Bao Shi

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-17β (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.

Molecular Characterization of Three Gonadotropin Subunits and Their Expression Patterns during Ovarian Maturation in Cynoglossus semilaevis

The endocrine regulation of reproduction in a multiple spawning flatfish with an ovary of asynchronous development remains largely unknown. The objectives of this study were to monitor changes in mRNA expression patterns of three gonadotropin hormone (GTH) subunits (FSHβ, LHβ and CGα) and plasma GTH levels during ovarian maturation of half-smooth tongue sole Cynoglossus semilaevis. Cloning and sequence analysis revealed that the cDNAs of FSHβ, LHβ and CGα were 541, 670 and 685 bp in length, and encode for peptides of 130, 158 and 127 amino acids, respectively. The number of cysteine residues and potential N-linked glycosylation sites of the flatfish GTHs were conserved among teleosts. However, the primary structure of GTHs in Pleuronectiformes appeared to be highly divergent. The FSHβ transcriptional level in the pituitary remained high during the vitellogenic stage while plasma levels of FSH peaked and oocyte development was stimulated. The LHβ expression in the pituitary and ovary reached the maximum level during oocyte maturation stages when the plasma levels of LH peaked. The brain GTHs were expressed at the different ovarian stages. These results suggested that FSH and LH may simultaneously regulate ovarian development and maturation through the brain-pituitary-ovary axis endocrine system in tongue sole.

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

&NA; 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. HighlightsKiss2 increased CRE‐luc activity in COS‐7 cells expressing its cognate receptor.Kiss2 stimulated SRE‐luc activity in COS‐7 cells transfected with its cognate receptor.These stimulatory effects were reduced by inhibitors of PKA and PKC pathways, respectively.LPXRFa‐2 inhibited Kiss2‐induced CRE‐luc activity in a dose‐dependent manner.

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.

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.

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 mPRβ 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 mPRβs (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.

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 17β-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.

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

Abstract 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.