Identification and functional characterization of relaxin-like gonad stimulating peptide in the sea cucumber, Stichopus horrens

Abstract

In the state of Hawaiʻi, there is rising interest in the culture of native sea cucumbers, such as Stichopus horrens; however, current production is hindered by a limited understanding of their reproductive biology. Originally described in starfish, relaxin-like gonad stimulating peptide (RGP) has been characterized and used to induce oocyte maturation and spawning in sea cucumbers. For the first time using S. horrens specifically, we identified the RGP (Sh-RGP) coding sequence through de novo transcriptomics, synthesized the mature peptide, and investigated its role in inducing oocyte maturation in vitro, and spawning in vivo. The Sh-RGP precursor gene encodes a single mature peptide composed of two amino acid chains. The B- and A-chains contain two and four cysteine residues, respectively, resulting in an intra-chain linkage on the A-chain and two disulfide connections between the two chains. A synthetic Sh-RGP was produced from solid-phase peptide synthesis, bonded, and isolated by reverse-phase HPLC. A tissue distribution analysis revealed that shrgp mRNA expression is highest (P < 0.001) in the neural ring. In vitro, Sh-RGP (0.1–10 μM) induced germinal vesicle breakdown in oocytes incubated with gonadal tissue in time and concentration-dependent fashions. When injected with Sh-RGP (∼4.5 μg/kg body weight), all mature male and female sea cucumbers spawned within 39 min of treatment. Overall, these results indicate that Sh-RGP induces oocyte maturation and spawning and provide a framework for integrating transcriptomics and functional assays to elucidate and validate endogenous species-specific reproductive hormones that can be used to address barriers in sea cucumber aquaculture.