Luteinizing Hormone-releasing Hormone Molecule Research Articles

A synthetic luteinizing hormone releasing hormone vaccine: I. Conjugation and specificity trials in BALB/c mice

The immunobiology of luteinizing hormone releasing hormone (LHRH) was explored, to provide a conceptual and practical basis for the use of LHRH in immunocastration. Cysteine substituted analogues of LHRH were synthesized including Cys 1-LHRH (C1-LHRH), Cys 6-LHRH (C6-LHRH) and Cys 10-LHRH (C10-LHRH). These were reacted to carrier molecules using the heterobifunctional cross-linking reagent m-maleimidobenzoylsulfosuccinimide ester (SMBS), producing peptide-carrier conjugates of known peptide content and conjugation orientation. This reaction regime was found to be rapid, efficient and allowed for easy control of peptide to carrier ratios. Conjugates were used in active immunization trials in BALB/c mice to characterize the murine immune response against LHRH. BALB/c mice were shown to have the capacity to recognise all three cysteine substituted LHRH analogues and to produce antibodies cross-reactive with native LHRH. The specificity of LHRH antisera generated was found to be dependent on the site of conjugation of the peptide to carrier molecule. C1-LHRH generated carboxy terminal directed antibodies, C10-LHRH generated amino terminal directed antibodies, while C6-LHRH could generate amino terminal directed or carboxy terminal directed antibodies, or both within a given animal. No intrinsically immunodominant epitopes were seen within the LHRH molecule.

Facilitation of lordosis in female rats by CNS-site specific infusions of an LH-RH fragment, Ac-LH-RH-(5–10)

Structural alterations of the luteinizing hormone-releasing hormone (LH-RH) molecule have been performed to yield analogs which are more potent than, or which complete with, the parent hormone to increase or decrease the release of LH from the pituitary gland. The effects of these analogs on mating behavior, however, do not always parallel their effects on LH release. The present study tested the effectiveness of a pituitary-inactive fragment of LH-RH, namely Ac-LH-RH-(5–10), in potentiating mating behavior in the ovariectomized, estrogen-primed female rat. This fragment, when infused bilaterally into the medial preoptic area (POA), the ventromedial hypothalamus (VMH), or the midbrain central gray (MCG), significantly enhanced lordosis. Infusion of the fragment into the cerebral cortex was ineffective. Elevated lordotic responding was first apparent in the POA at 15 min postinfusion and was maintained for the duration of the testing session (180 min). Ac-LH-RH-(5–10) infused into the VMH or MCG enhanced lordotic behavior at 90 and 180 min postinfusion. The results indicate that only a portion of the LH-RH molecule may be required for behavioral activity and suggest that degradation of the LH-RH molecule is physiologically relevant.

A new antiserum with conformational specificity for LHRH: Usefulness for radioimmunoassay and immunocytochemistry

We have produced and characterized a new high titer, highly specific antiserum for luteinizing hormone-releasing hormone (LHRH), and demonstrated its usefulness for radioimmunoassay (RIA) and immunocytochemistry. The antiserum can be used at a final dilution of 1:500,000 to 1:600,000 for RIAs with a sensitivity of 0.2 pg/tube. Both the amino and carboxy terminal ends of the LHRH molecule are required for antibody recognition, and the antigenic determinant appears to be part of a three-dimensional structure of LHRH. Fragments of LHRH and other brain peptides are not recognized by the antiserum. Using immunocytochemical techniques, we have localized LHRH-containing neurons in the medial basal hypothalamus of the rhesus monkey, guinea pig, and rat. Staining of LHRH fibers and cell bodies was eliminated by preabsorbtion of the immune serum with synthetic LHRH. This antiserum should be useful in studies that require quantification of very low amounts of LHRH and in studies that require correlation between immunocytochemical localization and tissue content or secretion of LHRH.

Luteinizing hormone-releasing hormone peptidase activities in the female rat: Characterization by an assay based on high-performance liquid chromatography

So far, the presence of two peptidase activities which degrade luteinizing hormone-releasing hormone (LHRH) have been found in hypothalamic tissue: an endopeptidase and a postproline cleaving enzyme (PPCE). Although it is possible that degradation of LHRH plays a physiological role in the control of gonadotropin secretion, evidence in favor of this hypothesis remains controversial, since only indirect measurements of peptidase activity have been reported. The present paper describes an assay that directly estimates LHRH degradation. This includes fractionation of the degradation products of LHRH by high-performance liquid chromatography (HPLC) and the use of digital integration to measure the disappearance of the LHRH molecule and account for the products deriving from its cleavage. The assay allows for measurement of total LHRH peptidase activity in discrete hypothalamic regions, such as median eminence (ME) and ventromedial preoptic area (POA), of individual animals. These peptidase activities are linear with respect to tissue concentration and time of incubation for up to 40 min for POA and anterior pituitary (AP) and for 2–2.5 h for ME. The major degradation product after HPLC separation and amino acid analysis is the LHRH 1–5 fragment whose appearance in the incubation medium is highly correlated with the disappearance of the LHRH molecule. With the use of this assay, various inhibitors of this activity were examined; the sulfhydryl reagents PCMS and ZnCl 2, the peptide antibiotic bacitracin, and the metalloendopeptidase inhibitor, 1,10-phenanthroline were the most effective.

Isolation and characterization of chicken hypothalamic luteinizing hormone-releasing hormone

A peptide having gonadotropin-releasing activity was isolated in a yield of 2.5 μg from an extract of 2,000 chicken hypothalami. The biopotency was monitored using rat anterior pituitary cell culture system. The peptide differs from mammalian Luteinizing Hormone-Releasing Hormone (LH-RH) in its behavior during chromatographic separation (ionexchange and high performance liquid chromatography) and in its reaction towards anti-LH-RH antiserum directed against the C-terminal region of the LH-RH molecule. The peptide (chicken LH-RH) stimulates secretion of both LH and FSH from rat anterior pituitary cells. The biological potency of this peptide was about 4 % of that of the authentic decapeptide estimated in the rat anterior pituitary system. The amino acid composition is (Ser, Pro, Glx 2, Gly 2, Leu, Tyr, His, Trp), which differs from mammalian LH-RH only in that one Arg residue is replaced by a Glx residue. Based on the behavior on CM cellulose chromatography and the reaction towards anti-LH-RH antiserum, one possible structural candidate for this peptide (chicken LH-RH) is [Gln 8]-LH-RH.

Inhibition of the Postovariectomy Depletion of Hypothalamic Luteinizing Hormone Releasing Hormone (LHRH) by Suckling1

A study was undertaken to further the understanding of the mechanism by which suckling inhibits the release of pituitary LH and depresses the postovariectomy rise of plasma LH in lactating mammals. To that end, the effect of suckling (10 pups/animal) for 1 or 3 weeks on the LHRH content of the hypothalamus and preoptic area (POA) in ovariectomized and intact rats was examined. Controls consisted of intact and 1 or 3 week ovariectomized, nonlactating animals. Following decapitation, the brains were rapidly removed and blocks containing the POA and the hypothalamus (with median eminence) were isolated. Tissue was extracted with acetic acid and LHRH quantitated via validated RIAs utilizing 2 antisera specific for different portions of the LHRH molecule. Ovariectomy of nonlactating, diestrous animals resulted in a significant decline in hypothalamic LHRH, reaching 50% of control levels by 3 weeks. During the same intervals, plasma LH increased dramatically to 20- and 60-fold over intact controls by 1 and 3 weeks, respectively. In contrast, LHRH levels were not decreased at 1 or 3 weeks in ovariectomized rats which were suckled, at which time plasma LH was greatly depressed. When intact animals were evaluated, the suckling stimulus failed to induce a detectable change in LHRH content of the hypothalamus of LHRH in the POA between any of the treatment or control groups. These data from ovariectomized rats suggest that suckling inhibits LHRH release from the hypothalamus and hence provides an explanation for the depression of plasma LH observed in suckled ovariectomized and intact animals.

Three luteinizing hormone-releasing hormone like substances in a teleost fish brain: None identical with the mammalian LH-RH decapeptide

Acetic acid extracts of codfish brain contain three different immunoreactive (IR) luteinizing hormone-releasing hormone (LH-RH) like materials which differ from the mammalian LH-RH decapeptide by their behavior on chromatographic separation (molecular sieving, ion exchange and high performance liquid chromatography), and their reaction with two well characterized antisera directed against different regions of the LH-RH molecule. One of the IR LH-RH like substances is a larger molecular weight compound (1800 to 4,000 daltons), which may be a precursor of LH-RH extended at the N-terminus. The other two are approximately the same size as mammalian LH-RH and their nature is best explained by the occurrence of single amino acid substitutions at the C-terminal region of the ‘authentic’ decapeptide.

Immunocytochemical analysis of the rat pineal gland using antisera generated against analogs of luteinizing hormone-releasing hormone (LHRH).

Antisera generated against synthetic luteinizing hormone-releasing hormone (LHRH) or analogs of LHRH, agonists and antagonist, were used to stain the rat pineal gland and the median eminence of the hypothalamus. Of the LHRH analogs used, two revealed immunoreactive material in median eminence and not pineal, two stained pineal only, and three stained median eminence and pineal. Our observations suggest that the immunoreactive material in the pineal gland may be an LHRH-like substance whose affinity characteristics are different from that of hypothalamic LHRH. The affinity properties of hypothalamic LHRH for antibody appear to reside in the C-terminus of the molecule, whereas binding of the antigen in the pineal to antibody appear to be influenced by an N-terminal modification of the LHRH molecule.

Enzymes involved in the degradation of thyrotropin releasing hormone (TRH) and luteinizing hormone releasing hormone (LH-RH) in bovine brain

As part of an investigation of neuropeptide inactivation mechanisms, we have resolved an enzymatic activity in bovine brain which catalyzes the deamidation of thyrotropin releasing hormone (TRH) and the hydrolysis of the Pro9--Gly10--NH2 bond of luteinizing hormone releasing hormone (LH-RH) from a second LH-RH degrading activity which does not degrade TRH. The former activity is similar, if not identical to, the post-proline cleaving enzyme in kidney as it is active toward the post-proline cleaving enzyme substrate CbzGly--Pro--Leu--Gly and inhibited by CbzPro--Phe and diisopropylfluorophosphate. In addition, products derived from the degradation of TRH and LH-RH by this activity show a specific cleavage on the carboxyl side of a proline residue. The latter activity has not yet been characterized with respect to its site of cleavage of the LH-RH molecule due to the presence of other contaminating peptidases.

On the function of arginine in luteinizing hormone-releasing hormone

Many analogs of luteinizing hormone-releasing hormone (LH-RH) have been recently synthesized in order to investigate the significance of the various constituent amino acids in hormonal activity [ 1 ] . Assessments of the capacity of analogs to stimulate release of LH and FSH have revealed that arginine in position 8 on the peptide chain is critical for eliciting full activity [2-51. It was suggested that the basic guanidine side-chain of arginine participates in the binding of LH-RH to its specific pituitary receptor site via an ionic interaction [5] and probably by a hydrogen-bond formation mechanism, as well [3]. It was also postulated, based on fluorescence studies, that the importance of arginine is due to its location in the tertiary structure of the LH-RH molecule itself, that is, in the assembly of the amino acids His’, Tyr’, and Arg8 which exist as a close-knit unit, probably playing an active role in the hormonal action of the peptide [6,7]. The structural function of Arg* was also suggested by binding studies of LH-RH and several of its analogs to plasma membranes of the anterior pituitary [8] . To gain further understanding of the function of

Quantitative evaluation and determination of the biological potency of iontophoretically applied luteinizing hormone-releasing hormone (LRH)

A series of experiments was performed to establish the rate of release for tritium-labelled luteinizing hormone-releasing hormone (LRH) and to establish its biological activity. The in vitro iontophoretic release of labelled LRH from glass micropipettes was determined by liquid scintillation counting. The release of LRH was found to have a non-linear relationship with charge. Much higher charges were needed to iontophorese labelled LRH in detectable quantities as compared to that needed to iontophorese norepinephrine. It is thought that because of the size and solubility of LRH. its release from micropipettes depends on electroosmosis. A 5 nA current was found to be the most effective means of retaining LRH in the glass pipettes. In order to assess the biological activity of the iontophoresed LRH molecule, oestrone-treated, ovaricctomized female rats were given intravenous injections of either 50 ng of LRH, samples of LRH collected from iontophoresis or saline. The injection of LRH or the iontophoresed LRH significantly increased luteinizing hormone levels in these animals, whereas saline administration had no effect. It is suggested that LRH can be reliably iontophoresed from glass micropipettes and that its biological activity is not altered by application of a charge and movement of the molecule through the micropipette orifice.