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

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Highlights

  • Relaxin-like gonad-stimulating peptide (RGP) is a starfish gonadotropic hormone.

  • A new RGP (AamRGP) was identified in starfish Asterias amurensis.

  • AamRGP is the ortholog of the ApeRGP previously identified in Asterina pectinifera.

  • ApeRGP could induce gamete spawning in both A. pectinifera and A. amurensis.

  • In contrast, AamRGP induced spawning in A. amurensis but not in A. pectinifera.

Abstract

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.

Introduction

In most marine invertebrates, fertilization occurs in seawater, outside the females’ body. Particularly in sea urchins, eggs and spermatozoa have long been used as excellent materials for the study of fertilization when eggs are artificially inseminated in vitro. Sea urchin oocytes have already accomplished meiotic maturation within the ovary during the breeding season long before spawning (Fuji, 1960). On the other hand, in starfish, oocytes in a ripe ovary remain arrested at the end of the first prophase stage of meiosis. These oocytes are immature and fail to undergo normal fertilization. Therefore, a hormonal substance is required for maturation and ovulation in starfish oocytes (Kanatani, 1985, Nagahama et al., 1995).

Chaet and McConnaughy (1959) first reported that an aqueous extract of starfish radial nerves could induce the shedding of gametes when injected into the coelomic cavity of ripe animals. The active substance contained in the nerve extract was considered to be a peptide hormone named gonad-stimulating substance (GSS) (Kanatani and Shirai, 1967, Kanatani and Shirai, 1969). GSS is the primary mediator of oocyte maturation in starfish. However, the effect of GSS on oocyte maturation is indirect. Resumption of meiosis in immature oocytes and release from the ovary are induced by a second mediator, maturation-inducing hormone (MIH), identified as 1-methyladenine (1-MeAde) in starfish (Kanatani et al., 1969, Kanatani, 1985). Thus, GSS plays an important role in 1-MeAde production in ovarian follicle cells (Hirai and Kanatani, 1971, Hirai et al., 1973) by way of activation of its receptor, G-protein and adenylyl cyclase (Mita et al., 1987, Mita et al., 1989, Mita and Nagahama, 1991). In this sense, GSS is functionally identical to vertebrate luteinizing hormone (LH), especially piscine and amphibian LHs, acting on ovarian follicle cells to produce MIH to induce the final maturation or meiotic resumption of the oocyte (Nagahama et al., 1995).

Fifty years since the initial finding of Chaet and McConnaughy (1959), GSS was finally purified from the radial nerves of starfish Asterina pectinifera (Mita et al., 2009). The purified hormone is a heterodimer composed of two different peptides, A- and B-chains with disulfide cross-linkages. Based on its cysteine motif, starfish GSS was classified as a member of the insulin/insulin-like growth factor (IGF)/relaxin superfamily and, more precisely, it belongs to a relaxin-like peptide family (Mita et al., 2009). Recently, GSS in starfish A. pectinifera was designated as relaxin-like gonad-stimulating peptide (RGP) (Haraguchi et al., in press).

Previous studies have shown that GSS is present in the radial nerves of all species in the Class Asteroidea tested so far (Kanatani, 1979). However, the ovary of A. pectinifera fails to respond to GSS of Asterias amurensis, although A. pectinifera GSS is active in A. amurensis (Noumura and Kanatani, 1962). A. amurensis belongs to the Order Forcipulatida in the Class Asteroidea, whereas A. pectinifera is the Order Valvatida. This suggested that the GSS molecule of A. amurensis (the Order Forcipulatida) is different from RGP of A. pectinifera (the Order Valvatida). However, the chemical structure of GSS in A. amurensis has not yet been identified. To elucidate whether GSS from A. amurensis is a relaxin-like peptide, we tried to identify the A. amurensis GSS.

Section snippets

Animals

Starfish A. amurensis were collected from Asamushi (Aomori Prefecture, Japan), Yokosuka, (Kanagawa Prefecture, Japan) and Tateyama (Chiba Prefecture, Japan). A. pectinifera were also collected from Asamushi, Yokosuka and Ushimado (Okayama Prefecture, Japan).

cDNA cloning

Total RNA was extracted from radial nerves of A. amurensis after homogenization with Sepasol (Nacalai Tesque, Kyoto, Japan) as the RNA extraction solution. A Poly(A)+ RNA fraction was obtained using Oligotex-dT30 (Nippon Gene, Tokyo, Japan).

Results

The starfish A. amurensis is an endemic Japanese species and inhabits inter-tidal zone in Japanese waters. Dramatic color polymorphism in A. amurensis is known in this local population. Adult A. amurensis with a purple color were observed in Asamushi (Aomori Prefecture, Japan) (Fig. 1A), but yellow A. amurensis were found in Yokosuka (Kanagawa Prefecture, Japan) (Fig. 1B). Because this color polymorphism might be related to genetic variations, cDNA sequences of GSS were examined in A. amurensis

Discussion

Gonadotropins play important regulatory roles in reproduction in both vertebrates and invertebrates. It has been shown that gonadotropic hormone purified from the starfish A. pectinifera is not a glycoprotein as is the case for vertebrate gonadotropins, but a relaxin-like peptide, RGP (Mita et al., 2009, Haraguchi et al., in press). Previously, relaxin was shown to be an important hormone for reproduction in mammals only, but starfish RGP has activities analogous to gonadotropins in vertebrates

Acknowledgments

The authors are grateful to Dr. K. Kyozuka and Mr. M. Washio (Research Center for Asamushi Marine Biology, Graduate School of Life Sciences, Tohoku University); Dr. M. Kiyomoto and Mr. M. Yamaguchi (Marine Coastal Research Center, Ochanomizu University); and Dr. R. Sakamoto and Mr. W. Godo (Ushimado Marine Laboratory, Okayama University) for their kind help in collecting starfish. We also thank Dr. S. Kanda (Department of Biological Sciences, Graduate School of Science, The University of Tokyo)

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