Molecular Forms Of Gonadotropin-releasing Hormone Research Articles

SUN-035 A Role for GNRH-II in the Control of Puberty?

Hypothalamic gonadotropin-releasing hormone (GnRH) neurons represent the primary neuroendocrine link between the brain and the reproductive system. Although they play a key role in stimulating the release of FSH and LH from the anterior pituitary gland, the underlying mechanism by which they trigger the onset of puberty is unclear. To address this issue, RT-PCR, in situ hybridization histochemistry, and Affymetrix gene arrays were used to profile hypothalamic GnRH gene expression in prepubertal and adult rhesus macaques (Macaca mulatta). Like humans, these nonhuman primates express two molecular forms of GnRH (GnRH-I and GnRH-II), both of which are highly effective at stimulating gonadotropin release via the same GnRHR1 receptor. However, only GnRH-II shows increased hypothalamic expression in the presence of elevated estrogen concentrations (i.e., positive feedback), whereas GnRH-I expression either remains the same or decreases (i.e., negative feedback). In the present study, the hypothalamic expression levels of GnRH-I and GnRHR1 were found to be no different between prepubertal and adult animals, despite marked differences in circulating sex-steroid hormone levels, whereas the hypothalamic expression level of GnRH-II was significantly higher in the adults than in the juveniles. Therefore, although the traditional GnRH-I neurons are likely to play a fundamental role in initiating FSH and LH release during the early stages of pubertal development, GnRH-II neurons may play an important role in maintaining elevated gonadotropin release during the final stages (i.e., at a time when the GnRH-I neurons are subjected to increasing negative sex-steroid feedback from the maturing gonads). Taken together, the data suggest that sexual maturation in primates is likely to be orchestrated by the concerted action of two distinct GnRH neuronal subtypes that respond differentially to sex-steroid feedback.

Selective targeting of GnRH-II neurons to block ovulation

BackgroundIn rhesus macaques, luteinizing hormone (LH) secretion appears to be regulated by two distinct gonadotropin-releasing hormone (GnRH) neuronal populations, which can be distinguished by their unique anatomical locations and because they express different molecular forms of GnRH (GnRH-I and GnRH-II). Study DesignThe effect of estradiol on GnRH gene expression was examined. ResultsEstradiol inhibited GnRH-I neurons but stimulated GnRH-II neurons, suggesting that GnRH-II neurons play the dominant role in mediating estradiol-positive feedback and triggering the midcycle preovulatory LH surge. ConclusionsSelective silencing of GnRH-II neurons in women could serve as a novel contraceptive, by blocking ovulation while leaving the rest of the reproductive axis relatively unperturbed.

Effect of 17beta-estradiol on hypothalamic GnRH-II gene expression in the female rhesus macaque.

The hypothalamus of rhesus macaques expresses two molecular forms of gonadotropin-releasing hormone (GnRH-I and GnRH-II). However, it is unclear whether these two neuropeptides play similar roles in the control of reproductive neuroendocrine function, especially in the context of positive and negative estrogen feedback. To address this issue, in situ hybridization histochemistry was used to compare the effect of 17beta-estradiol (E) on the expression of GnRH-I and GnRH-II mRNA in the medial basal hypothalamus (MBH) of adult female macaques. GnRH-I mRNA expression was found to be significantly (P<0.01) more abundant in ovariectomized (ovx) animals compared with ovariectomized E-treated (ovx+E) animals. In marked contrast, GnRH-II mRNA expression was found to be significantly (P<0.05) more abundant in ovx+E animals than in the ovx animals. To help elucidate how E exerts this stimulatory action on GnRH-II gene expression, hypothalamic sections were subsequently double labeled using a combination of immunohistochemisty for estrogen receptor (ER) -alpha or -beta and in situ hybridization histochemistry for GnRH-II. Approximately 50% of the GnRH-II positive cells in the MBH were found to express ERbeta, but none expressed ERalpha. Taken together, these data give credence to a novel pathway by which E may control the primate neuroendocrine reproductive axis, one that involves stimulation of GnRH-II release via an ERbeta-mediated mechanism.

Gonadotropin-Releasing Hormones (GnRHs) in a Primitive Teleost, the Arowana: Phylogenetic Evidence That Three Paralogous Lineages of GnRH Occurred Prior to the Emergence of Teleosts

Multiple molecular forms of gonadotropin-releasing hormone (GnRH) are present in a single vertebrate species. To extend the knowledge on GnRH evolution and the number of GnRH forms in one organism, GnRH cDNAs have been isolated and characterized from one of the most primitive teleosts, the arowana Scleropages jardini. This species had two molecular forms of GnRH: salmon-type GnRH (sGnRH) and chicken-II-type GnRH (cGnRH-II). Sequence comparison between the prepro-GnRHs of the arowana and those of other teleosts indicated that sGnRH represented a paralogue separate from any other forms of GnRH. Consistently, subsequent phylogenetic analysis showed that known forms of GnRH in teleosts fell into three paralogous lineages: sGnRH alone on one lineage, cGnRH-II on another, and many other forms on the other. These results suggest that an ancestral GnRH gene duplicated twice prior to the emergence of teleosts and, therefore, that teleosts, and probably also tetrapods, would possess three paralogous forms of GnRH in individual brains.

A Novel Form of Gonadotropin-Releasing Hormone in the Medaka, Oryzias latipes

The present study has identified three molecular forms of gonadotropin-releasing hormone (GnRH) in the brain of a teleost, the medaka, by isolation of their cDNAs. This species has a novel GnRH, which is here named medaka-type GnRH (mdGnRH), in addition to two characterized forms, chicken-II-type GnRH (cGnRH-II) and salmon-type GnRH (sGnRH). Phylogenetic analysis showed that mdGnRH is a medaka homolog of and seabream-type GnRH (sbGnRH) and mammalian-type GnRH (mGnRH) in other species, and suggested that all vertebrates have three distinct GnRHs. Furthermore, in situ hybridization revealed that the mdGnRH gene is expressed only in neurons clustered within the preoptic area as sbGnRH and mGnRH genes in other species are, while the genes for cGnRH-II and sGnRH are only in the midbrain tegmentum and nucleus olfactoretinalis, respectively. This result suggested that mdGnRH is a hypophysiotropic factor and the other two forms are involved in other physiological events as neuromodulators or neurotransmitters.

Molecular Cloning and Tissue-Specific Expression of a Gonadotropin-Releasing Hormone Receptor in the Japanese Eel

Gonadotropin-releasing hormone (GnRH) is a key regulatory neuropeptide involved in the control of reproduction in vertebrates. In the Japanese eel, one of the most primitive teleost species, two molecular forms of GnRH, mammalian-type GnRH and chicken-II-type GnRH (cGnRH-II), have been identified. This study has isolated a full-length cDNA for a GnRH receptor from the pituitary of the eel. The 3233-bp cDNA encodes a 380-amino acid protein which contains seven hydrophobic transmembrane domains and N- and C-terminal regions. The exon/intron organization of the open reading frame of the eel GnRH receptor gene was also determined. The open reading frame consists of three exons and two introns. The exon–intron splice site is similar to that of the GnRH receptor genes of mammals reported so far. Expression of the eel GnRH receptor was detected in various parts of the brain, pituitary, eye, olfactory epithelium, and testis. This result suggests that GnRH has local functions in these tissues in addition to its actions on gonadotropin synthesis and release in the pituitary. This tissue-specific expression pattern is similar to that of the eel cGnRH-II. Furthermore, the present eel receptor shows very high amino acid identity with the catfish and goldfish GnRH receptors, which are highly selective for the cGnRH-II. These results suggest that the cGnRH-II acts through binding to the present receptor in the eel.

Two molecular forms of gonadotropin-releasing hormone (GnRH-I and GnRH-II) are expressed by two separate populations of cells in the rhesus macaque hypothalamus

Gonadotropin-releasing hormone represents the primary neuroendocrine link between the brain and the reproductive axis, and at least two distinct molecular forms of this decapeptide (GnRH-I and GnRH-II) are known to be expressed in the forebrain of rhesus macaques ( Macaca mulatta). Although the distribution pattern of the two corresponding mRNAs is largely dissimilar, their expression appears to show some overlap in specific regions of the hypothalamus; this raises the possibility that some cells express both molecular forms of GnRH. To resolve this issue, double-label histochemistry was performed on hypothalamic sections from six male rhesus macaques, using a monoclonal antibody to GnRH-I and a riboprobe to monkey GnRH-II mRNA. In total, more than 2000 GnRH neurons were examined but in no instance were GnRH-I peptide and GnRH-II mRNA found to be coexpressed. This finding emphasizes that GnRH-I and GnRH-II are synthesized by two distinct populations of hypothalamic neurons, and suggests that they may be regulated by different neuroendocrine pathways.

GnRH-Systems in the Forebrain of Cichlid Fish

The neuronal systems that contain gonadotropin-releasing hormone (GnRH) were quantitatively examined in the forebrain of two cichlid species (Oreochromis niloticus Lin. and Tilapia maria Boulenger) using tracing methods and immunostaining. In both species the nucleus olfacto-retinalis and a population of neurons in the basal preoptic region contained GnRH. The nucleus olfacto-retinalis was divided into several neuronal subpopulations which differed with respect to cytology, projection pattern, and peptide content. GnRH cell number increased with body size, the rates being different for the different subpopulations. A sexual dimorphism was found in the basal preoptic region of Tilapia; males had significantly fewer GnRH-containing cells than females. Selective staining of the basal preoptic region demonstrated that this cell group is the main source of GnRH-innervation of the pituitary gland and indicate that the molecular forms of GnRH expressed in the two cell populations studied may be different.

Multiple gonadotropin‐releasing hormone neuronal systems in vertebrates

Gonadotropin‐releasing hormone (GnRH) was originally isolated as a hypo‐thalamic peptide that regulates reproduction by stimulating the release of gonadotropins. Using comparative animal models has led to the discovery that GnRH has a more ancient evolutionary origin. During evolution, GnRH peptide underwent gene duplication and structural changes to give rise to multiple molecular forms of GnRHs. Mammalian GnRH initially considered to be the sole molecular form, is now grouped as a family of peptides along with GnRH variants determined from representatives in all classes of vertebrates. Vertebrate species, including primates and humans have more than one GnRH variant in individual brains; a unique GnRH form in the forebrain and chicken II GnRH in the midbrain. Furthermore, several species of bony fish have three molecular variants of GnRH: salmon GnRH, sea‐bream GnRH and chicken II GnRH. Also, it has been shown that in addition to the olfactory placodes and the midbrain, there is a third embryonic source...

Gonadotropin-Releasing Hormone (GnRH) in Ancient Teleosts, the Bonytongue Fishes: Putative Origin of Salmon GnRH

The molecular forms of gonadotropin-releasing hormone (GnRH) were examined in the bonytongue fishes (Osteoglossomorpha), one of the most ancient living teleost groups. These fish represent a phylogenetic link between the early ray-finned fishes and the modern teleosts. Five representative species from four of six bonytongue families were examined for GnRH using high-performance liquid chromatography and radioimmunoassay techniques with antisera raised against salmon (s), chicken-II (c-II), and mammalian (m) forms of GnRH. Salmon GnRH and cGnRH-II were identified in four of the species (arawana, elephantnose, false featherfin, Asiatic featherfin) whereas in the butterfly fish, mGnRH and cGnRH-II were identified. Our data suggest that teleosts such as eels and butterfly fish, which have mGnRH like that of even earlier ray-finned fishes, may have evolved before fish with sGnRH. We also suggest that sGnRH first appeared in the Osteoglossomorpha. The phylogenetic relationship of the eels (Anguillidae), butterfly fish (Pantodontidae), and bonytongue fish among other teleosts needs to be reexamined using additional characteristics.

Characterization and localization of two forms of gonadotropin-releasing hormone (GnRH) in the spinal cord of the frog Rana ridibunda.

Two molecular variants of gonadotropin-releasing hormone (GnRH) have been previously characterized in the brain of amphibians, i.e., mammalian GnRH (mGnRH) and chicken GnRH-II (cGnRH-II). The aim of the present study was to identify the molecular forms of gonadotropin-releasing hormone and to localize gonadotropin-releasing hormone-containing elements in the spinal cord of the frog Rana ridibunda using highly specific antisera against mGnRH and cGnRH-II. High-performance liquid chromatography (HPLC) analysis combined with radioimmunoassay (RIA) detection revealed that frog spinal cord extracts contained both mGnRH and cGnRH-II. Immunohistochemical labeling revealed that the frog spinal cord was devoid of GnRH-containing cell bodies. In contrast, numerous GnRH-immunoreactive fibers were observed throughout the entire length of the cord. mGnRH immunoreactivity was only detected in the rostral region of the cord and consisted of varicose processes located in the vicinity of the central canal. cGnRH-II-positive fibers were found throughout the spinal cord, the density of immunoreactive processes decreasing gradually toward the caudal region. Two main cGnRH-II-positive fiber tracts with a rostrocaudal orientation were observed: a relatively dense fiber bundle surrounding the central canal, and a more diffuse plexus in the white matter. In addition, short, varicose cGnRH-II-positive processes with a radial orientation were present throughout the gray matter. These fibers were particularly abundant ventromedially and formed a diffuse network that ramified laterally to end in the vicinity of motoneurons. Taken together, these data indicate that the frog spinal cord, like the frog brain, contains two forms of GnRH. The presence of numerous cGnRH-II-immunoreactive fibers in the ventral horn suggests that cGnRH-II may influence the activity of a subpopulation of motoneurons.

Gonadotropin-releasing hormone gene expression in teleosts

Expression of multiple molecular forms of gonadotropin-releasing hormone (GnRH) mRNAs and GnRH peptides were examined in the brains of tilapia ( Oreochromis mossambicus) and sockeye salmon ( Oncorhynchus nerka), using in situ hybridization histochemistry and immunohistochemical techniques. After otherwise identical conditions, lesser background and stronger GnRH hybridization signals were observed on cryostat vs. paraffin sections. In both fresh and Bouin's-fixed paraffin-embedded tissues, there was a good correlation between the distribution of GnRH mRNA and GnRH peptide-containing cells. Although the brains of tilapia and the sockeye were immunoreactive to three forms of the GnRH molecule (salmon, mammal, chicken-II), GnRH mRNA expression was site-specific and species-specific. In the tilapia, ganglionic cells of the nucleus olfactoretinalis, basal telencephalon and the anteroventral preoptic area were immunoreactive to salmon-, and mammalian-GnRH peptide. Neurons of the nucleus olfactoretinalis expressed cichlid-GnRH I mRNA. The preoptic neurons, despite the immunoreactivity, expressed no hybridization signals. Midbrain neurons were immunoreactive to salmon-GnRH but expressed cichlid-GnRH IIβ (= chicken-GnRH II) mRNA hybridization signals. In the sockeye, ganglionic cells along the extracerebral course of the nervus terminalis were immunoreactive to mammalian-, chicken-II and salmon-GnRH. These neurons expressed only salmon-GnRH mRNA hybridization signals. Intracerebral GnRH expression in the sockeye was delayed till smoltification. The basal telecephalon and midbrain neurons immunoreactive to salmon-GnRH, formed no hybridization signals with GnRH antisense probes. Oligonucleotide probes complementary to chicken-GnRH I and mammalian-GnRH revealed no hybridization signals in the tilapia and in the sockeye brain. Fibers, immunoreactive to salmon-, mammalian-, and chicken II-GnRH were seen in close association with growth hormone cells. Chicken-GnRH II-immunoreactive fibers were also seen in close proximity to somatolactin cells in the sockeye salmon.

Distribution of two molecular forms of gonadotropin-releasing hormone (GnRH) in the central nervous system of the frog Rana ridibunda

Two molecular forms of gonadotropin-releasing hormone (GnRH) have been recently characterized in the brain of the frog Rana ridibunda i.e. mammalian GnRH (mGnRH) and chicken GnRH-II (cGnRH-II). Using highly specific antisera against each form of GnRH, we have investigated the distribution of these two neuropeptides in the frog brain by the indirect immunofluorescence and the peroxidase-antiperoxidase techniques. mGnRH-immunoreactive cell bodies were restricted to a well defined region corresponding to the septal-anterior preoptic area. mGnRH-containing fibers projected through the ventral diencephalon and ended in the median eminence. In contrast, cGnRH-II-immunoreactive structures were widely distributed in the frog brain. In the telencephalon cGnRH-II-positive elements formed a ventromedial column extending from the olfactory bulb to the septal area, a pathway which corresponds to the terminal nerve. A dense accumulation of eGnRH-II-immunoreactive cell bodies was also found in the septal-anterior preoptic area; these neurons sent processes towards the median eminence via the hypothalamus. Double immunostaining revealed that, in this area, mGnRH- and cGnRH-II-like immunoreactivity co-existed in the same neurons. In the mid-diencephalon, numerous cGnRH-II-immunoreactive perikarya were found, surrounding the third ventricle, in the posterior preoptic and infundibular areas. Many of these neurons sent processes towards the ventricular cavity. More caudally, a dense population of cGnRH-II-immunoreactive perikarya was also observed in the nucleus of the paraventricular organ and the posterior tubercle. Dorsally, the thalamus, the tegmentum, the tectum and the granular layer of the cerebellum were richly innervated by cGnRH-II-positive fibers. In the medulla oblongata, numerous cGnRH-II-immunoreactive perikarya were seen in several cranial nerve nuclei. Ventrally, a dense plexus of immunoreactive fibers projected rostrocaudally into the spinal cord. The occurrence of mGnRH- and cGnRH-II-like immunoreactivity in the septal-anterior preoptic area and the hypothalamo-pituitary pathway supports the view that both peptides act as hypophysiotropic neurohormones. The widespread distribution of cGnRH-II-immunoreactive elements in the central nervous system of the frog strongly suggests that this peptide may also exert neuromodulator and/or neurotransmitter activities.

Nature and Distribution of Gonadotropin-Releasing Hormone (GnRH) in the Brain, and GnRH and GnRH Binding Activity in Serum of the Spotted Dogfish Scyliorhinus canicula

The distribution of different molecular forms of gonadotropin-releasing hormone (GnRH) in the brain and serum of the spotted dogfish, Scyliorhinus canicula, was investigated by an indirect immunofluorescence method, using antisera against salmon (s-), chicken-II (cII-) and mammalian (m-) GnRHs, and by reverse-phase high-performance liquid chromatography (RP-HPLC) coupled with radioimmunoassays. Five GnRH molecular forms were demonstrated on the basis of the retention time in the RP-HPLC system. The characterestics of four of these GnRH peptides are consistent with those of m-, cII-, dogfish (df-), and sGnRH. The fifth form appears to be novel. Immunoreactive sGnRH structures were confined to the diencephalon; whereas cIIGnRH and mGnRH were found in the telecephalon and diencephalon, cIIGnRH-and dfGnRH-like molecules were detected in the serum. Moreover, specific, low-affinity GnRH binding protein (GnRH-BP) was found in the serum of the spotted dogfish. The binding of [ 125I]sGnRHA to the serum GnRH-BP was dependent on incubation time, equilibrium being reached within 1 hr at 4° binding was rapid and incompletely reversible. Scatchard analysis yielded a linear plot with a K d of 7.9 × 10 -7 M. The presence of a Gnrh-BP in spotted dogfish serum suggests a probable action of GnRH via the general circulation.

Immunocytochemical localization of mammalian GnRH (gonadotropin-releasing hormone) and chicken GnRH-II in the brain of the European silver eel ( Anguilla anguilla L.)

Using specific antibodies for the two molecular forms of gonadotropin-releasing hormone (GnRH) present in the European eel, Anguilla anguilla, (mammalian GnRH, mGnRH, and chicken GnRH II, cGnRH-II), we employed immunocytochemistry to determine the distribution of these two peptides in the brain and in the pituitary. The results indicate that mGnRH and cGnRH-II are localized in different neurons: mGnRH-immunoreactive (ir) perikaria were observed in the olfactory bulbs, the junction between olfactory bulbs and telencephalon (nucleus olfactoretinalis), the telencephalon, the preoptic region and the mediobasal hypothalamus. These cell bodies are located along a continuum of ir-fibers that could be traced from the olfactory nerve to the pituitary. Mammalian GnRH-ir fibers were detected in many parts of the brain (olfactory bulbs, ventral telencephalon, hypothalamus, optic tectum, mesencephalon) and in the pituitary. Chicken GnRH-II-ir cell bodies were detected in the nucleus of the medial longitudinal fasciculus of the midbrain tegmentum, but only scattered fibers could be detected in different parts of the brain. The pituitary exhibited very few cGnRH-II-ir fibers, contrasting with an extensive mGnRH innervation. These results are in agreement with our previous data obtained in the same species using specific radioimmunoassays for mGnRH and cGnRH-II. They demonstrate a differential distribution of the two forms of GnRH in the brain of the eel, as in the brain of some other vertebrate species, and suggest differential physiological roles for the two GnRH forms in the eel. They also provide information concerning the evolution of the GnRH systems in vertebrates.

Antibodies against different forms of GnRH distinguish different populations of cells and axonal pathways in a urodele amphibian, Taricha granulosa

Neurons immunoreactive to the peptide hormone gonadotropin-releasing hormone (GnRH) have been identified in the posterior diencephalon or anterior midbrain of diverse vertebrates. These cells are located caudal to the more well-characterized GnRH neurons in the nervus terminalis and septo-preoptic area, and are believed to express one or more of the nonmammalian forms of the GnRH. The present study utilized immunocytochemical techniques to determine whether the posterior GnRH group is present in a urodele amphibian, the newt Taricha granulosa. Antibodies directed against different molecular forms of GnRH were used to evaluate the immunological properties of GnRH-containing neurons in amphibians. An antibody selective for mammalian GnRH labeled perikarya in the nervus terminalis (terminal nerve) and septo-preoptic region, as described previously. Thick fibers that arise from terminal nerve and septo-preoptic neurons project mainly to the median eminence, medial pallium and habenula. An antibody selective for chicken GnRH II labeled cell bodies in the paraventricular organ and posterior tubercle of the caudal diencephalon, and thin fibers that project widely throughout the central nervous system. Region-specific staining with different GnRH antibodies supports the interpretation that different molecular forms of GnRH are expressed by neuroanatomically distinguishable systems.

Multiple molecular forms of gonadotropin-releasing hormone in the brain of an elasmobranch: Evidence for IR-lamprey GnRH

These studies investigated brains of skate, Raja erinacea (order Rajiformes, class Chondrichthyes), for gonadotropin-releasing hormone (GnRH) peptides by chromatography and immunoreactivity with region-specific antisera raised against mammalian GnRH and lamprey GnRH. The region-specific antibody to lamprey GnRH-I was produced following conjugation to bovine serum albumin using the bis-diazotized benzidine method. This antibody was characterized by assaying a range of increasing dilutions of the known vertebrate GnRHs, as well as analogs to lamprey GnRH-I. Two analogs, lamprey [Phe 2]GnRH-I and lamprey [Leu 7]GnRH-I, were synthesized by solid phase peptide synthesis using a benzhydrylamine resin as the supporting medium and purified by chromatography. This antibody demonstrated less than 0.01% cross-reactivity with all GnRH peptides tested, suggesting a highly specific antibody with a region of amino acids 2–8 that appears essential for binding. In the skate brain, five immunoreactive (IR) GnRH forms were detected, four of which eluted in the same positions as synthetic mammal and chicken GnRH-I (which coelute): lamprey GnRH-I, salmon and chicken GnRH-II, and one that was an unidentified form. A minor peak coeluted with lamprey GnRH-III. The major form in the skate brain is considered to have eluted with synthetic mammalian GnRH. These studies confirm an earlier report of an IR-mammalian GnRH peptide and provide new evidence for IR-lamprey GnRH in the brain of an elasmobranch.

Two molecular forms of gonadotropin-releasing hormone from the brain of the frog, Rana ribibunda: purification, characterization, and distribution

The molecular forms of GnRH in amphibia have not been characterized structurally. An antiserum to mammalian GnRH that shows appreciable cross-reactivity with chicken GnRH-I ([Gln8]GnRH), chicken GnRH-II ([His5,Trp7,Tyr8]GnRH), and salmon GnRH ([Trp7, Leu8]GnRH) was used in a RIA to detect GnRH-related peptides in an extract of the brain of the European green frog, Rana ridibunda. Two peptides were purified to apparent homogeneity, and determination of their primary structures showed that they are identical to mammalian GnRH and chicken GnRH-II. Salmon GnRH and lamprey GnRH-I ([Tyr3,Leu5,Glu6,Trp7,Lys8]GnRH) were not identified in this species of frog. Immunocytochemical analysis using specific antisera has identified regions of the frog diencephalon and telencephalon in which chicken GnRH-II and mammalian GnRH are localized to different populations of neurons. This differential distribution suggests distinct physiological roles for the two forms of GnRH in amphibia.

Immunohistochemical demonstration of the presence and localization of diverse molecular forms of gonadotropin-releasing hormone in the lizard ( Podarcis s. sicula) brain

The immunoshistochemical presence and the distribution pattern of four different molecular forms of gonadotropin-releasing hormone (GnRH) were investigated in the brain of both sexes of the lizard, Podarcis s. sicula. Animals used in this study were collected in November and April, representing two different periods of the reproductive cycle. The antisera used were those raised against synthetic mammalian GnRH, chicken GnRH-I and II, and salmon GnRH. Strong immunoreaction was obtained for salmon, chicken-I, and chicken-II GnRHs, whereas a very weak reaction was seen for the mammalian form of GnRH. The distribution of immunoreactive-GnRH perikarya and fibers did not vary with the sex, the reproductive condition of the animals, or the antiserum used. Also, the intensity of immunoreaction with any one antiserum was quite similar in both periods of the year and in all brains examined. The immunoreactive perikarya were seen as two distinct groups, one in the mesencephalon and the other in the infundibulum. Immunoreactive fiber endings were seen in the telencephalon, the optic tectum, the anterior preoptic area, the median eminence, the central grey matter, the rhombencephalon, and the cerebellum. No immunoreactive perikarya were seen in the telencephalon or the anterior preoptic area.

Activity of vertebrate gonadotropin-releasing hormones and analogs with variant amino acid residues in positions 5, 7 and 8 in the goldfish pituitary

All non-mammalian vertebrates as well as marsupial mammals have two or more forms of gonadotropin-releasing hormone (GnRH) in the brain. Goldfish brain and pituitary contains two molecular forms of GnRH, salmon GnRH ([Trp 7, Leu 8]m-GnRH; s-GnRH) and chicken GnRH-II ([His 5, Trp 7, Tyr 8]m-GnRH; cII-GnRH). Both sGnRH and cII-GnRH stimulate gonadotropin (GtH) as well as growth hormone (GH) release from the goldfish pituitary. The purpose of the present study was to study the activity of the five known forms of GnRHs as well as analogs of mammalian GnRH (m-GnRH) with variant amino acid residues in positions 5, 7 and 8 in terms of binding to GnRH receptors, and release of GTH and GH from the perifused fragments of goldfish pituitary in vitro. All five vertebrate GnRH peptides stimulated both GtH and GH release in a dose-dependent manner, although their potencies were very different. cII-GnRH was somewhat more active than s-GnRH in releasing GtH, whereas s-GnRH tended to have a greater potency than cII-GnRH in terms of GH release. Both chicken GnRH-I (cI-GnRH) and lamprey GnRH (l-GnRH) were significantly less potent than mGnRH, s-GnRH and cII-GnRH in releasing GtH and GH. cII-GnRH binds with higher affinity for the high affinity binding sites compared to all other native peptides. The activity of [Trp 7]-GnRH was similar to both s-GnRH and cII-GnRH in releasing GtH and GH. Substitution of His 5 resulted in a significant decrease in GtH releasing potencies compared to mGnRH, sGnRH and cII-GnRH. [His 5]-GnRH also had lower GH releasing potency than mGnRH and sGnRH. Tyr 8, His 8 and Leu 8 substitutions caused significant decreases in GtH releasing potencies compared to mGnRH, s-GnRH and cII-GnRH, but did not cause a significant change in GH releasing potency. The combination of [His 5, Trp 7]-GnRH had GtH and GH releasing activities similar to m-GnRH, s-GnRH and cII-GnRH. However, [His 5, Tyr 8]-GnRH had significantly lower GtH but not GH releasing potency compared to m-GnRH, s-GnRH and cII-GnRH. In terms of binding affinity, there were no correlations between binding affinities of the high or low affinity binding sites and GH or GtH releasing activities of the peptides used in the present study, suggesting that full molecular structure of native peptides are required for receptor recognition; in this context, previous studies demonstrated a good correlation between high affinity GnRH receptor binding and GTH release activity for GnRH analogs containing Trp 7, Leu 8 residues and various d-amino acids at position 6. Viewing all of the data together it is apparent that the presence of tryptophan in position 7 of the native GnRH peptides in goldfish, s-GnRH and cII-GnRH, is essential for the high potency of these peptides in releasing GtH from the goldfish pituitary. It is further evident that in goldfish the GH releasing activities of GnRH peptides are much less affected by position 8 substitutions than are the GtH releasing activities; position 8 substitutions in the m-GnRH molecule with histidine, leucine, methionine or tyrosine produces analogs that are significantly less potent in terms of GtH and are in this way more selective for the release of GH. These findings support the hypothesis that the GnRH receptors on somatotrophs and gonadotrophs in goldfish are different and have different requirements for optimal ligand binding and activity.