Molecular Identification, Characterization, and Expression Analysis of a Gonadotropin-Releasing Hormone Receptor (GnRH-R) in Pacific Abalone, Haliotis discus hannai

Abstract

A full-length cDNA sequence encoding a GnRH receptor was cloned from the pleuropedal ganglion of the Pacific abalone, Haliotis discus hannai. The cloned sequence is 1499-bp in length encoding a protein of 460 amino acid residues, with a molecular mass of 52.22 kDa and an isoelectric point (pI) of 9.57. The architecture of HdhGnRH-R gene exhibited key features of G protein-coupled receptors (GPCRs), including seven membrane spanning domains, putative N-linked glycosylation motifs, and phosphorylation sites of serine and threonine residues. It shared 63%, 52%, and 30% sequence identities with Octopus vulgaris, Limulus polyphemus, and Mizuhopecten yessoensis GnRH-R II sequences, respectively. Phylogenetic analysis indicated that HdhGnRH-R gene was clustered with GnRH-R II of O. vulgaris and O. bimaculoides. qPCR assay demonstrated that the mRNA expression level of this receptor was significantly higher in the pleuropedal ganglion than that in any other examined tissue. Transcriptional activities of this gene in gonadal tissues were significantly higher in the ripening stage. The mRNA expression of this gene was significantly higher in pleuropedal ganglion, testis, and ovary at higher effective accumulative temperature (1000 °C). In situ hybridization revealed that HdhGnRH-R mRNA was expressed in neurosecretory cells of pleuropedal ganglion. Our results suggest that HdhGnRH-R gene synthesized in the neural ganglia might be involved in the control of gonadal maturation and gametogenesis of H. discus hannai. This is the first report of GnRH-R in H. discus hannai and the results may contribute to further studies of GPCRs evolution or may useful for the development of aquaculture method of this abalone species.

Invertebrate Gonadotropin-Releasing Hormone Receptor Signaling and Its Relevant Biological Actions

“…Moreover, the expression of GnRH, GnRHR, and the serotonin receptor in the cognate male and female gonads were shown to be upregulated by the treatment with 250-ng/g BW Hdh-GnRH and buserelin [ 53 ]. These biological effects are compatible with the expression of the putative cognate receptor Hdh-GnRHR [ 46 ]. Similar to Has-GnRH in Haliotis asinine [ 40 ], the injection of 1000-ng/g BW Hdh-GnRH and buserelin was less effective than the aforementioned gonadal cell proliferation and gene expression effects in Haliotis discus hannai [ 53 ].…”

“…As shown in Table 1 , putative protostome GnRHR sequences were detected in several species [ 9 , 11 , 43 , 45 , 46 ], but only three protostome GnRHRs have been shown to interact with the cognate GnRH ligands. The first protostome GnRHR identified was oct-GnRHR in the octopus O. vulgaris [ 47 ].…”

Characterization of Insulin-Like Growth Factor Binding Protein 7 (Igfbp7) and Its Potential Involvement in Shell Formation and Metamorphosis of Pacific Abalone, Haliotis discus hannai

“…All samples were immediately frozen in liquid nitrogen and stored at −80 °C until total RNA extraction. For in situ hybridization, cryosections were prepared from mantle and gill tissues following a previously reported method by Sharker et al [ 49 , 50 , 51 ].…”

“…Ribosomal protein L-5 (JX002679.1) primer (forward: 5′-TGTCCGTTTCACCAACAAGG-3′ and reverse: 5′-AGATGGAATCAAGTTTCAATT-3′) from H. discus hannai was adopted as an internal control based on its expression stability [ 57 ]. In qRT-PCR, 1 μL cDNA from cerebral ganglion, shell muscle, gill, mantle, heart, digestive gland, hemocyte, testis, and ovary was used as a template for 20 µL reaction as described by Sharker et al [ 50 ]. PCR was carried out using the following cycling parameters: pre-incubation at 95 °C for 3 min, followed by three-step amplification at 94 °C for 2 min, 60 °C for 1 min, and 72 °C for 30 s for 40 cycles.…”

Characterization of Insulin-Like Growth Factor Binding Protein-5 (IGFBP-5) Gene and Its Potential Roles in Ontogenesis in the Pacific Abalone, Haliotis discus hannai

“…Primers (forward: 5′-GGTGCTGTCTTCGCTGACAA-3′ and reverse: 5′-CCAGTCTTCTATATGGACAGT-3′) were designed from IGFBP-5 nucleotide sequence, producing 548-bp PCR product. Amplified products were cloned into pGEM-T Easy vector (Enzynomics, Daejeon, Korea) and recombinant plasmids were used as template to synthesize antisense and sense RNA probes by in vitro transcription following a previous method [ 41 , 42 ]. The cerebral ganglion tissue sections were prehybridized with hybridization buffer and yeast total RNA (50 μL) for 2 h, followed by overnight hybridization with DIG-labeled antisense or sense RNA probe at 65 °C.…”