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.

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.

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.

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.

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 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.

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.

Mechanism of gamete shedding in starfish: Involvement of acetylcholine in extracellular Ca2+-dependent contraction of gonadal walls.

Starfish are suitable animals for the study of hormonal regulatory mechanism of oocyte maturation and ovulation. Although contraction of the gonadal walls is essential for the shedding gametes, little was known about the mechanism. When ovaries of starfish Patiria pectinifera were incubated in Ca2+-free seawater in the presence of 1-methyladenine (1-MeAde), the germinal vesicles in oocytes broke down, but no ovulation occurred. Verapamil, a potent inhibitor of voltage-dependent Ca2+ channels, inhibited 1-MeAde-induced ovulation. These results suggest that extracellular Ca2+ and its influx are indispensable for gamete shedding. Furthermore, acetylcholine (ACh) was involved in extracellular Ca2+-dependent contractions of gonadal walls. Although 1-MeAde failed to induce contraction of the gonadal walls in normal seawater containing L-glutamic acid, application of ACh or carbachol, an agonist for ACh receptor, could bring about shedding of mature oocytes. Atropine, a competitive antagonist of the muscarinic ACh receptor, inhibited 1-MeAde-induced ovulation, but a nicotinic ACh receptor antagonist mecamylamine had no effect. Furthermore, ACh was detected in the ovaries and testes in P. pectinifera. These findings suggest that ACh acts on muscarinic ACh receptors in gonadal walls to induce peristaltic contractions caused by Ca2+ influx via Ca2+ channels in the gonadal wall muscle for gamete shedding. The present study also provides new insight into the regulatory mechanism that 1-MeAde acts on secretion of ACh in ovaries and testes.

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.

A novel approach for preparing disulfide-rich peptide-KLH conjugate applicable to the antibody production.

To produce the antiserum against a small peptide, the target peptide-keyhole limpet hemocyanine (KLH) conjugate is generally used as an antigen, although the disulfide-rich peptide-KLH conjugate is still difficult to prepare. In our previous study, we have developed a preparation method of the disulfide-rich peptide-KLH conjugate, and this method was applied to produce the antiserum against a relaxin-like peptide. However, this method is limited to the synthetic peptide antigen, and is not applicable to a native or a recombinant peptide. In this study, to expand the applicability of this method to wide variety of peptides, we newly designed a novel thiol probe enabling the conjugation between various peptides and KLH, and applied it to produce the antiserum against relaxin-like peptide of a starfish Asterias amurensis. The antiserum obtained here showed high antibody-titer and good specificity, strongly suggesting that the method developed in this study is applicable to various peptides.

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.

Radioimmunoassay 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. An antiserum against P. pectinifera RGP (PpeRGP) was produced by immunizing rabbits with a PpeRGP sulfanyl-polyethylene glycol derivative conjugated with keyhole limpet hemocyanin (KLH) as the antigen. The antiserum was used for the development of a specific and sensitive radioimmunoassay (RIA) for the measurement of RGP. In binding experiments using radioiodinated PpeRGP and antiserum against PpeRGP, a displacement curve was obtained using radioinert PpeRGP. The sensitivity of the RIA, defined as the amount of PpeRGP that significantly decreased the counts by 2 SD from the 100% bound point, averaged 0.040±0.002pmol PpeRGP per 100µl assay buffer (0.40±0.02nM) in 10 assays. Intra-assay and inter-assay coefficients of variation were 6.1% and 2.7%, respectively. Serial dilution of whole homogenates from the radial nerve cords and circumoral nerve-rings of P. pectinifera produced displacement curves parallel to the PpeRGP standard. Thus, the amounts of PpeRGP were determined as 1.54±0.09pmol/mg wet weight of radial nerves and 0.87±0.04pmol/mg wet weight of nerve-rings, respectively. On contrary, pyloric stomach, pyloric caeca, tube-feet, ovaries, testes, and ovarian follicle cells did not react in the RIA system. Furthermore, the A- and B-chains of PpeRGP, Asterias amurensis RGP, bovine insulin, and human relaxin did not show cross-reactivity in the RIA. These results strongly suggest that the RIA system is a highly specific and sensitive with respect to PpeRGP.

A sulfanyl-PEG derivative of relaxin-like peptide utilizable for the conjugation with KLH and the antibody production.

A small peptide-keyhole limpet hemocyanin (KLH) conjugate is generally used as an antigen for producing specific antibodies. However, preparation of a disulfide-rich heterodimeric peptide-KLH conjugates is difficult. In this study, we developed a novel method for preparation of the conjugate, and applied it to the production of specific antibodies against the relaxin-like gonad-stimulating peptide (RGP) from the starfish. In this method, a sulfanyl group necessary for the conjugation with KLH was site-specifically introduced to the peptide after regioselective disulfide bond formation reactions. Using the conjugate, we could obtain specific antibodies with a high antibody titer. This method might also be useful for the production of antibodies against other heterodimeric peptides with disulfide cross-linkages, such as vertebrate relaxins.

Starfish gonadotropic hormone: Relaxin-like gonad-stimulating peptides.

Relaxin-like gonad-stimulating peptide (RGP) of starfish Patiria (= Asterina) pectinifera is the first identified invertebrate gonadotropin to trigger final gamete maturation. Recently, chemical structures of RGP were identified in several species of starfish. Three kinds of RGP molecules are found in the class Asteroidea. The chemical structure of P. pectinifera RGP (PpeRGP) is conserved among starfish of the order Valvatida beyond species. In contrast, the chemical structures of RGP identified in Asterias amurensis and Aphelasterias japonica of the order Forcipulatida are quite different from that of PpeRGP. The chemical structure of RGP in A. amurensis (AamRGP) is exactly the same as that in Asterias rubens (the order Forcipulatida), Astropecten scoparius (the order Paxillosida), Astropecten polyacanthus (the order Paxillosida), and Echinaster luzonicus (the order Spinulosida). The chemical structure of Coscinasterias acutispina RGP (the order Forcipulatida) is consistent with that of A. japonica RGP (AjaRGP). In cross-experiments using P. pectinifera, A. amurensis, and A. japonica ovaries, AamRGP and AjaRGP can induce each species of ovaries. Neither AamRGP nor AjaRGP induce oocyte maturation and ovulation in the ovary of P. pectinifera, although the PpeRGP is active in ovaries of A. amurensis and A. japonica. This suggests that the AamRGP and AjaRGP partly act species specificity.

A relaxin-like gonad-stimulating peptide from the starfish Aphelasterias japonica.

Relaxin-like gonad-stimulating peptide (RGP) in starfish is the first identified invertebrate gonadotropin responsible for final gamete maturation. In this study, a new ortholog RGP was identified from Aphelasterias japonica. The DNA sequence encoding A. japonica RGP (AjaRGP) consists of 342 base pairs with an open reading frame encoding a peptide of 113 amino acids (aa), including a signal peptide (26aa), B-chain (20aa), C-peptide (42aa), and A-chain (25aa). AjaRGP is a heterodimeric peptide with disulfide cross-linkages. Comparing with Asterias amurensis RGP (AamRGP) and Patiria (=Asterina) pectinifera RGP (PpeRGP), the amino acid identity levels of AjaRGP with respect to AamRGP and PpeRGP are 84% and 58% for the A-chain and 90% and 68% for the B-chain, respectively. This suggests that AjaRGP is closer to AmaRGP rather than PpeRGP. Although chemical synthetic AjaRGP can induce gamete spawning and oocyte maturation in ovarian fragments of A. japonica, the ovary of P. pectinifera fails to respond to AjaRGP. This suggests that AjaRGP acts species-specifically.

Nucleotide sequence and expression of relaxin-like gonad-stimulating peptide gene in starfish Asterina pectinifera.

Starfish gonad-stimulating substance (GSS) is the only known invertebrate peptide hormone responsible for final gamete maturation, rendering it functionally analogous to gonadotropins in vertebrates. Because GSS belongs to the relaxin-like peptide family, we propose renaming for starfish gonadotropic hormone as relaxin-like gonad-stimulating peptide (RGP). This study examined the primary structure and expression regulation of the RGP gene in starfish Asterina pectinifera. RGP consisted of 3896 base pairs (bp) divided over two exons, exon 1 of 208 bp and exon 2 of 2277 bp, and one intron of 1411 bp. Promoter sequences, CAAT and TATA boxes, were present in the 5'-upstream region of the coding DNA sequence of RGP. The transcript was 2485 bases (b) in length. The AAUAAA polyadenylation signal was found in 3'-untranslated region over 2kb away from the stop codon. This showed that only 14% of the RGP mRNA was translated into the peptide, because a size of the open-reading frame was 351 b. Furthermore, an analysis by using real-time quantitative PCR with specific primers for RGP showed that mRNA of RGP was expressed at high levels in the radial nerves. Expression was also observed in the cardiac stomachs, although the level was low, and trace levels were detected in the gonads, pyloric caeca and tube feet. This result suggests that the RGP gene is transcribed mainly in the radial nerves of A. pectinifera.

A new relaxin-like gonad-stimulating peptide identified in the starfish Asterias amurensis.

Relaxin-like gonad-stimulating peptide (RGP) of starfish Asterina pectinifera was the first invertebrate gonadotropin to have its chemical structure identified. However, it is unclear whether gonadotropic hormones in other species starfish are relaxin-like peptides. Thus, this study tried to identify the molecular structure of gonadotropic hormone in Asterias amurensis. As a result, we identified A. amurensis gonadotropic hormone as the RGP (AamRGP). The DNA sequence encoding AamRGP consisted of 330 base pairs with an open reading frame encoding a peptide of 109 amino acids (aa), including a signal peptide (26 aa), B-chain (20 aa), C-peptide (38 aa) and A-chain (25 aa). Comparing with A. pectinifera RGP (ApeRGP), the amino acid identity levels between AmaRGP and ApeRGP were 58% for the A-chain and 73% for the B-chain. Furthermore, chemical synthetic AamRGP induced gamete spawning and oocyte maturation in ovarian fragments of A. amurensis. In contrast, the ovary of A. pectinifera failed to respond to the AamRGP. This suggested that AamRGP is a new relaxin-like peptide.

Possible role of pineal allopregnanolone in Purkinje cell survival.

It is believed that neurosteroids are produced in the brain and other nervous systems. Here, we show that allopregnanolone (ALLO), a neurosteroid, is exceedingly produced in the pineal gland compared with the brain and that pineal ALLO acts on the Purkinje cell, a principal cerebellar neuron, to prevent apoptosis in the juvenile quail. We first demonstrated that the pineal gland is a major organ of neurosteroidogenesis. A series of experiments using molecular and biochemical techniques has further demonstrated that the pineal gland produces a variety of neurosteroids de novo from cholesterol in the juvenile quail. Importantly, ALLO was far more actively produced in the pineal gland than in the brain. Pinealectomy (Px) decreased ALLO concentration in the cerebellum and induced apoptosis of Purkinje cells, whereas administration of ALLO to Px quail chicks prevented apoptosis of Purkinje cells. We further found that Px significantly increased the number of Purkinje cells that expressed active caspase-3, a key protease in apoptotic pathway, and daily injection of ALLO to Px quail chicks decreased the number of Purkinje cells expressing active caspase-3. These results indicate that the neuroprotective effect of pineal ALLO is associated with the decrease in caspase-3 activity during the early stage of neuronal development. We thus provide evidence that the pineal gland is an important neurosteroidogenic organ and that pineal ALLO may be involved in Purkinje cell survival during development. This is an important function of the pineal gland in the formation of neuronal circuits in the developing cerebellum.

Involvement of Gαs-proteins in the action of relaxin-like gonad-stimulating substance on starfish ovarian follicle cells.

Gonad-stimulating substance (GSS) in starfish is the only known invertebrate peptide hormone responsible for final gamete maturation, rendering it functionally analogous to gonadotropins in vertebrates. In breeding season (stage V), GSS stimulates oocyte maturation to induce 1-methyladenine (1-MeAde) by ovarian follicle cells. The hormonal action of GSS is mediated through the activation of its receptor, G-proteins and adenylyl cyclase. It has been reported that GSS fails to induce 1-MeAde and cyclic AMP (cAMP) production in follicle cells of ovaries during oogenesis (stage IV). This study examined the regulatory mechanism how ovarian follicle cells acquire the potential to respond to GSS by producing 1-MeAde and cAMP. Because the failure of GSS action was due to G-proteins of follicle cells, the molecular structures of Gαs, Gαi, Gαq and Gß were identified in follicle cells of starfish Asterina pectinifera. The cDNA sequences of Gαs, Gαi, Gαq and Gß consisted of ORFs encoding 379, 354, 353 and 353 amino acids. The expression levels of Gαs were extremely low in follicle cells at stage IV, whereas the mRNA levels increased markedly in stage V. On contrary, the mRNA levels of Gαi were almost constant regardless of stage IV and V. These findings strongly suggest that de novo synthesis of Gαs-proteins is contributed to the action of GSS on follicle cells to produce 1-MeAde and cAMP.