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

Highlights

• Recombinant relaxin-like gonad stimulating peptide (RGP) is bioactive in vitro.

• Histidine linker does not affect recombinant RGP folded structure and bioactivity.

• Recombinant RGP induces Crown-of-Thorn Seastar oocyte maturation in vitro.

• Recombinant RGP induces Crown-of-Thorn Seastar ovulation in vitro.

Abstract

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.

Introduction

The Crown-of-Thorns Seastar (COTS), Acanthaster planci species-complex, is an echinoderm of enormous interest due to its impact on coral reef ecosystems around the world. COTS are a major cause of coral reef decline due to their requirement for coral as a source of food and with weaponry consisting of sharp toxic spines, they render themselves unpalatable to most animals (Dana et al., 1972, Kenyon, 2014, Coles et al., 2015). Approaches to limit population numbers are limited since we still have gaps in our understanding of their biology, including reproductive physiology (Johnson et al., 1990, De’ath et al., 2012, Hunt, 2013, Morello et al., 2014, GBRMPA, 2017). However, as an invertebrate deuterostome, the molecular components regulating physiological processes partly resemble that of both invertebrates and vertebrates (Tian et al., 2016, Semmens and Elphick, 2017). For example, echinoderm-like neuropeptides can be found regulating reproductive physiology in invertebrates and vertebrates (Semmens et al., 2016, Tian et al., 2016, Semmens and Elphick, 2017).

Neuropeptides are a class of signalling molecules derived from a larger precursor protein that are primarily produced and secreted from neural tissue (Taghert and Nitabach, 2012). One neuropeptide regarded as a key reproduction regulator in seastars is the relaxin-like gonad stimulating peptide (RGP). RGP was first reported over half a century ago and is responsible for final oocyte maturation (Chaet, 1959). It is heterodimeric, comprised of a two-chain structure including A-chain and B-chain, with disulphide cross-linkages. RGP is presumed to bind to gonad follicle cell G-protein coupled receptors (GPCRs) that stimulate internal Gαs and adenylate cyclase activity (Hirai and Kanatani, 1971, Mita and Nagahama, 1991, Mita et al., 2011a, Mita et al., 2011b, Mita, 2013a, Mita, 2013b). The end result is the release of the secondary messenger 1-Methyladenine (1-MeAde), which resumes meiosis of oocytes arrested in prophase (Hirai and Kanatani, 1971, Mita et al., 2011a, Mita et al., 2011b). RGP is only effective upon gonads with oocytes at the final maturation stage (oocyte diameter > 150 µm) (Takahashi and Kanatani, 1981) since the immature gonads lack an active Gαs (Mita, 2013a, Mita, 2013b).

In the Asterias rubens, in situ hybridization experiments have shown that RGP localises to solitary cells of the radial nerve cord (RNC) and sensory tentacles (Lin et al., 2017). Our previous investigation into neuropeptides present in the COTS has demonstrated that RGP is most abundant in the sensory tentacle (Smith et al., 2017), although this was observed on a single individual. The RGP is processed from a precursor protein, involving prohormone convertase processing at dibasic cleavage sites then complex folding due to the heterodimerisation of cysteine-rich chains. As such, the RGP may be difficult to accurately chemically synthesized or produced through heterologous expression systems [e.g. relaxin/insulin-like synthetics (Thalluri et al., 2018)]. An Asterina (=Patiria) pectinifera RGP has been synthesised, including disulphide bonded chains-A and -B, that could induce ovarian oocyte maturation and spawning in several seastar species, albeit some species-specificity was noted (Mita, 2013a, Mita, 2013b, Mita et al., 2015a, Mita et al., 2015b, Mita, 2016, Mita and Katayama, 2016). Also, a synthetic Asterias rubens and Asterias amurensis RGP has been produced and functionality determined through oocyte maturation (Mita et al., 2015a, Mita et al., 2015b, Lin et al., 2017). No recombinant RGP had been described.

In this study, we explored the differential expression of RGP in COTS RNC and sensory tissues, then generated a biologically active recombinant COTS RGP using a yeast expression system. The recombinant COTS RGP is capable of inducing oocyte maturation and ovulation in vitro.