Characterization and expression analysis of a GnRH-like peptide in the Pacific abalone, Haliotis discus hannai

Characterization and spatiotemporal expression of gonadotropin-releasing hormone in the Pacific abalone, Haliotis discus hannai

Effective accumulative temperature affects gonadal maturation by controlling expression of GnRH, GnRH receptor, serotonin receptor and APGWamide gene in Pacific abalone, Haliotis discus hannai during broodstock conditioning in hatcheries.

Gonadotropin-releasing hormone (GnRH) is an important regulator of reproductive function in various vertebrates and invertebrates. In the present study, we have identified the GnRH-like peptide cDNA and peptide from the cerebral ganglion (CG) of the Pacific abalone, Haliotis discus hannai. Pacific abalone GnRH-like peptide (hdhGnRH-like peptide) cDNA encodes precursor, which possesses the typical organization of the known mollusk GnRH-like peptide precursors, including a hydrophobic signal peptide, GnRH-like peptide, and a cleavage site followed by a GAP-like peptide region. Three hdhGnRH-like peptides, pQNYHFSNGWHAamide (hdhGnRH-11amide), pQNYHFSNGWHA (hdhGnRH-11OH), and pQNYHFSNGWHAG (hdhGnRH-12OH), were determined from the acid/acetone extract of the CG by mass spectrometry (LC-MS/MS) analysis. The hdhGnRH-like peptide mRNA expression was detected not only in the CG but also in gonads, and hdhGnRH-11amide was also detected in the extract of gonads. The mRNA expression of hdhGnRH-like peptide in the CG was lower in spawned males than in non-spawned animals, while no change in hdhGnRH-like peptide mRNA expression was shown in both ovulated and non-ovulated abalone. The hdhGnRH-11amide induces spawning and ovulation of both mature males and females in a concentration-dependent fashion following intramuscular injection. These results indicate that three hdhGnRH-like peptides are yielded from a single hdhGnRH-like peptide precursor, and that at least hdhGnRH-11amide is involved in the control of reproduction of the Pacific abalone.

Intron retention in a salmon gonadotropin releasing-hormone in three species of Chirostoma genus.

Molecular and structural analysis of Hdh-MIRP3 and its impact on reproductive regulation in female Pacific abalone, Haliotis discus hannai

A series of experiments was performed to monitor plasma luteinizing hormone (LH), follicle-stimulating hormone (FSH), and prolactin responses to human gonadotropin-releasing hormone (GnRH) associated peptide (GAP) and related peptides. Ovariectomized hypothalamo-pituitary disconnected (HPD) ewes were challenged with injections (1-10 micrograms i.v.) of GAP, or given, with and without estradiol, hourly 500- or 1,000-ng pulses of GAP for 5-7 days. In all cases GAP failed to cause the release of LH or FSH from the pituitary gland or to alter mean plasma prolactin concentrations. When the same HPD ewes were given hourly or 2-hourly pulses of 250 ng GnRH, LH responded in a a pulsatile manner, and FSH secretion was maintained, thus confirming the functional integrity of the pituitary gland after HPD. Fragments of the GAP molecule (pro-GnRH 14-36, 28-36, 38-49, and 51-66) and GAP dimer did not stimulate LH or FSH or inhibit prolactin release in HPD ewes. GAP and GAP dimer did not affect pituitary responsiveness to GnRH administration. GAP also failed to inhibit the thyrotropin-releasing hormone-induced rise in prolactin. Finally, GAP injections (100 micrograms i.v.) given to lactating ewes did not cause any change in plasma prolactin concentrations. These data show that human GAP, GAP dimer, or putative processed GAP peptides do not act on the sheep pituitary gland in a variety of physiological states to regulate gonadotropin or prolactin secretion.

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.

Insulin-like growth factor binding protein family is known to be involved in regulating biological actions of insulin-like growth factors (IGFs). In the present study, a full-length cDNA encoding the IGFBP-5 gene was cloned and characterized from the cerebral ganglion of Haliotis discus hannai. The 921-bp full-length sequence of Hdh IGFBP-5 cDNA transcript had an open reading frame of 411 bp encoding a predicted polypeptide of 136 amino acids, sharing high sequence identities with IGFBP-5 of H. diversicolor. The deduced Hdh IGFBP-5 protein contained a putative transmembrane domain (13-35 aa) in the N-terminal region. It also possessed a signature domain of IGFBP protein family (IB domain, 45-120 aa). Six cysteine residues (Cys-47, Cys-55, Cys-73, Cys-85, Cys-98, and Cys-118) in this cloned sequence could potentially form an intrachain disulfide bond. Phylogenetic analysis indicated that the Hdh IGFBP-5 gene was robustly clustered with IGFBP-5 of H. diversicolor. Tissue distribution analysis based on qPCR assay showed that Hdh IGFBP-5 was widely expressed in all examined tissues, with significantly (p < 0.05) higher expression in the cerebral ganglion. In male and female gametogenetic cycles, Hdh IGFBP-5 mRNA was expressed at all stages, showing significantly higher level at ripening stage. The expression level of Hdh IGFBP-5 mRNA was significantly higher in the polar body stage than in other ontogenic stages. In situ hybridization revealed that Hdh IGFBP-5 mRNA was present in the neurosecretory cells of the cerebral ganglion. This is the first study describing IGFBP-5 in H. discus hannai that might be synthesized in the neural ganglia. Our results demonstrate Hdh IGFBP-5 is involved in regulating ontogenic development and reproductive regulation of H. discus hannai.

In vivo effects of GnRH peptide on gonadal proliferation and related gene expression of Pacific abalone, Haliotis discus hannai

Gonadotropin-releasing-hormone (GnRH)-associated peptide (GnRH-AP) is a 56 amino acid neuropeptide derived from the GnRH prohormone. GnRH-AP corresponds to the C-terminal fragment flanking the GnRH peptide. Using an antiserum raised against human GnRH-AP [1-56], or against human GnRH, we have investigated the neuronal systems containing either peptide in the central nervous system of the frog Rana ridibunda by immunohistological techniques. A main group of GnRH-AP-containing perikarya was found in a dorsoventral orientation of the supra anterior preoptic area (SAPA) just in front of the preoptic recess. Fibers originating from these perikarya projected rostrally toward the medial septal nucleus and the diagonal band of Broca. A network of GnRH-AP-immunoreactive (ir) fibers runs caudally from the SAPA toward the ventral hypothalamus. A high density of GnRH-AP-ir terminals was found in the median eminence. A few positive fibers were detected in the neural lobe of the pituitary, particularly in the region bordering the pars intermedia. Labelling of consecutive sections by either GnRH-AP or GnRH antibodies showed that GnRH and GnRH-AP-like irs were contained in the same cells of the SAPA. The double-staining technique with electrophoretic elution confirmed the colocalization of GnRH and GnRH-AP within the same neurons. Such a coexistence indicates that frog GnRH originates from a high molecular weight precursor which is closely related to rat pro-GnRH. The relative preservation of the C-terminal sequence of the pro-GnRH during evolution suggests that GnRH-AP may possess intrinsic biological activity. The high density of GnRH-AP-containing neurons projecting through the external zone of the median eminence would support the concept that GnRH-AP is involved in the modulation of pituitary hormone secretion.

Molecular characterization of four genes encoding abalone insulin-related peptides and their roles in regulation of hemolymph glucose in the Pacific abalone Haliotis discus hannai

We examined whether gonadotropin-releasing hormone (GnRH)-like peptides are present in the neural ganglia of the gastropod Pacific abalone (Haliotis discus hannai) by reverse-phase high performance liquid chromatography (rpHPLC) combined with time-resolved fluoroimmunoassay (TR-FIA) analysis and by immunohistochemistry. Cerebral ganglion extracts showed a similar retention time to lamprey GnRH-II (lGnRH-II) in rpHPLC combined with TR-FIA analysis. GnRH-like-immunoreactive (ir) cell bodies (which reacted with a mouse monoclonal antibody raised against the common amino acid sequence of vertebrate GnRH) were detected in the peripheral region of the cerebral ganglion, and they were observed to send fibers into the neuropil. GnRH-like-ir fibers were also detected in the neuropil of the pedal ganglion, the visceral nerve, and the nerve originating from the pedal ganglion. Chicken GnRH-II (cGnRH-II)-like-ir fibers (which reacted with a rabbit polyclonal antibody raised against cGnRH-II) were also observed in the neuropil of the cerebral ganglion. GnRH-like-ir fibers and cGnRH-II-like-ir fibers were distinguishable in the neuropil of the cerebral ganglion by double-staining immunohistochemistry. These results suggest that multiple GnRH-like peptides exist in the neural ganglia of the Pacific abalone.

Temperature has crucial effects on gonadal development and reproduction of abalone. To understand the impact of thermal stress on molecular and physiological processes triggering the regulation of reproduction, changes in the mRNA expression of neuroendocrine genes encoding two abalone gonadotropin-releasing hormone (Hdh-GnRH, Hdh-GnRH-like peptide), GnRH receptor (Hdh-GnRH-R), Hdh-APGWamide, serotonin receptor (5-HThdh), and a heat shock protein HSP70 were examined in ganglia and testis of male Pacific abalone (Haliotis discus hannai). Abalone were exposed to low water temperature (LWT) and high water temperature (HWT) in early and peak breeding seasons for 7 days. Then, gonadosomatic index (GSI) was calculated, relative gene expression was measured by qRT-PCR, and levels of testosterone in hemolymph were also measured by ELISA during the peak breeding season. GSI did not show any significant changes during the early breeding season. However, it was significantly decreased in LWT- or HWT-exposed abalone compared to the normal water temperature (NWT) group during the peak breeding season. In the early breeding season, changes of mRNA expression of all five genes were significant between LWT and HWT groups on day-7. In the peak breeding season, compared to the NWT group, the mRNA expressions of different genes were significantly decreased in different tissues both in LWT and HWT groups of abalone, such as Hdh-GnRH-like peptide in the cerebral ganglion (CG) and testis; Hdh-GnRH in the pleuropedal ganglion (PPG) and branchial ganglion (BG); Hdh-GnRH-R in the CG, PPG, and testis; and Hdh-APGWamide in the PPG and testis. Interestingly, the expression of 5-HThdh was significantly increased in the HWT group but decreased in the LWT group. Expression of HSP70 was significantly increased in the testis after exposure to HWT. Hemolymph levels of testosterone were significantly decreased in the HWT group compared to those in the NWT group. Altogether, these results denote that thermal stress has a repressive effect on gonadal maturation and reproduction by regulating the expression of Hdh-GnRH-like peptide, Hdh-GnRH, Hdh-GnRH-R, Hdh-APGWamide, 5-HThdh, and HSP70 genes and levels of hemolymph testosterone.

The effects of gonadotropin-releasing hormone (GnRH) and GnRH-associated peptide (GAP) on cytosolic free calcium concentration ([Ca(2+)](i)) were investigated in 20 human nonfunctioning pituitary adenomas. We divided these tumors into three classes according to their response pattern to hypothalamic peptides. In type I adenomas (8 out of 20 adenomas), GnRH and GAP mobilized intracellular calcium ions stored in a thapsigargin (TG)-sensitive store. For the same concentration of agonist, two distinct patterns of GnRH-GAP-induced Ca(2+) mobilization were observed (1) sinusoidal oscillations, and (2) monophasic transient. The latter is followed by a protein kinase C (PKC)-dependent increase in calcium influx through L-type channels. In type II adenomas (7 out of 20 adenomas), GnRH and GAP only stimulate calcium influx through dihydropyridine-sensitive Ca(2+) channels by a PKC-dependent mechanism. TG (1 μM) did not affect [Ca(2+)](i) in these cells, suggesting that they do not possess TG-sensitive Ca(2+) pools. All the effects of GnRH and GAP were blocked by an inhibitor of phospholipase C (PLC), suggesting that they were owing to the activation of the phosphoinositide turnover. Type I and type II adenoma cells showed spontaneous Ca(2+) oscillations that were blocked by dihydropyridines and inhibition of PKC activity. GnRH and GAP had no effect on the [Ca(2+)](i) of type III adenoma cells that were also characterized by a low resting [Ca(2+)](i) and by the absence of spontaneous Ca(2+) fluctuations. K(+)-induced depolarization provoked a reduced Ca(2+) influx, whereas TG had no effect on the [Ca(2+)](i) of type III adenoma cells. The variety of [Ca(2+)](i) response patterns makes these cells a good cell model for studying calcium homeostasis in pituitary cells.

Palliative effect of abalone insulin-related peptide 2 on emersion stress-induced hyperglycemia in the Pacific abalone (Haliotis discus hannai)