Efthimia Antonopoulou

Molecular characterization of three peroxisome proliferator-activated receptors from the sea bass (Dicentrarchus labrax)

Peroxisome proliferator-activated receptors (PPAR) are nuclear hormone receptors that control the expression of genes involved in lipid homeostasis in mammals. We searched for PPAR in sea bass, a marine fish of particular interest to aquaculture, after hypothesizing that the physiological and molecular processes that regulate lipid metabolism in fish are similar to those in mammals. Here, we report the identification of complementary DNA and corresponding genomic sequences that encode three distinct PPAR from sea bass. The sea bass PPAR are the structural homologs of the mammalian PPARα, β/δ and γ isotypes. As revealed by RNase protection, the tissue expression profile of the fish PPAR appears to be very similar to that of the mammalian PPAR homologs. Thus, PPARα is mainly expressed in the liver, PPARγ in adipose tissue, and PPARβ in all tissues tested, with its highest levels in the liver, where it is also the dominant isotype expressed. Like mammalian PPAR, the sea bass isotypes recognize and bind to PPAR response elements of both mammalian and piscine origin, as heterodimers with the 9-cis retinoic acid receptor. Through the coactivator-dependent receptor ligand assay, we also demonstrated that natural FA and synthetic hypolipidemic compounds can act as ligands of the sea bass PPARα and β isotypes. This suggests that the sea bass PPAR act through similar mechanisms and perform the same critical lipid metabolism functions as mammalian PPAR.

Starvation and re-feeding affect Hsp expression, MAPK activation and antioxidant enzymes activity of European sea bass (Dicentrarchus labrax).

In the context of food deprivation in fish (wild and farmed), understanding of cellular responses is necessary in order to develop strategies to minimize stress caused by starvation in the aquaculture section. The present study evaluates the effects of long term starvation (1F-3S: one-month feeding-three-month starvation) and starvation/re-feeding (2S-2F: two-month starvation-two-month re-feeding) compared to the control group (4F-0S: four-month feeding-zero month starvation) on cellular stress response and antioxidant defense in organs, like the intestine, the liver, the red and white muscle of European sea bass Dicentrarchus labrax. Molecular responses were addressed through the expression of Hsp70 and Hsp90, the phosphorylation of stress-activated protein kinases and particularly p38 mitogen-activated protein kinase (p38 MAPK) and the extracellular signal-regulated kinases (ERK-1/2). For the determination of the effect of the oxidative stress caused by food deprivation and/or re-feeding, the (maximum) activities of antioxidant enzymes such as glutathione peroxidise (GPx), catalase (CAT) and superoxide dismutase (SOD) as well as the determination of thiobarbituric acid reactive substances (TBARS) were studied. The experimental feeding trials caused a tissue distinct and differential response on the cellular and antioxidant capacity of sea bass not only during the stressful process of starvation but also in re-feeding. Specifically, the intestine phosphorylation of ERKs and antioxidant enzymatic activities increased in the 2S-2F fish group, while in the 1F-3S group an increase was detected in the levels of the same proteins except for GPx. In the liver and the red muscle of 2S-2F fish, decreased Hsp70 and phosphorylated p38 MAPK levels and increased Hsp90 levels were observed. Additionally, SOD activity decreased in the red muscle of 2S-2F and 1F-3S groups. In the liver and red muscle of 1F-3S group Hsp70 levels increased, while the activation of p38 MAPK in the liver decreased. In the white muscle, Hsp90 levels decreased and the phosphorylation of p38 MAPK increased in both feeding regimes compared to control. In the same tissue, GPx and catalase levels were decreased in 2S-2F regime, while SOD levels were decreased in 1F-3S regime.

Effects of aromatase inhibitors on sexual maturation in Atlantic salmon, Salmo salar, male parr

Atlantic salmon, Salmo salar, male parr were implanted with Silastic capsules filled with different aromatase inhibitors: 1,4,6-androstatriene-3,17-dione (ATD), 4-hydroxy-4-androstene-3,17-dione (4OH), and the non-steroidal CGS16949 A, 4-benzonitrile monohydrochloride (CGS). Aromatization in brain homogenates were lower in salmon implanted with CGS and ATD than in controls. This was not the case for 4OH, but administration of 4OH to brain homogenates reduced the aromatase activity. All three aromatase inhibitors had effected gonadal weights in fish sampled in the summer, but the effects were markedly different among inhibitors. Plasma levels of the androgen 11-ketotestosterone (11KT) and the progestin 17α, 20β-dihydroxy-4-pregnen-3-one (17,20P) were measured by means of radioimmunoassay. CGS and ATD, but not 4OH, significantly decreased the plasma 17,20P levels in the autumn. Plasma levels of 11 KT were not influenced by ATD or CGS treatment, but 4OH had a lowering effect in one autumn sampling. ATD and 4OH (CGS not tested) increased the proportion of maturing males.These findings suggest that aromatization is of physiological importance in different mechanisms controlling reproduction in salmon.

A phenotypic trade-off between previous growth and present fecundity in round sardinella Sardinella aurita

The decision of how to allocate surplus energy to reproduction and growth can have important effects on fish population dynamics as well as on other life history traits. The natural examples on the interrelationship between maternal growth and number of offspring produced in fishes are scarce. We tested the hypothesis that these traits are competing for resources by estimating maternal previous growth decisions, using back-calculation, and present reproduction, expressed as absolute fecundity, of female round sardinella (Sardinella aurita) in the northeastern Mediterranean Sea. Despite the overall increasing trend of fecundity with age, individual fecundity was negatively related to individual specific growth rate between the most recent annulus formation and spawning within ages. A decreasing trend between previous growth rate and present fecundity emerged, showing that round sardinella allocate increasingly less energy to growth with age and more into reproduction and that the previous growth decisions determine present fecundity.

Effects of testosterone on gonadotropins, testes, and plasma 17α,20β-dihydroxy-4-pregnene-3-one levels in postbreeding mature Atlantic salmon, Salmo salar, male parr

Atlantic salmon, Salmo salar, mature male parr were implanted with testosterone (T) in small (T3) or large (T10) Silastic capsules in the breeding season or at its end in November or December, in order to find out whether the postbreeding decline in 17alpha, 20beta-dihydroxy-4-pregnene-3-one (17,20beta-P) and milt production is a consequence of declining T levels. In the first of three experiments, fish were sampled the following January and March, whereas in the second and in the third they were sampled in April. Pituitary and plasma concentrations of gonadotropic hormone (GTHs) I and II and plasma levels of T, 11-ketotestosterone (11-KT) and 17, 20beta-P were measured by radioimmunoassays, and the testes were examined histologically. Administration of T prolonged the period in which running milt was present, suppressed Sertoli cells, and prevented the postbreeding decline in testes weight in experiment two. The postbreeding decline in plasma 17,20beta-P levels was diminished by T10 in experiment one, and by both T3 and T10 in experiment two. The similar decline in 11-KT levels was not influenced by T treatment (only studied in experiment one). T treatment also prevented a decline in pituitary GTH II content (in experiments two and three) and in plasma GTH II levels (only studied in experiment three). However, pituitary GTH I content was not influenced (experiment two and three), whereas plasma GTH I levels (only studied in experiment three) were suppressed by T. To summarize, T treatment prevents postbreeding decline in 17,20beta-P levels, probably via a stimulation of GTH II secretion. J. Exp. Zool. 284:425-436, 1999.

Effects of aromatase inhibitors and different doses of testosterone on gonadotropins in one year old male Atlantic salmon (Salmo salar).

The effects of different doses of testosterone (T), the aromatase inhibitors 1,4,6-androstatriene-3,17-dione (ATD) and 4-hydroxy-4-androstene-3,17-dione (4OH), and the combined treatment of T and ATD on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) at the onset of puberty in juvenile Atlantic salmon males were investigated. T always increased pituitary LH. Also, ATD increased pituitary LH, though to a lesser extent than T. However, ATD combined with T diminished pituitary LH levels compared to T alone, indicating an aromatase-dependent positive feedback of T on LH in immature males. 4OH, which was less effective than ATD as an aromatase inhibitor, increased LH content. ATD treatment resulted in increased pituitary FSH levels, similar to those of mature controls. Positive effects of ATD on plasma FSH were found, indicating the presence of an aromatase-dependent negative feedback. The 4OH effects on FSH levels were inconsistent. T exerted both positive and negative effects on pituitary FSH and testes growth, depending on dose and season, with the positive effects being more pronounced with the low doses and the negative effects with the high doses. The treatment of T combined with ATD did not affect the positive effect of T alone on pituitary and plasma FSH, indicating the presence of an aromatase-independent positive feedback on FSH. There was a positive correlation between FSH and gonadosomatic index, especially during summer when gonadal development occurs.

Expression of developmental-stage-specific genes in the gilthead sea bream Sparus aurata L.

The mechanism of early fish development as well as the control of egg quality is of great importance for the ability of the oocyte to develop after fertilization. Embryonic development is initially regulated by maternally provided mRNAs and later by the zygotic genome. Maternal mRNAs have an important role in initiating processes crucial to patterning the developing fish embryo. Furthermore, it has been shown that maternal RNA plays an important role in egg quality. The identification and characterization of candidate maternal genes in non-model fish species with important aquaculture interest like the gilthead sea bream Sparus aurata L. is of importance for future studies related to egg quality. The broodstock of the gilthead sea bream produces large quantities of eggs with a high and non-controllable quality variation. In the present study, we have studied the gene expression of 16 genes (gapdh 1 and 2, cathepsin D, L, S and Z, erk1, jnk1, p38 alpha and p38 delta, ppar alpha, beta and gamma, tubulin beta, ferritin M, cyclinA2) of different functional categories in seven developmental stages. The 16 genes were chosen based on their putative involvement in egg quality and regulation of early development. In total, 11 showed a characteristic gene expression pattern pinpointing to the possible function as maternal genes and thus may function as molecular biomarker for egg quality.

Effects of aromatase inhibitors on reproduction in male three-spined sticklebacks, Gasterosteus aculeatus, exposed to long and short photoperiods

Three-spined stickleback Gasterosteus aculeatus, males were implanted with Silastic capsules filled with different aromatase inhibitors; 1,4,6-androstatriene-3,17-dione or the non-steroidal CGS16949 A, 4-(5,6,7,8-tetrahydrimidazol [1,5-a]pyridin-5-yl) benzonitrile monohydrochloride or empty capsules. The fish were then exposed to long or short photoperiod. Under the long photoperiod most fish in all treatments displayed a hypertrophied kidney (a secondary sexual character in sticklebacks) and completed, quiescent spermatogenesis, similar as in the natural spawning period. Under the short photoperiod the controls had unstimulated kidneys and an active spermatogenesis, whereas the males implanted with both aromatase inhibitors had stimulated kidneys, though not to the extent as in the long photoperiod, and completed, quiescent spermatogenesis. These findings suggest that aromatization is of importance for the inhibitory effects of short photoperiod on reproduction in the stickleback.

Dynamics of gene expression patterns during early development of the European seabass (Dicentrarchus labrax)

Larval and embryonic stages are the most critical period in the life cycle of marine fish. Key developmental events occur early in development and are influenced by external parameters like stress, temperature, salinity, and photoperiodism. Any failure may cause malformations, developmental delays, poor growth, and massive mortalities. Advanced understanding of molecular processes underlying marine larval development may lead to superior larval rearing conditions. Today, the new sequencing and bioinformatic methods allow transcriptome screens comprising messenger (mRNA) and microRNA (miRNA) with the scope of detecting differential expression for any species of interest. In the present study, we applied Illumina technology to investigate the transcriptome of early developmental stages of the European seabass (Dicentrarchus labrax). The European seabass, in its natural environment, is a euryhaline species and has shown high adaptation processes in early life phases. During its embryonic and larval phases the European seabass lives in a marine environment and as a juvenile it migrates to coastal zones, estuaries, and lagoons. Investigating the dynamics of gene expression in its early development may shed light on factors promoting phenotypic plasticity and may also contribute to the improvement and advancement of rearing methods of the European seabass, a species of high economic importance in European and Mediterranean aquaculture. We present the identification, characterization, and expression of mRNA and miRNA, comprising paralogous genes and differentially spliced transcripts from early developmental stages of the European seabass. We further investigated the detection of possible interactions of miRNA with mRNA.

In vitro assessment of the effects of cadmium and zinc on mammalian nerve fibres

Zinc and cadmium are environmental contaminants that have a wide range of effects on the nervous system, but zinc is also considered to be an important metal in the human body. In this study the effect of CdCl(2) and ZnCl(2), at concentrations of 50,150, 250 and 500 microM, on the nerve fibres of the sciatic nerve of the rat isolated in a three-chamber recording bath were studied. At the same concentrations, CdCl(2) and ZnCl(2) were found to have almost the same inhibitory effect on the compound action potential (CAP) of the nerve fibres. Their concentration-effect curves almost overlap and there was no significant difference in their EC(50) which for CdCl(2) is 250.1+/-18 microM (n=5) and for ZnCl(2) is 282.2+/-25 microM (n=5) correspondingly (P>0.05). The no-observed-effect concentration (NOEC) was estimated to be 50-100 microM for both metals. The identical inhibitory effect of both metals on the sciatic nerve fibres indicates a common mode of action which is related to their potential to generate reactive oxygen species (ROS).