対して, 私共は純化ブタプロラクチンを使用した.純化ブタプロラクチンのDose Respose Curveは稀釈ヒト血清と平行する事が証明された.Jacobs等の方法は標準血清をBioassayで力価を測定する必要があるが, 私共の改良法によれば直ちに国際単位で結果を表示できる.

HUMAN PITUITARY PROLACTIN DURING PREGNANCY AND POST PARTUM AS MEASURED IN SERUM BY A RADIOIMMUNOASSAY

Human pituitary prolactin (HPr) was measured using its cross reaction with sheep prolactin in a radioimmunoassay initially developed by Davis et al. (1971). The proposed system has been proved to be suitable for specific measurement of HPr contained in 200 μl of human serum (Midgley and L’Hermite; to be published). There was no inhibition of the binding of the tracer (highly purified ovine prolactin; LER-860-2) to the anti-ovine prolactin serum by as much as 2500 IU of human chorionic gonadotrophin (Commercial HCG, Roussel), 500 ng of human growth hormone (HGH-HS1394) and 10 μg of human chorionic somatomammotropin (HCS, Lederle 717.340). Immunoreactive HPr was measured in serum with reference to a pool of serum collected from pregnant women. This pool was temporarily used as laboratory standard. An immunological activity of 1.0 Unit (U) has been arbitrarily attributed to the amount of HPr contained in 1.0 ml of this pool.

A mixed heterologous radioimmunoassay for human prolactin.

A mixed heterologous radioimmuoassay for human prolactin (HPr) has been developed in which prolactin in serum or plasma competes with radio-iodinated porcine prolactin for binding sites on anti-ovine prolactin antibody. The system is unaffected by all pituitary and placental peptides tested, and allows specific measurement of HPr in unextracted plasma or serum throughout pregnancy and in acromegaly. Sensitivity is sufficient to detect HPr in serum of normal males and females, whose mean levels (± se) were 6.2 ± 0.6 and 9.0 ± 0.6 ng/ml, respectively. Mean HPr levels (± se) during normal pregnancy were 56.2 ± 5.6, 117.4 ± 14.0, and 251.4 ± 23.3 ng/ml in the three successive trimesters. Elevated levels were found in most patients with non-puerperal galactorrhea of various etiologies, and were very high in patients with HPr-secreting pituitary tumors.