Diversity of prolactin systems in the insect Leucophaea maderae: Use of antiserum polyclonality for immunocytochemical detection of neuropeptide heterogeneity

Summary The presence of prolactin-like neuropeptides was demonstrated immunocytochemically in the brain and affiliated neuroendocrine structures of the insect Leucophaea maderae. Use of the unlabelled peroxidase-antiperoxidase method of Sternberger revealed a rather widespread and differential distribution of reaction products resembling human (hPRL) and ovine (oPRL) prolactin. Tests with antirat PRL antibody were negative. The specificity of the antibodies used was established by liquid-phase absorptions and confirmed in tissue control systems. In L. maderae, anti-oPRL identifies part of an oPRL-like molecule different from human and rat PRL. Anti-hPRL reveals part of a human and ovine PRL-like molecule different from rat prolactin. These results indicate the occurrence, in the nervous tissue of one insect species, of at least two types of prolactin-like molecules.Supported in part by SNF grants 11-5082 and 11-6652 (G.N.H.) and NIH Grant NS 22344-02 (B.S.). The authors are indebted to Mrs. Bente Hershøj for skillful technical assistance     

Comparative immunocytochemical localization of prolactin and somatotropin in the pituitaries of Lepidosiren paradoxa,Rana temporaria and Ambystoma mexicanum

Summary The cellular binding sites of anti-oPRL IgG and anti-bSTH IgG were demonstrated in the pituitary glands of Lepidosiren paradoxa, Rana temporaria and Ambystoma mexicanum by means of the unlabeled antibody enzyme method by light and electron microscopy (the latter only in Lepidosiren). With the light microscope PRL or PRL-like substances and STH or STH-like substances were revealed in two different cell types in the distal lobe corresponding to the acidophils. However, as a result of the insufficient differentiation of the acidophils in Lepidosiren after staining with Brookes' procedure it was not possible to distinguish the two types of acidophils in this animal. Treatment with low dilutions of both anti-oPRL and anti-bSTH IgG revealed simultaneous immunocytochemical staining in both types of acidophils in Lepidosiren and in Rana. These results, indicating that there is antigenic cross-reaction between anti-oPRL and anti-bSTH IgG and both PRL and STH in these animals, are discussed.The electron microscopic investigations of Lepidosiren revealed that the specific anti-oPRL IgG reactive cells contain granules ranging in size from 200 to 300 nm, while the specific anti-bSTH IgG reactive cells contain smaller immunoreactive granules ranging from 80 to 160nm.     

Identification by immunofluorescence of prolactin-and somatotropin-producing cells in the pituitary gland of the Mexican axolotl (Ambystoma mexicanum,Shaw)

Summary The indirect immunofluorescence procedure was used to identify prolactin (LTH)-and somatotropin (STH)-producing cells in the pituitary of the Mexican axolotl. Histological staining techniques were employed to corroborate immunocytological results. The LTH cells are large, orange-staining cells (acidophils 1) distributed in the posterior two-thirds of the pars distalis. The STH cells are small, erythrosinophilic elements (acidophils 2) principally concentrated in the dorsal part of the pars distalis.     

Immunoreactive material resembling ovine prolactin in perikarya and nerve terminals of the rat hypothalamus

Summary The presence of prolactin (PRL)-like material is demonstrated in the brain of rats with the aid of anti-ovine PRL (oPRL) IgG as primary antibody in the unlabeled antibody-enzyme method. Immunoreactive deposits are visualized as an intraneuronal constituent with a widespread distribution in the hypothalamus and neural lobe of the pituitary. Dense networks of reactive nerve terminals derived from two prominent fibre tracts, a ventral (VHT) and a dorsal hypothalamo-neurohypophysial tract (DHT) are seen. The VHT is confined to the median eminence and pars oralis tuberis, the DHT to the pars caudalis tuberis. Both fibre tracts pass through the infundibular stalk into the neural lobe. The origin of the immunoreactive nerve terminals can be elucidated only to some extent. The VHT gives off beaded fibres entering the ependymal and glandular layer of the median eminence. Immunoreactive perikarya are observed in the supraoptic nucleus, the paraventricular nucleus, the anterior hypothalamic nucleus, the anterior commissural nucleus, the preoptic nucleus and the interstitial nucleus of the stria terminalis. A few of the immunoreactive perikarya are observed in close connection with brain vessels and the ependymal cells of the third ventricle. The results indicate that the anti-oPRL has a unique region specificity implying that only a segment of the mammalian PRL molecule is present in these nuclei of the brain. Fragments of PRL may function as neuromodulators or neurotransmitters in the rat brain.We are indebted to Dr. Mogens Hammer, Rigshospitalet, Copenhagen for the gift of Arg-VP and anti-VP, and to NIAMDD for the gift of ovine PRL, ratPRL, anti-rPRL, anti-hPRL and bovineSTH     

Large-Scale Preparation of Recombinant Ovine Prolactin and Determination of Its in Vitro and in Vivo Activity

Recombinant bovine Ala-prolactin (PRL) (GenBank Accession No. V00112) in prokaryotic expression plasmid pMON3401 was mutated using a mutagenesis kit, to prepare plasmid encoding ovine PRL (oPRL) (GenBank Accession No. M27057) Escherichia coli cells transformed with this latter plasmid overexpressed large amounts of oPRL upon induction with nalidixic acid. The expressed protein, found in inclusion bodies, was refolded and purified to homogeneity on a Q-Sepharose column, yielding an electrophoretically pure fraction composed of over 98% monomeric protein of the expected molecular mass of 23 kDa. The biological activity of the recombinant oPRL after proper renaturation was evidenced in vitro by its ability to stimulate proliferation of rat lymphoma Nb₂ cells possessing PRL receptors, to stimulate luciferase activity in HEK 293 cells transiently transfected with oPRL receptors, and to induce progesterone secretion in primary cultures of luteal cells obtained from midpregnant ewes. In contrast to ovine growth hormone or ovine placental lactogen, recombinant oPRL had no galactopoietic effect in lactating ewes.     

Immunocytochemical demonstration of mammalian lutropin-like material in the pituitary of the lungfish,Lepidosiren paradoxa

Summary The presence of lutropin (LH)-like material in the pituitary gland of the South-American lungfish, Lepidosiren paradoxa, has been demonstrated by means of the unlabeled antibody-enzyme method, by use of rabbit antiovine LH as first antibody. The LH-like substance was revealed in a single PAS-positive cell type primarily located in the anterior part of the distal lobe. Dot blot tests as well as conventional liquid-phase absorption experiments indicate that the anti-ovine LH antibodies possess specificity against the oLH/gb subunit. These observations indicate that dipnoans (Lepidosiren) share a number of antigenic determinants with those of mammalian LH/gb and support the concept that mammalian LH/gb, or part of it, was established early in evolution. The exact nature and physiological function of the substance detected remains to be defined.     

Induction of lactogenic receptors. I. In the liver of hypophysectomized female rats.

To identify the hormones which affect lactogenic receptors in the liver of chronically hypophysectomized female rats, hormones were injected s.c. for 7 days. Specific binding (%, SB) of labelled ovine prolactin (PRL) in liver membrane preparations (1000,000 X g pellet) of controls was 1%. Estradiol (E2), cortisone (Con), ACTH or bovine growth hormone (bGH) treatment did not induce hepatic binding sites for PRL. Human GH and a single dose of 2mg PRL (but not lower doses) increased SB of PRL. Treatment with oPRL plus ACTH was less effective than hGH plus ACTH (13 vs 28%); combinations of oPRL plus Con as well as administration of oPRL plus ACTH to hypophysectomized and adrenalectomized female rats did not induce SB for PRL. Therapy with oPRL plus hGH (26%) was more potent than oPRL plus bGH (2%). These studies suggest that PRL, GH, and ACTH induce and in concert with sex steroids, modulate the lactogenic receptors in the female rat liver. The effect of ACTH is not due to increased adrenal corticoid secretion.     

Monoclonal antibodies against rabbit mammary prolactin receptors. Specific antibodies to the hormone binding domain

Three monoclonal antibodies (M110, A82, and A917) were obtained by fusing myeloma cells and spleen cells from mice immunized with partially purified rabbit mammary gland prolactin (PRL) receptors. All 3 antibodies were capable of complete inhibition of 125I-ovine prolactin (oPRL) binding to rabbit mammary PRL receptors in either particulate or soluble form. M110 showed slightly greater potency than oPRL in competing for 125I-oPRL binding. These antibodies also inhibited PRL binding to microsomal fractions from rabbit liver, kidney, adrenal, ovary, and pig mammary gland, although A82 showed poor inhibition in pig mammary gland. There was no cross-reaction of any of the 3 monoclonal antibodies (mAbs) for the other species tested: human (T-47D breast cancer cells) and rat (liver, ovary). In order to confirm that these antibodies are specific to the binding domain, antibodies were purified, iodinated, and binding characteristics were investigated. 125I-M110 and 125I-A82 binding was completely inhibited by lactogenic hormones, whereas nonlactogenic hormones did not cross-react. Competition of 125I-M110 by oPRL (ID50 = 0.44 nM) was comparable to that of 125I-oPRL by unlabeled oPRL (ID50 = 0.35 nM), while 125I-A917 binding was only partially competed (30-60%) by lactogenic hormones. Tissue and species specificity of labeled antibody binding paralleled results of binding inhibition experiments using 125I-oPRL. In addition, A82 and A917 completely inhibited 125I-M110 binding. In contrast, 125I-A82 binding was stimulated by A917 and 125I-A917 binding was stimulated by A82. These findings indicate that monoclonal antibodies can be readily prepared from partially purified PRL receptors from rabbit mammary gland; two antibodies (M110 and A82) are hormone binding site specific while the other (A917) binds a domain partially but not entirely distinct from the hormone binding site, and that all three antibodies have strong species specificity.     

Effect of pituitary hormones on phosphodiesterase and adenyl cyclase activity of brain tissue in vitro

Effects of seiwhale somatotropin (STH), its biologically active fragment 77--107, porcine corticotropin (ACTH) and seiwhale prolactin on phosphodiesterase and adenylate cyclase activity of glial cells and synaptosomes isolated from the rat brain cortex were investigated. As compared with control, ACTH increased phosphodiesterase activity of glial cells by 392%, of synaptosomes by 123%, while STH by 49 and 77%, respectively, somatotropin fragment by 455 and 74%, and prolactin by 30 and 37%, respectively. Adenylate cyclase activity was significantly changed only by ACTH and only in synaptosomes (a 50% decrease). STH, its fragment and prolactin virtually failed to alter adenylate cyclase activity. The data obtained indicate that some of pituitary hormones, primarily ACTH and STH, may play the role of neuromodulators in some brain structures by decreasing the cyclic AMP level, by activating phosphodiesterase (STH and ACTH) and inhibiting adenylate cyclase (ACTH in synaptosomes).     

Immunocytochemical localization and immunochemical characterization of an insulin-related peptide in the pancreas of the urodele amphibian,Ambystoma mexicanum

Summary The pancreas of the axolotl, Ambystoma mexicanum, was investigated by immunocytochemical methods for the presence of immunoreactivity to a number of antisera raised against mammalian insulins. All anti-insulin antisera tested revealed substantial amounts of reaction products confined solely to the aldehyde-fuchsinophilic B cells of the endocrine pancreas. The reactive cell population was detected by use of one polyclonal antiserum against bovine insulin and eight different monoclonal antibodies against insulins from various mammalian species. Six of these antibody clones have known specificity to sub-regions of the insulin molecule. Additionally, fractions of an ethanol-HCl extract of pancreatic tissue from Ambystoma was studied in both conventional dot-blot tests by means of the same panel of antibodies and a two-site sandwich time-resolved immunofluorometric assay for human insulin involving two of the monoclonal antibodies. These experiments support the immunocytochemical observations by demonstrating the existence of an insulin-related peptide with a great deal of structural resemblance to mammalian insulins and displaying antigenic determinants in common at least with the amino acid residues A_8–10 and B_26–30. In conclusion, we interpret the findings as indicating that the immunocytochemically revealed tissue bound antigen in the Ambystoma pancreatic B-cells may be a peptide related to human insulin.Supported in part by SNF grant 11-5082 (G.N.H.). The authors are indebted to Dr. P. Rosenkilde for the gift of the Ambystoma material     

Prolactin control of growth and prolactin autoregulation in cultured human pituitary cells

A human pituitary cell line (18-54,SF) grows in serum-free medium and secretes prolactin (PRL). Autoregulation of pituitary cell growth and PRL production by exogenously supplied ovine PRL (oPRL) was investigated. Human PRL (hPRL) and oPRL stimulated pituitary cell growth up to 92% and 85%, respectively, at hPRL and oPRL additions of 100-1,000 ng/ml. Short-term (1 h) incubation of the cells with oPRL decreased hPRL secretion from the cells by 72% at 10 ng/ml addition. Intracellular hPRL was stimulated under the same conditions by 50-275% at oPRL concentrations of 10-1,000 ng/ml. Long-term (10 days) incubation of the cells with oPRL had no significant effect on extracellular or intracellular hPRL production. These data suggest that the pituitary gland can serve as a primary feedback site and that PRL can autoregulate its own production as well as affect the growth of pituitary cells.     

Down-regulation of prolactin receptors in the liver, mammary gland and kidney of female virgin rat, infused with ovine prolactin or human growth hormone

Down regulation of prolactin (PRL) receptors resulting from i.v. infusion of oPRL or human growth hormone (hGH) into female virgin rats was demonstrated. A decrease of over 85% in the number of free receptors was achieved within 15 - 30 min using infusion of oPRL or hGH at 25 micrograms/h and remained at this level until the end of infusion. Ovine growth hormone or recombinant bovine growth hormone at ten-fold higher concentration had no effect at all. The decrease in the specific binding resulted from a lower number of binding sites and not from change in the dissociation constants. The decrease in the total receptors in the liver was more gradual and leveled off at 40 - 50% of the initial value. Our results suggest that a change in blood PRL or hGH level may lead to a new steady state in the number, occupancy and distribution of prolactin receptors.     

Histochemical demonstration of prolactin binding sites

We have developed a new probe for histochemical demonstration of prolactin binding sites. Ovine prolactin (oPRL) was conjugated with the N-hydroxy-succinimide ester derivative of the fluorochrome 7-amino-4-methylcoumarin-3-acetic acid. Under mild reaction conditions the ester derivative reacted with available NH2 groups of the prolactin molecule to form stable bonds. The coupling reaction yielded products that co-migrated with oPRL but had slightly decreased isoelectric points. The receptor binding and bioactivity of the flurochrome-hormone derivatives were decreased essentially proportional to the extent of conjugation. The derivatives were further tested for their ability to label PRL binding sites, using frozen sections of mammary gland and brain tissue of lactating rats. The results presented in this report describe the validation of this probe with regard to labeling of PRL binding sites at the light microscopic level in known target organs (mammary gland and choroid plexus). In addition, this fluorescent probe was used to demonstrate the presence of PRL binding sites at a novel site, the ependymal lining of the third ventricle.     

A novel concatenated dimer of recombinant bovine somatotropin

A novel protein concatenated dimer structure was generated during the folding/oxidation of inclusion bodies of recombinant bovine somatotropin synthesized inEscherichia coli. The structure of this dimeric molecule was determined by peptide mapping with trypsin, and limited proteolysis by thrombin. Peptide mapping demonstrated that the two disulfide pairs in bovine somatotropin dimer were identical to those in monomer. Limited proteolysis with thrombin resulted in the cleavage of only a single peptide bond between arginine-132 and alanine-133 in bovine somatotropin dimer. This single peptide bond cleavage was sufficient to convert this dimer to a monomeric molecular weight species as analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and HPLC. Since the single cleaved peptide bond is present in the large disulfide loop of bovine somatotropin, these data demonstrate that the dimeric molecule exists as a novel concatenated structure through the interlocking of the disulfide loops of this protein.     

Immunocytochemical localization and immunochemical characterization of an insulin-related peptide in the insect Leucophaea maderae

Summary Immunocytochemical tests with eight monoclonal antibodies against either bovine or human insulin and seven polyclonal antibodies against bovine insulin were carried out to determine the presence of insulin-like neuropeptides in the brain and affiliated neuroendocrine structures of the insect Leucophaea maderae. Reaction products identified in the brain, subesophageal ganglion, and corpus cardiacum-corpus allatum complex indicate the presence of materials resembling mammalian insulins in its antigenic properties. The immunostaining observed with monoclonal antibodies appears to indicate the occurrence of an insulin-related peptide that shows sequential similarities with parts of both the A- and B-chains of mammalian insulin molecules. These suppositions are supported by the results of dot-blot and two-site time-resolved immunofluorometric assay (TR-IFMA) screenings of fractions of Leucophaea tissue extracts obtained by chromatography. The polyclonal antibodies yielded reaction products in some of the same areas and in additional parts of the neuroendocrine system not visualized by the monoclonal antibodies. Immunoreaction was observed in the following areas: the pars intercerebralis of the protocerebrum, the nervi corporis cardiaci I transporting insulin-like material to the corpus cardiacum, the dorsolateral protocerebral area and the optic lobes, the deutocerebrum, the tritocerebrum, and the subesophageal ganglion. In addition, smaller cell bodies with immunoreactive deposits occur at the border between proto- and deutocerebrum, and in the central area of the protocerebrum. The distribution of reactive material in the corpus cardiacum-corpus allatum complex after use of both groups of antibodies was the same. The fact that polyclonal and monoclonal antibodies yielded reaction products in different cells of the brain suggests either that the two groups of antibodies recognize different epitopes of the same molecule, or that they reveal two different types of immunoreactive molecules related to mammalian insulins. Together with the biochemical data reported by Nagasawa and coworkers (PNAS 83, 1986) the present immunocytochemical analysis has established a closer relationship between mammalian and insect insulins than was previously known.Supported in part by NIH grant NS 2344-02 (B.S.) and SNF grant 11-5082 and 11-7705 (G.N.H.)     

An investigation of sites that bind human somatotropin (growth hormone) in the liver of the pregnant rabbit.

The binding of 125I-labelled human somatotropin (growth hormone) to a crude membrane preparation from the liver of pregnant rabbit, and to receptors solubilized from this fraction by Triton X-100, was dependent on time, temperature and receptor concentration. At 4 degrees C a steady state was reached after 20 h, and maximum specific binding (as a percentage of total tracer added) was approx. 50% for both membrane-bound and solubilized receptors. Solubilization did not significantly affect the binding properties of the receptor at low concentrations of Triton X-100 (less than 0.05%, v/v, in the assay tube). However, at higher concentrations (approx. 0.1%, v/v), the detergent lowered the ability of some hormones, for example ovine prolactin, to displace 125I-labelled human somatotropin, but did not affect other hormones such as bovine somatotropin. Some somatogenic hormones, such as bovine somatotropin, and some lactogenic hormones, such as ovine prolactin, displaced 125I-labelled human somatotropin from membrane-bound and solubilized receptor preparations. Furthermore, 85% of 125I-labelled bovine somatotropin was displaced from membrane-bound receptors by ovine prolactin, and 125I-labelled ovine prolactin was almost completely displaced by bovine somatotropin. Scatchard analysis of the binding data for human somatotropin suggested a single class of binding sites in the membrane-bound receptor preparation, with an affinity (Ka) of 1.9 X 10(9) M-1 and a capacity of 1726 fmol/mg of protein; these values were slightly increased by solubilization (Ka = 3.2 X 10(9) M-1, capacity = 2103 fmol/mg of protein). Scatchard analysis of binding to membrane-bound receptors also indicated a single class of high-affinity binding sites for bovine somatotropin (Ka = 4.8 X 10(9) M-1, capacity = 769 fmol/mg) and for ovine prolactin (Ka = 6.1 X 10(9) M-1, capacity = 187 fmol/mg).     

PURIFICATION AND BIOLOGICAL CHARACTERIZATION OF BACTERIALLY EXPRESSED RECOMBINANT BUFFALO PROLACTIN

Recombinant prolactin (PRL) from water buffalo (Bubalus bubalis) has been cloned and expressed in a prokaryotic expression system. The hormone was also successfully refolded into a biologically active form. Total RNA was purified from buffalo pituitaries and the buPRL cDNA was synthesized using primers designed on bovine PRL sequence. This prolactin cDNA was cloned in a pET 28a vector and expressed in Escherichia coli strain BL21(DE3)pLysS. Most of the expressed protein was present as insoluble inclusion bodies. The inclusion bodies were solubilized and buPRL was purified by Ni-NTA column. The purified protein was refolded by gradually decreasing the concentration of denaturant during dialysis. Total yield of the refolded and soluble prolactin was 22 mg/L from 100 mL bacterial culture in LB medium. The recombinant prolactin was as active as native prolactin in stimulating growth of Nb2 lymphoma cells.     

Antibodies for Growth Hormone and Prolactin Using Multiple Antigen Peptide Immunogens

Antibodies elicited by novel synthetic peptide antigens derived from a highly conserved domain of the growth hormone (GH) and prolactin (PRL) of vertebrates were developed using the multiple antigen peptide approach. The sequence of the antigens is located near the carboxy-terminus in the D domain of the GH and PRL in a cluster of 11 and 10 conserved amino acids, respectively, within a sequence of 18 residues. The synthetic peptides were manually synthesized, purified by high-performance liquid chromatography, and the corresponding antibodies, elicited in rabbits, were cross-reacted with the GH and PRL of a variety of mammalian (human, bovine, ovine, pig, and equine) and nonmammalian (chicken, coho salmon, chum salmon, rainbow trout, catfish and striped bass) vertebrates. The cross-reactivity between the immunogen and its corresponding antigen was tested by immunobloting using either GH or PRL. The GH and PRL of the organisms tested cross-reacted specifically with the corresponding antibody. Chicken and fish GH and PRL showed stronger antibody cross-reactivity than that observed in mammalian sources. These results demonstrate the utility of peptide-derived polyclonal antibodies in the detection of native and recombinant GH and PRL of a variety of vertebrates. Received June 1, 1998; accepted November 13, 1998.