Relaxin-like Gonad-stimulating Peptide Research Articles

Total Chemical Synthesis of Aggregation-Prone Disulfide-Rich Starfish Peptides.

A relaxin-like gonad-stimulating peptide (RGP), Aso-RGP, featuring six cysteine residues, was identified in the Crown-of-Thorns Starfish (COTS, Acanthaster cf. solaris) and initially produced through recombinant yeast expression. This method yielded a single-chain peptide with an uncleaved C-peptide (His Tag) and suboptimal purity. Our objective was to chemically synthesize Aso-RGP in its mature form, comprising two chains (A and B) and three disulfide bridges, omitting the C-peptide. Furthermore, we aimed to synthesize a newly identified relaxin-like peptide, Aso-RLP2, from COTS, which had not been previously synthesized. This paper reports the first total chemical synthesis of Aso-RGP and Aso-RLP2. Aso-RGP synthesis proceeded without major issues, whereas the A-chain of Aso-RLP2, in its reduced and unfolded state with two free thiols, presented considerable challenges. These were initially marked by "messy" RP-HPLC profiles, typically indicative of synthesis failure. Surprisingly, oxidizing the A-chain significantly improved the RP-HPLC profile, revealing the main issue was not synthesis failure but the peptide's aggregation tendency, which initially obscured analysis. This discovery highlights the critical need to account for aggregation in peptide synthesis and analysis. Ultimately, our efforts led to the successful synthesis of both peptides with purities exceeding 95 %.

Gonadotropic activity of a second relaxin-type peptide in starfish

In starfish, a relaxin-like gonad-stimulating peptide (RGP) acts as a gonadotropin that triggers gamete maturation and spawning. In common with other relaxin/insulin superfamily peptides, RGP consists of an A- and a B-chain, with cross-linkages mediated by one intra- and two inter-chain disulfide bonds. In this study, a second relaxin-like peptide (RLP2) was identified in starfish species belonging to the orders Valvatida, Paxillosida, and Forcipulatida. Like RGP, RLP2 precursors comprise a signal peptide and a C-peptide in addition to the A- and B-chains. However, a unique cysteine motif [CC-(3X)-C-(10X)-C] is present in the A-chain of RLP2, which contrasts with the cysteine motif in other members of the relaxin/insulin superfamily [CC-(3X)-C-(8X)-C]. Importantly, in vitro pharmacological tests revealed that Patiria pectinifera RLP2 (Ppe-RLP2) and Asterias rubens RLP2 (Aru-RLP2) trigger shedding of mature eggs from ovaries of P. pectinifera and A. rubens, respectively. Furthermore, the potencies of Ppe-RLP2 and Aru-RLP2 as gonadotropic peptides were similar to those of Ppe-RGP and Aru-RGP, respectively, and the effect of RLP2 exhibited partial species-specificity. These findings indicate that two relaxin-type peptides regulate spawning in starfish and therefore we propose that RGP and RLP2 are renamed RGP1 and RGP2, respectively.

Localization of relaxin-like gonad-stimulating peptide expression in starfish reveals the gonoducts as a source for its role as a regulator of spawning.

Oocyte maturation and gamete release (spawning) in starfish are triggered by relaxin-like gonad-stimulating peptide (RGP), a neuropeptide that was first isolated from the radial nerve cords of these animals. Hitherto, it has generally been assumed that the radial nerve cords are the source of RGP that triggers spawning physiologically. To investigate other sources of RGP, here we report the first comprehensive anatomical analysis of its expression, using both in situ hybridization and immunohistochemistry to map RGP precursor transcripts and RGP, respectively, in the starfish Asterias rubens. Cells expressing RGP precursor transcripts were revealed in the ectoneural epithelium of the radial nerve cords and circumoral nerve ring, arm tips, tube feet, cardiac stomach, pyloric stomach, and, most notably, gonoducts. Using specific antibodies to A. rubens RGP, immunostaining was revealed in cells and/or fibers in the ectoneural region of the radial nerve cords and circumoral nerve ring, tube feet, terminal tentacle and other arm tip-associated structures, body wall, peristomial membrane, esophagus, cardiac stomach, pyloric stomach, pyloric caeca, and gonoducts. Our discovery that RGP is expressed in the gonoducts of A. rubens proximal to its gonadotropic site of action in the gonads is important because it provides a new perspective on how RGP may act as a gonadotropin in starfish. Thus, we hypothesize that it is the release of RGP from the gonoducts that triggers gamete maturation and spawning in starfish, while RGP produced in other parts of the body may regulate other physiological/behavioral processes.

Relaxin-like Gonad-Stimulating Peptides in Asteroidea.

Starfish relaxin-like gonad-stimulating peptide (RGP) is the first identified peptide hormone with gonadotropin-like activity in invertebrates. RGP is a heterodimeric peptide, comprising A and B chains with disulfide cross-linkages. Although RGP had been named a gonad-stimulating substance (GSS), the purified peptide is a member of relaxin-type peptide family. Thus, GSS was renamed as RGP. The cDNA of RGP encodes not only the A and B chains, but also signal and C-peptides. After the rgp gene is translated as a precursor, mature RGP is produced by eliminating the signal and C-peptides. Hitherto, twenty-four RGP orthologs have been identified or predicted from starfish in the orders Valvatida, Forcipulatida, Paxillosida, Spinulosida, and Velatida. The molecular evolution of the RGP family is in good accordance with the phylogenetic taxonomy in Asteroidea. Recently, another relaxin-like peptide with gonadotropin-like activity, RLP2, was found in starfish. RGP is mainly present in the radial nerve cords and circumoral nerve rings, but also in the arm tips, the gonoducts, and the coelomocytes. RGP acts on ovarian follicle cells and testicular interstitial cells to induce the production of 1-methyladenine (1-MeAde), a starfish maturation-inducing hormone. RGP-induced 1-MeAde production is accompanied by an increase in intracellular cyclic AMP levels. This suggests that the receptor for RGP (RGPR) is a G protein-coupled receptor (GPCR). Two types of GPCRs, RGPR1 and RGPR2, have been postulated as candidates. Furthermore, 1-MeAde produced by RGP not only induces oocyte maturation, but also induces gamete shedding, possibly by stimulating the secretion of acetylcholine in the ovaries and testes. Thus, RGP plays an important role in starfish reproduction, but its secretion mechanism is still unknown. It has also been revealed that RGP is found in the peripheral adhesive papillae of the brachiolaria arms. However, gonads are not developed in the larvae before metamorphosis. It may be possible to discover new physiological functions of RGP other than gonadotropin-like activity.

The C-terminally amidated relaxin-like gonad-stimulating peptide in the starfish Astropecten scoparius

A relaxin-like gonad-stimulating peptide (RGP) in starfish was the first identified invertebrate gonadotropin, consisting of A- and B-chain. Recently, an RGP ortholog (Asc-RGP) from Astropecten scoparius in the order Paxillosida was found to harbor an amidation signal (Gly-Arg) at the C-terminus of the B-chain (Mita et al., 2020a). Two cleavage sites were also predicted within the signal peptide of the Asc-RGP precursor. Thus, four kinds of analogs (Asc-RGP-NH2(S), Asc-RGP-GR(S), Asc-RGP- NH2(L), Asc-RGP-GR(L) were hypothesized as natural Asc-RGPs. To identify the natural Asc-RGP, an extract of radial nerve cords from A. scoparius was analyzed using reverse-phase high-performance liquid chromatography and MALDI-TOF-mass spectrometry. The molecular weight of Asc-RGP was 4585.3, and those of A- and B-chains were 2511.8 and 2079.8, respectively. This strongly suggests that natural RGP in A. scoparius is Asc-RGP-NH2(S). Asc-RGP-NH2(S) stimulated 1-methyladenine and cyclic AMP production in isolated ovarian follicle cells of A. scoparius. On the other hand, the concentrations of four synthetic Asc-RGP analogs required for the induction of spawning in 50% of ovarian fragments were almost the same. The size and C-terminal amidation of the B-chain might not be important for spawning-inducing activity. C-terminally amidated RGPs in the B-chain were also observed in other species of starfish belonging to the order Paxillosida, particularly the family Astropectinidae, but not the family Luidiidae.

A Relaxin-Like Gonad-Stimulating Peptide Appears in the Early Development of the Starfish Patiria pectinifera.

Relaxin-like gonad-stimulating peptide (RGP) is a hormone with gonadotropin-like activity in starfish. This study revealed that spawning inducing activity was detected in an extract of brachiolaria larvae of Patiria pectinifera. Spawning inducing activity in the extract was due to P. pectinifera RGP (PpeRGP), not 1-methyladenine. The expression of PpeRGP mRNA was also found in brachiolaria. Immunohistochemical observation with specific antibodies for PpeRGP showed that PpeRGP was distributed in the peripheral adhesive papilla of the brachiolaria arms. In contrast, PpeRGP was not detected in the adult rudiment or ciliary band regions, which are present in the neural system. These findings strongly suggest that RGP exists in the larvae before metamorphosis. Because gonads are not developed in starfish larvae, it seems likely that RGP plays another role other than gonadotropic action in the early development of starfish.

Sex-specific expression of pheromones and other signals in gravid starfish

BackgroundMany echinoderms form seasonal aggregations prior to spawning. In some fecund species, a spawning event can lead to population outbreaks with detrimental ecosystem impacts. For instance, outbreaks of crown-of-thorns starfish (COTS), a corallivore, can destroy coral reefs. Here, we examine the gene expression in gravid male and female COTS prior to spawning in the wild, to identify genome-encoded factors that may regulate aggregation and spawning. This study is informed by a previously identified exoproteome that attracts conspecifics. To capture the natural gene expression profiles, we isolated RNAs from gravid female and male COTS immediately after they were removed from the Great Barrier Reef. ResultsSexually dimorphic gene expression is present in all seven somatic tissues and organs that we surveyed and in the gonads. Approximately 40% of the exoproteome transcripts are differentially expressed between sexes. Males uniquely upregulate an additional 68 secreted factors in their testes. A suite of neuropeptides in sensory organs, coelomocytes and gonads is differentially expressed between sexes, including the relaxin-like gonad-stimulating peptide and gonadotropin-releasing hormones. Female sensory tentacles—chemosensory organs at the distal tips of the starfish arms—uniquely upregulate diverse receptors and signalling molecules, including chemosensory G-protein-coupled receptors and several neuropeptides, including kisspeptin, SALMFamide and orexin.ConclusionsAnalysis of 103 tissue/organ transcriptomes from 13 wild COTS has revealed genes that are consistently differentially expressed between gravid females and males and that all tissues surveyed are sexually dimorphic at the molecular level. This finding is consistent with female and male COTS using sex-specific pheromones to regulate reproductive aggregations and synchronised spawning events. These pheromones appear to be received primarily by the sensory tentacles, which express a range of receptors and signalling molecules in a sex-specific manner. Furthermore, coelomocytes and gonads differentially express signalling and regulatory factors that control gametogenesis and spawning in other echinoderms.

Characterization and localization of relaxin-like gonad-stimulating peptide in the crown-of-thorns starfish, Acanthaster cf. solaris

In starfish, a relaxin-like gonad-stimulating peptide (RGP) is the gonadotropin responsible for final gamete maturation. RGP comprises two different peptides, A- and B-chains with two interchain and one intrachain disulfide bonds. The existence of two isomers of RGP in the crown-of-thorns starfish, Acanthaster planci, has been reported previously, but it was recently shown that A. planci represents a species complex with four different species. Here we elucidated the authentic sequence of the Pacific species, Acanthaster cf. solaris, RGP (Aso-RGP). The Aso-RGP precursor encoded by a 354 base pair open reading frame was composed of 117 amino acids (aa). The amino acid identity of Aso-RGP to Patiria pectinifera RGP (Ppe-RGP) and Asterias amurensis RGP (Aam-RGP) was 74% and 60%, respectively. Synthetic Aso-RGP induced spawning of ovarian fragments from A. cf. solaris. Ppe-RGP and Aam-RGP also induced spawning by A. cf. solaris ovaries. In contrast, Ppe-RGP and Aso-RGP induced spawning by P. pectinifera ovaries, but Aam-RGP was inactive. Notably, anti-Ppe-RGP antibodies recognized Aso-RGP as well as Ppe-RGP. Localization of Aso-RGP was observed immunohistochemically using anti-Ppe-RGP antibodies, showing that Aso-RGP was mainly present in the radial nerve cords of A. cf. solaris. Aso-RGP was distributed not only in the epithelium of the ectoneural region but also in the neuropile of the ectoneural region. These results suggest that Aso-RGP is synthesized in the epithelium of the ectoneural region, then transferred to fibers in the neuropile of the ectoneural region in radial nerve cords.

Spawning induction of the high-value white teatfish sea cucumber, Holothuria fuscogilva, using recombinant relaxin-like gonad stimulating peptide (RGP)

We have previously shown in Holothuria leucospilota the maturation-inducing role of the neurohormone relaxin-like gonad-stimulating peptide (RGP). Here, we report the induction of oocyte maturation and spawning in another holothuroid, the white teatfish, H. fuscogilva, with a species-specific recombinant RGP produced in the yeast expression system. The recombinant H. fuscogilva RGP (rwt_RGP) was bioactive in vitro, as demonstrated in germinal vesicle breakdown (GVBD) assays, and in vivo, stimulating spawning in H. fuscogilva at three doses tested (50, 100 and 150 μg/kg). The spawned gametes were viable, and here we report their development until the early juvenile stage. The rwt_RGP was also bioactive in H. scabra and in H. leucospilota. We also investigated the bioactivity of two forms of synthetic RGP, whose sequences were synthesised according to that of the H. fuscogilva. The peptides have linked B- and A-chains with one form having an amidation at the carboxy terminal of the B-chain. We observed bioactivity of both synthetic peptides, as evidenced by induction of GVBD in in vitro assays and by induction of spawning in fully grown H. leucospilota. Altogether, our results provide further evidence of the gonad-stimulating role of RGP in echinoderms. We have shown that our recombinant and synthetic RGPs are applicable among the Holothuria species, providing a reliable breeding method for the highly valuable cultured species of this class. Our findings have significance both for the survival of threatened sea cucumbers and for their sustainable aquaculture.

A specific and sensitive enzyme-linked immunosorbent assay for measurement of relaxin-like gonad-stimulating peptide in the starfish Asterias rubens

A relaxin-like gonad-stimulating peptide (RGP) acts as a gonadotropic hormone in starfish. In this study, antibodies to Asterias rubens RGP (AruRGP) were used for the development of a specific and sensitive enzyme-linked immunosorbent assay (ELISA) to measure AruRGP. Biotin-conjugated RGP (biotin-AruRGP) that binds to peroxidase-conjugated streptavidin was synthesized chemically so that it could be specifically detected using 3, 3′, 5, 5′-tetramethylbenzidine (TMB)/hydrogen peroxide as a substrate. Similar to AruRGP, biotin-AruRGP bound to AruRGP antibodies. In binding experiments with biotin-AruRGP using wells coated with AruRGP antibodies, a displacement curve was obtained using serial dilutions of AruRGP. Using this ELISA system, AruRGP could be measured in the range 0.01–5.0 pmol per 50 µl test solution. Furthermore, 0.22 ± 0.03 and 0.20 ± 0.04 pmol AruRGP/mg wet weight tissue were detected in the radial nerve cords and circumoral nerve-rings of A. rubens, respectively. Smaller amounts of AruRGP were detected in tube feet, pyloric stomach and cardiac stomach but AruRGP was not detected in pyloric caeca, ovaries and testes. Analysis of the specificity of the AruRGP antibodies revealed that the A- and B-chains of AruRGP, Patiria pectinifera RGP, Aphelasterias japonica RGP, and human relaxin exhibit little or no cross-reactivity in the ELISA. We conclude, therefore, that we have successfully generated an ELISA system that is highly sensitive and specific for detection of AruRGP.

A relaxin-like gonad-stimulating peptide identified from the starfish Astropecten scoparius.

A relaxin-like gonad-stimulating peptide (RGP) in starfish was the first identified invertebrate gonadotropin responsible for final gamete maturation. An RGP ortholog was newly identified from Astropecten scoparius of the order Paxillosida. The A. scoparius RGP (AscRGP) precursor is encoded by a 354 base pair open reading frame and is a 118 amino acid (aa) protein consisting of a signal peptide (26 aa), B-chain (21 aa), C-peptide (47 aa), and A-chain (24 aa). There are three putative processing sites (Lys-Arg) between the B-chain and C-peptide, between the C-peptide and A-chain, and within the C-peptide. This structural organization revealed that the mature AscRGP is composed of A- and B-chains with two interchain disulfide bonds and one intrachain disulfide bond. The C-terminal residues of the B-chain are Gln-Gly-Arg, which is a potential substrate for formation of an amidated C-terminal Gln residue. Non-amidated (AscRGP-GR) and amidated (AscRGP-NH2 ) peptides were chemically synthesized and their effect on gamete shedding activity was examined using A. scoparius ovaries. Both AscRGP-GR and AscRGP-NH2 induced oocyte maturation and ovulation in similar dose-dependent manners. This is the first report on a C-terminally amidated functional RGP. Collectively, these results suggest that AscRGP-GR and AscRGP-NH2 act as a natural gonadotropic hormone in A. scoparius.

A novel G protein-coupled receptor for starfish gonadotropic hormone, relaxin-like gonad-stimulating peptide.

Gonadotropic hormones play important regulatory roles in reproduction. Relaxin-like gonad-stimulating peptide (RGP) is a gonadotropin-like hormone in starfish. However, a receptor for RGP remains to be identified. Here, we describe the identification of an authentic receptor for RGP (RGPR) in the starfish, Patiria pectinifera. A binding assay using radioiodinated P. pectinifera RGP (PpeRGP) revealed that RGPR was expressed in ovarian follicle cells. A RGPR candidate was identified by homology-searching of transcriptome data of P. pectinifera follicle cells. Based on the contig sequences, a putative 947-amino acid PpeRGPR was cloned from follicle cells. Like the vertebrate relaxin family peptide receptors (RXFP 1 and 2), PpeRGPR was a G protein-coupled receptor that harbored a low-density lipoprotein-receptor class A motif and leucine-rich repeat sequences in the extracellular domain of the N-terminal region. Sf9 cells transfected with Gαq16-fused PpeRGPR activated calcium ion mobilization in response to PpeRGP, but not to RGP of another starfish Asterias amurensis, in a dose-dependent fashion. These results confirmed the species-specific reactivity of RGP and the cognate receptor. Thus, the present study provides evidence that PpeRGPR is a specific receptor for PpeRGP. To the best of our knowledge, this is the first report on the identification of a receptor for echinoderm RGP.

Effect of chimeric relaxin-like gonad-stimulating peptides on oocyte maturation and ovulation in the starfish Asterias rubens and Aphelasterias japonica

A relaxin-like gonad-stimulating peptide (RGP), comprising two peptide chains (A- and B-chains) linked by two interchain bonds and one intrachain disulfide bond, acts as a gonadotropin in starfish. RGP orthologs have been identified in several starfish species, including Patiria pectinifera (PpeRGP), Asterias rubens (AruRGP) and Aphelasterias japonica (AjaRGP). To analyze species-specificity, this study examined the effects on oocyte maturation and ovulation in ovaries of A. rubens and A. japonica of nine RGP derivatives comprising different combinations of A- and B-chains from the three species. All nine RGP derivatives induced spawning in A. rubens and A. japonica ovaries. However, AruRGP, AjaRGP and their chimeric derivatives were more potent than peptides containing the A- or B-chain of PpeRGP. Three-dimensional models of the structures of the RGP derivatives revealed that residues in the B-chains, such as AspB6, MetB10 and PheB13 in PpeRGP and GluB7, MetB11, and TyrB14 in AruRGP and AjaRGP, respectively, are likely to be involved in receptor binding. Conversely, it is likely that ArgA18 in the A-chain of AruRGP and AjaRGP impairs binding of these peptides to the PpeRGP receptor in P. pectinifera. In conclusion, this study provides new insights into the structural basis of RGP bioactivity and RGP receptor activation in starfish.

A Crown-of-Thorns Seastar recombinant relaxin-like gonad-stimulating peptide triggers oocyte maturation and ovulation

The Acanthaster planci species-complex [Crown-of-Thorns Seastar (COTS)] are highly fecund echinoderms that exhibit population outbreaks on coral reef ecosystems worldwide, including the Australian Great Barrier Reef. A better understanding of the COTS molecular biology is critical towards efforts in controlling outbreaks and assisting reef recovery. In seastars, the heterodimeric relaxin-like gonad stimulating peptide (RGP) is responsible for triggering a neuroendocrine cascade that regulates resumption of oocyte meiosis prior to spawning. Our comparative RNA-seq analysis indicates a general increase in RGP gene expression in the female radial nerve cord during the reproductive season. Also, the sensory tentacles demonstrate a significantly higher expression level than radial nerve cord. A recombinant COTS RGP, generated in a yeast expression system, is highly effective in inducing oocyte germinal vesicle breakdown (GVBD), followed by ovulation from ovarian fragments. The findings of this study provide a foundation for more in-depth molecular analysis of the reproductive neuroendocrine physiology of the COTS and the RGP.

Starfish Gonadotropic Hormone: From Gamete-Shedding Substance to Relaxin-Like Gonad-Stimulating Peptide.

The first report of a gonadotropic substance in an invertebrate hot-water extract of radial nerve cords from starfish Asterias forbesi that induced the shedding of gametes when injected into the coelomic cavity in a ripe individual occurred in 1959. The active substance was named gamete-shedding substance (GSS) or radial nerve factor. GSS is the primary mediator of oocyte maturation and ovulation in starfish. However, the effect of GSS is indirect. Resumption of meiosis in immature oocytes and release from the ovary are induced by a second mediator, maturation-inducing hormone, identified as 1-methyladenine (1-MeAde) in starfish. The role of GSS is to induce 1-MeAde production by ovarian follicle cells. Thus, GSS was redesignated as gonad-stimulating substance (also GSS). Although GSS has been characterized biochemically as a peptide hormone, identification of the chemical structure had to wait until 2009. Fifty years after the initial finding, GSS was purified from the radial nerve cords of starfish Patiria pectinifera (P. pectinifera). The purified hormone was a heterodimer composed of A- and B-chains, with disulfide cross-linkages. Based on its cysteine motif, GSS is classified as a member of the insulin/insulin-like growth factor (IGF)/relaxin superfamily. More specifically, phylogenetic sequence analysis revealed that P. pectinifera GSS is a member of the relaxin-type peptide family. Therefore, GSS in starfish has been redesignated as relaxin-like gonad-stimulating peptide (RGP). Subsequently, orthologs of P. pectinifera RGP have been identified in other starfish species, including Asterias amurensis (A. amurensis), and Aphelasterias japonica (A. japonica).

Interaction of starfish gonadotropin with its receptor: Effect of chimeric relaxin-like gonad-stimulating peptides

A relaxin-like gonad-stimulating peptide (RGP) of starfish Patiria (Asterina) pectinifera is the first identified invertebrate gonadotropin for final gamete maturation. Recently, we found three orthologs of RGP in the class Asteroida; PpeRGP in P. pectinifera, AamRGP in Asterias amurensis, and AjaRGP in Aphelasterias japonica. In this study, nine kinds of RGP derivatives with exchanged each A- and B-chain were synthesized chemically to analyze the interaction of RGP with its receptor. Among these RGP derivatives, PpeRGP and its chimeric RGPs with B-chains from AamRGP or AjaRGP could induce oocyte maturation and ovulation in P. pectinifera ovaries. In contrast, other RGP derivatives were failed to induce spawning in P. pectinifera ovaries. Circular dichroism spectra of PpeRGP were similar to those of chimeric RGPs with the B-chains from AamRGP or AjaRGP. Furthermore, the predicted three-dimensional structure models of the B-chains from RGP derivatives have almost the same conformation. These findings suggest that the B-chain of PpeRGP is involved in binding to its receptor. Thus, it is likely that the A-chain of AamRGP or AjaRGP disturbs the binding of the PpeRGP B-chain to its receptor.

Aquaculture Breeding Enhancement: Maturation and Spawning in Sea Cucumbers Using a Recombinant Relaxin-Like Gonad-Stimulating Peptide.

Wild sea cucumber resources have been rapidly exhausted and therefore there is an urgent need to develop approaches that will help restocking. Currently, there is a lack of information regarding the genes involved in sea cucumber reproductive processes. The neurohormone relaxin-like gonad-stimulating peptide (RGP) has been identified as the active gonad-stimulating peptide in sea stars (Asteroidea), which could also be present in other echinoderm groups. In this study, a sea cucumber RGP was identified and confirmed by phylogenetic analysis. A recombinant Holothuria scabra RGP was produced in the yeast Pichia pastoris and confirmed by mass spectrometry. To assess bioactivity, four levels of purification were tested in an in vitro germinal vesicle breakdown (GVBD) bioassay. The most pure form induced 98.56 ± 1.19% GVBD in H. scabra and 89.57 ± 1.19% GVBD in Holothuria leucospilota. Cruder levels of purification still resulted in some GVBD. Upon single injection into female H. scabra, the recombinant RGP induced head waving behavior followed by spawning within 90–170 min. Spawned oocytes were fertilized successfully, larvae settled and developed into juveniles. Our results provide a key finding for the development of a break-through new artificial breeding approach in sea cucumber aquaculture.

Enzyme-linked immunosorbent assay of relaxin-like gonad-stimulating peptide in the starfish Patiria (Asterina) pectinifera

A relaxin-like gonad-stimulating peptide (RGP) from starfish Patiria (Asterina) pectinifera is the first identified invertebrate gonadotropin for final gamete maturation. Recently, we succeeded in obtaining specific antibodies against P. pectinifera RGP (PpeRGP). In this study, the antibodies were used for the development of a specific and sensitive enzyme-linked immunosorbent assay (ELISA) for the measurement of PpeRGP. A biotin-conjugated peptide that binds to peroxidase-conjugated streptavidin is specifically detectable using 3,3′,5,5′-tetramethylbenzidine (TMB)/hydrogen peroxide as a substrate; therefore, biotin-conjugated RGP (biotin-PpeRGP) was synthesized chemically. Similarly to PpeRGP, synthetic biotin-PpeRGP bound to the antibody against PpeRGP. In binding experiments with biotin-PpeRGP using wells coated with the antibody, a displacement curve was obtained using serial concentrations of PpeRGP. The ELISA system showed that PpeRGP could be measured in the range 0.01–10pmol per 50µl assay buffer. On the contrary, the B-chains of PpeRGP, Asterias amurensis RGP, Aphelasterias japonica RGP, and human relaxin showed minimal cross-reactivity in the ELISA, except that the A-chain of PpeRGP affected it slightly. These results strongly suggest that this ELISA system is highly specific and sensitive with respect to PpeRGP.

Inhibitory mechanism of l-glutamic acid on spawning of the starfish Patiria (Asterina) pectinifera.

l-Glutamic acid was previously identified as an inhibitor of spawning in the starfish Patiria (Asterina) pectinifera; this study examined how l-glutamic acid works. Oocyte release from ovaries of P. pectinifera occurred after germinal vesicle breakdown (GVBD) and follicular envelope breakdown (FEBD) when gonads were incubated ex vivo with either relaxin-like gonad-stimulating peptide (RGP) or 1-methyladenine (1-MeAde). l-Glutamic acid blocked this spawning phenotype, causing the mature oocytes to remain within the ovaries. Neither RGP-induced 1-MeAde production in ovarian follicle cells nor 1-MeAde-induced GVBD and FEBD was affected by l-glutamic acid. l-Glutamic acid may act through metabotropic receptors in the ovaries to inhibit spawning, as l-(+)-2-amino-4-phosphonobutyric acid, an agonist for metabotropic glutamate receptors, also inhibited spawning induced by 1-MeAde. Application of acetylcholine (ACH) to ovaries under inhibitory conditions with l-glutamic acid, however, brought about spawning, possibly by inducing contraction of the ovarian wall to discharge mature oocytes from the ovaries concurrently with GVBD and FEBD. Thus, l-glutamic acid may inhibit ACH secretion from gonadal nerve cells in the ovary. Mol. Reprod. Dev. 84: 246-256, 2017. © 2017 Wiley Periodicals, Inc.

Cellular localization of relaxin-like gonad-stimulating peptide expression in Asterias rubens: New insights into neurohormonal control of spawning in starfish.

Gamete maturation and spawning in starfish is triggered by a gonad‐stimulating substance (GSS), which is present in extracts of the radial nerve cords. Purification of GSS from the starfish Patiria pectinifera identified GSS as a relaxin‐like polypeptide, which is now known as relaxin‐like gonad‐stimulating peptide (RGP). Cells expressing RGP in the radial nerve cord of P. pectinifera have been visualized, but the presence of RGP‐expressing cells in other parts of the starfish body has not been investigated. Here we addressed this issue in the starfish Asterias rubens. An A. rubens RGP (AruRGP) precursor cDNA was sequenced and the A chain and B chain that form AruRGP were detected in A. rubens radial nerve cord extracts using mass spectrometry. Comparison of the bioactivity of AruRGP and P. pectinifera RGP (PpeRGP) revealed that both polypeptides induce oocyte maturation and ovulation in A. rubens ovarian fragments, but AruRGP is more potent than PpeRGP. Analysis of the expression of AruRGP in A. rubens using mRNA in situ hybridization revealed cells expressing RGP in the radial nerve cords, circumoral nerve ring, and tube feet. Furthermore, a band of RGP‐expressing cells was identified in the body wall epithelium lining the cavity that surrounds the sensory terminal tentacle and optic cushion at the tips of the arms. Discovery of these RGP‐expressing cells closely associated with sensory organs in the arm tips is an important finding because these cells are candidate physiological mediators for hormonal control of starfish spawning in response to environmental cues. J. Comp. Neurol. 525:1599–1617, 2017. © 2016 Wiley Periodicals, Inc.