Monoclonal antibodies to luteinizing hormone-releasing hormone: production, characterization, and immunocytochemical application

Luteinizing hormone-releasing hormone (LHRH) was conjugated to bovine thyroglobulin and used to immunize a BALB/c mouse. Spleen lymphocytes were subsequently fused to SP2/0 myeloma cells and two of the resulting hybridoma clones were found to produce high titer antibodies to LHRH (HU4H and HU11B); both belonged to the IgG1 subclass. Characterization of the monoclonal antibodies revealed that HU4H and HU11B have conformational and sequential specificity to LHRH, respectively, and that neither one shows significant immunoactivity with pro-LHRH. The value of these antibodies in immunocytochemical applications is demonstrated by their ability to cause intense specific staining of LHRH neuronal cell bodies and fibers in brain sections from several mammalian species.     

Receptors for luteinizing hormone-releasing hormone.

Antisera to luteinizing hormone-releasing hormone (LH-RH) confer on Araldite sections of occasional rat pituitaries moderate immunocytochemical staining to the large secretory granules of gonadotrophs. Treatment of the sections with LH-RH before anti-LH-RH yields strong staining in all animals, irrespective of presence or absence of staining without pretreatment. This enhancement of staining is specific for LH-RH and is a high affinity, saturable reaction. Staining with or without LH-RH pretreatment is absent when anti-LH-RH absorbed with insolubilized LH-RH is used. Staining is inhibited by carboxyterminally-deficient LH-RH, unaffected by aminoterminally deficient LH-RH.     

Relationship between neuropeptide Y and luteinizing-hormone-releasing hormone immunoreactivities in the hypothalamus and preoptic region

The purpose of this study was to examine the anatomical relationships of perikarya and fibers containing neuropeptide Y (NPY) and luteinizing-hormone-releasing hormone (LHRH) in the hypothalamus and preoptic region of female rats. In view of our previous report of stimulatory effects of estrogen on LHRH and NPY levels in the median eminence, animals were bilaterally ovariectomized and subsequently implanted subcutaneously with capsules containing estradiol benzoate in oil or vehicle. Following intracerebroventricular injection of colchicine, rats were perfused with fixative and their brains sectioned and processed for immunohistochemical visualization of NPY and LHRH in the same section and in consecutive sections. Estrogen treatment had no discernible effect on the distribution or relationship of these peptides. NPY-immunoreactive fibers were intimately associated with LHRH-labeled primary dendrites and perikarya in the medial preoptic region and horizontal limb of the diagonal band of Broca. Fibers containing NPY or LHRH overlapped extensively in the lateral palisade region of the median eminence and also in the subependymal and internal zones. The external zone of the median eminence displayed relatively less overlap of these peptide systems. LHRH-immunoreactive axons coursed among NPY-labeled perikarya in the arcuate nucleus and appeared to contact these cells. These results suggest that NPY-containing axons may influence LHRH-positive neurons at the cell body and also at the site of axon termination in the median eminence. LHRH-containing axons appear to contact NPY-immunoreactive perikarya in the arcuate nucleus and may interact with terminals in the median eminence. This arrangement may provide a mechanism for communication between NPY and LHRH neurons and for the neuroendocrine coordination of hypothalamic NPY and LHRH secretion before ovulation.     

Luteinizing hormone-releasing hormone I (LHRH-I) and its metabolite in peripheral tissues

Luteinizing hormone-releasing hormone (LHRH) was first isolated in the mammalian hypothalamus and shown to be the primary regulator of the reproductive system through its initiation of pituitary gonadotropin release. Since its discovery, this form of LHRH (LHRH-I) has been shown to be one of many structural variants with a variety of roles in both the brain and peripheral tissues. Enormous interest has been focused on LHRH-I and LHRH-II and their cognate receptors as targets for designing therapies to treat cancers of the reproductive system. LHRH-I is processed by a zinc metalloendopeptidase EC 3.4.24.15 (EP24.15) that cleaves the hormone at the fifth and sixth bond of the decapeptide (Tyr(5)-Gly(6)) to form LHRH-(1-5). We have previously reported that the autoregulation of LHRH gene expression can also be mediated by its processed peptide, LHRH-(1-5). Furthermore, LHRH-(1-5) has also been shown to be involved in cell proliferation. This review will focus on the possible roles of LHRH and its processed peptide, LHRH-(1-5), in non-hypothalamic tissues.     

The localization of oxytocin,vasopressin, somatostatin and luteinizing hormone releasing hormone in the rat neurohypophysis

Summary The hypothalamic hormones arginine-vasopressin (AVP), oxytocin (OXT), somatostatin (SOM), and luteinizing hormone-releasing hormone (LHRH) were localized in the rat neurohypophysis by the use of semithin serial sections and the unlabeled antibody enzyme method. Clusters of AVP fibres are present within the central region of the neural lobe, clusters of OXT fibres mainly in the peripheral part. The AVP fibres enter bilaterally into the neural lobe.The results call into question previous reports on the presence of AVP on receptors in the pars intermedia cells, since incubation with anti-AVP resulted in similar staining in the pars intermedia of the Wistar and homozygous Brattleboro rat, a mutant strain deficient in AVP. The same intermediate lobe cells are stained after incubation of serial sections with anti-AVP and anti--melanocyte-stimulating hormone (-MSH). This staining of anti-AVP could be removed by solid phase absorption to -MSH and is thus most probably due to cross reaction with -MSH. SOM fibres appear to be present in the peripheral parts of the proximal neurohypophysial stalk and mainly lateral in its more distal parts. In the neural lobe they rapidly decrease in number, although some fibres continue into the distal part of the neural lobe, running bilaterally and situated adjacent to the pars intermedia. The SOM staining within magnocellular elements, which has been reported in the literature, can most probably be explained by cross reaction of anti-SOM with neurophysins. LHRH fibres are very scarce in the neurohypophysial stalk and absent in the neural lobe.Supported by the Foundation for Medical Research FUNGOThe authors wish to thank Drs. J. De Mey (Beerse, Belgium), A. Arimura (New Orleans, U.S.A.), M.P. Dubois (Nouzilly, France), B.L. Baker (Ann Arbor, U.S.A.) and A.G.E. Pearse (London, U.K.) for their gifts of anti-somatostatin serum, Dr. B. Kerdelhué (Gif-sur-Yvette, France) for anti-LHRH serum, and Dr. F. Vandesande (Ghent, Belgium) for anti-neurophysin I and II serum and bovine neurophysin I and II. Dr. J.G. Streefkerk (Free University, Amsterdam) is acknowledged for critical comments and Mr. A.T. Potjer and Miss J. van der Velden for their skilled assistance     

Absence of specific luteinizing hormone releasing hormone receptors in ovine, bovine and porcine ovaries

Crude and membrane-enriched homogenates of unfrozen follicular and luteal tissue from cows, ewes and sows were assayed for the presence of specific luteinizing hormone releasing hormone (LHRH) receptors by one-point saturation analysis using [D-Ser-(TBU)6, des-Gly-NH2(10)] LHRH-EA as the labeled and unlabeled ligand. Pituitaries from cows, ewes, sows and rats, and rat ovaries served as positive controls and were assayed with each ovarian tissue assay. Scatchard analysis was used to determine binding affinity of pools of ovarian and pituitary tissue. Specific high-affinity LHRH receptors were found in the pituitaries of cows, ewes, sows and rats and in the rat ovary. In contrast, no specific LHRH binding was detected in follicular or luteal tissue of cows, ewes or sows. Thus, unlike the rat ovary which contains LHRH receptors, ovaries from these domestic species lack specific LHRH receptors.     

Combined light and electron microscope immunocytochemical localization of scattered peptidergic neurons in the central nervous system

A pre-embedding immunocytochemical technique is described for combined light and electron microscope study of peptidergic neurons in the central nervous system. The protocol is especially designed to overcome the sampling problems inherent in electron microscope study of structures, such as luteinizing hormone-releasing hormone (LHRH) neurons, that are scattered individually across large brain regions. The fixation methods outlined for several mammalian species include immersion and vascular perfusion with acrolein. Fine-structural preservation and LHRH immunoreactivity obtained with this fixative are compared to results with more conventional fixatives. Vibratome sectioning and a "pretreatment" regime, which prepare the tissues for immunocytochemistry, are described. Immunocytochemical labeling is done with free-floating sections and the peroxidase-antiperoxidase unlabeled antibody enzyme technique. Techniques are also described for the subsequent processing of immunoreacted sections for electron microscopy. These methods ensure that the processed sections are readily scanned by light microscopy, so that regions containing immunoreactive structures can be specifically chosen for electron microscope analysis. Sample electron micrographs are shown that illustrate some fine structural features of LHRH neurons in rats, bats, ferrets, and monkeys, as revealed with the techniques described.     

Processing of the luteinizing hormone-releasing hormone precursor in the preoptic area and hypothalamus of the rat

The LHRH precursor is known to contain the decapeptide and a 56 amino acid peptide termed gonadotropin-releasing hormone-associated peptide (GAP). The purpose of our study was to characterize the proLHRH and its processed products from the cell body and fiber region and from the nerve terminal region of LHRH neurons. The median eminence (ME) and a tissue block containing the preoptic area and hypothalamus (POH) were dissected separately. Tissues were homogenized and peptides were separated according to mol wt. Three different LHRH antisera bound to one immunoreactive (IR) substance which eluted at approximately 1200 mol wt. Subsequently, this material coeluted with synthetic LHRH on a reversed-phase column as a single peak. There was approximately 1.6-fold more LHRH-like IR in the ME than in the POH. The four different GAP antisera recognized multiple mol wt forms of GAP-like IR at approximately 16,000 to 14,000, 8,200, 6,500, 3,500, and 2,800 mol wt. There were more of the high mol wt materials and less of the 6500 and lower mol wt materials in the POH than in the ME. The most abundant species in both regions was the 6500 mol wt form. This IR substance coeluted with synthetic rat GAP1-56 on a reversed-phase column as a single peak. These experiments demonstrate 1) that multiple IR forms of the LHRH prohormone exist in the POH of the rat and 2) that nerve terminals of the LHRH neurons contain LHRH, GAP1-56, and some lower mol wt GAP-like substances. These results provide the first information concerning the processing scheme for the LHRH prohormone in the rat brain.     

Immunocytochemistry with osmium-fixed tissue. I. Light microscopic localization of growth hormone and prolactin with the unlabeled antibody-enzyme method.

Growth hormone and prolactin were localized on thin plastic sections of rat anterior pituitary gland and mammosomatotropic tumor MtTW15 that were fixed with osmium tetroxide (alone,mixed with aldehydes, or after aldehydes). Intense immunocytochemical staining for both antigens was obtained after plastic was removed from sections with an alcoholic solution of sodium hydroxide. The results indicated that antigenic determinants of rat prolactin and growth hormone were not completely destroyed or inactivated by fixation with osmium and embedment in epoxy resin, and that removal of the polymerized epoxy resin was necessary to obtain light microscopic postembedding immunocytochemical staining of these antigens. The results also demonstrated that tissues which have been conventionally processed for morphological evaluation by electron microscopy may be suitable for postembedding immunocytochemical staining of some antigens for light microscopy.     

Immunostaining of growth hormone and prolactin in paraffin-embedded and stored or previously stained materials.

In attempts to evaluate immunocytochemically autopsy and biopsy material previously obtained and processed for conventional histologic staining, we had to resort to immunostaining of tissues embedded years ago or even sections already stained with hematoxylin-eosin or aldehyde thionin-PAS-orange G. Hypophysial growth hormone and prolactin proved remarkably resistant to such prior treatment with regard to their antigenic properties, and could be readily immunostained in tissue embedded in paraffin 3-4 years earlier, and after destaining of sections prepared up to 7 years earlier. The results of such "retrospective" immunocytochemical evaluation of autopsy and biopsy materail is illustrated with the staining of "pregnancy cells" for prolactin in the hypophysis of a woman postpartum, the immunostaining for prolactin in the cells of adenomas associated with marked hyperprolactinemia, the staining for growth hormone in adenomas removed from children with gigantism, and the immunostaining for prolactin, growth hormone or both in several adenomas that were discovered at autopsy and not associated with a known clinical history of endocrine aberrations.     

Localization of luteinizing hormone-releasing hormone in the forebrain of the pig

The distribution of luteinizing hormone-releasing hormone (LHRH)-immunostained perikarya and processes was examined in the forebrains of six sexually mature female pigs by use of indirect biotin-avidin horseradish peroxidase immunocytochemistry. Two primary antisera (Drs. Y.F. Chen and V.D. Ramirez CRR11B73 and Miles-Yeda UZ-4) yielded positive staining. Adjacent sections treated either primary antiserum preabsorbed with LHRH or with normal rabbit serum substituted for primary antiserum lacked positive staining. The greatest proportion of LHRH-immunostained perikarya were found in the medial preoptic area adjacent to the organum vasculosum of the lamina terminalis. The LHRH-immunostained perikarya were also scattered rostrally in the diagonal band of Broca, and within the lateral hypothalamic area, paraventricular nucleus, periventricular zone, suprachiasmatic nucleus, and medial basal hypothalamus. LHRH-immunostained processes, which extended from the medial preoptic area, coursed either along the ventral surface to the median eminence or medially and ventrally along the third ventricular wall ventrally to the median eminence and caudally to the level of the mammillary bodies. Extrahypothalamic processes were located adjacent to the lateral ventricular floor and the third ventricle from the lateral septal area (stria terminalis) to the level of the habenular nucleus. LHRH-immunostained neurons were unipolar, bipolar, and multipolar. Close associations between individual LHRH-immunostained neurons were observed.     

Regulation of type II luteinizing hormone-releasing hormone (LHRH-II) gene expression by the processed peptide of LHRH-I, LHRH-(1-5) in endometrial cells

Luteinizing hormone-releasing hormone (LHRH) was first isolated in the mammalian hypothalamus and shown to be the primary regulator of the reproductive system through its initiation of pituitary gonadotropin release. Since its discovery, this form of LHRH (LHRH-I) has been shown to be one of many structural variants with a variety of roles in both the brain and peripheral tissues. Enormous interest has been focused on LHRH-I, LHRH-II, and their cognate receptors as targets for designing therapies to treat cancers of the reproductive system. LHRH-I is processed by a zinc metalloendopeptidase EC 3.4.24.15 (EP24.15) that cleaves the hormone at the Tyr(5)-Gly(6) bond. We have previously reported that the autoregulation of LHRH gene expression can also be mediated by its processed peptide, LHRH-(1-5). Given its importance in the brain, we have investigated the role of the specific processed peptide of LHRH-I, LHRH-(1-5), within Ishikawa cells, a human endometrial cell line. Using real-time polymerase chain reaction, we observed that LHRH-(1-5) upregulates LHRH-II mRNA expression in Ishikawa cells but does not exert any influence on LHRH-I mRNA levels. This is in contrast to the effects of LHRH-I, which affects the expression of LHRH-I mRNA. Our findings support a potential role for LHRH-(1-5) as a processed metabolite in the endometrium. Further investigations are needed to determine the role of this processed metabolite and to identify specific pathways involved in LHRH-(1-5) signaling.     

Establishment and clinical application of enzyme immunoassays for determination of luteinizing hormone releasing hormone and metastin.

Metastin, a 54-residue peptide, was identified as the cognate ligand of human G-protein-coupled receptor GPR54. Since metastin is a gene product of the human metastasis suppressor gene 'KiSS-1', early studies on metastin were focused on its activity as a tumor metastasis suppressor. Recently, there have been some reports that metastin is found in human plasma and is particularly abundant in the plasma of pregnant women. Dysfunction of the GPR54 receptor causes diseases that are characterized by an insufficient release of gonadotropin and lack or delay of pubertal maturation. This information strongly suggests that metastin is involved in the regulation of reproductive endocrine functions. In order to determine the plasma levels of metastin and luteinizing hormone releasing hormone (LHRH) in an isolated hypogonadotropic hypogonadism (IHH) patient, who received intermittent administrations of LHRH, we tried to establish a sensitive and specific enzyme immunoassay. The plasma LHRH levels of the patient were very high, while plasma metastin levels were at almost the same levels as circadian rhythms of healthy male humans. In the central nervous system, metastin stimulates the neuroendocrine reproductive axis. However, the effects of peripheral metastin are not known. Our result suggested that peripheral metastin had a genesis and activity different from central metastin.     

Immunocytochemical localization of luteinizing hormone-releasing hormone within the olfactory bulb of pigs

LHRH was immunocytochemically localized within the olfactory bulb of prepubertal (n = 3), ovariectomized (n = 3), and hypophyseal-stalk-transected (HST) female pigs (n = 3). Perikarya of LHRH-immunoreactive neurons of all pigs were sparsely distributed mostly in the rostral half of the olfactory bulb, along the ventromedial and ventrolateral edge of the olfactory nerve layer, or at its interace with the glomerular layer. Processes from these cells and other LHRH containing axons either entered individual glomeruli forming a network within its interior or coursed around glomeruli penetrating into the external granular layers. Additional fibers penetrated into similar regions of the accessory olfactory bulb. Irregularly shaped perikarya were also detected within the internal granular layer of the ventral olfactory bulb, but only in tissue from HST pigs. From analysis of serial sections, there was no evidence of LHRH projections across the olfactory peduncle that connects the olfactory bulb with adjacent brain regions. If olfactory LHRH neurons are involved in reproductive behavior and physiology in the pig, this pathway involves additional unidentified intervening neurons. Endocrine factors probably influence the expression of immunoreactive LHRH in the internal granule layer, since their presence was revealed only in HST pigs.     

Luteinizing hormone-releasing hormone and thyrotropin-releasing hormone in human and bovine milk

Two hypothalamic peptide hormones, luteinizing hormone-releasing hormone (LHRH) and thyrotropin-releasing hormone (TRH), have been isolated from human milk and bovine colostrum. Acidified methanolic extracts, prepared from human milk, bovine colostrum and rat hypothalami, as well as synthetic LHRH and TRH markers were subjected to high-pressure liquid chromatography (HPLC). The eluates were tested for the presence of LHRH and TRH by specific radioimmunoassays. It was found that milk extracts contain significant amounts of LHRH (3.9 - 11.8 ng/ml) and TRH (0.16 - 0.34 ng/ml), which comigrate with the corresponding marker hormones and with those of hypothalamic origin. The HPLC-purified LHRH from both human and bovine milk was bioactive in a dose-response manner similar to synthetic LHRH.     

Presence of luteinizing hormone-releasing hormone binding sites in cultured porcine lymphocytes.

Membrane-enriched homogenates of fresh and cultured (48 h) porcine lymphocytes were assayed for the presence of specific LHRH receptors by saturation and displacement analysis using [D-Ser-(TBU)6, des-Gly-NH2(10)] LHRH-EA as the labeled and unlabeled ligand. Membrane-enriched homogenates of porcine pituitaries served as positive controls while porcine granulosa cell membranes and crude liver homogenates served as negative controls. Specific high-affinity LHRH receptors were found in porcine pituitaries (Kd = 0.3 nM) and cultured lymphocytes (Kd = 13 nM) but not in fresh lymphocytes. No specific binding was observed in negative control tissues. Porcine lymphocytes have measurable high-affinity LHRH receptors after 48 h of culture.     

The effect of luteinizing hormone releasing hormone on the copulatory behavior of hyperprolactinemic male rats

The present study was undertaken to test the hypothesis that the deficits in copulatory behavior observed in hyperprolactinemic male rats may be related to a reduction in hypothalamic release of luteinizing hormone releasing hormone (LHRH). Adult male Fischer 344 rats were made hyperprolactinemic by ectopic pituitary grafts or were sham operated and 30 min prior to being tested for copulatory performance received a single subcutaneous injection of 500 ng LHRH, 100 ng LHRH, or saline. On different occasions, testosterone (T) levels were measured in plasma collected 30 min following identical treatments. Plasma prolactin (PRL) levels were determined in samples collected 30 min after injection of 500 ng LHRH. Pituitary grafting produced the expected, significant increase in plasma PRL levels and significant deficits in copulatory behavior. Treatment of hyperprolactinemic subjects with 500 ng LHRH significantly reduced both the time to first intromission and the time to ejaculation to times comparable with those of sham-operated subjects. The 100-ng dose produced a significant reduction in mount frequency. Plasma T levels were significantly elevated following either dose of LHRH. These results demonstrate that exogenous LHRH can restore normal copulatory performance in hyperprolactinemic male rats and support the hypothesis that a reduction in hypothalamic LHRH release is responsible for the behavioral deficits observed in those animals.     

Luteinizing hormone-releasing hormone in olfactory bulbs of primates

This immunohistochemical study of luteinizing hormone-releasing hormone (LHRH) in the olfactory bulbs in primates was undertaken in order to see whether there was an LHRH innervation in these species similar to that found in rodents. One old world (Macaca fascicularis) and two new world (Saimiri sciureus and Aotus trivirgatus) monkeys were studied. Aotus trivirgatus was of particular interest as it is noctural and so presumably more dependent upon olfactory cues. Animals were perfused with fixative, olfactory bulbs removed and sectioned, and tissues reacted immunocytochemically using LR1 (Benoit) antiserum to LHRH. Some LHRH innervation was found in the olfactory bulbs of all three species, comprising a few LHRH neurons and many fibers that ramified within the bulbs. The accessory bulb (not present as a distinct entity in old world primates) had more LHRH innervation than did the main olfactory bulb. Aotus trivirgatus had the greatest representation of LHRH of the three species. The layer of the olfactory bulb with the greatest number of LHRH fibers was the external plexiform layer. This is also true in rodents. There is evidence that LHRH has a role in the mediation of olfactory cues in reproductive behavior in rodents. It is not known how LHRH functions within the olfactory system in primates. However, the fact that it is distributed similarly in the two groups suggests that it may serve a similar function.     

Precursor and deaminated forms of both luteinizing hormone-releasing hormone (LHRH) and (hydroxyproline9)LHRH are present in the rat hypothalamus.

A naturally occurring analog of the decapeptide luteinizing hormone-releasing hormone ([Hyp9]LHRH) has been described previously in the hypothalamus of several mammals. It derives from post-translational hydroxylation of the LHRH proline9 residue. In the present work, intermediate LHRH precursors exhibiting both Pro9 or Hyp9 residues in the LHRH sequence were characterized in the rat hypothalamus. Hydroxylation of the Pro9 residue can thus be assumed to occur at an early stage of post-translational maturation. Deaminated, free acid forms of both native decapeptides were also detected. They correspond most likely to catabolites from incompletely processed precursors.     

Luteinizing hormone releasing hormone increases proliferation of meningioma cells in vitro

The fact that meningioma shows at least a 2:1 predilection for women over men is considered to be due to endocrinological and paracrine regulation of the development of this tumour. The presence of receptors for the luteinizing hormone releasing hormone (LHRH) in gynaecological cancer permits the use of LHRH agonistic or antagonistic analogues with a direct effect or by the gonado-pituitary axis suppression in the treatment of these tumours. Therefore, the effect of LHRH on meningioma cells is tested in this study. Meningioma cells from three female patients were cultured and LHRH (50 ng/ml) was added to the growth medium daily, for fourteen days. At the end of this period the cells were counted by means of a Coulter Counter. The stimulating effects of LHRH on the increase of the amount of cells in the meningioma monolayer culture were 146% (p < 0.01), 134% (p < 0.05) and 141% (p < 0.05) of the control, respectively, for the three patients.