Structure and biological activity of gonadotropin-releasing hormone isoforms isolated from rat and hamster brains.

Rat and hamster brain tissues were used to investigate the possible existence of a follicle stimulating hormone (FSH)-releasing factor with similar characteristics to the lamprey gonadotropin-releasing hormone III (lGnRH-III) form proposed in previous reports. The present studies involved isolation and purification of the molecule by high-performance liquid chromatography (HPLC), identification by radioimmunoassay, sequence analysis by automated Edman degradation, mass spectrometry and examination of biological activity. Hypothalamic extracts from both species contained an HPLC fraction that was immunoreactive to GnRH and coeluted with lGnRH-III and 9-hydroxyproline mGnRH ([Hyp(9)]GnRH). Determination of primary structure from purified total brain material demonstrated that the isolated molecule was [Hyp(9)]GnRH. This is the first report showing the presence of the posttranslationally modified form already known as [Hyp(9)]GnRH by primary sequence analysis. The biological activity of distinct GnRH peptides was also tested in vitro for gonadotropin release using rat pituitary primary cell cultures. The results showed that [Hyp(9)]GnRH stimulated both luteinizing hormone and FSH release, as already reported, whereas lGnRH-III had no action on the secretion of either gonadotropin.

The role of EC 3.4.24.15 in the post-secretory regulation of peptide signals.

In the present studies, we characterized the degradation of gonadotropin-releasing hormone (GnRH) by tissues of the ovine hypothalamo-pituitary axis. Membrane and soluble fractions of the medial basal hypothalamus, the pre-optic area, the median eminence and the anterior pituitary demonstrated greater GnRH-degrading activity than either hypophysial-portal or jugular plasma. The primary stable product of the membrane fractions was GnRH1-3, while the major product of the soluble fractions was GnRH1-5, both fragments were generated by plasma. Of all tissue fractions, the highest specific activity was observed in the soluble median eminence. Partial purification and characterization of soluble hypothalamic peptidase activity suggested that GnRH degradation by this tissue occurs via a two-step mechanism involving both post-proline cleaving enzyme and the metalloendopeptidase 3.4.24.15.

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.

Metabolism of pro-luteinizing hormone-releasing hormone in immortalized hypothalamic neurons.

An immortalized hypothalamic neuronal cell line was recently developed by genetically targeting the expression of the simian virus-40 large T-antigen in LHRH neurons. These GT1 cells were subcloned to GT1-1, GT1-3, and GT1-7 cells, and they have been shown to express the mRNA for pro-LHRH and secrete LHRH-like immunoreactive (IR) materials into the media. The purpose of our study was to biochemically and immunologically characterize the IR materials within and secreted from these cells. Both LHRH- and GnRH-associated peptide (GAP)-like IR materials were present and were secreted from these four cell lines. Up to 3% of the total cellular protein was composed of LHRH and GAP materials. When materials from the cell lysate and media were separated according to mol wt (Mr), at least three different pro-LHRH species were detected. These precursors contained both LHRH- and GAP-like IR determinants, and they eluted in the void volume and at approximately 10,000-12,000 and 8,400-8,500 Mr. A material that contained GAP-like IR eluted at approximately 6,500-6,800 Mr. This species is probably mouse GAP-(1-56) because it eluted on a reverse phase column in the approximate position of rat GAP-(1-56). Cell lysates contained a single LHRH-like IR form which coeluted on a size-exclusion column with synthetic LHRH. This material stimulated secretion of LH from anterior pituitary cells in a dose-response manner. By comparison, two different molecular forms of LHRH were detected in media at approximately 1,500 and 540 Mr. HPLC analyses revealed these peaks to be heterogeneous and to contain at least (Gln1)LHRH-(Gly11,Lys12,Arg13), (Gln1)LHRH-(Gly1,Lys12), LHRH-(Gly11), and LHRH. These experiments demonstrate that the cells contain and secrete multiple molecular forms of the pro-LHRH and that processing of the prohormone must involve 1) cleavage by an endopeptidase to give GAP-(1-56) and a C-terminally extended LHRH, 2) removal of C-terminal basic amino acids by a carboxypeptidase, 3) amidation of LHRH-(Gly11) to LHRH, and 4) cyclization of glutamine to pyroglutamate at the N-terminal of LHRH. These results provide the first evidence for intermediates in the metabolic pathway of pro-LHRH to LHRH.

A second form of gonadotropin-releasing hormone (GnRH), with chicken GnRH II-like properties, occurs together with mammalian GnRH in marsupial brains.

GnRH peptides in the hypothalami of marsupials (tammar wallaby, short-nosed bandicoot, and eastern quoll) and a monotreme (echidna) were investigated by reverse phase HPLC and RIA with region-specific antisera. In the wallaby hypothalamic extract, a single form of GnRH was present, which eluted in the same position as synthetic mammalian GnRH on HPLC and was recognized by antibodies directed against the NH2- and COOH-termini of mammalian GnRH as well as by antibodies to the middle region. Two GnRH molecular forms were demonstrated in the bandicoot and quoll hypothalamic extracts. One form eluted in the same position as synthetic mammalian GnRH on HPLC and was quantified equally by two mammalian GnRH antisera. The second form eluted in the same position as synthetic chicken GnRH II and was recognized by specific antibodies to this molecule. Quantification of this immunoreactive peak with two chicken GnRH II antisera was not equal, suggesting that the peptide has similar properties to, but may not be identical to, chicken GnRH II. Immunoreactive GnRH was also detected in the echidna hypothalamic extract. These findings demonstrate that in some mammals more than one form of GnRH is present in the brain of a single species, as has previously been found in species from all nonmammalian vertebrate classes. The finding in marsupial brain of a peptide with properties of chicken GnRH II, which has previously been reported in species of Aves, Reptilia, Amphibia, Osteichthyes, and Chondrichthyes, supports our hypothesis that this widespread structural variant may represent an early early evolved and conserved form of GnRH.

Effect of mammalian luteinizing hormone-releasing hormone on plasma testosterone in male alligators, with observations on the nature of alligator hypothalamic gonadotropin-releasing hormone.

Mature and subadult male alligators (30-50 kg body wt) were injected with a single dose (500 micrograms) of synthetic LH-RH. A blood sample was taken immediately before the hormone was injected and additional samples taken either at 24-hr or at 2-hr intervals up to 8 hr followed by a 24-hr sample. Testosterone concentrations in the blood were then determined by radioimmunoassay. LH-RH caused an increase in plasma testosterone in all animals by 2 hr. Plasma testosterone was still significantly higher in the LH-RH-injected animals than in the saline-injected group at 24 hr. Alligators, therefore, differ from other reptiles in that they respond to the mammalian hormone. Hypothalamic tissue from immature alligators was extracted and tested for the presence of immunoreactive LH-RH-like material using two different antisera. One of the antisera (IJ-29) did not cross-react with the tissue extract, whereas a good parallel displacement curve was obtained when the extract was tested against the other antiserum (R-42). Based on the known specificities of the antisera we conclude that alligator hypothalamic tissue contains an LH-RH-like peptide that differs from the mammalian hormone in at least the 8 position, and that alligator LH-RH may be similar to chicken LH-RH.

Isolation and structural characterization of chicken hypothalamic luteinizing hormone releasing hormone.

Studies on partially purified chicken hypothalamic luteinizing hormone releasing hormone (LHRH) utilizing chromatography, radioimmunoassay with region-specific antisera, enzymic inactivation, and chemical modification established that the peptide is structurally different from mammalian hypothalamic LHRH. These studies demonstrated that arginine in position 8 is substituted by a neutral amino acid. On the basis of conformational criteria and evolutionary probability of amino acid interchange for arginine, the most likely substitution was glutamine. We therefore synthesized [Gln8]-LHRH and established that it had identical chromatographic, immunologic, and biological properties to the natural chicken peptide. In concurrent studies, purification of 17 micrograms of an LHRH from 249,000 chicken hypothalami was achieved using acetic acid extraction, immuno-affinity chromatography, and cation exchange and reverse phase high performance liquid chromatography. Amino acid composition and sequence analyses confirmed the structure of this form of chicken LHRH as pGlu-His-Trp-Ser-Tyr-Gly-Leu-Gln-Pro-Gly-NH2.

Luteinizing hormone-releasing hormone and thyrotropin-releasing hormone in the hypothalamus of women: effects of age and reproductive status.

In the study of the effects of age and reproductive status on LHRH and TRH content in the hypothalamus of women, we found that the amount of LHRH (58 +/- 5.5 ng; mean +/- SE) in the hypothalamus of young women (16-29 yr) was significantly greater (P less than 0.001) than that (28 +/- 3.0 ng) in postmenopausal women (50-78 yr). The hypothalamic content of LHRH (18 +/- 2.4 ng) of bilaterally ovariectomized women (39-47 yr) was significantly less (P less than 0.001) than that (60 +/- 12.6 ng) in younger ovulatory women (30-39 yr) or that (56 +/- 13.5 ng) in ovulatory women of comparable age (40-49 yr). In contrast, the hypothalamic content of TRH (121.4 +/- 32.8 ng) in postmenopausal women were similar to that (122.3 +/- 12.5 ng) in young women. Although aging in women is associated with a significant reduction in the amount of LHRH in the hypothalamus, such a reduction appears to be a consequence of ovarian failure and not of aging per se.

Purification of a bioactive FSH-releasing factor (FSHRF).

Before the advent of radioimmunoassay (RIA), FSH-releasing factor (FSHRF) appeared to be separable from LH-releasing hormone (LHRH) by chromatography followed by bioassay for FSH. In this study, we re-examined hypothalamic extracts for the existence of an FSHRF distinct from LHRH, utilizing the Steelman-Pohley bioassay as well as RIA for identification of FSH. Acid extracts of rat hypothalamic fragments were chromatographed on Sephadex G-25. LH- and FSH-releasing activities of each fraction were assessed by bio- and immunoassay of FSH and immunoassay of LH released after incubation with hemipituitaries from adult male rats. The immunoreactive LHRH(IR-LHRH) concentration of each fraction was also measured by RIA. In order to evaluate the FSH-releasing activity of LHRH, three doses of synthetic LHRH were tested and FSH-releasing activity determined by bio- and immunoassay. By RIA, the FSH-releasing activity of each column fraction could be accounted for by IR-LHRH contamination. However, greater FSH-releasing activity than could be predicted by IR-LRH contamination was detected by Steelman-Pohley assay in fractions eluted prior to the LHRH peak in 2 separate fractionations. These fractions from the second fractionation were pooled and eluted from a CMC column with ammonium acetate buffers. Again greater FSH-releasing activity than could be accounted for by IR-LHRH was detected prior to the IR-LHRH peak by Steelman-Pohley assay. These results agree with early work from our laboratory and suggest the presence of a bioactive FSHRF in hypothalamic extracts.

Structure of chicken hypothalamic luteinizing hormone-releasing hormone. II. Isolation and characterization.

Avian luteinizing hormone-releasing hormone (LH-RH) has been isolated from 249,000 chicken hypothalami and shown to differ structurally from mammalian hypothalamic LH-RH. Purification was achieved by acetic acid extraction, anti-LH-RH affinity chromatography, and cation exchange and reverse phase high performance liquid chromatography. The isolated peptide eluted as a single peak on reverse phase high performance liquid chromatography. Acid hydrolysis of the peptide yielded integral molar ratios of amino acids and a composition identical with that of mammalian decapeptide LH-RH, except for the presence of an additional glutamic acid residue and the absence of arginine. The isoelectric point of chicken LH-RH (7.3) is consistent with the glutamic acid representing a glutamine residue. We therefore synthesized [Gln8]LH-RH and established that it has chromatographic properties identical with natural chicken LH-RH. These studies indicate that the structure of chicken hypothalamic LH-RH is: pGlu-His-Trp-Ser-Tyr-Gly-Leu-Gln-Pro-Gly-NH2.

Decapeptide luteinizing hormone releasing hormone in ovine pineal gland.

Ovine pineal gland extracts were examined throughout the year for content and molecular form of immunoreactive (IMR) LH releasing hormone (LH-RH). The content of IMR LH-RH in pineal glands collected in spring and summer was 160-2230 pg/gland, while the content in pineal glands collected in autumn and winter was lower (31-39 pg/gland). Pineal gland IMR LH-RH, purified by affinity and Sephadex G-25 chromatography, yielded displacement curves parallel to those of hypothalamic IMR LH-RH and synthetic LH-RH in radioimmunoassays employing four antisera which require different regions of the decapeptide for effective binding, suggesting considerable similarities in the structure of the molecule. The majority of pineal gland IMR LH-RH behaved identically to hypothalamic IMR LH-RH and synthetic LH-RH on gel filtration, cation exchange chromatography, high-pressure liquid chromatography and cellulose thin-layer chromatography. However, a small amount of a less positively charged LH-RH species was also present in all pineal gland extracts. Our findings indicate that hypothalamic decapeptide LH-RH occurs in the ovine pineal gland.

Occurrence of higher molecular forms of LHRH in fractionated extracts from rat hypothalamus, cortex and placenta.

Separation of higher molecular forms of LHRH-like immunoreactive material was attempted in homogenates and subcellular fractions of hypothalamus, cerebral cortex and placenta. Physiological activity was checked by means of antibodies directed against different sequences of LHRH and shown to recognize synthetic LHRH analogs extended on either C- or N-terminal portions of the molecule. After molecular-sieve filtration, 2 peaks of immunoreactive material corresponding apparently to sequences of LHRH extended on the C terminus were recovered. Peak I, with a molecular weight of about 26 000 dalton, was found exclusively in a cytoplasmic and axoplasmic supernatant (S2), where it migrated alone with microsomes. Peak II (1800 dalton) was present both in S2 and in a synaptosomal fraction (P2) corresponding to nerve endings. Native LHRH was almost exclusively recovered from the synaptosomal fraction. Extracts from placenta or cerebral cortex contained little or no native LHRH; in contrast, a small amount of immunoreactive material corresponding to peak II was detected in the cerebral cortex, and fairly large amounts of both putative precursors were found in the placenta. Chromatography of tissues containing no LHRH, such as cerebellum or liver, yielded no immunoreactive material at either elution site, thus suggesting specific detection of LHRH-like material under our experimental conditions. The present data suggest that 2 higher molecular forms, one slightly heavier than the native peptide itself, and another corresponding to a much larger protein, could represent LHRH precursors and are present in the hypothalamus as well as the cerebral cortex and the placenta.

A model system for the study of the release of luteinizing hormone-releasing hormone from isolated storage granules.

We have developed an in vitro system for the study of the release of luteinizing hormone-releasing hormone (LH-RH) from its storage granules. In this system, homogenates of hypothalamic tissue are subjected to hypoosmotic shock, and the LH-RH-containing granules are isolated by means of differential centrifugation. The isolated granules are then incubated in a buffered medium, and the incubation is terminated by passing the incubation mixture through LH-RH affinity columns. The LH-RH associated with the granules passes freely through the columns, whereas the LH-RH released into the medium binds to the columns and is subsequently eluted with an acid solution. LH-RH is quantified by radioimmunoassay (RIA). We tested the effects of various concentrations of KCl on LH-RH release, which was found to be dependent on the concentration of KCl in the medium over the range 40-160 mM. We then studied the effects of pH on the release of LH-RH. Incubation of granules at pH 7.8 in the presence of 160 mM-KCl resulted in the release from the granules of 14% of the stored LH-RH, whereas incubation at pH 6.2 resulted in the release of approximately 30% of the LH-RH. In addition, granules were incubated at pH 7.8 with MgATP and KCl. MgATP elicited a marked release of LH-RH that was approximately twice that seen in the absence of MgATP. In summary, in this in vitro system, granules containing LH-RH are stable under defined biochemical conditions, and LH-RH release from these granules is stimulated by ions and MgATP.