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1.
Sci Rep ; 6: 19480, 2016 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-26806354

RESUMO

Despite identification of several sex-determining genes in non-mammalian vertebrates, their detailed molecular cascades of sex determination/differentiation are not known. Here, we used a novel RNAi to characterise the molecular mechanism of Dmy (the sex-determining gene of medaka)-mediated masculinity in XY fish. Dmy knockdown (Dmy-KD) suppressed male pathway (Gsdf, Sox9a2, etc.) and favoured female cascade (Rspo1, etc.) in embryonic XY gonads, resulting in a fertile male-to-female sex-reversal. Gsdf, Sox9a2, and Rspo1 directly interacted with Dmy, and co-injection of Gsdf and Sox9a2 re-established masculinity in XY-Dmy-KD transgenics, insinuating that Dmy initiates masculinity by stimulating and suppressing Gsdf/Sox9a2 and Rspo1 expression, respectively. Gonadal expression of Wt1a starts prior to Dmy and didn't change upon Dmy-KD. Furthermore, Wt1a stimulated the promoter activity of Dmy, suggesting Wt1a as a regulator of Dmy. These findings provide new insights into the role of vertebrate sex-determining genes associated with the molecular interplay between the male and female pathways.


Assuntos
Proteínas de Peixes/genética , Regulação da Expressão Gênica no Desenvolvimento , Oryzias/genética , Processos de Determinação Sexual/genética , Sequência de Aminoácidos/genética , Animais , Animais Geneticamente Modificados , Feminino , Proteínas de Peixes/biossíntese , Gônadas/crescimento & desenvolvimento , Masculino , Mutação , Oryzias/crescimento & desenvolvimento , Fatores de Transcrição SOX9/genética , Trombospondinas/genética , Fator de Crescimento Transformador beta/genética , Proteínas WT1/genética , Cromossomo Y/genética
2.
Sci Rep ; 3: 2862, 2013 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-24096556

RESUMO

The sexual plasticity of the gonads is not retained after the completion of sex differentiation in vertebrates, except in some hermaphroditic species. Here, we report that the depletion of estradiol-17ß (E2) by aromatase inhibitors (AI) for up to six months resulted in a functional female-to-male sex reversal in sexually-mature adults of two gonochoristic fish species, Nile tilapia and medaka. The sex-reversed fish showed a typical male pattern of E2 and androgen levels, secondary sexual characteristics, and male-like sex behavior, producing fertile sperm. Conversely, co-treatment of E2 inhibited AI-induced sex reversal. In situ hybridization of medaka gonads during AI-induced sex reversal indicated that cysts on the dorsal side of the adult ovaries are the origin of germ cells and Sertoli cells in the newly formed testicular tissue. Gonochoristic fish maintain their sexual plasticity until adulthood and E2 plays a critical role in maintaining the female phenotype.


Assuntos
Estrogênios/metabolismo , Peixes/genética , Peixes/metabolismo , Gônadas/citologia , Gônadas/metabolismo , Diferenciação Sexual/fisiologia , Animais , Comportamento Animal , Biomarcadores , Diferenciação Celular , Estradiol/metabolismo , Estradiol/farmacologia , Estrogênios/farmacologia , Feminino , Células Germinativas/citologia , Células Germinativas/metabolismo , Hormônios Esteroides Gonadais/biossíntese , Gônadas/efeitos dos fármacos , Células Intersticiais do Testículo/citologia , Células Intersticiais do Testículo/metabolismo , Masculino , Fenótipo , Reprodução , Caracteres Sexuais , Diferenciação Sexual/efeitos dos fármacos , Testículo/fisiologia
3.
Sex Dev ; 7(1-3): 115-25, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-22948719

RESUMO

Among vertebrates, fishes show an exceptional range of reproductive strategies regarding the expression of their sexuality. Fish sexualities were categorized into gonochorism, synchronous/sequential hermaphrodite, or unisexual reproduction. In gonochoristic fishes, sex is determined genetically or by environmental factors. After sex determination, the gonads are differentiated into ovary or testis, with the sex remaining fixed for the entire life cycle. In contrast, some sequential hermaphrodite fishes can change their sex from male to female (protandrous), female to male (protogynous), or serially (bi-directional sex change) in their life cycle. In many cases, sex change is cued by social factors such as the disappearance of a male or female from a group. This unique diversity in fishes provides an ideal animal model to investigate sex determination and differentiation in vertebrates. This review first discusses genetic-orientated sex determination mechanisms. Then, we address the gonadal sex differentiation process in a gonochoristic fish, using an example of the Nile tilapia. Finally, we discuss various types of sex change that occur in hermaphrodite fishes.


Assuntos
Peixes/fisiologia , Processos de Determinação Sexual/fisiologia , Diferenciação Sexual/fisiologia , Animais , Feminino , Hormônios Esteroides Gonadais/metabolismo , Gônadas/citologia , Gônadas/crescimento & desenvolvimento , Gônadas/metabolismo , Organismos Hermafroditas/fisiologia , Masculino
4.
Gene ; 509(1): 68-76, 2012 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-22835697

RESUMO

Teleostean 20ß-hydroxysteroid dehydrogenase (20ß-HSD) is involved in final oocyte maturation and steroid hormone metabolism. It has structural and functional similarities to mammalian carbonyl reductases that are involved in the metabolism of endogenous carbonyl and xenobiotic compounds. To understand the transcriptional regulation of 20ß-HSD, here we report the cloning of 20ß-HSD promoter from two fish species, rainbow trout and air-breathing catfish. Analysis of the promoter motifs, in silico identified the presence of several sites for transcription factor binding including cAMP, xenobiotic and steroid hormone responsive elements. Luciferase reporter assays with progressive deletion constructs demonstrated that 20ß-HSD type B of trout has no promoter activity while 20ß-HSD type A of trout and catfish 20ß-HSD promoters showed basal promoter activity. A TATA box flanked by a CAAT box is important for basal transcription. Deletion of cAMP responsive element in the promoter decreased basal promoter activity significantly. Reporter assays with forskolin and IBMX, drugs that increase intracellular cAMP induced the promoter activity over the basal level. Intriguingly, ß-nafthoflavone, an arylhydrocarbon receptor ligand, induced the 20ß-HSD promoter activity and is further evidenced by the induction of 20ß-HSD expression in the livers of catfish, in vivo. These results demonstrate for the first time that 20ß-HSD expression is not only modulated by cAMP but also by xenobiotics and further studies may provide significance to the ubiquitous distribution and broad substrate specificity of this enzyme.


Assuntos
Peixes-Gato/genética , Cortisona Redutase/genética , Oncorhynchus mykiss/genética , Regiões Promotoras Genéticas , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Sequência de Bases , Peixes-Gato/metabolismo , Colforsina/farmacologia , AMP Cíclico/metabolismo , DNA/genética , DNA/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Dados de Sequência Molecular , Oncorhynchus mykiss/metabolismo , Ovário/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteínas Recombinantes/genética , Especificidade da Espécie , Xenobióticos/metabolismo , beta-Naftoflavona/farmacologia
5.
Cytogenet Genome Res ; 136(2): 123-30, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22286236

RESUMO

Using the genome sequence of the medaka, Oryzias latipes, we examined the genomic complement of Fox genes in this organism to gain insight into the evolutionary relationships and expression patterns of this gene family. We identified 31 Fox genes by searching for Forkhead domains in the medaka genome and by polymerase chain reaction (PCR) using primers designed from protein alignments. All the medaka Fox genes are encoded in 18 subclasses as follows: 5 Fox genes in subclass D; 3 Fox genes in subclass O; 2 Fox genes in each of subclass A, B, E, F, G, I, P, and Q, respectively; 1 Fox gene in each of subclass C, H, J, K, M, N, and R. The gene structures and general features are also discussed. In addition, we examined the expression patterns of some of these genes in different adult tissues and during embryonic development in medaka using quantitative PCR and in situ hybridization. The present study shows that Forkhead transcription factors play an important role during early embryonic development in medaka, and the results will enhance our knowledge in terms of Fox family evolution in different taxa.


Assuntos
Proteínas de Peixes/genética , Fatores de Transcrição Forkhead/genética , Família Multigênica , Oryzias/genética , Animais , Mapeamento Cromossômico , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Evolução Molecular , Feminino , Fatores de Transcrição Forkhead/classificação , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Hibridização In Situ , Masculino , Dados de Sequência Molecular , Oryzias/embriologia , Oryzias/crescimento & desenvolvimento , Filogenia , Isoformas de Proteínas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Fatores de Tempo
6.
Cytogenet Genome Res ; 134(4): 283-94, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21720154

RESUMO

Genes of the Sox family encode evolutionarily conserved high-mobility group box containing transcription factors, which play key roles in various events of developmental contexts. In this study, we identified 15 sox genes by searching for the high-mobility group domain in the medaka genome and by polymerase chain reaction using primers designed from the results obtained from homology protein alignment. All medaka sox genes except a novel sox gene, Olsox32, are encoded in 5 groups as follows: 4 sox genes in group B; 3 sox genes in group D and F, respectively; 2 sox genes in group C and E, respectively, while no sox genes were found in groups A, G, H, I, and J. The medaka Olsox32 does not fall within any of the previously defined groups A-J. Here we have assigned it to a new group K. Together with the Sox protein sequences of other species, the phylogenetic relationship was analyzed and compared. Our findings point to recent sox gene loss, duplication and divergence occurring during the evolution of tetrapod and teleost lineages. The expression pattern shows that sox genes play a variety of roles in the early embryonic development of medaka.


Assuntos
Proteínas de Peixes/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Genoma/genética , Oryzias/genética , Fatores de Transcrição SOX/genética , Sequência de Aminoácidos , Animais , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Feminino , Proteínas de Peixes/classificação , Masculino , Dados de Sequência Molecular , Oryzias/embriologia , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOX/classificação , Homologia de Sequência de Aminoácidos
7.
J Neuroendocrinol ; 23(5): 412-23, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21332842

RESUMO

The brain of teleost fish exhibits a significant degree of sexual plasticity, even in adulthood. This unique feature is almost certainly attributable to a teleost-specific sexual differentiation process of the brain, which remains largely unknown. To dissect the molecular basis of sexual differentiation of the teleost brain, we searched for genes differentially expressed between both sexes in the medaka brain. One gene identified in the screen, cyp19a1b, which encodes the steroidogenic enzyme aromatase, was selected for further analysis. As opposed to the situation in most vertebrates, medaka cyp19a1b is expressed at higher levels in the adult female brain than the male brain. The female-biased expression in the brain is consistent regardless of reproductive or diurnal cycle. Medaka cyp19a1b is expressed throughout the ventricular zones in wide areas of the brain, where, in most regions, females have a greater degree of expression compared to males, with the optic tectum exhibiting the most conspicuous predominance in females. Contrary to what is known in mammals, cyp19a1b expression exhibits neither a transient elevation nor a sex difference in medaka embryos. It is not until just before the onset of puberty that cyp19a1b expression in the medaka brain is sexually differentiated. Finally, cyp19a1b expression in the medaka brain is not under the direct control of sex chromosome genes but relies mostly, if not solely, on oestrogen derived from the gonad. These unique properties of aromatase expression in the brain probably contribute substantially to the less rigid sexual differentiation process, thus ensuring remarkable sexual plasticity in the teleost brain.


Assuntos
Aromatase/genética , Aromatase/metabolismo , Encéfalo/fisiologia , Expressão Gênica , Oryzias/fisiologia , Caracteres Sexuais , Animais , Encéfalo/anatomia & histologia , Embrião não Mamífero/anatomia & histologia , Embrião não Mamífero/fisiologia , Feminino , Masculino , Oryzias/anatomia & histologia
8.
Sex Dev ; 5(2): 89-101, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21325793

RESUMO

The process of morphological development of a differentiated gonad from an undifferentiated primordium is a very important step of gonadogenesis. Studies on sexually dimorphic gene expression are important to increase our understanding of this process and to investigate how environmental factors such as temperature can regulate gonadal development. The aim of this study was to identify putative genes involved in sex differentiation in pejerrey (Odontesthes bonariensis) reared at male- and female-producing temperatures (MPT and FPT, respectively) using a microarray heterologous from the medaka (Oryzias latipes), a closely phylogenetic species. Genes related to numerous processes presented higher expression at MPT, including those involved in muscular contraction, metabolic pathways, developmental processes, and reproduction. Genes induced by FPT were classified under the gene ontology terms of response to stimulus, transport and proteolysis. From genes selected for validation, at MPT ndrg3 expression was observed in the somatic cells, whereas pen-2 was detected in germ cells in the caudal portion of the gonads, where no apoptotic signals were observed. Finally, hsp90 was highly expressed in somatic cells of the gonads at the FPT. The results suggest that the interplay of pro-apoptotic and anti-apoptotic genes is important during the masculinization process and for the prevention of sterility following exposure to warm temperatures.


Assuntos
Gônadas/crescimento & desenvolvimento , Gônadas/metabolismo , Smegmamorpha/crescimento & desenvolvimento , Smegmamorpha/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Feminino , Gônadas/citologia , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Organogênese/genética , Organogênese/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
9.
Gen Comp Endocrinol ; 171(1): 94-104, 2011 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-21156177

RESUMO

Tryptophan hydroxylase (tph) is the key regulator in serotonin (5-HT) biosynthesis that stimulates the release of GnRH and gonadotropins by acting at the level of hypothalamo-hypophyseal axis. In brain, 5-HT is expressed predominantly in preoptic area-hypothalamus (POA-HYP) region in teleosts. Therefore, in the present study we isolated tph2 from catfish brain to evaluate its expression pattern in male and female brains during early development. Tph2 cloned from catfish brain is 2.768 Kb in length which encodes predicted protein of 488 amino acid residues. The characterization of recombinant tph2 was done by transient transfection in CHO cells. Tissue distribution of tph2 revealed ubiquitous expression except ovary. Real time PCR analysis in discrete regions of adult male brain revealed that tph2 mRNA was abundant in the POA-HYP and optic tectum+cerebellum+thalamus (OCT) regions. Differential expression of tph2 was observed at mRNA and protein levels in the POA-HYP and OCT regions of male and female brains during development that further correlate with the 5-hydroxytryptophan (5-HTP) and 5-HT levels measured using HPLC method in these regions of male and female brains. Tph2 immunoreactive neurons were observed in different regions of brain at 50 days post hatch using catfish specific tph2 antibody. Changes in tph2 mRNA expression, 5-HTP, and 5-HT levels in the POA-HYP+OCT region of brains of methyltestosterone and para-chlorophenylalanine treated fishes during development further endorse our results. Based on our results, we propose that the serotonergic system is involved in brain sex differentiation in teleosts.


Assuntos
5-Hidroxitriptofano/metabolismo , Encéfalo/metabolismo , Serotonina/metabolismo , Diferenciação Sexual/fisiologia , Triptofano Hidroxilase/genética , Animais , Western Blotting , Encéfalo/enzimologia , Células CHO , Peixes-Gato , Cromatografia Líquida de Alta Pressão , Cricetinae , Cricetulus , Feminino , Imuno-Histoquímica , Masculino , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Diferenciação Sexual/genética
10.
Gen Comp Endocrinol ; 166(2): 320-9, 2010 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-19925800

RESUMO

Serotonin (5-HT) is well known for modulating the release of GnRH and gonadotropin in teleosts. Reports on increased female:male ratio after the blockade of 5-HT biosynthesis proposed a role for 5-HT in brain sex differentiation. Two types of tryptophan hydroxylase (Tph), rate-limiting enzyme in the biosynthesis of 5-HT were cloned from vertebrates. In the present study, we cloned Tph from brain and evaluated its importance during early development of XX and XY Nile tilapia. Tph cloned from tilapia brain is 1888 bp in length and it encodes predicted protein of 462 amino acid residues. Tph activity of tilapia was confirmed by demonstrating the conversion of L-tryptophan to 5-hydroxy tryptophan by the recombinant protein after transient transfection of this cDNA clone in COS-7 cells. Northern blot identified single transcript around 2kb in male brain. Tissue distribution of Tph revealed high abundance in brain, kidney, liver and testis. Semi-quantitative RT-PCR revealed exclusive expression of Tph in the male brain from 5 to 20 days post hatch (dph) while in the female brain, it was from 25 dph. These results were authenticated by localization of Tph transcripts in olfactory bulb-telencephalon region of 11 dph male brain using in situ hybridization. Tph immunoreactivity (-ir) was also evident in the nucleus preopticus-periventricularis area of male brain as early as 12 dph. However, Tph-ir was observed in several regions of both male and female brain without any distinction from 30 dph. Dimorphic expression pattern of Tph during early brain development around the critical period (7-21 dph) of gonadal sex determination and differentiation may implicate a role for Tph in brain sex differentiation of tilapia.


Assuntos
Encéfalo/enzimologia , Expressão Gênica , Caracteres Sexuais , Tilápia/crescimento & desenvolvimento , Tilápia/metabolismo , Triptofano Hidroxilase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Células COS , Chlorocebus aethiops , Clonagem Molecular , DNA Complementar/genética , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Bulbo Olfatório/enzimologia , RNA Mensageiro/análise , Proteínas Recombinantes/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Telencéfalo/enzimologia , Triptofano Hidroxilase/análise , Triptofano Hidroxilase/química
11.
Heredity (Edinb) ; 105(3): 247-56, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19997127

RESUMO

Chromosomal sex determination is widely used by vertebrates, however, only two genes have been identified as master sex-determining genes: SRY/Sry in mammals and DMY in the teleost medaka. Transfer of both genes into genetically female (XX) individuals can induce male development. However, transgenic strains have not been established in both cases because of infertility of the transgenic founders in mammals and low germline transmission rates in medaka. In this study, we used a BAC clone containing DMY in a 117 kb genomic region and two types of fluorescent marker to establish two DMY-transgenic medaka strains. In these strains, exogenous DMY is completely linked to a male phenotype and early gonadal development is not different from that of the wild-type strain. Sex-linkage analysis showed that the exogenous DMY was located on linkage group (LG) 23 in one strain and on LG 5 in the other strain, whereas the sex chromosome in medaka is on LG 1. Real-time PCR analysis indicated that these strains have multiple copies of DMY and higher DMY expression levels than the wild-type strain. These results showed that LGs 23 and 5 function as sex chromosomes in the two strains, respectively. This is not only the first example of the artificial generation of heritable sex chromosomes in vertebrates but also the first evidence showing plasticity of homomorphic sex chromosomes. This plasticity appears to be a characteristic of lower vertebrates and the underlying cause of frequent sex chromosome switching, recently reported in several fish and frog species.


Assuntos
Cromossomos Artificiais , Proteínas de Peixes/genética , Genes sry/genética , Oryzias/genética , Cromossomos Sexuais/genética , Animais , Animais Geneticamente Modificados , Dosagem de Genes , Oryzias/crescimento & desenvolvimento
12.
Sex Dev ; 3(2-3): 108-17, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19684456

RESUMO

In contrast to many developmental processes, sex-determining mechanisms show no clear evolutionary conservation among phyla. However, recent studies indicate that some downstream products of sex determination genes are functionally similar in diverse species. To date, numerous conserved genes involving gonadal sex differentiation have been examined in the teleost fish Nile tilapia (Oreochromis niloticus). Morphogenesis during gonadal differentiation is also conserved, as is evident in the differentiation and development of parenchyma/medullary cells (testis cord) and follicles. Therefore, it is important to understand the mechanisms of gonadal sex differentiation from the perspective of the relationship between conserved gene expression cascades and morphogenesis during gonadal sex differentiation. This article reviews the expression profiles of male- and female-related genes involved in histogenesis during sex differentiation in tilapia and discusses gene function in gonadal sex differentiation, especially the role of endogenous estrogens for ovarian differentiation.


Assuntos
Gônadas/fisiologia , Rios , Diferenciação Sexual/genética , Tilápia/fisiologia , Animais , Gônadas/embriologia , Morfogênese , Tilápia/genética
13.
Fish Physiol Biochem ; 35(3): 325-32, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19578939

RESUMO

DNA methylation is an important biochemical epigenetic determinant of gene expression in cells and therefore actively involved in gene regulation, chromosomal conformation, and protein activity. Protein arginine methyltransferases (PRMTs) play a major role in the methylation of proteins that have an arginine residue, catalyzing both the asymmetric dimethylation of arginine (aDMA) and symmetric dimethylation of arginine (sDMA). PRMT5, a type II PRMT which catalyzes sDMA, has been shown to have a pivotal role in pole plasm assembly and germ cell development in Drosophila and also to be an associate factor of Blimp1 for germ cell development in mouse. Here, we report a homolog of prmt5 identified in medaka, Oryzias latipes, which was detected in the brain, gill, muscle, heart, liver, spleen, intestine, testis and ovary of adult fish by reverse transcriptase-PCR. The expression of prmt5 in the gonads is restricted to oocytes of the ovary, spermatogonia, and spermatocytes of testis. The prmt5 transcripts were detected as early as the one-cell stage and in all the tissues of embryos during embryogenesis. In summary, prmt5 is a maternal determinant factor of embryogenesis of medaka, possibly playing an important role in oogenesis and spermatogenesis in adult medaka.


Assuntos
Metilação de DNA , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Oryzias/embriologia , Oryzias/metabolismo , Filogenia , Proteína-Arginina N-Metiltransferases/metabolismo , Fatores Etários , Animais , Sequência de Bases , Análise por Conglomerados , Primers do DNA/genética , Embrião não Mamífero/metabolismo , Perfilação da Expressão Gênica , Hibridização In Situ , Dados de Sequência Molecular , Proteína-Arginina N-Metiltransferases/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA
14.
Gen Comp Endocrinol ; 160(1): 47-58, 2009 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-18992749

RESUMO

In order to elucidate regulatory mechanisms during puberty final oocyte maturation and spawning, full-length sequences coding for the receptors for follicle-stimulating hormone (FSH-R) and luteinizing hormone (LH-R) were isolated from female Atlantic cod (Gadus morhua) by a RACE-PCR based strategy. The nucleotide and amino acid sequences showed high homologies with the corresponding sequences of other fish species but contained some distinct differences. Conserved features important for functionality, such as a long N-terminal extracellular domain (ECD), seven transmembrane domains and a short C-terminal intracellular domain, were identified in both predicted proteins. Partial genomic sequences for these genes were also determined, allowing the design of mRNA-specific quantitative PCR assays. Due to suspected alternative splicing during expression of these genes, additional real-time PCR assays detecting variants containing the membrane-anchoring domain were established. Besides the expected expression of FSH-R and LH-R mRNA in the gonads similarly strong signals for LH-R were also obtained in male gill, and in female and male brain. When relative expression was analysed at different stages of sexual maturation, levels for FSH-R increased moderately during gonadal growth whereas those of LH-R showed a high peak at spawning.


Assuntos
Receptores do FSH/genética , Receptores do LH/genética , Animais , Feminino , Gadus morhua , Masculino , Reação em Cadeia da Polimerase Via Transcriptase Reversa
15.
Mol Cell Endocrinol ; 299(2): 146-52, 2009 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-19084575

RESUMO

Multiple forms of 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) and their differential tissue expression pattern have not been shown in any lower vertebrates. In the present study, we report cloning of two novel 3beta-HSDs and two variants from gonads of the Nile tilapia. 3beta-HSD cDNAs encode two peptides of 375 (3beta-HSD type-I/variant 1) and 367 (3beta-HSD type-II/variant 1) amino acid residues that share 31.9% homology. 3beta-HSD type-I/variant 1 shared high homology with other piscine counterparts while 3beta-HSD type-II/variant 1 exhibited homology to mammalian DeltaC27-3beta-HSD and multifunctional viral 3beta-HSD. The latter seems to be ancient form among vertebrates. Transiently transfected 3beta-HSDs' open reading frames in COS-7 cells converted exogenous pregnenolone/androsta-5-ene-3beta-17beta-diol to progesterone/testosterone. Tissue distribution pattern of 3beta-HSDs by RT-PCR revealed varied expression pattern. Northern blot analysis of 3beta-HSDs demonstrated steady or gradual rise in transcripts level at different gonadal stages. These data revealed the importance of novel 3beta-HSDs in teleosts and also provided phylogenetic significance.


Assuntos
17-Hidroxiesteroide Desidrogenases/metabolismo , Ciclídeos/metabolismo , Ovário/enzimologia , Filogenia , Reprodução , Testículo/enzimologia , 17-Hidroxiesteroide Desidrogenases/química , 17-Hidroxiesteroide Desidrogenases/genética , Sequência de Aminoácidos , Animais , Northern Blotting , Células COS , Chlorocebus aethiops , Clonagem Molecular , Feminino , Perfilação da Expressão Gênica , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção
16.
J Exp Zool A Ecol Genet Physiol ; 309(7): 419-26, 2008 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-18521911

RESUMO

Seabream gonadotropin-releasing hormone (sbGnRH)-the chief preoptic area-hypothalamus (POA-H) form of GnRH in tilapia is involved in sexual maturation. In this study, we investigated the qualitative changes in ontogeny of sbGnRH immunoreactivity (ir-), between sexes to understand its impending role during sex differentiation. For this, the differences in immunocytochemical localization of sbGnRH in genetically male (XY) and female (XX) fish were studied from 1 day after hatching (dah), through the critical period of sex differentiation (7-21 dah) to 40 dah and mature Nile tilapia. Specific antisera against sbGnRH were used for immunolocalization. SbGnRH ir- neurons were observed in POA-H as early as 5 and 15 dah in XY fish and XX fish, respectively. Higher ir- was detected in the POA-H of XY tilapia compared with XX population till 10 dah. There was a qualitative drop in sbGnRH ir- neurons/cell bodies in POA-H around 20 dah till 30 dah in XY population compared with other durations. SbGnRH ir- cells were detected in pituitary of XX fish by 15 dah and in XY fish around 10 dah but seemed to drop down by 20 dah in XY whereas it continued to remain steady in XX fish. The sbGnRH ir- in XY fish showed a rise from 35 dah and thence till 40 dah. This study revealed subtle differences in POA-H and pituitary sbGnRH ir- during early development between genetic male and female fish with possible implications in sex differentiation.


Assuntos
Encéfalo/metabolismo , Ciclídeos/crescimento & desenvolvimento , Ciclídeos/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , Hipófise/metabolismo , Animais , Feminino , Masculino , Diferenciação Sexual/fisiologia , Maturidade Sexual/fisiologia
17.
Acta Physiol (Oxf) ; 193(1): 3-15, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18284378

RESUMO

The neuropeptide gonadotropin-releasing hormone (GnRH) has a central role in the neural control of vertebrate reproduction. This review describes an overview of what is currently known about GnRH in vertebrates in the context of its structural and functional evolution. A large body of evidence has demonstrated the existence of three paralogous genes for GnRH (GnRH1, GnRH2 and GnRH3) in the vertebrate lineage. They are most probably the products of whole-genome duplications that occurred early in vertebrate evolution. Although GnRH3 has been identified only in teleosts, comparative genomic analyses indicated that GnRH3 has not arisen from a teleost-specific genome duplication, but has been derived from an earlier genome duplication in an ancestral vertebrate, followed by its loss in the tetrapod lineage. A loss of other paralogous genes has also occurred independently in different vertebrate lineages, leading to species-specific differences in the organization of the GnRH system. In addition to the GnRH3 gene, the GnRH2 gene has been deleted or silenced in certain mammalian species, while some teleosts seem to have lost the GnRH1 or GnRH3 gene. The duplicated GnRH genes have undergone subfunctionalization during the evolution of vertebrates; GnRH1 has become the major stimulator of gonadotropins and probably other pituitary hormones as well, whereas GnRH2 and GnRH3 would have functioned as neuromodulators, affecting reproductive behaviour. Conversely, in cases where a paralogous gene for GnRH has been lost, one of the remaining paralogues appears to have adopted its role.


Assuntos
Evolução Molecular , Hormônio Liberador de Gonadotropina/genética , Vertebrados/genética , Sequência de Aminoácidos , Animais , Duplicação Gênica , Hormônio Liberador de Gonadotropina/fisiologia , Dados de Sequência Molecular , Reprodução/genética , Reprodução/fisiologia , Especificidade da Espécie , Relação Estrutura-Atividade , Vertebrados/fisiologia
18.
J Mol Endocrinol ; 39(4): 319-28, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17909270

RESUMO

In order to better quantify the molecular mechanisms regulating final oocyte maturation and spawning, complete coding sequences with partially or fully untranslated regions for the steroidogenic enzymes, cytochrome P450 aromatase and 20 beta-hydroxysteroid dehydrogenase, were cloned from ovaries of Atlantic cod (Gadus morhua). The nucleotide and amino acid sequences showed high homologies with the corresponding sequences of other fish species, and conserved features important for functionality were identified in both predicted proteins. The sequences of the corresponding genomic loci were also determined, allowing the design of mRNA-specific quantitative PCR assays. As a reference gene for the real-time RT-PCR assays, eukaryotic elongation factor 1 alpha was chosen, and the mRNA as well as the genomic sequence was determined. In addition, a real-time quantitative PCR assay for the 18S rRNA was adapted to be used in cod. Analysis of immature and maturing female cod from July to January respectively showed that the enzyme genes showed the expected quantitative changes associated with physiological regulation. However, mRNA for eukaryotic elongation factor 1 alpha, and to a lesser extent even 18S rRNA, showed variable expression in these samples as well. To find accurate standards for real-time PCR in such a dynamic organ as the cod ovary is not an easy task, and several possible solutions are discussed.


Assuntos
Aromatase/genética , Cortisona Redutase/genética , Gadus morhua/genética , Regulação Enzimológica da Expressão Gênica , Maturidade Sexual/genética , Sequência de Aminoácidos , Animais , Aromatase/metabolismo , Sequência de Bases , Cortisona Redutase/metabolismo , Fator de Iniciação 1 em Eucariotos/genética , Fator de Iniciação 1 em Eucariotos/metabolismo , Feminino , Masculino , Dados de Sequência Molecular , Estações do Ano , Homologia de Sequência de Aminoácidos , Distribuição Tecidual
19.
J Mol Endocrinol ; 35(1): 103-16, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16087725

RESUMO

In order to elucidate the roles of 17beta-HSDs in fish gonadal steroidogenesis, three types of 17beta-HSDs (17beta-HSD1, 17beta-HSD8 and putative 17beta-HSD12) were cloned and characterized from the Nile tilapia, Oreochromis niloticus. The cloned cDNAs of 17beta-HSD type 1, 8 and 12 were 1504, 1006 and 1930 bp long, with open reading frames encoding proteins of 289, 256 and 314 aminoacids, respectively. Tissue distribution pattern analyzed by RT-PCR and Northern blot showed that 17beta-HSD1 was dominantly expressed in the ovary, while the putative 17beta-HSD12, one of the two duplicates found in fish, is a male specific enzyme and expressed exclusively in testis (detected by RT-PCR only). On the other hand, 17beta-HSD8 was expressed in the brain, gill, heart, liver, intestine, gonad, kidney and muscle of both male and female. Enzymatic assays of the three types of 17beta-HSDs were performed using recombinant proteins expressed in E. coli or HEK 293 cells. Tilapia 17beta-HSD1 expressed in E. coli had the preference for NADP(H) as cofactor and could catalyze the inter-conversion between estrone and estradiol efficiently as well as the inter-conversion between androstenedione and testosterone, but less efficiently. Tilapia 17beta-HSD8 recombinant protein expressed in HEK 293 cells could catalyze the conversion of testosterone to androstenedione, as well as the inter-conversion between estrone and estradiol. However, the putative 17beta-HSD12 expressed in E. coli or in HEK 293 cells showed no conversion to any of the four substrates tested in this study. Based on enzyme characterization and tissue distribution, it is plausible to attribute crucial roles to 17beta-HSDs in the gonadal steroidogenesis of teleosts.


Assuntos
17-Hidroxiesteroide Desidrogenases/genética , 17-Hidroxiesteroide Desidrogenases/metabolismo , Ciclídeos/genética , Ciclídeos/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , DNA Complementar/genética , Escherichia coli/enzimologia , Feminino , Expressão Gênica , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Dados de Sequência Molecular , Ovário/enzimologia , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Esteroides/biossíntese , Testículo/enzimologia , Distribuição Tecidual
20.
J Neurol Neurosurg Psychiatry ; 76(2): 169-75, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15654026

RESUMO

OBJECTIVES: To explore the neural substrates corresponding to the perseverative errors in the Wisconsin Card Sorting Test (WCST). METHODS: The study examined the correlations between the WCST performances and the SPECT measurements of regional cerebral blood flow (rCBF) in subjects with neurodegenerative dementia. Negative non-linear correlations between the rCBF and the two different types of the perseverative errors ("stuck-in-set" and "recurrent" perseverative errors) were calculated on a voxel basis and volume-of-interest basis in the mixed groups of 72 elderly and dementia patients. RESULTS: The stuck-in-set perseverative error was associated with the reduced rCBF in the rostrodorsal prefrontal cortex, whereas the recurrent perseverative error was related to the left parietal activity but not to the prefrontal activity. CONCLUSIONS: These findings augment evidence that the rostrodorsal prefrontal cortex crucially mediates attentional set shifting, and suggest that the stuck-in-set perseverative errors would be a true pathognomonic sign of frontal dysfunction. Moreover, this study shows that the recurrent perseverative errors may not be associated closely with the prefrontal function, suggesting that this error and the stuck-in-set error should be differentially estimated in the WCST.


Assuntos
Transtornos Cognitivos/diagnóstico , Transtornos Cognitivos/fisiopatologia , Demência/diagnóstico , Demência/fisiopatologia , Entrevista Psiquiátrica Padronizada , Córtex Pré-Frontal/irrigação sanguínea , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Córtex Pré-Frontal/patologia , Psicometria , Fluxo Sanguíneo Regional , Tomografia Computadorizada de Emissão de Fóton Único
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