GnRH-like Peptides Research Articles

Identification of GnRH-like peptide and its potential signaling pathway involved in the oocyte meiotic maturation in the Chinese mitten crab, Eriocheir sinensis

Gonadotropin-releasing hormone (GnRH) plays a pivotal role in reproductive regulation in vertebrates. However, GnRH was rarely isolated and its function remains poorly characterized in invertebrates. The existence of GnRH in ecdysozoa has been controversial for a long. Here, we isolated and identified two GnRH-like peptides from brain tissues in Eriocheir sinensis. Immunolocalization showed that the presence of EsGnRH-like peptide in brain, ovary and hepatopancreas. Synthetic EsGnRH-like peptides can induce germinal vesicle breakdown (GVBD) of oocyte. Similar to vertebrates, ovarian transcriptomic analysis revealed a GnRH signaling pathway in the crab, in which most genes exhibited dramatically high expression at GVBD. RNAi knockdown of EsGnRHR suppressed the expression of most genes in the pathway. Co-transfection of the expression plasmid for EsGnRHR with reporter plasmid bearing CRE-luc or SRE-luc response element into 293T cells showed that EsGnRHR transduces its signal via cAMP and Ca2+ signaling transduction pathways. In vitro incubation of the crab oocyte with EsGnRH-like peptide confirmed the cAMP-PKA cascade and Ca2+ mobilization signaling cascade but lack of a PKC cascade. Our data present the first direct evidence of the existence of GnRH-like peptides in the crab and demonstrated its conserved role in the oocyte meiotic maturation as a primitive neurohormone.

The in-vitro effect of gonadotropin-releasing hormones, GnRH-I and GnRH-II, on the motility, vitality and acrosome integrity of Vervet monkey (Chlorocebus aethiops) spermatozoa.

Two isoforms of the gonadotropin-releasing hormone (GnRH), GnRH-I and GnRH-II, are expressed in mammals, and the presence of one or more GnRH-like peptides has been demonstrated in the male reproductive tract. GnRH and its receptors (GnRHR) are present in human and non-human primate testis, prostate, epididymis, seminal vesicle, spermatozoa and seminal human plasma. GnRH-II is site-specific and acts directly in an inhibitory or stimulatory fashion. Previous studies speculated that GnRH-II could disrupt specific sperm processes, such as sperm motility or capacitation and could be utilized as an effective contraceptive agent. Our study aimed to investigate the in-vitro effects of GnRH-I and GnRH-II on Vervet monkey sperm function. Electro-ejaculated semen samples from 10 Vervet monkeys (Chlorocebus aethiops) were used to select motile sperm populations. Sperm aliquots were incubated with GnRH-I and GnRH-II at different concentrations for 1 h, where after sperm motility and kinematic parameters were assessed using the automated Sperm Class Analyser. Additional sperm aliquots were incubated with two 10-amino acid control peptides, a non-related peptide and an inactive peptide to exclude the possible influence on sperm motility from other peptides with a structure similar to GnRH. Additionally, a GnRHR-I antagonist (GnRHR-A), Cetrorelix, was tested to establish its antagonistic capability on GnRH. The effect of selected concentrations of GnRH-I and GnRH-II on sperm vitality and acrosome intactness was also evaluated after 10- and 60min exposure. Analysis of the percentage total sperm motility revealed that different concentrations for GnRH-I and GnRH-II inhibited sperm motility significantly. While sperm progressiveness was also notably affected and a trend of decreased sperm kinematics were evident, no effect was found on sperm vitality or acrosome intactness. The non-related and inactive peptides had no impact on sperm motility. The GnRHR-A demonstrated no effect on sperm motility and effectively blocked the inhibitory outcome on the motility of both GnRH isoforms. While GnRH-I or GnRH-II at low-dose concentrations resulted in in-vitro inhibition of sperm motility, it appears to have no adverse effects on other sperm functional parameters evaluated. These collective observations possibly indicate an essential role for GnRH in the in-vivo process of sperm selection in the female reproductive tract.

Effective accumulative temperature affects gonadal maturation by controlling expression of GnRH, GnRH receptor, serotonin receptor and APGWamide gene in Pacific abalone, Haliotis discus hannai during broodstock conditioning in hatcheries.

Water temperature is a crucial environmental factor that influences reproductive function of abalone. Broodstock conditioning exposed to effective accumulative temperature (EAT) is a common practice in abalone hatcheries. To understand the molecular mechanism underlying the regulation of gonadal maturation and reproduction of Haliotis discus hannai exposed to EAT and induced spawning period, changes in expression of neuroendocrine genes encoding two gonadotropin releasing hormone (Hdh-GnRH, GnRH-like peptide), GnRH receptor (HdhGnRH-R), serotonin receptor (5-HTHdh) and Hdh-APGWamide in neural ganglia and gonadal tissues were examined. Gonadosomatic index (GSI) was significantly increased with increasing EAT °C-days. Expression levels of Hdh-GnRH, GnRH-like peptide, HdhGnRH-R, 5-HTHdh and Hdh-APGWamide mRNA were significantly increased with increasing EAT °C-days in ganglion (where the gene synthesized) and gonadal tissues. The significant increase in mRNA expression of each examined gene started from EAT 500 to 750°C-days, reached an initial peak at 1000°C-days, suggesting gonadal maturation started from the onset of EAT and slowly continued until 750°C-days, then at 1000°C-days reached to initial peak developmental period. The maturation reached to spawning state at 1000°C-days and peaked at 1500°C-days. Hdh-GnRH showed significantly higher mRNA expression in pleuropedal ganglion and branchial ganglion, whereas GnRH like peptide showed higher expression in cerebral ganglion, and HdhGnRH-R, 5-HTHdh and Hdh-APGWamide showed higher expression in pleuropedal ganglion. All genes were expressed higher at higher EAT °C-days. During induced spawning period, higher mRNA expression of examined genes was observed at the time of spawning; however, a sharp decrease occurred after spawning, suggesting that these genes are involved in spawning activities. Taken together, these results indicate that an increase of EAT °C-days can increase expression of neuroendocrine genes and enhance gonadal maturation. Besides all these genes are involved in the process of spawning induction, and increase of GSI has a positive correlation with the increase of gene expression.

Expression profile of GnRH-like peptide during gonadal sex differentiation in the cephalopod kisslip cuttlefish, Sepia lycidas

Gonadotropin-releasing hormone (GnRH) is one of the most important neuroendocrine regulators for animal reproduction. GnRH-like peptide (GnRH-like) has recently been shown to play a critical reproductive role mainly in gametogenesis or steroidogenesis in the gonads of some molluscs, including cephalopods. However, its involvement in gonadal sex differentiation remains unknown. Here, we show the expression profile of GnRH-like in the brain of the cephalopod kisslip cuttlefish, Sepia lycidas, throughout gonadal sex differentiation, by quantitative real time RT-PCR and immunohistochemistry. We found that GnRH-like could be detected in the brain at a sexually undifferentiated stage, and its expression level significantly increased upon initiation of gonadal sex differentiation. However, no significant difference in GnRH-like expression levels was observed between sexes during gonadal sex differentiation. Additionally, we demonstrated immunoreactivity of GnRH-like in glial cells or immature neurons, which are mainly distributed in the non-reproductive related area of the cephalopod brain, suggesting the immature function of the reproductive endocrine axis during early ontogenesis. Our results demonstrate for the first time, the expression profile of GnRH-like during early ontogenesis in cephalopods.

Invertebrate Gonadotropin-Releasing Hormone Receptor Signaling and Its Relevant Biological Actions.

Gonadotropin-releasing hormones (GnRHs) play pivotal roles in reproduction via the hypothalamus-pituitary-gonad axis (HPG axis) in vertebrates. GnRHs and their receptors (GnRHRs) are also conserved in invertebrates lacking the HPG axis, indicating that invertebrate GnRHs do not serve as “gonadotropin-releasing factors” but, rather, function as neuropeptides that directly regulate target tissues. All vertebrate and urochordate GnRHs comprise 10 amino acids, whereas amphioxus, echinoderm, and protostome GnRH-like peptides are 11- or 12-residue peptides. Intracellular calcium mobilization is the major second messenger for GnRH signaling in cephalochordates, echinoderms, and protostomes, while urochordate GnRHRs also stimulate cAMP production pathways. Moreover, the ligand-specific modulation of signal transduction via heterodimerization between GnRHR paralogs indicates species-specific evolution in Ciona intestinalis. The characterization of authentic or putative invertebrate GnRHRs in various tissues and their in vitro and in vivo activities indicate that invertebrate GnRHs are responsible for the regulation of both reproductive and nonreproductive functions. In this review, we examine our current understanding of and perspectives on the primary sequences, tissue distribution of mRNA expression, signal transduction, and biological functions of invertebrate GnRHs and their receptors.

Characterization and expression analysis of a GnRH-like peptide in the Pacific abalone, Haliotis discus hannai

Gonadotropin-releasing hormone (GnRH) is a key neuropeptide of vertebrates, involved in gonadal maturation and primarily regulated by the hypothalamic-pituitary-gonadal axis. The full length GnRH-like cDNA isolated from the cerebral ganglion of Haliotis discus hannai, encodes a deduced protein of 216 amino acids with a theoretical molecular mass of 23.36 kDa and an isoelectric point of 7.72. The GnRH transcript comprises a putative signal peptide, a GnRH dodecapeptide, a proteolytic processing site, and a GnRH associated peptide (GAP). Comparative structural analysis revealed that the cloned sequence shares relatively high homology with other molluscan species and a lower degree of amino acid identity with GnRH of piscine vertebrates. Phylogenetic comparison with other known GnRH genes revealed that the GnRH-like mRNA of H. discus hannai is most closely related to the marine gastropod, H. laevigata. Three-dimensional homology structure of H. discus hannai GnRH and GAP exhibited a helix-loop-helix structure. Quantitative PCR assay demonstrated wide expression of GnRH in all ganglia, among them cerebral ganglion exhibited the highest expression level. Significantly higher expression was observed in cerebral ganglion and gonadal tissues at higher effective accumulative temperature (1000 °C). In situ hybridization showed that the GnRH expressing neurosecretory cells distributed throughout the cortex of the cerebral ganglion. These results suggest that GnRH synthesized from the cerebral ganglia may be involved in gonadal maturation and regulating the secretion of other reproductive hormones.

Identification and Function of GnRH-like Peptide in the Pacific Abalone, Haliotis discus hannai.

Gonadotropin-releasing hormone (GnRH) is an important regulator of reproductive function in various vertebrates and invertebrates. In the present study, we have identified the GnRH-like peptide cDNA and peptide from the cerebral ganglion (CG) of the Pacific abalone, Haliotis discus hannai. Pacific abalone GnRH-like peptide (hdhGnRH-like peptide) cDNA encodes precursor, which possesses the typical organization of the known mollusk GnRH-like peptide precursors, including a hydrophobic signal peptide, GnRH-like peptide, and a cleavage site followed by a GAP-like peptide region. Three hdhGnRH-like peptides, pQNYHFSNGWHAamide (hdhGnRH-11amide), pQNYHFSNGWHA (hdhGnRH-11OH), and pQNYHFSNGWHAG (hdhGnRH-12OH), were determined from the acid/acetone extract of the CG by mass spectrometry (LC-MS/MS) analysis. The hdhGnRH-like peptide mRNA expression was detected not only in the CG but also in gonads, and hdhGnRH-11amide was also detected in the extract of gonads. The mRNA expression of hdhGnRH-like peptide in the CG was lower in spawned males than in non-spawned animals, while no change in hdhGnRH-like peptide mRNA expression was shown in both ovulated and non-ovulated abalone. The hdhGnRH-11amide induces spawning and ovulation of both mature males and females in a concentration-dependent fashion following intramuscular injection. These results indicate that three hdhGnRH-like peptides are yielded from a single hdhGnRH-like peptide precursor, and that at least hdhGnRH-11amide is involved in the control of reproduction of the Pacific abalone.

Invertebrate Gonadotropin-Releasing Hormone-Related Peptides and Their Receptors: An Update.

Gonadotropin-releasing hormones (GnRHs) play pivotal roles in reproductive functions via the hypothalamus, pituitary, and gonad axis, namely, HPG axis in vertebrates. GnRHs and their receptors (GnRHRs) are likely to be conserved in invertebrate deuterostomes and lophotrochozoans. All vertebrate and urochordate GnRHs are composed of 10 amino acids, whereas protostome, echinoderm, and amphioxus GnRH-like peptides are 11- or 12-residue peptide containing two amino acids after an N-terminal pyro-Glu. In urochordates, Halocynthia roretzi GnRH gene encodes two GnRH peptide sequences, whereas two GnRH genes encode three different GnRH peptides in Ciona intestinalis. These findings indicate the species-specific diversification of GnRHs. Intriguingly, the major signaling pathway for GnRHRs is intracellular Ca2+ mobilization in chordates, echinoderms, and protostomes, whereas Ciona GnRHRs (Ci-GnRHRs) are endowed with multiple GnRHergic cAMP production pathways in a ligand-selective manner. Moreover, the ligand-specific modulation of signal transduction via heterodimerization among Ci-GnRHR paralogs suggests the species-specific development of fine-tuning of gonadal functions in ascidians. Echinoderm GnRH-like peptides show high sequence differences compared to those of protostome counterparts, leading to the difficulty in classification of peptides and receptors. These findings also show both the diversity and conservation of GnRH signaling systems in invertebrates. The lack of the HPG axis in invertebrates indicates that biological functions of GnRHs are not release of gonadotropins in current invertebrates and common ancestors of vertebrates and invertebrates. To date, authentic or putative GnRHRs have been characterized from various echinoderms and protostomes as well as chordates and the mRNAs have been found to be distributed not only reproductive organs but also other tissues. Collectively, these findings further support the notion that invertebrate GnRHs have biological roles other than the regulation of reproductive functions. Moreover, recent molecular phylogenetic analysis suggests that adipokinetic hormone (AKH), corazonin (CRZ), and AKH/CRZ-related peptide (ACP) belong to the GnRH superfamily but has led to the different classifications of these peptides and receptors using different datasets including the number of sequences and structural domains. In this review, we provide current knowledge of, and perspectives in, molecular basis and evolutionary aspects of the GnRH, AKH, CRZ, and ACP.

Characterization and spatiotemporal expression of gonadotropin-releasing hormone in the Pacific abalone, Haliotis discus hannai

Gonadotropin-releasing hormone (GnRH) is a key neuropeptide regulating reproduction in humans and other vertebrates. Recently, GnRH-like cDNAs and peptides were reported in marine mollusks, implying that GnRH-mediated reproduction is an ancient neuroendocrine system that arose prior to the divergence of protostomes and deuterostomes. Here, we evaluated the reproductive control system mediated by GnRH in the Pacific abalone Haliotis discus hannai. We cloned a prepro-GnRH cDNA (Hdh-GnRH) from the pleural-pedal ganglion (PPG) in H. discus hannai, and analyzed its spatiotemporal gene expression pattern. The open reading frame of Hdh-GnRH encodes a protein of 101 amino acids, consisting of a signal peptide, a GnRH dodecapeptide, a cleavage site, and a GnRH-associated peptide. This structure and sequence are highly similar to GnRH-like peptides reported for mollusks and other invertebrates. Quantitative polymerase chain reaction demonstrated that Hdh-GnRH mRNA was more strongly expressed in the ganglions (PPG and cerebral ganglion [CG]) than in other tissues (gonads, gills, intestine, hemocytes, muscle, and mantle) in both sexes. In females, the expression levels of Hdh-GnRH mRNA in the PPG and branchial ganglion (BG) were significantly higher at the ripe and partial spent stages than at the early and late active stages. In males, Hdh-GnRH mRNA levels in the BG showed a significant increase in the partial spent stage. Unexpectedly, Hdh-GnRH levels in the CG were not significantly different among the examined stages in both sexes. These results suggest that Hdh-GnRH mRNA expression profiles in the BG and possibly the PPG are tightly correlated with abalone reproductive activities.

Adipokinetic hormones and their G protein-coupled receptors emerged in Lophotrochozoa

Most multicellular animals belong to two evolutionary lineages, the Proto– and Deuterostomia, which diverged 640–760 million years (MYR) ago. Neuropeptide signaling is abundant in animals belonging to both lineages, but it is often unclear whether there exist evolutionary relationships between the neuropeptide systems used by proto- or deuterostomes. An exception, however, are members of the gonadotropin-releasing hormone (GnRH) receptor superfamily, which occur in both evolutionary lineages, where GnRHs are the ligands in Deuterostomia and GnRH-like peptides, adipokinetic hormone (AKH), corazonin, and AKH/corazonin-related peptide (ACP) are the ligands in Protostomia. AKH is a well-studied insect neuropeptide that mobilizes lipids and carbohydrates from the insect fat body during flight. In our present paper, we show that AKH is not only widespread in insects, but also in other Ecdysozoa and in Lophotrochozoa. Furthermore, we have cloned and deorphanized two G protein-coupled receptors (GPCRs) from the oyster Crassostrea gigas (Mollusca) that are activated by low nanomolar concentrations of oyster AKH (pQVSFSTNWGSamide). Our discovery of functional AKH receptors in molluscs is especially significant, because it traces the emergence of AKH signaling back to about 550 MYR ago and brings us closer to a more complete understanding of the evolutionary origins of the GnRH receptor superfamily.

Humoral immune responses against gonadotropin releasing hormone elicited by immunization with phage-peptide constructs obtained via phage display

Phage display is based on genetic engineering of phage coat proteins resulting in fusion peptides displayed on the surface of phage particles. The technology is widely used for generation of phages with novel characteristics for numerous applications in biomedicine and far beyond. The focus of this study was on development of phage-peptide constructs that stimulate production of antibodies against gonadotropin releasing hormone (GnRH). Phage-peptide constructs that elicit production of neutralizing GnRH antibodies can be used for anti-fertility and anti-cancer applications. Phage-GnRH constructs were generated via selection from a phage display library using several types of GnRH antibodies as selection targets. Such phage constructs were characterized for sequence similarities to GnRH peptide and frequency of their occurrence in the selection rounds. Five of the constructs with suitable characteristics were tested in mice as a single dose 5×1011 virions (vir) vaccine and were found to be able to stimulate production of GnRH-specific antibodies, but not to suppress testosterone (indirect indicator of GnRH antibody neutralizing properties). Next, one of the constructs was tested at a higher dose of 2×1012 vir per mouse in combination with a poly(lactide-co-glycolide) (PLGA)-based adjuvant. This resulted in multifold increase in GnRH antibody production and significant reduction of serum testosterone, indicating that antibodies produced in response to the phage-GnRH immunization possess neutralizing properties. To achieve optimal immune responses for desired applications, phage-GnRH constructs can be modified with respect to flanking sequences of GnRH-like peptides displayed on phage. Anticipated therapeutic effects also might be attained using optimized phage doses, a combination of several constructs in a single treatment, or application of adjuvants and advanced phage delivery systems.

Oct-GnRH, the first protostomian gonadotropin-releasing hormone-like peptide and a critical mini-review of the presence of vertebrate sex steroids in molluscs

In protostome and deuterosome invertebrates, neurosecretory cells play major roles in the endocrine system. The optic glands of cephalopods are indicators of sexual maturation. In mature octopuses, optic glands enlarge and secrete a gonadotropic hormone. A peptide with structural features similar to that of vertebrate gonadotropin-releasing hormone (GnRH) was isolated from the octopus, Octopus vulgaris, and was named oct-GnRH. The discovery of oct-GnRH has triggered structural determinations and predictions of other mollusc GnRH-like peptides in biochemical and in silico studies. Interestingly, cephalopods studied so far are characterized by a single molecular form of oct-GnRH with a C-terminal -Pro-Gly-NH2 sequence, which is critical for gonadotropin-releasing activity in vertebrates. Other molluscan GnRH-like peptides lack the C-terminal -Pro-Gly-NH2 sequence but have -X-NH2 or -Pro-Gly although all protostome GnRH-like peptides have yet to be sequenced.In marine molluscs, relationships between GnRH-like peptides and sex steroids have been studied to verify the hypothesis that molluscs have vertebrate-type sex steroid system. However, it is currently questionable whether such sex steroids are present and whether they play endogenous roles in the reproductive system of molluscs. Because molluscs uptake and store steroids from the environment and fishes release sex steroids into the external environment, it is impossible to rule out the contamination of vertebrate sex steroids in molluscs. The function of key enzymes of steroidogenesis within molluscs remains unclear. Thus, evidence to deny the existence of the vertebrate-type sex steroid system in molluscs has been accumulated. The elucidation of substances, which regulate the maturation and maintenance of gonads and other reproductive functions in molluscs will require rigorous and progressive scientific study.

Characterization of GnRH-like peptides from the nerve ganglia of Yesso scallop, Patinopecten yessoensis

There is yet no firm experimental evidence that the evolutionary ancient gonadotropin-releasing hormone GnRH (i.e., GnRH1) also acts in invertebrate gametogenesis. The objective of this paper is to characterize candidate invGnRH peptides of Yesso scallop Patinopecten yessoensis (i.e., peptide identification, immunohistochemical localization, and immunoquantification) in order to reveal their bioactive form in bivalves. Using mass spectrometry (MS), we identified two invGnRH (py-GnRH) peptides from the scallop nerve ganglia: a precursor form of py-GnRH peptide (a non-amidated dodecapeptide; py-GnRH12aa-OH) and a mature py-GnRH peptide (an amidated undecapeptide; py-GnRH11aa-NH2). Immunohistochemical staining allowed the localization of both py-GnRH peptides in the neuronal cell bodies and fibers of the cerebral and pedal ganglia (CPG) and the visceral ganglion (VG). We found that the peptides showed a dimorphic distribution pattern. Notably, the broad distribution of mature py-GnRH in neuronal fibers elongating to peripheral organs suggests that it is multi-functional. Time-resolved fluorescent immunoassays (TR-FIA) enabled the quantification of each py-GnRH form in the single CPG or VG tissue obtained from one individual. In addition, we observed greater abundance of mature py-GnRH in VG compared with its level in CPG, suggesting that VG is the main producing organ of mature py-GnRH peptide and that py-GnRH may play a central regulatory role in neurons of scallops. Our study provides evidence, for the first time, for the presence of precursor and mature forms of invGnRH peptides in the nerve ganglia of an invertebrate.

Molecular cloning, characterization, and expression analysis of a gonadotropin-releasing hormone-like cDNA in the clam, Ruditapes philippinarum.

Gonadotropin-releasing hormone (GnRH) is a key neuropeptide regulating reproduction in vertebrates. In the present study, we have cloned and identified the cDNA sequence of the prepro-GnRH (rp-GnRH) in the Manila clam Ruditapes philippinarum. The open reading frame encodes a protein of 94 amino acids, which consists of a signal peptide, a GnRH dodecapeptide, a cleavage site, and a GnRH-associated peptide. These deduced peptides were highly homologous to that reported for other molluscs. We used temperature control to promote gonadal development of the clams. The mRNA expression of rp-GnRH in the visceral ganglia from clams at different reproductive stages was determined by quantitative RT-PCR. The levels of steroids progesterone (P), testosterone (T), and estradiol-17β (E2) in the hemolymph of the corresponding clam were measured by ELISA. The rp-GnRH mRNA was highly expressed at the gonadal early development stage. Concentrations of P, T, and E2 also increased at the early development stage in both sexes. Positive significant correlations between rp-GnRH expression and P content as well as between rp-GnRH expression and T content were observed throughout the gonadal maturation. The results from this study may help to better understand the physiological functions of the native forms of GnRH-like peptides and the actions of GnRH on sex steroids release in bivalve molluscs.

GnRH receptors and peptides: Skating backward

Gonadotropin-releasing hormone (GnRH) and its receptor are essential for reproduction in vertebrates. Although there are three major types of GnRH peptides and two major types of receptors in vertebrates, the pattern of distribution is unusual. Evidence is presented from genome mining that type I GnRHRs are not restricted to mammals, but can be found in the lobe-finned and cartilaginous fishes. This implies that this tail-less GnRH receptor emerged early in vertebrate evolution, followed by several independent losses in different lineages. Also, we have identified representatives from the three major GnRH peptide types (mammalian GnRH1, vertebrate GnRH2 and dogfish GnRH3) in a single cartilaginous fish, the little skate. Skate and coelacanth are the only examples of animals with both type I and II GnRH receptors and all three peptide types, suggesting this was the ancestral condition in vertebrates. Our analysis of receptor synteny in combination with phylogeny suggests that there were three GnRH receptor types present before the two rounds of whole genome duplication in early vertebrates. To further understand the origin of the GnRH peptide-receptor system, the relationship of vertebrate and invertebrate homologs was examined. Our evidence supports the hypothesis of a GnRH superfamily with a common ancestor for the vertebrate GnRHs, invertebrate (inv)GnRHs, corazonins and adipokinetic hormones. The invertebrate deuterostomes (echinoderms, hemichordates and amphioxus) have derived GnRH-like peptides, although one amphioxus GnRH with a syntenic relationship to human GnRHs has been shown to be functional. Phylogenetic analysis suggests that gene duplications in the ancestral bilaterian produced two receptor types, one of which became adipokinetic hormone receptor/GnRHR and the other corazonin receptor/invGnRHR. It appears that the ancestral deuterostome had both a GnRHR and invGnRHR, and this is still the case in amphioxus. During the transition to vertebrates both the invertebrate-type peptide and receptor were lost, leaving only the vertebrate-type system that presently exists.

Immunohistochemical detection of GnRH-like peptides in the neural ganglia and testis of Haliotis asinina

Gonadotropin releasing hormone (GnRH) is a peptide that is conserved in both vertebrate and invertebrate species. In this study, we have demonstrated the distribution pattern of two isoforms of GnRH-like peptides in the neural ganglia and testis of reproductively mature male abalone, H. asinina, by immunohistochemistry and whole mount immunofluorescence. We found octopus (oct) GnRH and tunicate-I (t) GnRH-I immunoreactivities (ir) in type 1 neurosecretory cells (NS1) and they were expressed mostly within the ventral horn of the cerebral ganglion, whereas in pleuropedal ganglia they were localized primarily in the dorsal horn. Furthermore, tGnRH-I-ir were strongly detected in fibers at the caudal part of the cerebral ganglia and both ventral and dorsal horns of the pleuropedal ganglia. In the testis, only octGnRH-ir was found primarily in the granulated cell and central capillaries within the trabeculae. These results suggest that multiple GnRH-like peptides are present in the neural ganglia which could be the principal source of their production, whereas GnRH may also be synthesized locally in the testis and act as the paracrine control of testicular maturation.

At the Transition from Invertebrates to Vertebrates, a Novel GnRH-Like Peptide Emerges in Amphioxus

Gonadotropin-releasing hormone (GnRH) is a critical reproductive regulator in vertebrates. Homologous peptides are also found in invertebrates, with a variety of characterized functions. In the amphioxus, an invertebrate that provides the best model for the transition to vertebrates, four GnRH receptors (GnRHRs) were previously described, but their native ligands were not identified. Using a more sensitive search methodology with hidden Markov models, we identified the first GnRH-like peptide confirmed in the amphioxus Branchiostoma floridae. This peptide specifically activated one of the four GnRHRs. Although the primary structure of this peptide was divergent from any previously isolated GnRH peptide, the minimal conserved residues found in all other GnRH superfamily members were retained. The peptide was immunolocalized in proximity of the central canal of the anterior nerve cord, a region where other neuropeptides and receptors have been found. Additionally, the amphioxus GnRH-like gene was positioned in a locus surrounded by syntenic homologs of the human GnRH paralogon. The amphioxus GnRH-like peptide, with its distinct primary structure, activated a receptor with equal potency to multiple ligands that span the GnRH superfamily.

Primary structure of a novel gonadotropin-releasing hormone (GnRH) in the ovary of red swamp crayfish Procambarus clarkii

Gonadotropin-releasing hormone (GnRH) is a neuropeptide known to regulate and maintain reproductive functions and conserved in both vertebrate and invertebrate species. In this study, a GnRH peptide was isolated from the ovaries of red swamp crayfish, Procambarus clarkii, by using chromatographic and immunological methods. The form was proven to be a novel form of GnRH by determining its primary structure by chemical sequencing and mass spectrometry, and was designated as P. clarkii GnRH (pcGnRH). The primary structure of pcGnRH is pQSYHFSLGWKP-NH2, which is different from the known forms of the vertebrate and invertebrate GnRH family. Synthetic pcGnRH was biologically active, as it showed stimulative effects on ovarian maturation to crayfish. Our data supported the hypothesis that in crustacean there is GnRH synthesized locally in the ovary and playing an important role in the regulation of oocyte development. This is the first report on the isolation and characterization of GnRH-like peptide in crustacean.

Histological organization of the central nervous system and distribution of a gonadotropin-releasing hormone-like peptide in the blue crab, Portunus pelagicus

We present a detailed histological description of the central nervous system (CNS: brain, subesophageal ganglion, thoracic ganglia, abdominal ganglia) of the blue crab, Portunus pelagicus. Because the presence of gonadotropin-releasing hormone (GnRH) in crustaceans has been disputed, we examine the presence and localization of a GnRH-like peptide in the CNS of the blue crab by using antibodies against lamprey GnRH (lGnRH)-III, octopus GnRH (octGnRH) and tunicate GnRH (tGnRH)-I. These antibodies showed no cross-reactivity with red-pigment-concentrating hormone, adipokinetic hormone, or corazonin. In the brain, strong lGnRH-III immunoreactivity (-ir) was detected in small (7-17 μm diameter) neurons of clusters 8, 9 and 10, in medium-sized (21-36 μm diameter) neurons of clusters 6, 7 and 11 and in the anterior and posterior median protocerebral neuropils, olfactory neuropil, median and lateral antenna I neuropils, tegumentary neuropil and antenna II neuropil. In the subesophageal ganglion, lGnRH-III-ir was detected in medium-sized neurons and in the subesophageal neuropil. In the thoracic and abdominal ganglia, lGnRH-III-ir was detected in medium-sized and small neurons and in the neuropils. OctGnRH-ir was observed in neurons of the same clusters with moderate staining, particularly in the deutocerebrum, whereas tGnRH-I-ir was only detected in medium-sized neurons of cluster 11 in the brain. Thus, anti-lGnRH-III shows greater immunoreactivity in the crab CNS than anti-octGnRH and anti-tGnRH-I. Moreover, our functional bioassay demonstrates that only lGnRH-III has significant stimulatory effects on ovarian growth and maturation. We therefore conclude that, although the true identity of the crab GnRH eludes us, crabs possess a putative GnRH hormone similar to lGnRH-III. The identification and characterization of this molecule is part of our ongoing research.

Molluscan GnRH associated with reproduction

Gonadotropin-releasing hormone (GnRH) is a neuropeptide that has an essential role in the neural regulation of vertebrate reproduction. Over the past two decades there has been increasing evidence strongly indicating that members of the GnRH superfamily, which includes GnRH, adipokinetic hormone (AKH), corazonin (Crz) and adipokinetic hormone/corazonin-related peptides (ACP), are almost ubiquitous amongst bilateral animals. Moreover GnRH possibly has origins in even more ancient, non-bilateral ancestors. Current knowledge about molluscan GnRH has been accumulated regarding immunological identification, physiological function and sequence analysis. In the present review we summarized a current status of molluscan GnRH research and focus on its role in the reproduction of the molluscs. In cephalopods and gastropods the presence of a GnRH-like peptide was detected with heterologous antibodies and the identified GnRH was suggested to be involved with behavior and reproduction. Reproductive roles for GnRH have been confirmed in both bivalve and cephalopod molluscs. These findings will provide useful insights into the evolution of reproductive endocrinology.