Cloning, phylogenetic and expression analysis of two MyoDs in yellowtail kingfish (Seriola lalandi).

Yellowtail kingfish (Seriola lalandi) is a pelagic piscivore distributed circumglobally. Owing to its great market value, the growth mechanism of S. lalandi, including muscle development and growth, is a hot research topic. The myoblast determination protein (MyoD) gene has been shown to play an important role in formation of myoblasts and the function of somites in fish. The open reading frame (ORF) sequences of MyoD1 and MyoD2 in S. lalandi encoded 298 and 263 amino acids possessing three common characteristic domains, respectively, containing a myogenic basic domain, a bHLH domain, and a ser-rich region (helix III). S. lalandi MyoDs shared the highest identity with the MyoDs of S. dumerili. MyoDs are highly expressed in white muscle (P < 0.05) in S. lalandi. The expression level of MyoD1 mRNA was higher than that of MyoD2 mRNA during embryonic and early developmental stages, indicating that the two MyoD isoforms may have different roles in muscle formation. Moreover, the mRNA expression of MyoDs in the brain, pituitary, liver and muscle of endocrine growth axis were analyzed in the various sizes and ages stages. The expression levels of MyoDs in the different sizes and ages of S. lalandi showed that expression of both these genes was particularly high in 400-g fish and 2-year-old fish (P < 0.05). Moreover, the increases in the mRNA expression and plasma levels of growth hormone (GH) and insulin-like growth factor (IGF-I) were accompanied by an increase in mRNA expression of MyoDs, indicating the roles of GH and IGF-I in muscle development and growth of S. lalandi. Overall, the expression profiles of genes associated with muscle development are the first step taken towards deciphering fast growth mechanism in this important Seriola fish.

Food deprivation differentially modulates gene expression of LPXRFa and kisspeptin systems in the brain-pituitary axis of half-smooth tongue sole (Cynoglossus semilaevis).

LPXRFa, also known as gonadotropin-inhibitory hormone (GnIH), and kisspeptin (Kiss) are two major hypothalamic peptides that modulate the reproductive axis of vertebrates, including teleosts. However, little information is available regarding the actions of nutritional status on the regulation of these two neuroendocrine systems in fish. Herein, we assessed the effects of starvation and refeeding on the expression of lpxrfa, kiss2 and their receptors (lpxrfa-r and kiss2r respectively) at the brain-pituitary level of half-smooth tongue sole (Cynoglossus semilaevis). Food deprivation for 4 weeks induced a rise in brain lpxrfa as well as brain and pituitary lpxrfa-r mRNA levels, and refeeding restored brain lpxrfa and lpxrfa-r expression back to normal. However, pituitary lpxrfa-r mRNA levels still remained high after 1 week of refeeding. Neither lpxrfa nor kiss2 transcripts in the pituitary were altered by fasting, but their mRNA levels increased significantly after 1 week of refeeding, and declined back to the control levels after 2 weeks of refeeding. None of brain kiss2 and kiss2r along with pituitary kiss2r transcripts were modified by the nutritional status. In summary, our results revealed an interaction between energy status and the elements of LPXRFa and Kiss systems in the brain-pituitary axis of half-smooth tongue sole. Food deprivation and refeeding differentially regulated the two systems, which provided additional evidence for the involvement of the LPXRFa and Kiss systems in the regulation of reproduction by energy balance in non-mammalian species.

New evidence for SPX2 in regulating the brain-pituitary reproductive axis of half-smooth tongue sole (Cynoglossus semilaevis).

Spexin (SPX) is an evolutionarily conserved neuropeptide, which was first identified in human proteome by data mining. Two orthologs (SPX1 and SPX2) are present in some non-mammalian species, including teleosts. It has been demonstrated that SPX1 is involved in reproduction and food intake, whereas the functional role of SPX2 is still absent in any vertebrate. The aim of the current study was to evaluate the actions of intraperitoneal injection of endogenous SPX2 peptide on the expression levels of some key reproductive genes of the brain-pituitary axis in half-smooth tongue sole. Our data showed an inhibitory action of SPX2 on brain gnih, spx1, tac3 and pituitary gthα, lhß mRNA levels. However, SPX2 had no significant effect on brain gnihr, gnrh2, gnrh3, kiss2, kiss2r, spx2 expression or pituitary gh expression. On the other hand, SPX2 induced an increase in pituitary fshß expression. Taken together, our results provide initial evidence for the involvement of SPX2 in the regulation of reproduction in vertebrates, which is in accordance with previous studies on SPX1.

Comprehensive CircRNA Profiling and Selection of Key CircRNAs Reveal the Potential Regulatory Roles of CircRNAs throughout Ovarian Development and Maturation in Cynoglossus semilaevis.

CircRNAs are novel endogenous non-coding small RNAs involved in the regulation of multiple biological processes. However, little is known regarding circRNAs in ovarian development and maturation of fish. Our study, for the first time, provides the genome-wide overview of the types and relative abundances of circRNAs in tongue sole tissues during three ovarian developmental stages. We detected 6790 circRNAs in the brain, 5712 in the pituitary gland, 4937 in the ovary and 4160 in the liver. Some circRNAs exhibit tissue-specific expression, and qRT-PCR largely confirmed 6 differentially expressed (DE) circRNAs. Gene Ontology and KEGG pathway analyses of DE mRNAs were performed. Some DE circRNA parental genes were closely associated with biological processes in key signalling pathways and may play essential roles in ovarian development and maturation. We found that the selected circRNAs were involved in 10 pathways. RNase R digestion experiment and Sanger sequencing verified that the circRNA had a ring structure and was RNase R resistant. qRT-PCR results largely confirmed differential circRNA expression patterns from the RNA-seq data. These findings indicate that circRNAs are widespread in terms of present in production-related tissues of tongue sole with potentially important regulatory roles in ovarian development and maturation.

LPXRFa and its receptor in yellowtail kingfish (Seriola lalandi): Molecular cloning, ontogenetic expression profiles, and stimulatory effects on growth hormone and gonadotropin gene expression.

Despite its functional significance in mammals and birds, the biological role of gonadotropin-inhibitory hormone (GnIH) in reproduction is still far from being fully understood in teleosts. In the current study, we have identified LPXRFa, the piscine ortholog of GnIH, and its cognate receptor (LPXRFa-R) in yellowtail kingfish (YTK), which is considered as a promising species for aquaculture industry worldwide. The YTK cDNA sequence of lpxrfa was 534 base pair (bp) in length and encoded a 178-amino acids (aa) preprohormone. The LPXRFa precursor comprised three putative peptide sequences that included -MPMRF, -MPQRF, or -LPERL motifs at the C-termini, respectively. The YTK lpxrfa-r cDNA sequence was composed of 1265 bp that gave rise to a LPXRFa-R of 420 aa, encompassing the characteristic seven hydrophobic transmembrane domains. In males, both lpxrfa and lpxrfa-r transcripts could be detected at high levels in the brain and testis. In females, a noteworthy expression of lpxrfa was observed in the brain and ovary, while the expression of lpxrfa-r was especially evident only in the brain. To study the ontogeny of LPXRFa system, transcript levels were also investigated during early life stages. Variable expression of the LPXRFa system was observed during all stages of YTK embryogenesis. The highest expression of lpxrfa and lpxrfa-r were noticed at 7 dph and 15 dph, respectively. Furthermore, LPXRFa peptides stimulated growth hormone (gh), luteinizing hormone (lhß) and follicle-stimulating hormone (fshß) gene expression from the pituitary. Taken together, our results provide initial evidence for the existence of the LPXRFa system in yellowtail kingfish and suggest its possible involvement at early development and reproductive functions.

A Chromosome-Level Genome Assembly of Yellowtail Kingfish (Seriola lalandi).

Yellowtail kingfish (Seriola lalandi) is a pelagic marine piscivore with a circumglobal distribution. It is particularly suitable for open ocean aquaculture owing to its large body size, fast swimming, rapid growth, and high economic value. A high-precision genome is of great significance for future genetic breeding research and large-scale aquaculture in the open ocean. PacBio, Illumina, and Hi-C data were combined to assemble chromosome-level reference genome with the size of 648.34 Mb (contig N50: 28.52 Mb). 175 contigs was anchored onto 24 chromosomes with lengths ranging from 12.28 to 34.59 Mb, and 99.79% of the whole genome sequence was covered. The BUSCOs of genome and gene were 94.20 and 95.70%, respectively. Gene families associated with adaptive behaviors, such as olfactory receptors and HSP70 gene families, expanded in the genome of S. lalandi. An analysis of selection pressure revealed 652 fast-evolving genes, among which mkxb, popdc2, dlx6, and ifitm5 may be related to rapid growth traits. The data generated in this study provide a valuable resource for understanding the genetic basis of S. lalandi traits.

Molecular identification and developmental expression patterns of growth hormone and its receptors in yellowtail kingfish (Seriola lalandi).

In fish and other vertebrates, growth hormone (GH) is an essential polypeptide required for normal growth and development. In an attempt to understand growth regulation in yellowtail kingfish (YTK), the full-length cDNA sequences encoding gh and its receptors (ghr1 and ghr2) were cloned, characterized and the expression profiles of these three genes were investigated during embryonic development. The full-length cDNA sequences of GH and its receptors were obtained by RT-PCR combined with RACE methord. YTK gh cDNA sequence was 852 base pairs (bp) that comprised an open reading frame (ORF) of 615 bp encoding a 204-amino acids (aa) precursor. The preprohormone compassed a signal peptide (17 aa) and the mature peptide (187 aa). YTK GHR1 protein consisted of a signal peptide (28 aa), an extracellular domain (222 aa), a single transmembrane domain (23 aa) and an intracellular domain (361 aa). GHR2 protein included 18 aa, 223 aa, 23 aa, and 321 aa, respectively. Tissue distribution analysis showed that the maximal level of gh expression was observed in the pituitary, and ghr1 mRNA was mainly detected in the liver, while ghr2 transcripts were most abundant in the gonad. Moreover, both ghr1 and ghr2 mRNAs were expressed in all embryonic stages and displayed different gene expression profiles. Overall, these results provide initial evidences for the involvement of the GH/GHR system in the early ontogeny of yellowtail kingfish.

Temporal expression profiles of leptin and its receptor genes during early development and ovarian maturation of Cynoglossus semilaevis.

Leptin (Lep) plays a key role in the regulation of food intake and energy homeostasis in vertebrates. Our previous studies have provided evidence for the existence of two leptin genes (lepa and lepb) and one leptin receptor (lepr) gene in a flatfish, the half-smooth tongue sole (Cynoglossus semilaevis). However, the spatial-temporal expression patterns and possible roles of the leptin system during early development and ovarian maturation are still poorly understood in teleosts. In the current study, we evaluated dynamic expression profiles of lepa, lepb, and lepr mRNAs during various developmental stages in this species. Quantitative RT-PCR analysis indicated that both ligand (lepa and lepb) and receptor (lepr) genes were detected in unfertilized eggs and during embryogenesis but with different expression profiles. In addition, lepa, lepb, and lepr transcripts levels increased significantly during larval development, reaching the peak at 10, 25, and 30 days post-hatching (dph), respectively. On the other hand, changes in mRNA expression of these three genes at the brain-pituitary-gonad (BPG) axis were also investigated during ovarian maturation, and lepa, lepb, and lepr mRNAs varied greatly. Taken together, our results encompass the first study reporting the dynamic expression patterns of leptin and its receptor mRNAs in the order Pleuronectiformes, providing evidence that the leptin system could be functional and play important roles during early development and ovarian maturation in tongue sole.

In vitro effects of tongue sole LPXRFa and kisspeptin on relative abundance of pituitary hormone mRNA and inhibitory action of LPXRFa on kisspeptin activation in the PKC pathway.

Results of previous studies indicated the existence of LPXRFa, the piscine ortholog of gonadotropin-inhibitory hormone (GnIH), and kisspeptin (Kiss2) in tongue sole (Cynoglossus semilaevis), and that LPXRFa exerts an inhibitory effect on Kiss2 activation in the protein kinase A (PKA) pathway. The functions in the control of reproduction and whether LPXRFa antagonizes the action of Kiss2 by inhibiting the protein kinase C (PKC) pathway, however, are still unknown. In the present study, there was an initial investigation of the direct effects of LPXRFa and Kiss2 on relative abundance of pituitary hormone mRNA transcripts using a whole pituitary culture system. Results indicated that LPXRFa-1 specifically functioned to increase relative abundance of lhß mRNA when there were comparisons with the control, without any effect on relative abundance of gh, gthα and fshß mRNA. Treatment with LPXRFa-2 resulted in a reduction in relative abundance of gthα and lhß mRNA, and did not alter relative abundance of fshß mRNA. Treatment of LPXRFa-2 resulted in a greater relative abundance of gh mRNA. Treatment with Kiss2, however, resulted in an increase in relative abundance of gthα and fshß mRNA transcripts, without altering relative abundances of gh and lhß mRNA. Subsequently, there was valuation of the potential interaction between LPXRFa and kisspeptin in COS-7 cells transfected with the cognate receptors. Both LPXRFa-1 and LPXRFa-2 suppressed serum responsive element-dependent luciferase (SRE-luc) activity when compared to stimulation with Kiss2 alone, indicating an inhibitory effect of LPXRFa on kisspeptin activation on the PKC pathway. Overall, data from the present study provide novel evidence for differential actions of LPXRFa and kisspeptin on pituitary hormone synthesis as well as for the interaction between LPXRFa and kisspeptin systems in teleosts.

Molecular characterization and expression profiles of insulin-like growth factors in yellowtail kingfish (Seriola lalandi) during embryonic development.

In this study, to understand the role of the insulin-like growth factor (IGF) system in the regulation of early development in yellowtail kingfish (YTK, Seriola lalandi), an economically important marine fish species with a high potential for aquaculture, we first cloned the full-length cDNAs for igf1 and igf2 from the liver. YTK igf1 cDNA was 1946 base pairs (bp) in length with an open reading frame (ORF) of 558 bp encoding preproIGF1 of 185 amino acids (aa). The preproIGF1 consisted of 44 aa for the signal peptide, 68 aa for the mature peptide comprising B, C, A, and D domains, and 73 aa for the E domain. YTK igf2 cDNA had an ORF of 648 bp that encoded a total of 215 aa spanning the signal peptide (47 aa), the mature peptide (70 aa), and the E domain (98 aa). At the protein level, both YTK IGF1 and IGF2 exhibited high sequence identities with their corresponding fish counterparts, respectively. Subsequently, quantitative RT-PCR analysis indicated that the highest level of igf1 mRNA expression was recorded in the gonad and liver, while the igf2 mRNA expression was most abundant in the gill and liver. In addition, both igf1 and igf2 were detected in all stages of embryonic development and exhibited different gene expression patterns, supporting that IGF1 and IGF2 could be functional and play important roles during YTK embryogenesis. Overall, this initial study of IGF1 and IGF2 provides an insight into the endocrine mechanism involved in the early development of yellowtail kingfish.

Recent studies of LPXRFa receptor signaling in fish and other vertebrates.

The hypothalamo-pituitary-gonadal (HPG) axis plays a major role in coordinating the reproduction of fish and other vertebrates. Gonadotropin-releasing hormone (GnRH) is the primary stimulatory factor responsible for the hypothalamic control of gonadotropin secretion. In 2000, a previously unidentified hypothalamic neuropeptide was isolated from the brain of Japanese quail and termed gonadotropin-inhibitory hormone (GnIH) based on its ability to directly inhibit gonadotropin release from the cultured quail anterior pituitary gland. One year later, the cDNA sequence that encodes the quail GnIH precursor polypeptide was cloned and was found to encompass two further peptides (GnIH-related peptide (RP)-1 and GnIH-RP-2) besides GnIH. To date, GnIH orthologous have been detected in a variety of vertebrates from fish to humans. These peptides possess a characteristic-LPXRFa (X = L or Q) motif at the C-terminus and are designated as LPXRFa peptides. It is generally accepted that LPXRFa peptides act on GnRH neurons in the hypothalamus to inhibit gonadotropin synthesis and release in addition to affecting the pituitary function in birds and mammals. However, the exact physiological role of LPXRFa is still uncertain in fish and dual actions of LPXRFa on the HPG axis have been observed. Research aiming to elucidate the detailed signaling pathways mediating the actions of LPXRFa on target cells may contribute to understanding the functional divergence of the LPXRFa system in teleosts. Accordingly, this review will discuss the recent advances in LPXRFa receptor signaling, as well as the potential interactions on cell signaling induced by other factors, such as GnRH and kisspeptin.

Leptin and leptin receptor genes in tongue sole (Cynoglossus semilaevis): Molecular cloning, tissue distribution and differential regulation of these genes by sex steroids.

Leptin (Lep) is a key factor for the regulation of food intake and energy homeostasis in mammals. To date, a number of studies have provided evidence for the existence of multiple leptin genes in teleosts, but not much information is available in fish regarding the regulation of leptin genes by sex steriods. As a first step, two leptin genes (lepa and lepb) and a leptin receptor (lepr) gene were cloned from the half-smooth tongue sole (Cynoglossus semilaevis), a representative species of the order Pleuronectiformes. The full-length cDNAs of tongue sole lepa and lepb were 1265 bp and 1157 bp in length, encoding for proteins of 160 aa and 158 aa, respectively. The three-dimensional structures modeling of tongue sole LepA and LepB showed strong conservation of tertiary structure with other vertebrates. The full-length cDNA of tongue sole lepr was 4576 bp, encoding a protein of 1133 aa which contained all functionally important domains conserved among vertebrate LepRs. Tissue distribution analysis showed that tongue sole lepa mRNA was highly detectable in the ovary and brain, while lepb mRNA was ubiquitously expressed in various tissues. Notably, the tongue sole lepr mRNA was most abundant in the ovary. Using a primary hepatocyte culture system, we evaluated the effects of sex steroids on lep/lepr gene expression. Both 17ß-estradiol (E2) and testosterone (T) inhibited hepatic lepa and lepr mRNAs without affecting lepb mRNA levels. In addition, T also suppressed growth hormone receptor 1 (ghr1), ghr2, and insulin-like growth factor 2 (igf-2) mRNA levels, and stimulated expression of igf-1 gene. On the other hand, none of these four genes were altered by E2. To the best of our knowledge, this is the first description of a direct and differential regulation of lep/lepr gene expression by sex steroids at the hepatocyte level of a flatfish, supporting that individual leptin peptide may possess different biological roles in teleosts.

Characterization of LPXRFa receptor in the half-smooth tongue sole (Cynoglossus semilaevis): Molecular cloning, expression profiles, and differential activation of signaling pathways by LPXRFa peptides.

Gonadotropin-inhibitory hormone (GnIH), a novel hypothalamic neuropeptide, serves as a key player in the regulation of reproduction across vertebrates, acting on the brain and pituitary to modulate reproductive physiology and behavior. However, little information is available in teleosts regarding the intracellular signal transduction pathway in response to GnIH. To this end, we first cloned the gene of LPXRFa (the piscine ortholog of GnIH) receptor in the half-smooth tongue sole (Cynoglossus semilaevis), a representative species of the order Pleuronectiformes. The full-length cDNA of LPXRFa receptor was 2201 bp in size with an open reading frame (ORF) of 1365 bp that encoded 454 amino acids. Tissue distribution showed that LPXRFa receptor transcripts could be detected at high levels in the brain, to a lesser extent in the pituitary, and at low levels in the ovary and other peripheral tissues. In vitro functional analysis revealed that putative tongue sole LPXRFa-1 and LPXRFa-2 peptides significantly stimulated serum responsive element-dependent luciferase (SRE-luc) activity in COS-7 cells transfected with the novel receptor, and these stimulatory effects were evidently reduced by two inhibitors of the PLC/PKC pathway. In addition, neither LPXRFa-1 nor LPXRFa-2 altered the cAMP-responsive element (CRE)-luc activity, but only LPXRFa-2 could markedly decrease forskolin-induced CRE-luc activity in COS-7 cells expressing its cognate receptor. Taken together, our results encompass the first study reporting the existence of LPXRFa receptor in the order Pleuronectiformes and provide novel evidence of differential activation of signaling pathways by LPXRFa peptides in fish.

Molecular characterization and expression profiles of LPXRFa at the brain-pituitary-gonad axis of half-smooth tongue sole (Cynoglossus semilaevis) during ovarian maturation.

Gonadotropin-inhibitory hormone (GnIH) has been characterized by its ability to inhibit either basal or gonadotropin-releasing hormone (GnRH)-induced gonadotropin synthesis and release in birds and mammals. However, the physiological role of GnIH on the reproductive axis in fish remains inconclusive, with most studies focusing on the orders Cypriniformes and Perciformes. To gain insight into the role of GnIH in the regulation of reproduction in the order Pleuronectiformes, we first cloned the LPXRFa gene, the piscine ortholog of GnIH, in the half-smooth tongue sole. The full-length cDNA of LPXRFa was 918bp in size with an open reading frame (ORF) of 585bp that encoded a 194 amino acids preprohormone with a calculated molecular mass and isoelectric point of 21.73kDa and 6.52, respectively. The LPXRFa precursor encoded two putative peptide sequences that included -MPMRF or -MPQRF motifs at the C-terminal. Tissue distribution analysis showed that LPXRFa transcripts could be detected at high levels in the brains of both sexes and to a lesser extent in the ovary, heart and stomach of females, while a noteworthy expression was observed in the kidney and muscle of males. Furthermore, the expression patterns of LPXRFa mRNA during ovarian maturation were also investigated. In the brain, the mRNA expression of LPXRFa increased significantly at stage III, declined at stage V and reached a maximum at stage VI. In the pituitary, the levels of LPXRFa mRNA remained stable during ovarian maturation and increased significantly to the top level at stage V and then declined back to basal levels. In contrast, the ovarian LPXRFa mRNA levels declined sharply at stage III and remained depressed over the course of ovarian maturation. Taken together, our results provide further evidence for the existence of LPXRFa in the order Pleuronectiformes and suggest its possible involvement in the regulation of reproduction in the female tongue sole.

Molecular characterization of kiss2 and differential regulation of reproduction-related genes by sex steroids in the hypothalamus of half-smooth tongue sole (Cynoglossus semilaevis).

Kisspeptin (Kiss) plays a critical role in mediating gonadal steroid feedback to the gonadotropin-releasing hormone (GnRH) neurons in mammals. However, little information regarding the regulation of kisspeptin gene by sex steroids is available in teleosts. In this study, we examined the direct actions of estradiol (E2) and testosterone (T) on hypothalamic expression of kisspeptin and other key factors involved in reproductive function of half-smooth tongue sole. As a first step, a partial-length cDNA of kiss2 was identified from the brain of tongue sole and kiss2 transcript levels were shown to be widely expressed in various tissues, notably in the ovary. Then, the actions of sex steroids on kiss2 and other reproduction-related genes were evaluated using a primary hypothalamus culture system. Our results showed that neither kiss2 nor its receptor kiss2r mRNA levels were significantly altered by sex steroids. Moreover, sex steroids did not modify hypothalamic expression of gonadotropin-inhibitory hormone (gnih) and its receptor gnihr mRNAs, either. However, E2 markedly stimulated both gnrh2 and gnrh3 mRNAs levels. Overall, this study provides insights into the role of sex steroids in the reproductive function of Pleuronectiform teleosts.

Inhibitory action of tongue sole LPXRFa, the piscine ortholog of gonadotropin-inhibitory hormone, on the signaling pathway induced by tongue sole kisspeptin in COS-7 cells transfected with their cognate receptors.

Kisspeptin (Kiss) acts as a positive regulator of reproduction by acting on gonadotropes and gonadotropin-releasing hormone (GnRH) neurons. Despite its functional significance, the intricate web of intracellular signal transduction pathways in response to Kiss is still far from being fully understood in teleosts. Accordingly, we investigated the molecular mechanism of Kiss action and its possible interaction with LPXRFa signaling in this study. In vitro functional analysis revealed that synthetic tongue sole Kiss2 decapeptide increased the cAMP responsive element-dependent luciferase (CRE-luc) activity in COS-7 cells transfected with its cognate receptor, while this stimulatory effect was markedly reduced by two inhibitors of the adenylate cyclase (AC)/protein kinase A (PKA) pathway. Similarly, Kiss2 also significantly stimulated serum responsive element-dependent luciferase (SRE-luc) activity, whereas this stimulatory effect was evidently attenuated by two inhibitors of the phospholipase C (PLC)/protein kinase C (PKC) pathway. In addition, LPXRFa-2 suppressed Kiss2-elicited CRE-luc activity in a dose-dependent manner. Taken together, Kiss2 utilizes both AC/PKA and PLC/PKC pathways to exert its functions via its cognate receptor and LPXRFa may antagonize the action of Kiss2 by inhibiting kisspeptin signaling. As far as we know, this study is the first to characterize the half-smooth tongue sole kisspeptin and LPXRFa signaling pathway in COS-7 cells transfected with their cognate receptors and provides novel information on the interaction between LPXRFa system and kisspeptin system in teleosts.

Molecular characterization of Kiss2 receptor and in vitro effects of Kiss2 on reproduction-related gene expression in the hypothalamus of half-smooth tongue sole (Cynoglossus semilaevis).

Kisspeptin (Kiss) and its receptor, KissR (previously known as GPR54), play a critical role in the control of reproduction and puberty onset in mammals. Additionally, a number of studies have provided evidence of the existence of multiple Kiss/KissR systems in teleosts, but the physiological relevance and functions of these kisspeptin forms (Kiss1 and Kiss2) still remain to be investigated. To this end, we examined the direct actions of Kiss2 on hypothalamic functions in the half-smooth tongue sole (Cynoglossus semilaevis), a representative species of the order Pleuronectiformes. As a first step, the full-length cDNA for kiss2r was identified and kiss2r transcripts were shown to be widely expressed in various tissues, notably in the brain of tongue sole. Then, the effects of Kiss2 decapeptide on reproduction-related gene expression were evaluated using a primary hypothalamus culture system. Our results showed that neither gnrh2 nor gnrh3 mRNA levels were altered by Kiss2. However, Kiss2 significantly increased the amounts of gnih and kiss2 mRNAs. In contrast, Kiss2 elicited an evident inhibitory effect on both gnihr and kiss2r mRNA levels. To the best of our knowledge, this is the first description of a direct and differential regulation of reproduction-related gene expression by Kiss2 at the hypothalamus level of a teleost fish. Overall, this study provides novel information on the role of Kiss2/Kiss2R system in the reproductive function of teleosts.

Evidences for involvement of growth hormone and insulin-like growth factor in ovarian development of starry flounder (Platichthys stellatus).

Although gonadotrophins are major regulators of ovarian function in teleosts and other vertebrates, accumulating evidence indicates that the growth hormone (GH)-insulin-like growth factor (IGF) axis also plays an important role in fish reproduction. As a first step to understand the physiological role of the GH-IGF system in the ovarian development of starry flounder (Platichthys stellatus), the expression profiles of GH and IGF messenger RNAs (mRNAs) and plasma GH, IGF-I, estradiol-17ß (E2), and testosterone (T) levels during the ovarian development were investigated. The developmental stages of ovaries were divided into five stages (II, III, IV, V, and VI) by histological analysis. The hepatosomatic index (HSI) and gonadosomatic index (GSI) values increased and peaked at stage IV and stage V, respectively, and then declined at stage VI. Pituitary GH mRNA levels decreased sharply at stage III and raised to top level at stage VI. The hepatic IGF-I mRNA levels ascended to maximum value at stage V and then declined significantly at stage VI. However, the hepatic IGF-II mRNA levels remained stable and increased significantly at stage VI. In contrast, the ovarian IGF-I mRNA levels increased gradually and peaked at stage VI. The ovarian IGF-II mRNA levels were initially stable and increased significantly at stage V until the top level at stage VI. Consistent with the pituitary GH mRNA levels, plasma GH levels reduced sharply at stage III and remained depressed until stage V and then raised remarkably at stage VI. Plasma IGF-I level peaked at stage V and then declined to initial level. Plasma E2 level peaked at stage IV and then dramatically descended to the basal level. Plasma T level peaked at stage V and then declined significantly back to the basal level. Based on statistical analysis, significant positive correlations between hepatic IGF-I mRNA and GSI, ovarian IGF-II mRNA and hepatic IGF-II mRNA, ovarian IGF-I mRNA and ovarian IGF-II mRNA, and plasma IGF-I and plasma T were observed, respectively. These results suggest that the GH-IGF system may be involved in the ovarian development of starry flounder; GH and IGFs appear to play distinct roles in the regulation of the ovarian development in paracrine/autocrine manners. These findings extend our knowledge of the roles of the GH-IGF axis on reproduction regulation in fish.

The complete mitochondrial genome of the marbled flounder Pseudopleuronectes yokohamae (Pleuronectiformes, Pleuronectidae).

We sequenced the complete mitochondrial genome of the marbled flounder Pseudopleuronectes yokohamae collected from the Yellow Sea off China. The mitogenome comprised a 16 864-bp circular DNA molecule containing 37 genes and an AT-rich control region known as the D-loop. Phylogenetic analysis based on the mitochondrial genomes of marbled flounder indicated that P. yokohamae and Verasper variegatus are the most closely related species, which strongly supports their close phylogenetic affinity.

Identification and characterization of a progestin and adipoQ receptor (PAQR) structurally related to Paqr7 in the ovary of Cynoglossus semilaevis and its potential role in regulating oocyte maturation.

Membrane progestin receptors (mPRs) play an important role in the regulation of oocyte meiotic maturation in fish. However, details of the molecular endocrine mechanism regulating oocyte maturation in multiple spawning fish with asynchronous ovarian development remain unclear. The cDNA encoding a novel progestin and adipoQ receptor with structural similarity to mPRα (Paqr7), herein called Paqr7b, was cloned and sequenced from the ovary of half-smooth tongue sole Cynoglossus semilaevis. Phylogenetic analysis showed that Paqr7b represents an evolutionary intermediate between mPRα and mPRß and shares high homology with other similar Paqr proteins in other teleost species. However, the tongue sole Paqr7b protein showed much greater homology to teleost mPRαs (average 52%) than mPRßs (average 40%), suggesting it may have arisen from gene duplication of mPRα. paqr7b and paqr7 mRNA exhibited similar patterns of tissue expression. The mRNA and protein of Paqr7b were ubiquitously detected in all tissues analyzed, including the ovary. Moreover, in situ hybridization results revealed that paqr7b was expressed in stage V oocytes, as well as in scattered cells in the pituitary. The expression of paqr7b mRNA in brain and ovary significantly increased from ovarian development stage II to stage V (P<0.05), and was maximal at stage V, and then sharply decreased at stage VI. The transcript level of paqr7b mRNA in the pituitary also peaked at stage V (P<0.05). Treatment of tongue sole ovarian follicles with gonadotropin consistently increased the expression level of Paqr7b protein and mRNA in both a dose- and stage-dependent manner. Microinjection of tongue sole oocytes with a morpholino antisense oligonucleotide to Paqr7b blocked the progestin induction of oocyte maturation. Our findings demonstrate an important role of Paqr7b in the regulation of oocyte maturation in tongue sole and suggest the receptor may also influence other aspects of reproduction, such as pituitary function.