Neuropeptide expression and action in the reproductive system of the starfish Asterias rubens.

Reproductive processes are regulated by a variety of neuropeptides in vertebrates and invertebrates. In starfish (phylum Echinodermata), relaxin-like gonad-stimulating peptide triggers oocyte maturation and spawning. However, little is known about other neuropeptides as potential regulators of reproduction in starfish. To address this issue, here, we used histology and immunohistochemistry to analyze the reproductive system of the starfish Asterias rubens at four stages of the seasonal reproductive cycle in male and female animals, investigating the expression of eight neuropeptides: the corticotropin-releasing hormone-type neuropeptide ArCRH, the calcitonin-type neuropeptide ArCT, the pedal peptide-type neuropeptides ArPPLN1b and ArPPLN2h, the vasopressin/ocytocin-type neuropeptide asterotocin, the gonadotropin-releasing hormone-type neuropeptide ArGnRH, and the somatostatin/allatostatin-C-type neuropeptides ArSS1 and ArSS2. The expression of five neuropeptides, ArCRH, ArCT, ArPPLN1b, ArPPLN2h, and asterotocin, was detected in the gonoducts and/or gonads. For example, extensive ArPPLN2h expression was revealed in the coelomic epithelial layer of the gonads throughout the seasonal reproductive cycle in both males and females. However, seasonal and/or sexual differences in the patterns of neuropeptide expression were also observed. Informed by these findings, the in vitro pharmacological effects of neuropeptides on gonad preparations from male and female starfish were investigated. This revealed that ArSS1 causes gonadal contraction and that ArPPLN2h causes gonadal relaxation, with both neuropeptides being more effective on ovaries than testes. Collectively, these findings indicate that multiple neuropeptide signaling systems are involved in the regulation of reproductive function in starfish, with some neuropeptides exerting excitatory or inhibitory effects on gonad contractility that may be physiologically relevant when gametes are expelled during spawning.

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.