Characterization of GnRH-like peptides from the nerve ganglia of Yesso scallop, Patinopecten yessoensis

Highlights

• We identified a precursor and mature form of invGnRH peptides in Yesso scallop.

• Both py-GnRH peptides exhibit dimorphic localization patterns in CPG and VG.

• They do not co-localize in the neural cells of CPG but co-localize in those of VG.

• The mature py-GnRH is abundantly present in VG rather than CPG in both sexes.

• VG is the major producing site of the mature py-GnRH peptide.

Abstract

There is yet no firm experimental evidence that the evolutionary ancient gonadotropin-releasing hormone GnRH (i.e., GnRH1) also acts in invertebrate gametogenesis. The objective of this paper is to characterize candidate invGnRH peptides of Yesso scallop Patinopecten yessoensis (i.e., peptide identification, immunohistochemical localization, and immunoquantification) in order to reveal their bioactive form in bivalves. Using mass spectrometry (MS), we identified two invGnRH (py-GnRH) peptides from the scallop nerve ganglia: a precursor form of py-GnRH peptide (a non-amidated dodecapeptide; py-GnRH12aa-OH) and a mature py-GnRH peptide (an amidated undecapeptide; py-GnRH11aa-NH₂). Immunohistochemical staining allowed the localization of both py-GnRH peptides in the neuronal cell bodies and fibers of the cerebral and pedal ganglia (CPG) and the visceral ganglion (VG). We found that the peptides showed a dimorphic distribution pattern. Notably, the broad distribution of mature py-GnRH in neuronal fibers elongating to peripheral organs suggests that it is multi-functional. Time-resolved fluorescent immunoassays (TR-FIA) enabled the quantification of each py-GnRH form in the single CPG or VG tissue obtained from one individual. In addition, we observed greater abundance of mature py-GnRH in VG compared with its level in CPG, suggesting that VG is the main producing organ of mature py-GnRH peptide and that py-GnRH may play a central regulatory role in neurons of scallops. Our study provides evidence, for the first time, for the presence of precursor and mature forms of invGnRH peptides in the nerve ganglia of an invertebrate.

Introduction

Gonadotropin-releasing hormones (GnRHs) are neuropeptides that regulate reproductive function in vertebrates and are thought to be derived from a common evolutionary origin of Proto- and Deuterostomia about 700 million years ago, as is the case with the GnRH receptor superfamily [6]. Most vertebrates possess two GnRH forms (i.e., GnRH1 and GnRH2) and several teleost species possess three forms (i.e., GnRH1, GnRH2, and GnRH3): GnRH1 acts to stimulate the release of gonadotropins while the other two forms (i.e., GnRH2 and GnRH3) are thought to play a neuromodulatory role in extra hypothalamus [13]. In invertebrates, it has been thought that members of GnRH superfamily (i.e., invertebrate GnRH (invGnRH), corazonin (Crz), adipokinetic hormone (AKH), and AKH/Crz related peptide (ACP)) arose through gene duplication from a common ancestor in Bilaterians [15]. Intriguingly, synteny of limpet AKH paralogon with amphioxus GnRH showed diversified gene evolution in GnRH, suggesting functional divergence among the members of GnRH superfamily in invertebrates [16]. Therefore, whether the evolutionary related forms of GnRH in invertebrates can stimulate gametogenesis has yet to be determined even though invGnRHs have been cloned in a number of invertebrate species [18].

Due to the great interest in invGnRH function, several studies investigated whether invGnRH possesses reproductive function in mollusks. One study reported that octopus invGnRH (oct-GnRH) acts as a multifunctional modulator, including the ability to induce contraction of oviduct during egg laying [7]. In addition, oct-GnRH was found to be active in steroid biosynthesis including progesterone (P), testosterone (T), and 17β-estradiol (E₂), within the gonads [9]. Non-reproductive roles were also suggested by showing that of oct-GnRH also localizes in neuronal cells projecting to the non-reproductive organs, and is therefore possibly involved in feeding, memory, movement, and autonomic functions [7]. Moreover, in vivo administration in Aplysia revealed that invGnRH (ap-GnRH) did not act as a reproductive regulator [19] but as a multifunctional modulator of muscular contraction in peripheral tissues involved in locomotion [20]. Because physiological experiments with purified or synthetic invGnRH peptide remains the only way to confirm its biological function in invertebrates it is important to first characterize the native peptide form of invGnRH. However, there have been very few studies that identified the mature peptide form of invGnRH by mass spectrometry (MS) in mollusks (i.e., Octopus vulgaris [8] and Crassostrea gigas [2]) its characterization in other mollusks is therefore important.

The Yesso scallop Patinopecten yessoensis is one of the most economically important marine bivalve mollusks in the Far Eastern Asian coast (i.e., China and Japan, FAO website; http://www.fao.org/fishery/culturedspecies/Patinopecten_yessoensis/en). In addition, because of its well-known life history and the established rearing methods for aquaculture production we have studied this species as a model for understating neuroendocrinological regulation of bivalve reproduction [12], [21], [22], [23], [25]. Previously, we cloned invGnRH cDNA sequences from Yesso scallop (py-GnRH) [23]. We now aim to confirm the bioactive form of py-GnRH peptide in vivo. In the present study, we performed MS identification of precursor and the mature forms of py-GnRH peptides. In addition, we performed immunological analyses that revealed the localization pattern and abundance of each py-GnRH peptide in cerebral and pedal ganglia (CPG) and visceral ganglion (VG) that constitute the central nervous system of Yesso scallop.