Huapu Chen

Molecular cloning and functional characterization of spexin in orange-spotted grouper (Epinephelus coioides).

Spexin is a newly discovered neuropeptide in vertebrates. Comprehensive comparative studies are required to unveil its biological functions. In order to ascertain the neuroendocrine function of spexin in orange-spotted grouper, its full-length cDNA and genomic DNA sequences were cloned and analyzed. Sequence analyses showed that the spexin gene structure is composed of six exons and five introns, and the amino acids of mature peptide (spexin-14) in grouper are identical to that of other fish. Tissue expression analysis found that grouper spexin is highly expressed in the brain, liver and ovary. Real time-PCR analysis demonstrated that the hypothalamic expression of spexin declined gradually during the ovarian development, and was up-regulated by food deprivation. Intraperitoneal administration of spexin-14 peptides to grouper significantly elevated the mRNA levels of proopiomelanocortin (pomc) and suppressed the orexin expression in the hypothalamus, but could not change the hypothalamic expression of gonadotropin releasing hormone 1 (gnrh1). Both in vivo and in vitro administration of spexin could not significantly influence the expression of follicle-stimulating hormone β (fshβ) and luteinizing hormone β (lhβ) in the pituitary with the exception of an inhibition of gh expression. Our data suggested that the spexin has a significant role in the regulation of energy metabolism and food intake in orange-spotted grouper.

Molecular cloning, tissue distribution, and pharmacological characterization of melanocortin-4 receptor in spotted scat, Scatophagus argus

Melanocortin-4 receptor (MC4R) plays an important role in the regulation of food intake and energy expenditure in mammals. The functions of the MC4R in fish have not been investigated extensively. We herein reported on the cloning, tissue distribution, and pharmacological characterization of spotted scat (Scatophagus argus) MC4R (SAMC4R). It consisted of a 984bp open reading frame predicted to encode a protein of 327 amino acids. Sequence analysis revealed that SAMC4R was highly homologous (>80%) at amino acid levels to several teleost MC4Rs. Phylogenetic analyses showed that SAMC4R was closely related to piscine MC4R. Using RT-PCR, we showed that in addition to brain, pituitary, and gonads, mc4r mRNA was also widely expressed in peripheral tissues of spotted scat in sexually divergent pattern. With human MC4R (hMC4R) as a control, several agonists including α-melanocyte stimulating hormone (α-MSH), [Nle(4), D-Phe(7)]-α-MSH (NDP-MSH), adrenocorticotropic hormone (ACTH) and THIQ (N-[(3R)-1,2,3,4-tetrahydroisoquinolinium3-ylcarbonyl]-(1R)-1-(4-chlorobenzyl)-2-[4-cyclohexyl-4-(1H-1,2,4-triazol-1-ylmethyl)piperidin-1-yl]-2-oxoethylamine), were used to investigate the binding and signaling properties of SAMC4R. The results showed that SAMC4R bound NDP-MSH with the highest affinity followed by ACTH (1-24) and α-MSH. Similar ranking was also found for hMC4R, although SAMC4R had two to five-fold higher affinities for these ligands. THIQ did not displace NDP-MSH from SAMC4R, different from hMC4R. α-MSH, NDP-MSH, and ACTH (1-24) were identified as potent agonists to stimulate cAMP generation followed by THIQ in SAMC4R. The availability of SAMC4R and its pharmacological characteristics will facilitate the investigation of its function in regulating diverse physiological processes in spotted scat.

Structural and functional characterization of neuromedin S in the teleost fish, zebrafish (Danio rerio)

Neuromedin S (NMS) has been demonstrated to have important roles in many vertebrate physiological processes. However, the function of NMS in teleost fishes remains unclear. We explored the physiological roles of the NMS gene in the zebrafish model. An NMS cDNA was cloned from zebrafish brain tissue, and the full-length cDNA sequence was 521 bp in length and encoded a precursor of 110 amino acid residues. Interestingly, fish prepro-NMS is predicted to generate a short 34-residue peptide, designated as NMS-related peptide (NMSRP). Zebrafish prepro-NMS does not contain the NMS peptide which is found in the NMS precursors of mammals, and just retains the MNSRP peptide. A multiple-species sequence alignment showed that NMSRPs are conserved among the other sampled vertebrates. Zebrafish NMS mRNA was detected by RT-PCR revealing a tissue-specific distribution with high levels of expression in the brain, spleen, ovary, pituitary, and muscle. Furthermore, the locations of NMS-expressing cells in the zebrafish brain were detected by in situ hybridization in the parvocellular preoptic nucleus (PPa), the ventral zone of the periventricular hypothalamus (Hv), and lateral hypothalamic nucleus (LH). The levels of NMS mRNA in the hypothalamus were significantly increased after three days of food deprivation. Administration of zebrafish NMSRP by intraperitoneal injection significantly promoted the expression of neuropeptide Y (NPY) and orexin, suggesting an orexigenic role for NMSRP in zebrafish. The present study offers a new understanding of the NMS gene in vertebrates and increases our knowledge of the neuroendocrine regulation of feeding.

Cloning, expression and functional characterization on vitellogenesis of estrogen receptors in Scatophagus argus

Estrogen receptors (Er) play a critical role in vitellogenesis. Three ers (erα, erβ1 and erβ2) and vitellogenins (vtg-A, vtg-B and vtg-C) subtypes were isolated in various fish species, while the contribution of each Er to the regulation of vtgs expression was not analyzed in detail. Here, erα, erβ1 and erβ2 were cloned and all were found to be expressed in female liver in Scatophagus argus. During proteic vitellogenesis stage, erα was simultaneously up-regulated, while erβ1 and erβ2 were not, with three vtgs in female liver. The effects of 17β-estradiol (E2) alone or combined with Er antagonists on ers, vtgs mRNA expressions and Vtg protein content in incubated male liver were examined by real-time PCR and enzyme-linked immunosorbent assay (ELISA), respectively. The expressions of erα, erβ1, vtgs mRNA and Vtg protein increased significantly after 24h incubation with E2 (0.1, 1 and 10μM), while Er nonselective antagonist ICI 182 780 (0.01, 0.1 and 1μM) significantly attenuated the up-regulation effects of E2 on ers, vtgs mRNA and Vtg protein in a dose-dependent manner. Erα selective antagonist Methyl-piperidinopyrazole (MPP) (0.01, 0.1 and 1μM) significantly attenuated the up-regulation effects of E2 on erα, vtg-B, vtg-C mRNA and Vtg protein, while promoted the expression of erβ1 and vtg-A. Erβ selective antagonist Cyclofenil (0.01, 0.1 and 1μM) attenuated the up-regulation effects of E2 on erβ1, erβ2, vtg-A, vtg-C mRNA and Vtg protein while promoted the expression of erα and vtg-B. Our results suggest that the regulation of Ers on different vtgs was divergent in S. argus.

Characterization of the complete mitochondrial genome of Priacanthus tayenus (Perciformes: Priacanthidae) with phylogenetic consideration

Abstract The complete mitochondrial genome of the Priacanthus tayenus was sequenced and analyzed in this study. The mitochondrial genome is 16,866 bp long and consists of 13 protein-coding genes, two rRNA genes, 22 tRNA genes and a control region. The gene order and the composition of P. tayenus mitochondrial genome were similar to that of most other vertebrates. The nucleotide composition of the light strand in descending order is 30.91% of G, 26. 35% of T, 25.61% of A and 17.13% of C. The NADH dehydrogenase subunit 6 (ND6) and eight tRNA genes were localized in the light strand, and all other mitochondrial genes were encoded on the heavy strand. The phylogenetic analysis by maximum-likelihood (ML) method revealed that the P. tayenus showed the closer relationship to the Sciaenops ocellatus.

Molecular cloning of the insulin-like growth factor 3 and difference in the expression of igf genes in orange-spotted grouper (Epinephelus coioides).

Insulin-like growth factor (Igf) is the key regulator for development, growth, and reproduction. In most vertebrate species, the Igf family has two forms: Igf1 and Igf2. A novel form of Igf, termed Igf3, was recently discovered in fish. In the present study, we isolated igf3 from the orange-spotted grouper (Epinephelus coioides). The orange-spotted grouper igf3 consisted of a full-length cDNA of 1014 nucleotides with an open reading frame (ORF) of 597 bp, encoding for proteins of 199 amino acid residues in length. Tissue distribution analysis showed that igf1 widely expressed with the highest expression in the pituitary and liver. igf2 was expressed highly in all the tissues except the olfactory bulb, while igf3 showed the highest expression in the ovary, and moderate expression in brain areas. The expression profiles of three igf genes during the ovarian development and growth hormone (Gh) and human chorionic gonadotropin (hCG) treatment were also investigated. Three igf genes exhibited different expression patterns during the ovarian development, and showed different responses to the Gh and hCG treatments, appearing to play distinct roles in ovarian development. The present study provides further evidence for the existence of an intraovarian Igf system in orange-spotted grouper.

Sequencing, de novo assembly and characterization of the spotted scat Scatophagus argus (Linnaeus 1766) transcriptome for discovery of reproduction related genes and SSRs

Spotted scat (Scatophagus argus) is an economically important farmed fish, particularly in East and Southeast Asia. Because there has been little research on reproductive development and regulation in this species, the lack of a mature artificial reproduction technology remains a barrier for the sustainable development of the aquaculture industry. More genetic and genomic background knowledge is urgently needed for an in-depth understanding of the molecular mechanism of reproductive process and identification of functional genes related to sexual differentiation, gonad maturation and gametogenesis. For these reasons, we performed transcriptomic analysis on spotted scat using a multiple tissue sample mixing strategy. The Illumina RNA sequencing generated 118 510 486 raw reads. After trimming, de novo assembly was performed and yielded 99 888 unigenes with an average length of 905.75 bp. A total of 45 015 unigenes were successfully annotated to the Nr, Swiss-Prot, KOG and KEGG databases. Additionally, 23 783 and 27 183 annotated unigenes were assigned to 56 Gene Ontology (GO) functional groups and 228 KEGG pathways, respectively. Subsequently, 2 474 transcripts associated with reproduction were selected using GO term and KEGG pathway assignments, and a number of reproduction-related genes involved in sex differentiation, gonad development and gametogenesis were identified. Furthermore, 22 279 simple sequence repeat (SSR) loci were discovered and characterized. The comprehensive transcript dataset described here greatly increases the genetic information available for spotted scat and contributes valuable sequence resources for functional gene mining and analysis. Candidate transcripts involved in reproduction would make good starting points for future studies on reproductive mechanisms, and the putative sex differentiation-related genes will be helpful for sex-determining gene identification and sex-specific marker isolation. Lastly, the SSRs can serve as marker resources for future research into genetics, marker-assisted selection (MAS) and conservation biology.

Leptin receptor overlapping transcript: characterization, tissue distribution and changes in gene expression in response to eyestalk ablation in the Pacific white shrimp Litopenaeus vannamei

The leptin receptor overlapping transcript (LEPROT), which arose evolutionarily before the leptin receptor, is involved in the nutritional regulation of metabolism and reproduction. Eyestalk ablation is performed on brood stock to induce rapid gonad development in the Pacific white shrimp Litopenaeus vannamei. To clarify the role of L. vannamei leprot (Lvleprot) in gonadal development, Lvleprot cDNA was isolated and a phylogenetic tree was constructed based on the LEPROT amino acid sequences of shrimp, other invertebrates and vertebrates. The resulting topology clearly reflected the taxonomic relationships of the selected organisms. Alignment of LEPROT amino acid sequences showed that there was very high similarity between LvLEPROT and LEPROT from other invertebrates. Quantitative polymerase chain reaction showed that Lvleprot expression was highest in the gonads and intestine, moderate in the hepatopancreas, androgenic gland and muscle, and lowest in gill, heart and stomach, without a sexually dimorphic pattern difference between males and females. After eyestalk ablation, the gonadosomatic index increased significantly, but the hepatosomatic index decreased significantly. In addition, Lvleprot transcript levels in the gonads and intestine decreased significantly, but were significantly increased in the hepatopancreas after eyestalk ablation. These results indicate the significant roles of Lvleprot in the nutritional regulation of reproduction during the period of rapid development in Pacific white shrimp.

Molecular cloning, characterization and functional analysis of QRFP in orange-spotted grouper (Epinephelus coioides).

The peptide QRFP plays an important role in the regulation of vertebrate feeding behavior. In this study, we cloned the full length cDNA of a QRFP precursor in a teleost fish, the orange-spotted grouper (Epinephelus coioides). Sequence analysis has shown that the functional regions of QRFP in other vertebrates (QRFP-25 and QRFP-7) are conserved in orange-spotted grouper. RT-PCR demonstrated that the pre-processed mRNA of QRFP is widely expressed in orange-spotted grouper. Three days of food deprivation did not change the hypothalamic pre-processed QRFP expression. However, QRFP expression significantly increased when the fish were reefed after three days of fasting. Intraperitoneal injection of QRFP-25 peptide to orange-spotted grouper suppressed expression of orexin, but elevated expression of pro-opiomelanocortin (POMC) in the hypothalamus. We also investigated the effects of QRFP-25 on the expression of reproductive genes. The peptide suppressed the expression of seabream-type gonadotropin-releasing hormones (sbGnRH), luteinizing hormone beta subunit (LHβ) and follicle-stimulating hormone beta subunit (FSHβ) in vivo, as well as inhibited the expression of LHβ and FSHβ in pituitary cells in primary culture. Our results indicate that QRFP may play an inhibitory role in the regulation of feeding behavior and reproduction in orange-spotted grouper.

Neurokinin B signaling in hermaphroditic species, a study of the orange-spotted grouper (Epinephelus coioides)

Neurokinin B (NKB) plays important roles in the mammalian reproductive axis by modulating the release of gonadotropin-releasing hormone (GnRH) and gonadotropins. In the present study, the tac3 cDNA was cloned from a hermaphroditic species, the orange-spotted grouper. Sequence analysis showed that the grouper Tac3 precursor encoded two tachykinin peptides, NKB and NKB-related peptide (NKBRP). Expression analysis in different tissues revealed that tac3 mRNA was highly expressed in the brain of the orange-spotted grouper. In situ hybridization further revealed that it was localized in some hypothalamic nuclei associated with reproductive regulation. During ovarian development, an increase of tac3 expression in the hypothalamus was observed at vitellogenesis stage. Intraperitoneal administration of NKB could increase the gnrh1 and lhβ mRNA levels, and enhance the serum estrogen levels, but did not significantly influence lhβ expression in cultured pituitary cells, indicating that NKB does not directly exert its actions on the pituitary gland. However, it was found that NKBRP had no effect on the expression of two gnrhs and two gths in vivo and in vitro. Effects of sex steroids on tac3 expression were further investigated. During the 17-methyltestosterone-induced sex change in the orange-spotted grouper, hypothalamic tac3 expression showed no significant change. Interestingly, ovariectomy greatly stimulated tac3 expression, while the 17β-estradiol treatment reversed this effect. In general, our data highly indicated that NKB signaling could activate the reproductive axis in the orange-spotted grouper. Our study is the first description of the NKB signaling in the hermaphroditic species.