Hypothalamic pro-GnRH-GAP, GnRH-I and GnRH-II during the onset of photorefractoriness in the white-crowned sparrow (Zonotrichia leucophrys gambelii).

Gambel's white-crowned sparrow is a long distance migrant that undergoes spontaneous gonadal regression as a result of long day exposure. This termination of breeding is caused by the development of photorefractoriness and the birds become insensitive to long days, including continuous light. The present study investigated its possible mechanisms by examining the activity of the gonadotrophin-releasing hormone (GnRH) system under different photoperiodic regimes. We investigated the localisation and distribution of GnRH-I, its precursor pro-GnRH-GAP and GnRH-II in Gambel's white-crowned sparrow brain using immunocytochemistry with specific antibodies during photostimulation and the development of photorefractoriness. The study revealed that photoperiodic treatment, including the onset of photorefractoriness, had no significant effect on the size or number of GnRH-I, pro-GnRH-GAP or GnRH II immunoreactive cells, or the density of the GnRH-I, pro-GnRH-GAP immunoreactive fibres at the median eminence. GnRH-II was not found in the median eminence, suggesting that it does not regulate pituitary gonadotrophin secretion. GnRH-I measurement in hypothalamic extracts by radioimmunoassay did not reveal any significant difference between birds that were photostimulated or in the early stages of photorefractoriness. Furthermore, the action of the excitatory amino acid glutamate agonist N-methyl-D-aspartate on GnRH neurones in photorefractory birds was demonstrated by the significant blockade of luteinising hormone release with a specific GnRH antagonist. Taken together, these results suggest that, in Gambel's white-crowned sparrow, a decrease in GnRH-I secretion is the initial step for the onset of photorefractoriness and not a decrease in GnRH-I biosynthesis.

Role of aspartate7.32(302) of the human gonadotropin-releasing hormone receptor in stabilizing a high-affinity ligand conformation.

Mammalian gonadotropin-releasing hormone (GnRH) receptors preferentially bind mammalian GnRH, which has Arg in position eight. The Glu(7.32(301)) residue, which determines selectivity of the mouse GnRH receptor for Arg(8)-containing GnRH, is Asp(7.32(302)) in the human GnRH receptor. We have confirmed that Asp(7.32(302)) confers selectivity of the human GnRH receptor for Arg(8) of GnRH and investigated the mechanism of this specificity using site-directed mutagenesis and ligand modification. We find that although Arg(8) and Asp(7.32(302)) are required for high-affinity binding of GnRH, conformationally constrained peptides, with D-amino acid substitutions in position six or with a 6,7 gamma-lactam, bind the human GnRH receptor with high affinity, which is independent of the presence of Asp(7.32(302)) in the receptor or Arg(8) in the ligand. The ability of the ligand constraints to compensate for the absence of both Arg(8) and Asp(7.32(302)) indicates that these residues both have roles in stabilizing a high affinity ligand conformation and that their roles are complementary. This suggests that the Arg(8) and Asp(7.32(302)) side chains interact to induce a high affinity conformation of native GnRH. Thus, Asp(7.32(302)) of the human GnRH receptor determines selectivity for mammalian GnRH by its ability to induce a high affinity conformation of its native ligand. However, this initial interaction seems not to contribute to the final ligand-receptor complex. We propose that Arg(8) interacts transiently with Asp(7.32(302)) to induce a high-affinity ligand conformation of GnRH, which then interacts with a binding pocket that is common for both constrained and unconstrained analogs of GnRH.

Gonadotropin-releasing hormone receptor in the teleost Haplochromis burtoni: structure, location, and function.

GnRH acts via GnRH receptors (GnRH-R) in the pituitary to cause the release of gonadotropins that regulate vertebrate reproduction. In the teleost fish, Haplochromis burtoni, reproduction is socially regulated through the hypothalamus-pituitary-gonadal axis, making the pituitary GnRH-R a likely site of action for this control. As a first step toward understanding the role of GnRH-R in the social control of reproduction, we cloned and sequenced candidate GnRH-R complementary DNAs from H. burtoni tissue. We isolated a complementary DNA that predicts a peptide encoding a G protein-coupled receptor that shows highest overall identity to other fish type I GnRH-R (goldfish IA and IB and African catfish). Functional testing of the expressed protein in vitro confirmed high affinity binding of multiple forms of GNRH: Localization of GnRH-R messenger RNA using RT-PCR revealed that it is widely distributed in the brain and retina as well as elsewhere in the body. Taken together, these data suggest that this H. burtoni GnRH receptor probably interacts in vivo with all three forms of GNRH:

Three distinct types of GnRH receptor characterized in the bullfrog.

It has been proposed recently that two types of GnRH receptors (GnRHR) exist in a particular species. Here we present data demonstrating that at least three types of GnRHR are expressed in a single diploid species, the bullfrog. Three different cDNAs, encoding distinct types of bullfrog GnRHR (bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3), were isolated from pituitary and hindbrain of the bullfrog. BfGnRHR-1 mRNA was expressed predominantly in pituitary, whereas bfGnRHR-2 and -3 mRNAs were expressed in brain. The bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3 proteins have an amino acid identity of approximately 30% to approximately 35% with mammalian GnRHRs and approximately 40% to approximately 50% with nonmammalian GnRHRs. Interestingly, bfGnRHR-2 has an 85% amino acid homology with Xenopus GnRHR. Less than 53% amino acid identity was observed among the three bfGnRHRs. All isolated cDNAs encode functional receptors because their transient expression in COS-7 cells resulted in a ligand-dependent increase in inositol phosphate production. Notably, all three receptors exhibited a differential ligand selectivity. For all receptors, cGnRH-II has a higher potency than mGnRH. In addition, salmon GnRH also has a strikingly high potency to stimulate all three receptors. In conclusion, we demonstrated the presence of three GnRHRs in the bullfrog. Their expression in pituitary and brain suggests that bfGnRHRs play an important role in the regulation of reproductive functions in the bullfrog.

Molecular cloning, distribution and pharmacological characterization of a novel gonadotropin-releasing hormone ([Trp8] GnRH) in frog brain.

To date nine structural variants of GnRH have been identified in vertebrates and two additional forms have been isolated from a tunicate. In amphibians only mammalian GnRH ([Arg8] GnRH) and type II GnRH (chicken GnRH II, [His5, Trp7, Tyr8] GnRH) have been identified. In the present study, a full-length cDNA encoding a novel type of GnRH was isolated from pituitary of Rana dybowskii. The GnRH gene encodes a GnRH peptide ([Trp8] GnRH) in which tryptophan is substituted for arginine of mammalian GnRH Northern blot analysis revealed the presence of a single 500 bp transcript for the [Trp8] GnRH precursor in forebrain but its absence in testis, ovary, kidney and liver. Restriction digests of genomic DNA demonstrated a single copy of the gene. The [Trp8] GnRH immunoreactive cells were identified in the preoptic area of the frog brain. Synthetic [Trp8] GnRH was tested for its ability to stimulate inositol phosphate production by COS-1 cells transfected with the cloned Xenopus pituitary GnRH receptor and the cloned human GnRH receptor. [Trp8] GnRH had a potency of about 60% compared with mammalian GnRH ([Arg8] GnRH) for the Xenopus receptor, whereas the potency of [Trp8] GnRH was approximately 5% compared with mammalian GnRH for the human receptor. Both mammalian GnRH and [Trp8] GnRH were 1000-fold less potent than type II GnRH for the Xenopus GnRH receptor. The similar potency of [Arg8] GnRH and the novel [Trp8] GnRH for the Xenopus pituitary receptor indicates that, unlike the human receptor, the Xenopus receptor does not discriminate between these amino acids in position eight thereby allowing substitution of the arginine in the mammalian GnRH.

Complementary deoxyribonucleic acid cloning, gene expression, and ligand selectivity of a novel gonadotropin-releasing hormone receptor expressed in the pituitary and midbrain of Xenopus laevis.

We have cloned the full-length complementary DNA (cDNA) for a GnRH receptor from Xenopus laevis pituitary cDNA and determined its gene structure. The cDNA encodes a 368-amino acid protein that has a 46% amino acid identity to the human GnRH receptor. The X laevis GnRH receptor has all of the amino acids identified in the mammalian GnRH receptors as sites of interaction with the GnRH ligand. However, this receptor cDNA shares the same distinguishing structural features of the GnRH receptor that have been characterized from other nonmammalian vertebrates. These include the pair of aspartate residues in the transmembrane domains II and VII compared with the aspartate/asparagine arrangement in mammalian receptors, the amino acid PEY motif in extracellular loop III (SEP in mammals), and the presence of a carboxyl-terminal tail. Previous studies have reported that mammalian GnRH was equipotent to other naturally occurring GnRH subtypes in stimulating LH release from the amphibian pituitary. However, in this study we show that the X. laevis GnRH receptor has ligand selectivity for the naturally occurring GnRHs similar to other nonmammalian GnRH receptors. The order of potency of the GnRHs in stimulating inositol phosphate production in COS-1 cells transiently transfected with the X. laevis GnRH receptor cDNA was chicken GnRH II>salmon GnRH>mammalian GnRH. Transcripts of this GnRH receptor are expressed in the pituitary and midbrain of X. laevis.

Immunocytochemical localization of GnRH precursor in the hypothalamus of European starlings during sexual maturation and photorefractoriness.

Immunocytochemistry with quantitative image analysis, for both GnRH and its precursor proGnRH-GAP, was used in male European starlings (Sturnus vulgaris) to investigate four stages of a photoperiodically-induced reproductive cycle. Four different groups of birds were examined: photosensitive buy sexually immature, sexually mature, undergoing gonadal regression, and after the completion of regression and fully photorefractory. The size of cells staining for GnRH and proGnRH-GAP increased during gonadal maturation. A reduction in the number of cells staining for GnRH and the size of cells staining for both GnRH and proGnRH-GAP occurred during gonadal regression, though staining for GnRH and proGnRH-GAP in the median eminence remained high at this stage. Birds examined after completion of regression showed significantly reduced staining for both GnRH and its precursor. These observations suggest that photorefractoriness is promoted by a reduction in proGnRH-GAP production and in GnRH synthesis, rather than requiring inhibition of release of GnRH at the median eminence.

Evolutionary aspects of gonadotropin-releasing hormone and its receptor.

1. Gonadotropin-releasing hormone (GnRH) was originally isolated as a hypothalamic peptide hormone that regulates the reproductive system by stimulating the release of gonadotropins from the anterior pituitary. However, during evolution the peptide was subject to gene duplication and structural changes, and multiple molecular forms have evolved. 2. Eight variants of GnRH are known, and at least two different forms are expressed in species from all vertebrate classes: chicken GnRH II and a second, unique, GnRH isoform. 3. The peptide has been recruited during evolution for diverse regulatory functions: as a neurotransmitter in the central and sympathetic nervous systems, as a paracrine regulator in the gonads and placenta, and as an autocrine regulator in tumor cells. 4. Evidence suggests that in most species the early-evolved and highly conserved chicken GnRH II has a neurotransmitter function, while the second form, which varies across classes, has a physiologic role in regulating gonadotropin release. 5. We review here evolutionary aspects of the family of GnRH peptides and their receptors.

Identification of the molecular forms of and steroid hormone response to gonadotropin-releasing hormone in the Australian lungfish, Neoceratodus forsteri.

Gonadotropin-releasing hormone (GnRH) peptides in the hypothalamus of the lungfish, Neoceratodus forsteri, were investigated by reverse-phase HPLC and RIA with region-specific antisera. Both chicken GnRH II and mammalian GnRH were identified, the latter being present in greater concentration. The steroidogenic response to a single intracardiac injection of synthetic mammalian GnRH was investigated in early and late spring (beginning and end of spawning season) and in early autumn. In early spring, both sexes responded with rapid and transient elevation of circulating steroid hormones. Testosterone in males showed the greatest response by elevating from 120 to 240 nmol/liter within 2 min of the injection and returning to approximately 100 nmol/liter within 15 min. Female lungfish showed a similar but slightly less dramatic response in circulating estradiol and testosterone. The responses of both males and females were reduced in late spring and abolished in early autumn, which is indicative of a period of seasonal refractoriness.

Absence of rapid desensitization of the mouse gonadotropin-releasing hormone receptor.

Desensitization of gonadotropin release by the pituitary gland in response to gonadotropin-releasing hormone (GnRH) agonists has clinical applications in the treatment of gonadal-hormone-dependent disorders. We therefore investigated possible desensitization of inositol phosphate (IP) responses of GNRH receptors. No short-term homologous desensitization of the IP response to GnRH was observed in either alpha T3 gonadotrope cells line or GH3 cells transfected with GnRH receptor cDNA. The absence of homologous desensitization is unusual among G-protein-coupled receptors, and may be due to the absence of a C-terminal cytoplasmic tail, a unique feature of the GnRH receptor. Several potential protein kinase C phosphorylation sites which might mediate heterologous desensitization are present on the GnRH receptor. In both alpha T3 cells and GnRH-receptor-transfected Cos-1 cells, activation of protein kinase C by pretreatment with phorbol ester caused a 35-53% decrease in the IP response to GnRH. However, phorbol ester also inhibited guanosine 5'-[gamma-thio]triphosphate-stimulated IP production in permeabilized Cos-1 cells, suggesting that this inhibition is mediated at a post-receptor site.

A reciprocal mutation supports helix 2 and helix 7 proximity in the gonadotropin-releasing hormone receptor.

Activation of the pituitary gonadotropin-releasing hormone receptor, a member of the seven-transmembrane G protein-coupled receptor (GPCR) family, triggers a cascade of events leading to gonadotropin release and stimulation of the reproductive system. An unusual feature of this receptor, observed in mice, rats, and humans, is the presence of Asn87 in the second putative transmembrane helix at the location of a highly conserved aspartate in the GPCR family and of Asp318 in the putative seventh transmembrane helix where nearly all other GPCRs have asparagine. The possibility that these residues interact was suggested by this reciprocal pattern and by a three-dimensional model of the gonadotropin-releasing hormone receptor and was investigated by site-directed mutagenesis. Replacing Asn87 in the second transmembrane domain by aspartate eliminated detectable ligand binding. A second mutation, generating the double-mutant receptor Asp87Asn318, recreated the arrangement found in other GPCRs and re-established high affinity agonist and antagonist binding. The restoration of binding by a reciprocal mutation indicates that these two specific residues in helices 2 and 7 are adjacent in space and provides an empirical basis to refine the model of the transmembrane helix bundle of the receptor.

Failure of commercial oral amino acid supplements to increase serum growth hormone concentrations in male body-builders.

Amino acids are commonly ingested as ergogenic acids in the belief that they enhance protein synthesis and stimulate growth hormone release. The aim of this study was to determine the acute effect that amino acid supplements have on serum growth hormone (GH) concentration. Seven male body-builders reported to the laboratory on four occasions after an 8-hr fast and ingested, in random order, either a placebo, a 2.4-g arginine/lysine supplement, a 1.85-g ornithine/tyrosine supplement, or a 20-g BovrilR drink. Blood was collected before each treatment and again every 30 minutes for 3 hours for the measurement of serum GH concentration. On a separate occasion, subjects had an intravenous infusion of 0.5 microgram GH-releasing hormone.kg-1 body weight to confirm that GH secretory response was normal. The main finding was that serum GH concentrations were not altered consistently in healthy young males following the ingestion of the amino acid supplements in the quantities recommended by the manufacturers.

Prepubertal increases in gonadotropin-releasing hormone mRNA, gonadotropin-releasing hormone precursor, and subsequent maturation of precursor processing in male rats.

Changes in gonadotropins and gonadal steroids during sexual maturation in rats and humans are well documented but little is known about hypothalamic gonadotropin-releasing hormone (GnRH) gene expression in relation to these events. This study measured hypothalamic proGnRH mRNA, GnRH precursor, and fully processed GnRH from postnatal day 8 until day 62 in male rats. GnRH precursor increased on day 22, reached a peak on day 24, declined on day 25 and returned to infantile levels by day 28. A secondary rise in precursor occurred at about day 40 when testosterone levels increased. GnRH mRNA increased on day 22 and remained elevated over the study period to day 26. GnRH increased on day 24 and remained at this level until a secondary rise occurred coincident with the testosterone rise at about day 40. The ratio of GnRH precursor to GnRH was high until day 24 and was low from day 26 onwards, reflecting a maturation of the processing enzyme system between these 2 d. Thus, an abrupt increase in GnRH gene transcription (mRNA) occurs early in juvenile male rats (day 22), well before the onset of puberty. An increase in GnRH precursor accompanies these early changes and this is followed by the maturation of processing as evidenced by the rapid decline of precursor and increase in GnRH from day 24 onward.

Light and electron microscopic immunocytochemical analysis of antibodies directed against GnRH and its precursor in hypothalamic neurons.

A battery of antibodies directed against different portions of the precursor to gonadotropin-releasing hormone (GnRH), as well as to the mature decapeptide, were characterized immunocytochemically in two ways. Absorption experiments were used to determine the epitope recognized by each antiserum. Electron microscopic immunocytochemistry was then used to define the subcellular organelles that contained reaction product when tissue was incubated with these reagents. These latter observations helped to determine if the antibody recognized the epitope as part of the intact precursor or only after it had been cleaved from parent protein. Our results demonstrate that the GnRH precursor is routed from the rough endoplasmic reticulum through the Golgi apparatus to the secretory vesicles. Furthermore, we show that initial cleavage and processing of the GnRH precursor begin in the cell soma. These antibodies should be useful in the future in determining changes in processing of precursor in animals that differ in endocrine function.

Chicken GnRH II occurs together with mammalian GnRH in a South American species of marsupial (Monodelphis domestica).

Two molecular forms of gonadotropin-releasing hormone (GnRH) were demonstrated in hypothalamic extracts of M. domestica using high performance liquid chromatography and radioimmunoassay with specific GnRH antisera. One form eluted in the same position as synthetic mammalian GnRH and was quantified equally by two mammalian GnRH antisera, while the second form coeluted with synthetic chicken GnRH II and was quantified equally with two chicken GnRH II antisera. The finding of chicken GnRH II in a South American species of marsupial, which has previously been reported in some Australian species of marsupial and in species of Aves, Reptilia, Amphibia, Osteichthyes and Chondrichthyes, supports our hypothesis that this widespread structural variant may represent an early evolved and conserved form of GnRH.

[The GnRH precursor is present in the anterior pituitary gland and an important increase of its content precedes serum LH surge induced by estradiol-17 beta in female primates]. / Le précurseur du GnRH est présent dans l'antéhypophyse et une augmentation importante de son contenu précède le pic de la LH sérique induit par l'estradiol-17 beta chez la femelle du primate.

Estradiol-17 beta (E2 17 beta) is well known to evoke a preovulatory-like LH surge in ovariectomized monkeys even in the absence of the integrity of the hypothalamo-pituitary connections. LH release from the anterior pituitary (AP) is reliant on stimulation by hypothalamic GnRH which is derived from proteolytic cleavage of a precursor (designated Pro-GnRH-GAP) which also results in the production of an associated peptide (GAP). The present study examined the effects of E2 17 beta on the hypothalamic content of Pro-GnRH-GAP, GnRH and GAP while incidental observations revealed the presence of Pro-GnRH-GAP and its products in the AP. Changes in GnRH and GAP were closely related at all times after E2 17 beta treatment. However, the pattern of change in the hypothalamus and AP was inversely related. Pro-GnRH-GAP levels remained unchanged in the hypothalamus whereas in the AP the peptide increased markedly (48 hrs. post E2 17 beta) prior to the LH surge and declined to low levels (72 hrs. post E2 17 beta) at the time of the LH surge. The increase in Pro-GnRH-GAP in the AP that precedes the rise in GnRH and accompanying LH surge by 24 hrs. strongly indicates that AP GnRH is more important than hypothalamic GnRH for the mediation of the E2 17 beta-induced LH surge in female primate.

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.

Gonadotropin-releasing hormone molecular forms in mammalian hypothalamus.

Multiple forms of GnRH have been detected in brain tissue of species from all nonmammalian vertebrate classes, but in mammals it is generally believed a single molecular form of GnRH is present. We have investigated the possibility that additional structural variants of GnRH are present in mammalian (sheep, rat, and human) hypothalamus. Hypothalami were extracted with acetic acid and subjected to gel filtration chromatography and reverse phase HPLC systems specifically designed to separate GnRH analogs. Column fractions were assayed for immunoreactive GnRH using a library of specific antisera raised against the five known vertebrate GnRHs. Biological activity of the fractions was assessed by measuring their ability to release LH and FSH from cultured rat pituitary cells and/or LH release from dispersed chicken pituitary cells. Receptor binding activity was also measured in fractions from the human extract, using rat pituitary membranes. Several immunoreactive and biologically active forms of GnRH were found in sheep, rat, and human hypothalami. The major immunoreactive peptide consistently coeluted with mammalian GnRH. The other forms were not identifiable as any of the other known vertebrate GnRHs. Control experiments suggest these are modified forms of mammalian GnRH, which are artifacts generated during HPLC purification. Chromatographic and immunological studies indicate these forms of GnRH include peptides eluting both earlier and later than mammalian GnRH and which appear to be modified in the middle region and/or at the COOH-terminus of the molecule. Novel immunoreactive forms of GnRH, distinct from modified mammalian GnRH, were not apparent in any of the species. In chicken and rat pituitary cell bioassays and in rat receptor binding studies, the mammalian form of GnRH in HPLC fractions of the sheep and human hypothalamus displayed activity appropriate for this immunoreactive peak being mammalian GnRH. Some of the additional immunoreactive peaks (thought to be modified forms of mammalian GnRH) also displayed LH-releasing activity in the chicken and rat systems. Gonadotropin-releasing activity or receptor binding activity due to a second, novel, GnRH-like substance in HPLC fractions of the sheep and human hypothalamus was not detected. These data provide evidence for a single form of GnRH in sheep, rat, and human hypothalamus, unlike species from other vertebrate classes where two or more GnRHs are present within a single tissue.

Processing of luteinizing hormone-releasing hormone precursor in rat neurons.

Results of previous immunocytochemical studies indicate that in the rat brain proteolytic cleaving of LHRH precursors to generate the physiologically active decapeptide takes place within neuronal fibers and terminals and not within perikarya. A 69-amino acid (aa) LHRH precursor comprised of the decapeptide, a 3-aa cleavage and amidation site, and a 56-aa C-terminal extension has recently been characterized. Two antisera generated to specific aa sequences of the C-terminal extension (RM 8/5, anti aa 14-26; PS 39A, anti aa 40-53) and two directed to specific regions of the LHRH decapeptide (RM 1076, anti aa 4-8; A 422 generated to the N-terminal pGlu and C-terminal amidated Gly) were used to further examine intraneuronal sites of precursor processing. Patterns of immunoreactivity revealed with antisera directed to non-LHRH sequences of LHRH precursor paralleled those observed with antisera to the decapeptide. Immunopositive perikarya, processes, and neurovascular terminals were observed with PS 39A. Antiserum PS 39A binds to an internal aa sequence of the C-terminal extension and would, therefore, be expected to detect intact precursor LHRH as well as products of proteolytic cleavage. In contrast, only immunopositive processes and neurovascular terminals were observed with RM 8/5, an antiserum directed to an initial aa sequence of the C-terminal extension. The pattern of immunoreactivity revealed with RM 8/5 resembled that observed with an antiserum that binds the fully processed decapeptide (A 422), indicating that proteolytic cleavage of the decapeptide from the C-terminal extension is required for binding by this antiserum. Furthermore, the restricted distribution of reaction product observed with RM 8/5 relative to A 422 suggests that additional processing of the C-terminal extension may be required for binding. Such additional processing appears to occur in neurovascular terminals of the median eminence.

Localization of a peptide sequence contained in the precursor to gonadotropin releasing hormone (GnRH).

The precursor protein that contains the sequence for the neurohormone gonadotropin releasing hormone (GnRH) also contains an additional fragment (amino acids 14-26, designated pHGnRH14-26) that can release luteinizing hormone (LH) and follicle stimulating hormone (FSH) in vitro. An immunocytochemical study was carried out to determine if this sequence could be found in its processed form in hypothalamic nerve terminals. In rats, ewes and rhesus monkeys pHGnRH14-26 was demonstrated in both neuronal cell bodies and in axon terminals. In mice, immunoreactivity was present in terminals only. No reaction was found in the hamster hypothalamus. Double label immunocytochemical studies for GnRH and pHGnRH14-26 showed that both sequences could be found in the rat septal-preoptic-hypothalamic continuum in some but not all neuronal cell bodies. Control experiments strongly suggest that the 14-26 immunoreactivity represents the fragment after it has been cleaved from the precursor protein and that this peptide could be available for release into the hypophysial portal system.