Shuisheng Li

Structural and functional multiplicity of the kisspeptin/GPR54 system in goldfish (Carassius auratus)

To ascertain the neuroendocrine function of the kisspeptin/GPR54 system in non-mammalian species, full-length cDNAs encoding for Kiss1 and Kiss2 as well as their putative cognate receptors GPR54a and GPR54b, were isolated from goldfish (Carassius auratus). The deduced protein sequences between Kiss1 and Kiss2 in goldfish share very low similarity, but their putative mature peptides (kisspeptin-10) are relatively conserved. RT-PCR analysis demonstrated that the goldfish kiss1 gene (gfkiss1) is highly expressed in the optic tectum-thalamus, intestine, kidney, and testis, while the goldfish kiss2 gene (gfkiss2) is mainly detected in the hypothalamus, telencephalon, optic tectum thalamus, adipose tissue, kidney, heart, and gonads. The two receptor genes (gfgpr54a and gfgpr54b) are highly expressed in the brain regions including telencephalon, optic tectum thalamus, and hypothalamus. Both mature goldfish kisspeptin-10 peptides (gfKiss1-10 and gfKiss2-10) are biologically active as they could functionally interact with the two goldfish receptors expressed in cultured eukaryotic cells to trigger the downstream signaling pathways with different potencies. The actions of gfKiss1-10 and gfKiss2-10 on LH secretion were further investigated in vitro and in vivo. Intraperitoneal administration of gfKiss1-10 to sexually mature female goldfish could increase the serum LH levels. However, this peptide does not significantly influence LH release from goldfish pituitary cells in primary culture, indicating that the peptide does not exert its actions at the pituitary level. On the other hand, gfKiss2-10 appears to be a much less potent peptide as it exhibits no significant in vivo bioactivity and is also inactive on the primary pituitary cells.

The draft genome of the grass carp (Ctenopharyngodon idellus) provides insights into its evolution and vegetarian adaptation

The grass carp is an important farmed fish, accounting for ∼16% of global freshwater aquaculture, and has a vegetarian diet. Here we report a 0.9-Gb draft genome of a gynogenetic female adult and a 1.07-Gb genome of a wild male adult. Genome annotation identified 27,263 protein-coding gene models in the female genome. A total of 114 scaffolds consisting of 573 Mb are anchored on 24 linkage groups. Divergence between grass carp and zebrafish is estimated to have occurred 49–54 million years ago. We identify a chromosome fusion in grass carp relative to zebrafish and report frequent crossovers between the grass carp X and Y chromosomes. We find that transcriptional activation of the mevalonate pathway and steroid biosynthesis in liver is associated with the grass carps adaptation from a carnivorous to an herbivorous diet. We believe that the grass carp genome could serve as an initial platform for breeding better-quality fish using a genomic approach.

Structural diversity of the GnIH/GnIH receptor system in teleost: its involvement in early development and the negative control of LH release.

Gonadotropin inhibitory hormone (GnIH), via binding to GnIH receptor (GnIHR), plays a negative role on the avian and mammalian reproductive axis by inhibiting luteinizing hormone (LH) release. However, the biological significance of the GnIH/GnIHR system in other vertebrates is controversial. To demonstrate the presence of such a system in teleost, we have identified the orthologous gnih genes in zebrafish, stickleback, medaka and Takifugu. Three orthologous genes (gnihr1, gnihr2 and gnihr3) for the gnihr were also identified in zebrafish. The zebrafish gnih precursor contains three putative LPXRFamide peptides. The three zebrafish gnihrs are typical seven transmembrane G protein-coupled receptors sharing high sequence homology with the mammalian and avian GnIHRs (GPR147). Tissue expression studies revealed that zebrafish gnih is mainly expressed in the brain, eye, testis, ovary and spleen, corroborating largely with the tissue expression patterns of the gnihrs in zebrafish. The expression patterns of gnih and its receptors at different developmental stages of zebrafish were also studied. Gnih expression first appeared in the prim-5 stage, and thereafter maintained at a relatively constant level. The three gnihrs could be detected at all embryonic stages of zebrafish and also during early development after hatching. The biological action of the teleost gnih on LH release was further investigated in goldfish in vivo. Intraperitoneal administration of the mature zebrafish gnih peptide (LPXRFa peptide-3) could significantly reduce the basal serum LH level in goldfish. These results provided the first evidence that gnih plays an important role in the negative regulation of LH release in teleost.

Molecular Identification of the Kiss2/Kiss1ra System and Its Potential Function During 17Alpha-Methyltestosterone-Induced Sex Reversal in the Orange-Spotted Grouper, Epinephelus coioides

The Kiss1/Kiss1r system is a component of the hypothalamus-pituitary-gonadal (HPG) axis, which plays a crucial role in regulating gonadotropins and gonadotropin-releasing hormone. The sex reversal process is a special reproductive phenomenon regulated by the HPG axis. To better understand the neuroendocrine mechanisms of sex reversal, cDNAs encoding kiss2 and kiss1ra have been cloned and functionally characterized from the orange-spotted grouper Epinephelus coioides, a protogynous hermaphroditic teleost. The core mature peptide (Kiss2-10) of grouper Kiss2 shared high similarity to other KISS orthologs. In phylogenetic analyses, the grouper Kiss was clustered with the teleost Kiss2 clade and termed grouper kiss2. The predicted amino acid sequence of grouper kiss1ra contained three putative glycosylation sites at its N-terminus, showing high similarity to that of other teleosts. Synthesized Kiss2-10 was able to functionally interact with Kiss1ra in cultured COS-7 cells to trigger downstream signaling. Both kiss2 and kiss1ra mRNAs were expressed in all tissues examined, with highest levels in the olfactory bulb and moderate levels in the hypothalamus among brain areas and highest levels in ovary among peripheral tissues. Intraperitoneal injection of Kiss2-10 significantly increased gnrh1 mRNA levels in hypothalamus and follicle-stimulating hormone beta (fshb) mRNA levels in the pituitary at 6 and 12 h postinjection. During the process of sex reversal induced by 17 alpha-methyltestosterone (MT), kiss2 and kiss1ra mRNA expression were significantly decreased in the first week, but kiss2 increased in the fourth week, in accordance with the expression pattern of gnrh1 mRNA in the grouper hypothalamus. This is the first description of the Kiss2/Kiss1ra system during MT-induced sex reversal in orange-spotted grouper.

Identification of a Membrane Estrogen Receptor in Zebrafish with Homology to Mammalian GPER and Its High Expression in Early Germ Cells of the Testis

Abstract To study the rapid action of estrogen on the male reproductive system in teleost, a full-length cDNA homologous to the seven-transmembrane receptor GPER of humans and rodents was cloned from the testis of zebrafish. Biological characterization of this cloned zebrafish gper was performed based on its functional expression in cultured eukaryotic cells. Saturation analysis and Scatchard plotting of [3H]-estradiol binding to plasma membranes of gper-transfected COS-7 cells and cAMP response element transactivation assay demonstrated the biological function of the cloned gper as an estrogen receptor. In addition, treatment of gper-transfected COS-7 cells with 17beta-estradiol increased the phosphorylation of MAPK3/MAPK1. However, the inactivity of Gper in the FOS promoter transactivation study indicated some functional difference between the zebrafish and human receptors. We found gper to be highly expressed in the brain and testis by RT-PCR analysis. Results of in situ hybridization demonstrated the localization of gper in specific brain regions and in early germ cells of the testis, including the spermatogonia, spermatocytes, and somatic cells such as Sertoli cells in adult male zebrafish. Subsequent RT-PCR analysis in cells derived from laser capture microdissection microscopy further confirmed the high expression of gper in early germ cells of the testis. The present study demonstrates the existence of a functionally active Gper in zebrafish and suggests a putative role in mediating the rapid action of estrogen in male reproduction.

The kiss/kissr Systems Are Dispensable for Zebrafish Reproduction: Evidence From Gene Knockout Studies

The kiss1/gpr54 signaling system is considered to be a critical regulator of reproduction in most vertebrates. However, this presumption has not been tested vigorously in nonmammalian vertebrates. Distinct from mammals, multiple kiss1/gpr54 paralogous genes (kiss/kissr) have been identified in nonmammalian vertebrates, raising the possibility of functional redundancy among these genes. In this study, we have systematically generated the zebrafish kiss1(-/-), kiss2(-/-), and kiss1(-/-);kiss2(-/-) mutant lines as well as the kissr1(-/-), kissr2(-/-), and kissr1(-/-);kissr2(-/-) mutant lines using transcription activator-like effector nucleases. We have demonstrated that spermatogenesis and folliculogenesis as well as reproductive capability are not impaired in all of these 6 mutant lines. Collectively, our results indicate that kiss/kissr signaling is not absolutely required for zebrafish reproduction, suggesting that the kiss/kissr systems play nonessential roles for reproduction in certain nonmammalian vertebrates. These findings also demonstrated that fish and mammals have evolved different strategies for neuroendocrine control of reproduction.

Evidences for the regulation of GnRH and GTH expression by GnIH in the goldfish, Carassius auratus.

Gonadotrophin-inhibitory hormone (GnIH) plays an important role in regulating of reproduction in teleosts. To clarify the mode of action of GnIH on the synthesis of gonadotropin releasing hormone (GnRH) and gonadotrophin (GtH), three GnIHR cDNAs were cloned from the goldfish brain. In situ hybridization results showed that GnIHRs were localized to the hypothalamus and pituitary. In the hypothalamus, GnIHRs were found in the NPP, NPO and NLT, whereas sGnRH neurons were reported to be located, and potentially regulated by GnIH. In the pituitary, only two GnIHRs were observed and they were localized to the PI instead of the adenohypophysis where GtH-expressing cells are localized, suggesting indirect regulation of GtH by GnIH. In vivo, intraperitoneal (i.p.) injections of synthetic goldfish GnIH-II peptide and GnIH-III peptide significantly decreased sGnRH and FSHβ mRNA levels. Only GnIH-II decreased LHβ mRNA levels significantly. In vitro, both GnIH-II and GnIH-III showed no effect on GtH synthesis, but an inhibition of GnRH-stimulated LHβ and FSHβ synthesis was observed when GnIH-III was applied to primary pituitary cells in culture. Thus, GnIH could contribute to the regulation of gonadotropin in the brain and pituitary in teleosts.

Orange-spotted grouper (Epinephelus coioides) toll-like receptor 22: Molecular characterization, expression pattern and pertinent signaling pathways

The toll-like receptors (TLRs) are an important gene family in host innate immunologic surveillance. The TLR22 gene is an essential member of the TLRs that is only found in aquatic animals and has been detected in some bony fish. Here, a TLR22 homolog, EcTLR22, was characterized in the orange-spotted grouper (Epinephelus coioides) via homology cloning. The 3321 bp full-length cDNA sequence of EcTLR22 was obtained, which included an open reading frame of 2880 bp encoding a putative peptide of 960 amino acids containing three highly typical domains with the characteristics of TLR family members. The deduced amino acid sequence of EcTLR22 showed a relatively high similarity to flounder TLR22. Phylogenetic analysis showed that the orange-spotted grouper TLR22 sequence was clustered with those of Perciforme, such as flounder and croaker. Real-time quantitative PCR analysis revealed broad expression of EcTLR22, with relatively high expression detected in the head kidney, trunk kidney, spleen, peripheral blood leukocytes (PBLs) and heart of orange-spotted grouper. After injection with Vibrio alginolyticus, there was significant up-regulation of the expression of EcTLR22 in the spleen. In evaluating unstimulated/stimulated head kidney leukocytes and spleen leukocytes, a significant increase in EcTLR22 mRNA expression was detected, which implied a sensitive immune response. Furthermore, four important molecules for signal transduction, MyD88, TRIF, TNF-α and IRF3, were chosen to analyze the role of the EcTLR22 signaling pathway in anti-pathogen responses. Upon LPS or Poly I:C challenge, expression of the four genes was induced, with an increasing tendency detected in head kidney leukocytes, suggesting that the four genes might work with EcTLR22 in host defense against pathogenic microbes.

The evolution of somatostatin in vertebrates.

Somatostatins (SS) play important roles in the regulation of growth in vertebrates. In the present study, we identified six SS genes in zebrafish and named them SS1, SS2, SS3, SS4, SS5 and SS6. We subsequently found that five SS genes (SS1, SS2, SS3, SS4 and SS5) also existed in stickleback, medaka, Takifugu and Tetraodon. Phylogenetic analysis showed that vertebrate SS genes were grouped into five clades. Using a comparative genomic approach, we further investigated the evolutionary origin of these SS genes in vertebrates, and the results revealed that: (1) SS1, SS2 and SS5 were generated by two rounds of genome duplications (2R) that happened during the early stages of vertebrate evolution; (2) SS4 is an SS1 paralog generated by a third genome duplication (3R) that occurred to most teleost fish; and (3) SS3 and SS6 were produced by tandem duplication of SS1 and SS2 in teleost fish. RT-PCR analysis revealed that all six SS genes were functionally expressed in different zebrafish tissues. These data indicate that both genome-wide duplication and local duplication contribute to the expansion of SS genes in vertebrates.

The complete mitochondrial genome of the Trachinotus ovatus (Teleostei, Carangidae)

Abstract We present the complete mitochondrial genome of the Trachinotus ovatus in this study. The mitochondrial genome is 16,563 bp long and consists of 13 protein-coding genes, two rRNA genes, 22 tRNA genes and a control region. The gene order and composition of T. ovatus mitochondrial genome was similar to that of most other vertebrates. The nucleotide compositions of the light strand are 29.03% of A, 28.86% of C, 26.23% of T and 15.88% of G. With the exception of ND6 and eight tRNA genes, all other mitochondrial genes are encoded on the heavy strand. Two copies of tandem repeat sequence (56 bp) was observed in the 5′ end of the control region.