Danqi Lu

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.

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.

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.

Molecular cloning and functional characterization of the first non-mammalian 26RFa/QRFP orthologue in Goldfish, Carassius auratus.

In this study, we used data mining approach to predict 26RFa/QRFP precursors from fish, amphibian, reptile and avian species and subsequently cloned a 26RFa/QRFP precursor cDNA from goldfish brain based on the predicted sequences information. The goldfish 26RFa/QRFP precursor cDNA encoded a propeptide of 168 amino acids (aa) with predicted signal peptide of 30 aa at N-terminal and putative mature peptides, including 26RFa (26 aa) and 7RFa (7 aa) located at the C-terminal. Multiple sequence alignment showed almost all of the 26RFa/QRFP mature peptides possessed KGGFXFRF-amide motifs (X=G, S, A or N) at their C-terminus, and the last three residues FRF were fully conserved across vertebrates, indicating that the evolutionary pressure has exerted to conserve several C-terminal amino acid residues among the known and predicted 26RFa/QRFP precursors. Real-time PCR revealed that 26RFa/QRFP gene was expressed abundantly in goldfish hypothalamus, optic tectum-thalamus and testis. The regulation of goldfish hypothalamic 26RFa/QRFP gene expression by negative energy balance and putative role of goldfish 26RFa/QRFP in the control of luteinizing hormone (LH) release were studied. Hypothalamic 26RFa/QRFP gene expression was pronouncedly increased at 4 days after food deprivation. Furthermore, intraperitoneal (IP) injection of synthesized goldfish 26RFa/QRFP at a dose of 1 microg/g bodyweight significantly increased serum LH levels at 1h. However, LH levels were not significantly changed by IP injection of goldfish 26RFa/QRFP at lower dosage or at other time points (3 and 6 h), or by incubation of goldfish primary cell cultures. These results suggested that goldfish 26RFa/QRFP shared some similar features with its mammalian counterparts and partly exerted the regulatory function in energy homeostasis and hypothalamic-pituitary-gonadal (HPG) axis as observed in mammalian species.

Discovery of four estrogen receptors and their expression profiles during testis recrudescence in male Spinibarbus denticulatus

Estrogen plays an important role in male reproduction. Most of the actions are mediated by estrogen receptor (ER). To investigate the profile of estrogen affecting male fertility, we firstly cloned four ERs from the male Spinibarbus denticulatus, a local economically important cyprinid fish in China. Phylogenetic tree analysis ranked the four sdERs as two distinct groups of ERalpha and beta, which could be further divided into duplicated isoforms 1 and 2, respectively. High score identities were shared between each of the duplicated isoforms. All of the four sdERs distributed in central nervous system of male fish with a quite broad spectrum. However, distribution diversity became evident between sdERalpha and sdERbeta subtypes in the peripheral tissues. Both of the two isoforms of ERbeta were detected in all seven tissues examined, while expression of sdERalpha1 was mainly limited to liver, kidney, testis and intestine and sdERalpha2 was confined to liver, heart, kidney, testis and gill. During the testis recrudescing stages, serum concentration of luteinizing hormone (LH), testosterone (T) and estradiol-17beta (E(2)) were increasing. T and LH levels in the circulation were high until the later fully recrudesced phase, while serum E(2) level was low all the time. Quantitative real-time RT-PCR analysis determined the most abundance of sdERs in pituitary where the two sdERalpha isoforms positively expressed with testis development, while sdERbeta isoforms expressed with a reverse pattern. sdERalpha1 and sdERbeta1 were the primary forms in testis. sdERalpha1 gradually increased during the recrudescence process while sdERbeta1 firstly decreased during the recrudescing stage and then positively expressed in fully recrudesced stage. Little or no signal was detected in brain. The present work provided evidence of four sdERs in male reproductive system and suggested an important role of sdERalpha1 during testis recrudescence. Pituitary contained duplicates forms of sdERalpha which may play a role in the feedback effects of estrogen on LH secretion.

Molecular cloning, characterization and expression profiles of three estrogen receptors in protogynous hermaphroditic orange-spotted grouper (Epinephelus coioides)

Estrogen plays key roles in vertebrate reproductive system via estrogen receptors (ERs) as mediating pathways. In the present study, three full-length ERs cDNA sequences were isolated from a protogynous teleost, the orange-spotted grouper (Epinephelus coioides), and were 2235bp for gERα, 1967bp for gERβ1 and 2158bp for gERβ2, respectively. Phylogenetic and amino acid alignment analyses showed that each gER was clustered in the corresponding taxonomic groups of the perciformes and exhibited high evolutional conservation in functional domains. RT-PCR revealed that gERs expressed at different levels in all the obtained tissues. gERα highly expressed in mature ovaries, gERβ1 mainly expressed in immature ovaries and gERβ2 varied greatly during ovarian development. During female to male sex reversal induced by 17α-methyltestosterone (MT) implantation, gERα decreased gradually, gERβ1 increased gradually, and gERβ2 decreased firstly and recovered subsequently in male stage. The present study speculated the potential roles of gERs during female maturation and female to male sex reversal induced by MT in the protogynous grouper E. coioides.