Browsing by Author "Martins, Rute S. T."
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- Alternative transcripts of DMRT1 in the European sea bass: expression during gonadal differentiationPublication . Deloffre, Laurence A. M.; Martins, Rute S. T.; Mylonas, Constantinos C.; Canario, Adelino V. M.DMRT1 is conserved from invertebrates to human and has been implicated in sex differentiation and testis function in many organisms.We report the cloning of two DMRT1 transcripts, DMRT1a and DMRT1b, encoded by a single gene in the European sea bass, Dicentrarchus labrax, a teleost fish with polygenic sex determination influenced by temperature. DMRT1a and DMRT1b are specific to the testis and are present as transcripts of 2.7 and 4.6 kb, as determined by Northern blot, although the cloned cDNAs are 1.2 and 1.9 kb. DMRT1a, encodes a protein of 306-amino acids, of similar size to what has been found in other teleosts, while the DMRT1b encodes a unique protein which differ from DMRT1a by having a 26 amino acids insertion which separates the S and Y domains. This insertion is the result of an extra exon (exon 4), present in the genomes of teleosts but not in other vertebrate genomes. Furthermore, unlike in the European sea bass, the putative product of DMRT1b in other teleost species is a truncated peptide. In European sea bass, the two transcripts are expressed at a similar level and have a similar expression profile in developing gonads. They are first detected by reverse transcription-polymerase chain reaction at 150 days post-hatch coinciding with the start of sex differentiation. After 200 days, expression increases in testis and decreases in ovary. Although it is not clear whether the two transcripts have differential roles it is suggested that both are required for testicular function.
- Ca2+-Calmodulin regulation of testicular androgen production in Mozambique tilapia (Oreochromis mossambicus)Publication . Martins, Rute S. T.; Fuentes, J.; Almeida, O. G.; Power, Deborah; Canario, Adelino V. M.The Ca2+-Calmodulin (CaM) signaling pathway has previously been shown to be involved in the regulation of teleost fish ovarian steroidogenesis. However, a putative role of CaM in testicular steroidogenesis and potential targets has not been examined. To examine whether basal steroidogenesis is modulated by Ca2+ and CaM levels in the testis of Mozambique tilapia (Oreochromis mossambicus) we have incubated testicular fragments in vitro under different conditions and analyzed steroid output. Calcium-free medium with or without EGTA did not affect testicular basal 11-ketotestosterone (11-KT) and testosterone (T) secretion. However, addition of 80 lM the CaM inhibitor W7 significantly reduced basal 11-KT, T and androstenedione secretion. Interestingly, the decreased androgen production by 80 lM of W7 was accompanied by increased 11-desoxicortisol output and by the activation of cortisol synthesis in the testis, the latter undetected in untreated tissues. However, production of 17,20a-dihydroxy-4-pregnen- 3-one was unaltered by W7. This suggests that C17,20 desmolase, 21-hydroxylase and possibly 11bhydroxysteroid dehydrogenase are targets for CaM. In addition, androgen production was also found to be regulated by the level of cAMP since incubations with forskolin (FK) significantly increased 11-KT and T output. A cross-talk between the cAMP and Ca2+-CaM signaling pathways was detected since W7 administration also decreased FK stimulated androgen production. Altogether, these data show that both basal and cAMP stimulated androgen levels were modulated by intracellular Ca2+-dependent CaM and that possibly Ca2+-CaM determines the shift in steroidogenesis from C21 steroids to androgens.
- A cDNA for European sea bass (Dicentrachus labrax) 11 beta-hydroxylase: Gene expression during the thermosensitive period and gonadogenesisPublication . Socorro, S.; Martins, Rute S. T.; Deloffre, Laurence A. M.; Mylonas, Constantinos C.; Canario, Adelino V. M.Steroid P450 11 -hydroxylase, encoded by the CYP11B gene, is a key mitochondrial enzyme for the production of 11-oxygenated androgens, which have been shown to be potent masculinising steroids in several Wsh species. In this study we have isolated a CYP11B cDNA of 1903 base pairs from the testis of European sea bass (Dicentrarchus labrax) encoding a predicted protein of 552 amino acids. The amino acid identities to other vertebrate 11 -hydroxylase proteins ranged from 66% to 72% to other Wsh; 45% to amphibian and 35–39% to mammalian. Southern blot indicated that a single CYP11B gene is present. Northern blot analysis detected two transcripts in testis and head kidney, one of the same size of the isolated clone and the other of 3.9 kb. Reverse transcriptase-polymerase chain reaction showed abundant mRNA expression only in testis and head kidney, being residual in a range of other tissues. Expression of CYP11B and CYP19A (which encodes for ovarian aromatase) was detected from at least 4 days post-hatching and did not appear to be aVected by rearing temperature (15 and 20 °C) during the Wrst 60 days, a period in which high temperatures promote masculinisation in European sea bass. Throughout, gonadogenesis (60–300 dph), a highly dimorphic pattern of CYP11B expression was consistent with a role of this gene in testicular development.
- Changes in the gene expression profiles of the brains of male European eels (Anguilla anguilla) during sexual maturationPublication . Churcher, Allison; Pujolar, Jose M.; Milan, Massimo; Hubbard, Peter; Martins, Rute S. T.; L. Saraiva, João; Huertas, Mar; Bargelloni, Luca; Patarnello, T.; Marino, Ilaria A. M.; Zane, Lorenzo; Canario, Adelino V. M.Background: The vertebrate brain plays a critical role in the regulation of sexual maturation and reproduction by integrating environmental information with developmental and endocrine status. The European eel Anguilla anguilla is an important species in which to better understand the neuroendocrine factors that control reproduction because it is an endangered species, has a complex life cycle that includes two extreme long distance migrations with both freshwater and seawater stages and because it occupies a key position within the teleost phylogeny. At present, mature eels have never been caught in the wild and little is known about most aspects of reproduction in A. anguilla. The goal of this study was to identify genes that may be involved in sexual maturation in experimentally matured eels. For this, we used microarrays to compare the gene expression profiles of sexually mature to immature males. Results: Using a false discovery rate of 0.05, a total of 1,497 differentially expressed genes were identified. Of this set, 991 were expressed at higher levels in brains (forebrain and midbrain) of mature males while 506 were expressed at lower levels relative to brains of immature males. The set of up-regulated genes includes genes involved in neuroendocrine processes, cell-cell signaling, neurogenesis and development. Interestingly, while genes involved in immune system function were down-regulated in the brains of mature males, changes in the expression levels of several receptors and channels were observed suggesting that some rewiring is occurring in the brain at sexual maturity. Conclusions: This study shows that the brains of eels undergo major changes at the molecular level at sexual maturity that may include re-organization at the cellular level. Here, we have defined a set of genes that help to understand the molecular mechanisms controlling reproduction in eels. Some of these genes have previously described functions while many others have roles that have yet to be characterized in a reproductive context. Since most of the genes examined here have orthologs in other vertebrates, the results of this study will contribute to the body of knowledge concerning reproduction in vertebrates as well as to an improved understanding of eel biology.
- Characterization of estrogen receptor ßb in sea bream (Sparus auratus): phylogeny, ligand-binding, and comparative analysis of expressionPublication . Pinto, Patricia IS; Passos, A. L.; Martins, Rute S. T.; Power, Deborah; Canario, Adelino V. M.Estrogens control many physiological processes in both female and male vertebrates, mostly mediated by speciWc nuclear estrogen receptors (ER). Two ER subtypes (ER and ER ) are present in most vertebrates, including the sea bream (Sparus auratus) a hermaphrodite teleost Wsh. In the present study several variant cDNAs encoding a second sea bream ER (sbER b) is reported. Phylogenetic and Southern blot analysis indicate that sbER b and the previously cloned sbER a (formerly sbER ) are encoded by diVerent genes, which may have arisen by duplication of an ancestral ER gene. Competitive binding assays show that sbER b has high aYnity for 17 -estradiol (KdD1 nM) and speciWcally binds estrogen agonists (diethylstilbestrol and ethynylestradiol) and antagonists (ICI 182,780). In Northern blot sbER , sbER a, sbER b produce several diVerent transcripts in a variety of tissues. RT-PCR showed a partially overlapping but diVerential tissue distribution in both male and female sea bream.
- DAX1 regulatory networks unveil conserved and potentially new functionsPublication . Martins, Rute S. T.; Power, Deborah; Fuentes, J.; Deloffre, Laurence A. M.; Canario, Adelino V. M.DAX1 is an orphan nuclear receptorwith actions in mammalian sex determination, regulation of steroidogenesis, embryonic development and neural differentiation. Conserved patterns of DAX1 gene expression frommammals to fish have been taken to suggest conserved function. In the present study, the European sea bass, Dicentrarchus labrax, DAX1 promoter was isolated and its conserved features compared to other fish and mammalian DAX1 promoters in order to derive common regulators and functional gene networks. Fish andmammalian DAX1 promoters share common sets of transcription factor frameworkswhichwere also present in the promoter region of another 127 genes. Pathway analysis clustered these into candidate gene networks associated with the fish and mammalian DAX1. The networks identified are concordantwith described functions for DAX1 in embryogenesis, regulation of transcription, endocrine development and steroid production. Novel candidate gene network partners were also identified, which implicate DAX1 in ion homeostasis and transport, lipid transport and skeletal development. Experimental evidence is provided supporting roles for DAX1 in steroid signalling and osmoregulation in fish. These results highlight the usefulness of the in silico comparative approach to analyse gene regulation for hypothesis generation. Conserved promoter architecture can be used also to predict potentially newgene functions. The approach reported can be applied to genes from model and non-model species.
- Developmental expression of DAX1 in the European sea bass, Dicentrarchus labrax: lack of evidence for sexual dimorphism during sex differentiationPublication . Martins, Rute S. T.; Deloffre, Laurence A. M.; Mylonas, Constantinos C.; Power, Deborah; Canario, Adelino V. M.DAX1 (NR0B1), a member of the nuclear receptors super family, has been shown to be involved in the genetic sex determination and in gonadal differentiation in several vertebrate species. In the aquaculture fish European sea bass, Dicentrarchus labrax, and in the generality of fish species, the mechanisms of sex determination and differentiation have not been elucidated. The present study aimed at characterizing the European DAX1 gene and its developmental expression at the mRNA level.
- European sea bass genome and its variation provide insights into adaptation to euryhalinity and speciationPublication . Tine, Mbaye; Kuhl, Heiner; Gagnaire, Pierre-Alexandre; Louro, Bruno; Desmarais, Erick; Martins, Rute S. T.; Hecht, Jochen; Knaust, Florian; Belkhir, Khalid; Klages, Sven; Dieterich, Roland; Stueber, Kurt; Piferrer, Francesc; Guinand, Bruno; Bierne, Nicolas; Volckaert, Filip A. M.; Bargelloni, Luca; Power, Deborah M.; Bonhomme, Francois; Canario, Adelino V. M.; Reinhardt, RichardThe European sea bass (Dicentrarchus labrax) is a temperate-zone euryhaline teleost of prime importance for aquaculture and fisheries. This species is subdivided into two naturally hybridizing lineages, one inhabiting the north-eastern Atlantic Ocean and the other the Mediterranean and Black seas. Here we provide a high-quality chromosome-scale assembly of its genome that shows a high degree of synteny with the more highly derived teleosts. We find expansions of gene families specifically associated with ion and water regulation, highlighting adaptation to variation in salinity. We further generate a genome-wide variation map through RAD-sequencing of Atlantic and Mediterranean populations. We show that variation in local recombination rates strongly influences the genomic landscape of diversity within and differentiation between lineages. Comparing predictions of alternative demographic models to the joint allele-frequency spectrum indicates that genomic islands of differentiation between sea bass lineages were generated by varying rates of introgression across the genome following a period of geographical isolation.
- Novel galanin receptors in teleost fish: identification, expression and regulation by sex steroidsPublication . Martins, Rute S. T.; Pinto, Patricia IS; Guerreiro, P. M.; Zanuy, Silvia; Carrillo, Manuel; Canario, Adelino V. M.In fish, the onset of puberty, the transition from juvenile to sexually reproductive adult animals, is triggered by the activation of pituitary gonadotrophin secretion and its timing is influenced by external and internal factors that include the growth/adiposity status of the animal. Kisseptins have been implicated in the activation of puberty but peripheral signals coming from the immature gonad or associated to the metabolic/nutritional status are also thought to be involved. Additionally, there is evidence that the galaninergic system in the brain and testis of pre-pubertal male sea bass is a possible mediator involved in the translation of somatic signals leading to gonadal maturation. Here, the transcripts for four galanin receptors (GALR), named GALR1a, 1b, 2a and 2b, were isolated from European sea bass, Dicentrarchus labrax. Phylogenetic analysis confirmed the previously reported duplication of GALR1 in teleost fish, and unravelled the duplication of GALR2 in teleost fish and in some tetrapod species. Comparison with human showed that the key amino acids involved in ligand binding are present in the corresponding GALR1 and GALR2 orthologues. Transcripts for all four receptors are expressed in brain and testes of adult fish with GALR1a and GALR1b abundant in testes 15 and hardly detected in ovaries. In order to investigate whether GALR1 dimorphic expression was dependent on steroid context we evaluated the effect of 11-ketotestosterone and 17β-estradiol treatments on the receptor expression in brain and testes of pre-pubertal males. Interestingly, steroid treatments had no effect on the expression of GALRs in the brain while in the testes, GALR1a and GALR1b were significantly up regulated by 11KT. Altogether, these results support a role for the galaninergic system, in particular the GALR1 paralog in fish reproductive function.
- Photoperiodic Modulation of Circadian Clock and Reproductive Axis Gene Expression in the Pre-Pubertal European Sea Bass BrainPublication . Martins, Rute S. T.; Gomez, Ana; Zanuy, Silvia; Carrillo, Manuel; Canario, Adelino V. M.The acquisition of reproductive competence requires the activation of the brain-pituitary-gonad (BPG) axis, which in most vertebrates, including fishes, is initiated by changes in photoperiod. In the European sea bass long-term exposure to continuous light (LL) alters the rhythm of reproductive hormones, delays spermatogenesis and reduces the incidence of precocious males. In contrast, an early shift from long to short photoperiod (AP) accelerates spermatogenesis. However, how photoperiod affects key genes in the brain to trigger the onset of puberty is still largely unknown. Here, we investigated if the integration of the light stimulus by clock proteins is sufficient to activate key genes that trigger the BPG axis in the European sea bass. We found that the clock genes clock, npas2, bmal1 and the BPG genes gnrh, kiss and kissr share conserved transcription factor frameworks in their promoters, suggesting co-regulation. Other gene promoters of the BGP axis were also predicted to be co-regulated by the same frameworks. Co-regulation was confirmed through gene expression analysis of brains from males exposed to LL or AP photoperiod compared to natural conditions: LL fish had suppressed gnrh1, kiss2, galr1b and esr1, while AP fish had stimulated npas2, gnrh1, gnrh2, kiss2, kiss1rb and galr1b compared to NP. It is concluded that fish exposed to different photoperiods present significant expression differences in some clock and reproductive axis related genes well before the first detectable endocrine and morphological responses of the BPG axis.