Mast cells in the rat brain synthesize gonadotropin-releasing hormone

Mast cells occur in the brain and their number changes with reproductive status. While it has been suggested that brain mast cells contain the mammalian hypothalamic form of gonadotropin-releasing hormone (GnRH-I), it is not known whether mast cells synthesize GnRH-I de novo. In the present study, mast cells in the rat thalamus were immunoreactive to antisera generated against GnRH-I and the GnRH-I associated peptide (GAP); mast cell identity was confirmed by the presence of heparin, a molecule specific to mast cells, or serotonin. To test whether mast cells synthesize GnRH-I mRNA, in situ hybridization was performed using a GnRH-I cRNA probe, and the signal was identified as being within mast cells by the binding of avidin to heparin. GnRH-I mRNA was also found, using RT-PCR, in mast cells isolated from the peritoneal cavity. Given the function of GnRH-I in the regulation of reproduction, changes in the population of brain GnRH-I mast cells were investigated. While housing males with sexually receptive females for 2 h or 5 days resulted in a significant increase in the number of brain mast cells, the proportion of mast cells positive for GnRH-I was similar to that in males housed with a familiar male. These findings represent the first report showing that mast cells synthesize GnRH-I and that the mast cell increase seen in a reproductive context is the result of a parallel increase in GnRH-I positive and non-GnRH-I positive mast cells.     

Localization of immunoreactive lamprey gonadotropin-releasing hormone in the rat brain

A highly specific antiserum against lamprey gonadotropin-releasing hormone (GnRH) was used to localize 1-GnRH in areas of the rat brain associated with reproductive function. Immunoreactive 1-GnRH-like neurons were observed in the ventromedial preoptic area (POA), the region of the diagonal band of Broca and the organum vasculosum lamina terminalis, with fiber projections to the rostral wall of the third ventricle and the organum vasculosum lamina terminalis. Another population of 1-GnRH-like neurons was localized in the dorsomedial and lateral POA, with nerve fibers projecting caudally and ventrally to terminate in the external layer of the median eminence. Other fibers apparently projected caudally and circumventrically to terminate around the cerebral aqueduct in the mid-brain central gray. By using a highly specific antiserum directed against mammalian luteinizing hormone-releasing hormone (m-LHRH), the localization of the LHRH neuronal system was compared to that of the 1-GnRH system. There were no LHRH neurons in the dorsomedial or the lateral region of the POA that contained the 1-GnRH neurons. As expected, there was a large population of LHRH neurons in the ventromedial POA associated with the diagonal band of Broca and organum vasculosum lamina terminalis. In both of these regions, there were many more LHRH neurons than 1-GnRH neurons and the LHRH neurons extended more dorsally and laterally than the 1-GnRH neurons. The LHRH neurons seemed to project to the median eminence in the same areas as those that were innervated by the 1-GnRH neurons. Absorption studies indicated that 1-GnRH cell bodies were eliminated by adding 1 microg of either 1-GnRH-I or 1-GnRH-III, but not m-LHRH to the antiserum before use. Fibers were largely eliminated by the addition of 1 microg 1-GnRH-III to the antiserum. No chicken GnRH-II neurons or nerve fibers could be visualized by immunostaining. Because the antiserum recognized GnRH-I and GnRH-III equally, we have visualized an 1-GnRH system in rat brain. The results are consistent with the presence of either one or both of these peptides within the rat hypothalamus. Because 1-GnRH-I has only weak nonselective gonadotropin-releasing activity, whereas 1-GnRH-III is a highly selective releaser of follicle-stimulating hormone, and because 1-GnRH neurons are located in areas known to control follicle-stimulating hormone release selectively, our results support the hypothesis that 1-GnRH-III, or a closely related peptide, may be mammalian follicle-stimulating hormone-releasing factor.     

The delineation of a decapeptide gonadotropin-releasing sequence in the carboxyl-terminal extension of the human gonadotropin-releasing hormone precursor

The human gonadotropin-releasing hormone (GnRH) precursor consists of the GnRH sequence followed by a 59-amino acid carboxyl-terminal extension. A 56-amino acid peptide within this extension has been shown to stimulate gonadotropin release, and this activity has been localized to its amino-terminal region. A series of seven overlapping peptide fragments corresponding to the first 24 amino acids of the carboxyl-extension of the GnRH precursor were synthesized and tested for their ability to stimulate luteinizing hormone and follicle-stimulating hormone release from cultured human anterior pituitary cells. All active peptide fragments were found to incorporate the decapeptide sequence Asn-Leu-Ile-Asp-Ser-Phe-Gln-Glu-Ile-Val which is regarded as a minimal structural requirement delineated for gonadotropin-releasing activity. A further flanking sequence extending this active region from its carboxyl terminus was found to enhance gonadotropin-releasing activity although the flanking sequence itself was inactive. The gonadotropin release stimulated by the active peptides wa shown to be a dose- dependent, specific, and calcium-dependent phenomenon which occurred independently of the GnRH receptor on the pituitary gonadotrophs as a GnRH antagonist did not inhibit activity.     

Identification of a third isoform of Na+, K+-ATPase activity in rat brain synaptosomes

³H-ouabain binding and ouabain-inhibitable ⁸⁶Rb⁺ (K⁺) uptake were investigated as a means to identify a third isoform of Na⁺, K⁺-ATPase in crude synaptosome preparations. The specific binding of low concentrations (10 nM and 1 uM) of ³H-ouabain, in crude synaptosome preparations, was markedly inhibited by K⁺ (0.5–5 mM). Accordingly, ⁸⁶Rb⁺ (K⁺) uptake, in the presence of 5 mM K⁺ was not sensitive to inhibition by low concentrations (10⁻¹¹–10⁻⁷ M) of ouabain. Higher concentrations (10⁻⁶–10^−2.6 M) of ouabain resulted in a biphasic inhibition of K⁺ uptake, which distinguished the activities of the presumed alpha 2 and alpha 1 isozymes of Na⁺, K⁺-ATPase. Reduction of K⁺ (1.25 mM and 0.5 mM) in the incubation, resulted in the observation of a third component of ouabain- sensitive K⁺ uptake. This Na⁺, K⁺-ATPase activity, which was defined, pharmacologically, as very sensitive (VS) to ouabain, exhibited IC₅₀s of 3.6 nM and 92 nM at 1.25 mM K⁺ and 0.5 mM K⁺, respectively. Inhibition of ouabain binding and VS-dependent K⁺ uptake, at a high, physiological cocentration (5 mM) of K⁺, suggests that VS may be an inactive isoform of brain Na⁺, K⁺-ATPase under resting conditions.     

The polyphenol oxidase gene family in poplar: phylogeny,differential expression and identification of a novel,vacuolar isoform

Polyphenol oxidases (PPOs) are oxidative enzymes that convert monophenols and o-diphenols to o-quinones using molecular oxygen. The quinone products are highly reactive following tissue damage and can interact with cellular constituents and cause oxidative browning and cross-linking. The induction of PPO in some plants as a result of wounding, herbivore attack, or pathogen infection has implicated them in defense. However, PPO-like enzymes that act as specific hydroxylases, for example in lignan and pigment biosynthesis, have also been discovered. Here, we present the first genome-enabled analysis of a PPO gene family. The Populus trichocarpa genome was found to contain a minimum of nine complete PPO genes, and seven of these were characterized further. The PPO gene family includes both recently duplicated and divergent sequences that are 36–98% identical at the amino acid level. Gene expression profiling in poplar tissues and organs revealed that the PPO genes are all differentially expressed during normal development, but that only a small subset of PPO genes are significantly upregulated by wounding, methyl jasmonate or pathogen infection. Our studies also identified PtrPPO13, a novel PPO gene that is predicted to encode an N-terminal signal peptide. Transient expression of green fluorescent protein fusions demonstrated its localization to the vacuolar lumen. Together, our findings show that the poplar PPO family is diverse and is likely linked to diverse physiological functions.     

Relaxation of bovine, porcine and human brain arteries by parathyroid hormone

We have studied the relaxant effect of bovine parathyroid hormone (bPTH) on helical strips of branches of bovine and human middle cerebral arteries and bovine and porcine basilar arteries. All arteries were studied after contraction with prostaglandin (PG) F2 alpha or KCl. In the case of all arteries contracted with PGF2 alpha, the ED50 of PTH vasorelaxation related to maximal vasorelaxation induced by papaverine ranged from 9 to 14 nM for bPTH-(1-34) and 100 to 220 ng/ml for native bPTH-(1-84). The PTH inhibitor, [Nle8, Nle18, Tyr34]bPTH-(3-34) amide, attenuated the vasorelaxant effect of both bPTH-(1-34) and bPTH-(1-84). The vasorelaxant effects of PTH which we have observed in this study are consistent with the stimulatory effects of PTH on vascular adenylate cyclase which we had previously reported.     

The identification of gonadotropin-releasing hormone (GnRH) in hypothalamic and extrahypothalamic loci of the human nervous system.

Immunoreactive-like GnRH activity has been identified in 24 of 26 separate loci of the human central nervous system. Tissues, secured from 5 brains at autopsy, were dissected, extracted sequentially with 2N and glacial acetic acid, lyophilized, and eluted in buffered saline for GnRH determinations by specific radioimmunoassay. GnRH concentrations (ng/mg protein) ranged from 8.96 (infundibulum) to 0.001 (cerebellum. middle lobe). Highest extrahypothalamic concentrations of GnRH were found in mamillary body (0.076) and thalamus (0.002). Extrahypothalamic GnRH was identical to synthetic and hypothalamic GnRH by criteria of immunoidentity. No post-mortem GnRH peptidolysis, evaluated experimentally in rats, was evident between 0 and 16 hrs in intact tissues maintained at 4 degrees C. These data suggest that GnRH is distributed throughout regions of the human brain outside the hypothalamus and suggest new, non-endocrine functions for GnRH in the human CNS, analogous the those reported recently for GnRH in experimental animals.     

Polyphosphoinositide metabolism in rat brain: effects of neuropeptides, neurotransmitters and cyclic nucleotides

This study describes effects of various peptides, neurotransmitters and cyclic nucleotides on brain polyphosphoinositide metabolism in vitro. The interconversion of the polyanionic inositol phospholipids was studied by incubation of a lysed crude mitochondrial/synaptosomal fraction with [gamma-32P]-ATP. The reference peptide ACTH1-24 stimulated the formation of radiolabelled phosphatidylinositol 4,5-diphosphate (TPI) and inhibited that of phosphatidic acid (PA). Substance P inhibited both TPI and PA labelling, whereas beta-endorphin inhibited that of PA without any effect on TPI. Morphine had no effect at any concentration tested, whereas high concentrations of naloxone inhibited the labelling of both PA and TPI. Naloxone did not counteract the effects of ACTH1-24. The other peptides tested (lysine 8-vasopressin and angiotensin II) were without any effect. Under the conditions used, adrenaline, noradrenaline and acetylcholine did not affect the labelling of the (poly)phosphoinositides. Both dopamine and serotonin, however, dose-dependently inhibited the formation of radiolabelled TPI and PA. Low concentrations of cAMP stimulated TPI, but higher concentrations had an overall inhibitory effect on the labelling of TPI, PA and especially phosphatidylinositol 4-phosphate (DPI). The cyclic nucleotide did not mediate or counteract the effects of ACTH, and cGMP was without any effect. These results are discussed in the light of current ideas on the mechanism of action of neuropeptides.     

Cholinergic afferents to gonadotropin-releasing hormone neurons of the rat

Gonadotropin-releasing hormone-synthesizing neurons represent the final common pathway in the hypothalamic regulation of reproduction and their secretory activity is influenced by a variety of neurotransmitters and neuromodulators acting centrally in synaptic afferents to gonadotropin-releasing hormone neurons. The present study examined the anatomical relationship of cholinergic neuronal pathways and gonadotropin-releasing hormone neurons of the preoptic area. The immunocytochemical detection of choline acetyltransferase or vesicular acetylcholine transporter revealed a fine network of cholinergic fibers in this region. At the light microscopic level, the cholinergic axons formed appositions to the gonadotropin-releasing hormone immunoreactive cell bodies and dendrites. Results of electron microscopic studies confirmed the absence of glial interpositions in many of these neuronal contacts. Classical cholinergic synapses, which belonged to the asymmetric category, were only observed rarely on gonadotropin-releasing hormone neurons. The lack of synaptic density in most contacts corroborates previous observations on the cholinergic system elsewhere in the brain. Further, it suggests a dominantly non-synaptic route also in this cholinergic neuronal communication. This study provides direct neuromorphological evidence for the involvement of the cholinergic system in the afferent neuronal regulation of gonadotropin-releasing hormone neurons. The sources of cholinergic afferents and the receptorial mechanisms underlying this interaction will require further clarification.     

Effects of a systematic application of human chorionic gonadotropin, gonadotropin-releasing hormone analog and bovine anterior pituitary gonadotropin in cows with cystic ovarian disease

Fourty-one cows with ovarian follicular cysts (cysts) diagnosed by rectal palpation and observation of estrus behavior were injected intramuscularly (i.m.) with 20,000 IU of human chorionic gonadotropin (HCG). Ten days after this treatment all the cows were examined per rectum for changes in the ovaries with special regard to luteinization of cysts. Cows not responding to HCG were administered 500(10)mug of an analog of gonadotropin-releasing hormone (Gn-RH), Des-Gly-LH-RH-ethylamide, i.m.. If the cysts remained unchanged 10 days subsequent to the second treatment, a third treatment, 400 KE of bovine anterior pituitary gonadotropin (APG), was given i.m. HCG was clinically effective in only 8 cows (20 %). All of the 8 cows conceived. Of 33 cows not responding to HCG, 18 cows (55 %) responded to Gn-RH analog and 12 cows (36 %) conceived. Eleven (73 %) of 15 cows failing to respond to Gn-RH analog were successfully treated with APG and 6 cows (40 %) conceived. In 37 cases, treatment effects were also evaluated by determining serum levels of progesterone prior to and 10 days subsequent to each treatment. When effects of treatment were judged by 1.0 ng/ml or more increase of serum progesterone levels 10 days after treatment, HCG was effective in 13 of 37 cows (35 %), retreatment with Gn-RH analog was successful in 8 of 16 cows (50 %) and APG was ineffective in 3 cows not responding to both HCG and Gn-RH analog. It may be concluded that the therapeutic effect of HCG is disappointing and about half of the cases not responding to HCG were successfully treated with Gn-RH analog. If the cows did not respond to both HCG and Gn-RH analog, they may not respond to APG either.     

Amino-terminal sequences of a novel heparin-binding protein from human,bovine, rat,and chick brain: High interspecies homology

Recently, the partial structural characterization of a novel bovine brain protein was reported (1). Because of its mitogenic activity for vascular endothelial cells and its ability to strongly bind heparin it was termed heparin-binding brain mitogen (HBBM). Although HBBM shares these properties with members of the fibroblast growth factor (FGF) family of growth factors, its aminoterminal sequence is not homologous to that of the FGFs. Now, we report the isolation and partial structural characterization of HBBMs from human, rat and chick brain. Proteins were isolated by tissue extraction at pH 4.5, ammonium sulfate precipitation, cation exchange chromatography, heparin-Sepharose affinity chromatography and reverse-phase HPLC. The amino-terminal sequences of the HBBMs from human, bovine and rat brain are identical, whereas that of chick HBBM reveals a single amino acid substitution. The high sequence homology among the HBBMs from different species suggests an important biological role of the protein.Special issue dedicated to Dr. Sidney Udenfriend.     

Multiple molecular forms of gonadotropin-releasing hormone in teleost fish brain

Gonadotropin-releasing hormone (GnRH) immunoreactive peptides in extracts of hake (Merluccius capensis) and tilapia (Tilapia sparrmanii) brain were investigated by high performance liquid chromatography (HPLC) and radioimmunoassay with region-specific antisera. In hake brain, content and concentration of GnRH was higher in the pituitary gland than in the hypothalamic lobes or extrahypothalamic brain. Hake pituitary gland GnRH was purified by six consecutive HPLC systems. The major GnRH molecular form co-eluted with salmon brain GnRH (Trp7, Leu8-GnRH) in four different HPLC systems which were specifically designed to separate the four natural vertebrate GnRHs (mammalian, salmon, chicken I and II). The immunoreactive peak in the final purification step had a retention time identical to that of Trp7, Leu8-GnRH and an UV absorbance (280 nm) peak appropriate for two tryptophan residues in the peptide, as in Trp7, Leu8-GnRH. Six additional less hydrophobic forms of GnRH were detected. Tilapia brain extract contained two major GnRH molecular forms which had identical retention times to chicken GnRH I (Gln8-GnRH) and Trp7, Leu8-GnRH in an HPLC system which separates the natural vertebrate GnRHs. The immunological properties of these two immunoreactive peaks, determined by relative interaction with four region-specific GnRH antisera raised against vertebrate GnRHs, were identical to those of Gln8-GnRH and Trp7, Leu8-GnRH. Additional GnRH molecular forms were also detected. In summary, these findings indicate that a major GnRH molecule in hake pituitary gland is Trp7, Leu8-GnRH, while tilapia brain contains both Trp7, Leu8-GnRH and Gln8-GnRH. Additional GnRH molecular forms were detected in both species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solubilization and purification of rat pituitary gonadotropin-releasing hormone receptor

Gonadotropin-releasing hormone (GnRH) receptors were solubilized from rat pituitary membrane preparations in an active form by using the zwitterionic detergent CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid). The solubilized receptor exhibits high affinity, saturability, and specificity. The soluble supernatant retained 100% of the original binding activity when stored at 4 or -20 degrees C in the presence of 10% glycerol. The receptors were resolved into two components on the basis of chromatography on wheat germ agglutinin-agarose. Homogeneous receptor preparation was obtained by two cycles of affinity chromatography on immobilized avidin column coupled to [biotinyl-D-Lys6]GnRH. The overall recovery of the purified receptor was 4-10% of the initial activity in the CHAPS extract, and the calculated purification -fold was approximately 10,000 to 15,000. Analysis of iodinated purified GnRH receptors by autoradiography indicated the presence of two bands, Mr = 59,000 and 57,000. This was confirmed by photoaffinity labeling of the partially purified receptors and suggests that both components can specifically bind the hormone.     

Oxytocin and vasopressin in the human brain: social neuropeptides for translational medicine

The neuropeptides oxytocin (OXT) and arginine vasopressin (AVP) are evolutionarily highly conserved mediators in the regulation of complex social cognition and behaviour. Recent studies have investigated the effects of OXT and AVP on human social interaction, the genetic mechanisms of inter-individual variation in social neuropeptide signalling and the actions of OXT and AVP in the human brain as revealed by neuroimaging. These data have advanced our understanding of the mechanisms by which these neuropeptides contribute to human social behaviour. OXT and AVP are emerging as targets for novel treatment approaches--particularly in synergistic combination with psychotherapy--for mental disorders characterized by social dysfunction, such as autism, social anxiety disorder, borderline personality disorder and schizophrenia.     

Presence of two neuropeptides in the fusiform ganglion and reproductive ducts of Octopus vulgaris: FMRFamide and gonadotropin-releasing hormone (GnRH)

We have found evidence of FMRFamide-like and cGnRH-I-like immunoreactivity in the central nervous system (CNS) and in the reproductive ducts of both female and male cephalopod Octopus vulgaris. Cell bodies and fibers were immunolocalized in the fusiform ganglion from which the nerves that reach the female and male reproductive ducts arise. FMRFamide-like and cGnRH-I-like immunoreactive nerve endings were present in the oviduct, and in the oviducal gland of the female and in the seminal vesicle of the male. The GnRH-like peptide from the reproductive ducts has been partially characterized by HPLC. The retention time of the Octopus vulgaris GnRH-like peptide was similar to the retention time of cGnRH-I. Based on these observations we suggest that FMRFamide-like and a novel GnRH-like peptide are involved in the control of reproductive ducts of Octopus vulgaris. One possibility is that the peptides affect gamete transport. Another possibility is that they regulate secretory products such as mucus and mucilaginous substances from the oviducal gland and the seminal vesicle. Our data provide further evidence to support the hypothesis of the existence of a central and peripheral peptidergic control of reproduction of Octopus vulgaris.     

Induction of apoptosis by a gonadotropin-releasing hormone agonist during early pregnancy in the rat

We have demonstrated that continuous administration of a GnRH-agonist (GnRH-Ag) in the rat during early pregnancy suppressed plasma progesterone levels within 8 h after the commencement of treatment. The purpose of the present study is to evaluate the hypothesis that in vivo GnRH-Ag treatment induces apoptotic cell death during early pregnancy. Rats were treated individually on day 8 of pregnancy with 5 g/day of GnRH-Ag via an osmotic minipump. Sham-controlled rats received no treatment. Rats were killed at 4, 8 or 24 h after the treatment. At autopsy, blood samples were obtained and ovaries were removed. The corpora lutea (CL) from one ovary were removed for DNA laddering and RNA studies. As early as 8 h after the commencement of treatment, GnRH-Ag suppressed serum progesterone levels as expected, and increased the degree of DNA degradation in the CL that was time-dependent. At 24 h after the treatment, GnRH-Ag increased the Bax, a cell death gene, expression in the CL. Collectively, these data suggest that GnRH-Ag treatment induces apoptosis during early pregnancy in the rat.     

Distribution of gonadotropin-releasing hormone immunoreactive systems in the brain of the Senegalese sole, Solea senegalensis

The present paper reports the immunohistochemical distribution of the gonadotropin-releasing hormone (GnRH) structures in the brain of the Senegalese sole, Solea senegalensis. In this study, we have used two antibodies against the salmon GnRH and chicken GnRH-II forms and the streptavidin–biotin-peroxidase complex method. Immunoreactive cell bodies are observed at the junction between the olfactory bulbs and the telencephalon (terminal nerve ganglion cells), in the ventral telencephalon, in the preoptic parvocellular nucleus, and in the synencephalic nucleus of the medial longitudinal fasciculus. GnRH-immunoreactive fibres were found extensively throughout the brain, located in the telencephalon, preoptic area, hypothalamus, hypophysis, optic tectum, midbrain and rhombencephalon. The antisera used in this study against the two GnRH forms exhibited cross-reactivity on the same cell masses and did not allow cell populations expressing different GnRH forms to be discriminated clearly. However, anti-salmon GnRH immunostained the GnRH cells and fibres of the forebrain much more intensely, whereas the anti-chicken GnRH antiserum shows a higher immunoreactivity on synencephalic cells of the medial longitudinal fasciculus.