Evidence for 5S rDNA Horizontal Transfer in the toadfish Halobatrachus didactylus (Schneider, 1801) based on the analysis of three multigene families

Abstract Background The Batrachoididae family is a group of marine teleosts that includes several species with more complicated physiological characteristics, such as their excretory, reproductive, cardiovascular and respiratory systems. Previous studies of the 5S rDNA gene family carried out in four species from the Western Atlantic showed two types of this gene in two species but only one in the other two, under processes of concerted evolution and birth-and-death evolution with purifying selection. Here we present results of the 5S rDNA and another two gene families in Halobatrachus didactylus, an Eastern Atlantic species, and draw evolutionary inferences regarding the gene families. In addition we have also mapped the genes on the chromosomes by two-colour fluorescence in situ hybridization (FISH). Results Two types of 5S rDNA were observed, named type α and type β. Molecular analysis of the 5S rDNA indicates that H. didactylus does not share the non-transcribed spacer (NTS) sequences with four other species of the family; therefore, it must have evolved in isolation. Amplification with the type β specific primers amplified a specific band in 9 specimens of H. didactylus and two of Sparus aurata. Both types showed regulatory regions and a secondary structure which mark them as functional genes. However, the U2 snRNA gene and the ITS-1 sequence showed one electrophoretic band and with one type of sequence. The U2 snRNA sequence was the most variable of the three multigene families studied. Results from two-colour FISH showed no co-localization of the gene coding from three multigene families and provided the first map of the chromosomes of the species. Conclusions A highly significant finding was observed in the analysis of the 5S rDNA, since two such distant species as H. didactylus and Sparus aurata share a 5S rDNA type. This 5S rDNA type has been detected in other species belonging to the Batrachoidiformes and Perciformes orders, but not in the Pleuronectiformes and Clupeiformes orders. Two hypotheses have been outlined: one is the possible vertical permanence of the shared type in some fish lineages, and the other is the possibility of a horizontal transference event between ancient species of the Perciformes and Batrachoidiformes orders. This finding opens a new perspective in fish evolution and in the knowledge of the dynamism of the 5S rDNA. Cytogenetic analysis allowed some evolutionary trends to be roughed out, such as the progressive change in the U2 snDNA and the organization of (GATA)n repeats, from dispersed to localized in one locus. The accumulation of (GATA)n repeats in one chromosome pair could be implicated in the evolution of a pair of proto-sex chromosomes. This possibility could situate H. didactylus as the most highly evolved of the Batrachoididae family in terms of sex chromosome biology.This work was supported by grants from the Junta de Andalucía (Spain) to the PAI BIO-219 group (LR), CACYTMAR and the “Proyecto de excelencia” RNM-03074 (CS). MUM holds a CSIC JAE fellowship.Peer Reviewe

Cloning and characterization of Vasa gene expression pattern in adults of the Lusitanian toadfish Halobatrachus didactylus

The Vasa gene is essential for germ cell development in eukaryotes. It encodes a RNA helicase, a member of the DEAD box protein family. Using the RACE method, we cloned the Vasa cDNA of the Lusitanian toadfish Halobatrachus didactylus, and analyzed quantitative and qualitative Vasa expression and its protein immunolocalization. We reported a main product of about 2.4 kb which encodes a protein of 615 amino acids, but other minority Vasa products were also identified by RACE-PCR. This gene is predominantly expressed in the ovaries and testes, although some relatively low extragonadal expression levels have also been identified. In situ hybridization and immunolocalization analysis during gametogenesis in the testes showed that toadfish Vasa mRNA was detected in spermatogonia, spermatocytes and spermatids. However, in the ovaries, Vasa mRNA was detected in early vitellogenic oocytes and in more advanced vitel-logenic stages, showing a very weak signal in oogonia, whereas the Vasa protein was evidenced in the cytoplasm of oogonia and previtellogenic oocytes, becoming weaker as the vitellogenic and maturation processes progress. These results suggest that toadfish Vasa homologues can play an important role in gametogenesis and germ cell development, but it could also be functionally implicated in other processes that are not as well known. © The authors 2014.This work was financed by Proyecto de Excelencia (RNM-03074; Consejería de Inno- vación, Ciencia y Empresa, Junta de Andalucía, Spain). M.Ú-M. was supported by a CSIC JAE-Predoc fellowship.Peer Reviewe

Cloning and sequencing of Tert gene in gilthead seabream, Sparus aurata, and European seabass, Dicentrarchus labrax: Expression patterns in germ and somatic cells

Tert gene encodes a catalytic subunit of the enzyme telomerase, which protects telomeres from abnormal folds and their degradation. In mammals, the activity of telomerase in tissues of adults is limited to stem cells with high potential for proliferation, finding expression in the cells of the germline, tumors and neoplastic cells, although the Tert gene seems to be ubiquitous in fish. To gain insight on Tert implication for fish gonad cell differentiation and gametogenesis progress, we cloned the Tert cDNA of two reared marine fish species, gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax), and their quantitative and qualitative Tert mRNA expression were analyzed. Two Tert transcripts encoding proteins which differ at their functional C-terminal end were isolated from gilthead seabream, whereas only one Tert transcript was identified from European seabass. The qPCR assays showed that Tert genes are expressed ubiquitously in both fish species, and the highest expression levels were found in gonads and particularly in differentiating or maturating germ cells, which could suggest an important role of Tert genes in gametogenesis and cell-tissue development of fish species.Financial support from Spanish Projects (AGL2008-02172/AGL2014-52906R, and CSIC-Germoplasm2014-2015).Peer reviewe

Effects of the isoflavone daidzein in Senegalese sole, Solea senegalensis: Modulation of the oestrogen receptor-β, apoptosis and enzymatic signalling pathways

Phytochemicals are widely present in the aquatic environment and they are derived from many anthropogenic activities. The isoflavone daidzein is a natural compound that is found in the soya products used as habitual constituents of aquafeeds. Nevertheless, this isoflavone possesses oestrogenic and apoptotic properties. The present study determined the effects of daidzein (at 20 mg/L) during the first month and a half of life (from 7 to 44 days post-hatching -dph-) of the flatfish Senegalese sole, Solea senegalensis, focusing at the metamorphosis. We have analysed different gene expression levels and immunohistochemical protein patterns implicated in some oestrogenic, apoptotic and enzymatic pathways. In general, the oestrogen receptor (ERβ) and stimulating apoptosis death receptor factor (Fas) transcript levels showed similar baseline patterns and transcriptional responses induced by daidzein. Both ERβ and Fas were up-regulated by this isoflavone at the pre-metamorphosis and metamorphosis, and they were down-regulated in post-metamorphosed stages. The expression pattern of the apoptotic effector caspase (Casp6) was exclusively up-regulated at the premetamorphic phase. The Birc5 transcripts (i.e. antiapoptosis, Survivin) were down-regulated by daidzein during certain metamorphic and post-metamorphosed stages. Besides, daidzein showed an up-regulating effect on both enzymatic complexes, the haemoprotein CYP1A and the acetylcholinesterase (AChE), except for a temporary AChE down-regulation in some postmetamorphosed stages. Immunostaining analysis only showed increased CYP1A signals in the liver of daidzein exposed fish. Overall, a majority of the transcriptional oestrogenic and apoptotic imbalances could be gradually and/or temporarily stabilised. Most controls and exposed larvae (70-80%) developed and grew following normal ontogenetic developmental patterns

Cloning, sequenting and expression og genes Brdt and Tert in seabream, Sparus aurata and seabass, Dicentrarchus labrax

Trabajo presentado en el 10º Congreso de la Asociación Ibérica de Endocrinología Comparada, celebrado en Castellón del 23 al 25 de septiembre de 2015.Applying molecular amplification and cloning techniques obtained cDNA sequences of Brdt and Tert genes, which has allowed to analyze levels of differential expression in gonads, brain and somatic tissues, by q-PCR quantitative, comparatively, using two reference genes (β-actin and 18S rRNA) At the same time, its cellular-tissue distribution will be analyzed, and phylogenetic analyses are performed.Tert gene encodes a catalytic subunit of the enzyme telomerase reverse transcriptase, which is which keeps the telomeric ends of chromosomes, protecting them from abnormal folds and their degradation. In the majority of cells, telomeres will shorten progressively as this divides, and after a certain number of successive divisions, the telomeres are so short that stops cell division or cell apoptosis is induced. In mammals, the activity of telomerase in tissues of adults is limited to stem cells with high potential for proliferation, finding expression in the cells of the germline, tumors and neoplastic cells. However, in fish the Tert gene seems to express themselves, so ubiquitous, in different gonad, brain and somatic tissues. On the other hand, the Brdt gene encodes a protein regulating the transcription, which in mammals is expressed almost exclusively in the testis, and is essential for the proper sexual function of males. BRDT protein control reorganization post-meiotic of the genome that occurs after the hiperacetilation of histones in the phase of elongation of the spermatids, to be crucial in the development of spermatogenesis and therefore, determinants of male fertility. The results for both genes showed relative expression levels significantly higher in testis, in relation to ovarian, and other tissues, corroborating their great importance during the reproductive process in general, and in the spermatogenesis in particular. It was shown expression in postmeiotic stages, as in spermatocytes and spermatids, and negative expression in gametes.Peer reviewe

Potenciales nuevos marcadores sexuales en peces: clonaje, secuenciación y expresión de genes en dorada y lubina

Trabajo presentado en el XV Congreso Nacional de Acuicultura y el I Congreso Ibérico de Acuicultura, celebrados en Huelva del 13 al 16 de octubre de 2015.[ES] Se han identificado los genes Tert y Brdt de la lubina y se ha analizado su expresión en diferentes órganos y tejidos. La expresión del gen Tert presentó una distribución ubicua, en consonancia con el continuo crecimiento de los peces y con su gran capacidad para la regeneración. Sin embargo, la expresión del gen Brdt era significativamente más elevada en ovario y testículo, con niveles bajos en cerebro, lo que sugiere que en la lubina la expresión del gen Brdt juega un papel importante en la gametogénesis, pero también podría participar en otros procesos que no son bien conocidos. En mamíferos, se ha descrito que la subunidad enzimática de la telomerasa (Tert) es un componente de los telómeros de las células germinales, que se mantiene durante toda la espermatogénesis como un componente estructural sin afectar el alargamiento de los telómeros. Por otro lado, la proteína BRDT es esencial para el progreso normal de la espermatogénesis.[EN] We have identified the seabass Tert and Brdt genes and analyzed their expression in different organs systems and tissues. Tert gene expression showed ubiquitous distribution, in agreement with continuous fish growth and high capability for regeneration. Nevertheless, Brdt gene expression was significantly higher in testis and ovary, with lower levels in brain. These results suggest that in seabass, the Brdt gene expression can play an important role in gametogenesis, but it could also be implicated in other processes that are not as well known. In mammalian, the telomerase enzymatic subunit (Tert) is a component of telomeres of germ cells, which is maintained throughout spermatogenesis as a structural component without affecting telomere elongation. On the other hand, bromodomain testis-specific (BRDT) protein is essential for the normal process of spermatogenesis.N

Soya isoflavones, genistein and daidzein, induce differential transcriptional modulation in the ovary and testis of zebrafish Danio rerio

Most research into the effects of phytochemical isoflavones has focussed on endocrine disruptions, and especially on oestrogenic imbalances; however, little is known about their effects on other molecular signals such as transcriptional coregulators and choriolytic enzymatic pathways, which are also important in reproductive processes. In male and female zebrafish Danio rerio, the soya isoflavones genistein and daidzein (provided at 10 mg l-1 for 15 d) modulated the basal expression levels of oestrogen receptor transcripts (ERβ) in variable and differential ways. Exposure to genistein resulted in decreased levels of ERβ in the zebrafish ovary; conversely, this isoflavone increased the basal expression levels of the hatching enzyme (HE1) in both gonads. On the other hand, daidzein increased the basal expression levels of the bromodomain testis-specific gene (BRDT) in the male gonad, but not in the ovary. Both isoflavones also differentially modulated (up-down regulations) the basal expression patterns of the 3 molecular signals studied in other regions of the body (e.g. head, digestive system, skeletal musculature). Despite all these transcriptional imbalances, neither of the phytoestrogens modified gonadal histomorphology or the baseline histochemical pattern of proteins, carbohydrates and glycoconjugates distributed in either the vitelline structures or in the developing and maturing germ cells of Danio rerio.This study was financed by Spanish Projects (MINECO-Feder, AGL2014-52906R; RTI2018-0939-B-100). Grant contract-CSIC (M.U.M)

Effects of the isoflavone genistein in early life stages of the Senegalese sole, Solea senegalensis: role of the Survivin and proliferation versus apoptosis pathways

[Background] Phytochemical flavonoids are widely distributed in the environment and are derived from many anthropogenic activities. The isoflavone genistein is a naturally occurring compound found in soya products that are habitual constituents of the aquafeeds. This isoflavone possesses oestrogenic biological activity and also apoptotic properties. The present study has been performed to determine the effects of the genistein in the early life stages of the flatfish Senegalese sole during the first month of larval life, and it is focused especially at the metamorphosis, analysing the expression transcript levels and the immunohistochemical protein patterns implicated in the cell proliferation and apoptosis pathways (proliferation cellular/PCNA, anti-apoptosis Survivin/BIRC-5, death receptors/Fas, and Caspases).[Results] The isoflavone genistein induced some temporal disrupting effects in several pro-apoptotic signalling pathways (Fas, CASP-6) at both genistein doses (3 mg/L and 10 mg/L), with increased Fas transcripts and also decreasing CASP-6 mRNA expression levels during metamorphic and post-metamorphic stages of the Senegalese sole. On the other hand, the anti-apoptotic BIRC-5 expression levels were weakly down-regulated with both the highest and lowest doses, but all of these imbalances were stabilised to the baseline levels. In early life stages of the controls, the constitutive basal transcript levels were temporarily and differentially expressed, reaching the highest levels at the pre-metamorphosis phase, as especially in endotrophic larvae (i.e. BIRC-5 mRNA), as well as in the metamorphic (i.e. CASP-6 mRNA) and post-metamorphic stages (i.e. Fas mRNA). In general, through development, continuous and progressive increases in the protein patterns of cell proliferation-PCNA (e.g. mitotic nuclei), anti-apoptotic Survivin (e.g. haematopoietic system, brain, digestive system, gills) and CASP-2 and -6 (e.g. brain, gills, kidney, digestive system, vascular systems, among others) have been immunohistochemically detected. Besides, both the controls and genistein exposed larvae displayed parallel immunostaining protein patterns in the different organ-systems and tissues.[Conclusions] The transcriptional imbalances observed in the studied genes (BIRC-5, CASP-6, Fas) were only temporarily induced, and apparently no changes in the immunohistochemical protein patterns were detected. Thus, the isoflavone genistein caused not harmful effects in the development and metamorphosis of the Senegalese sole exposed to chronic environmentally relevant concentrations (3 and 10 mg/L).This study was supported by MINECO-Feder-UE (Project AGL2014- 52906R; Spain). MUM, contract financed by CSIC