Tissue explants as tools for studying the epigenetic modulation of the GH-IGF-I axis in farmed fish.

Somatic growth in vertebrates is mainly controlled by the growth hormone (GH)/insulin-like growth factor I (IGF-I) axis. The role of epigenetic mechanisms in regulating this axis in fish is far from being understood. This work aimed to optimize and evaluate the use of short-term culture of pituitary and liver explants from a farmed fish, the gilthead seabream Sparus aurata, for studying epigenetic mechanisms involved in GH/IGF-I axis regulation. Our results on viability, structure, proliferation, and functionality of explants support their use in short-term assays. Pituitary explants showed no variation in gh expression after exposure to the DNA methylation inhibitor decitabine (5-Aza-2'-deoxycytidine; DAC), despite responding to DAC by changing dnmt3bb and tet1 expression, and TET activity, producing an increase in overall DNA hydroxymethylation. Conversely, in liver explants, DAC had no effects on dnmt s and tet s expression or activity, but modified the expression of genes from the GH-IGF-I axis. In particular, the expression of igfbp2a was increased and that of igfbp4, ghri and ghrii was decreased by DAC as well as by genistein, which is suggestive of impaired growth. While incubation of liver explants with S-adenosylmethionine (SAM) produced no clear effects, it is proposed that nutrients must ensure the methylation milieu within the liver in the fish to sustain proper growth, which need further in vivo verification. Pituitary and liver explants from S. aurata can be further used as described herein for the screening of inhibitors or activators of epigenetic regulators, as well as for assessing epigenetic mechanisms behind GH-IGF-I variation in farmed fish.

Characterisation of ChE in Solea solea and exposure of isoflavones in juveniles of commercial flatfish (Solea solea and Solea senegalensis).

The isoflavones genistein and daidzein are flavonoid compounds mainly found in legumes, especially in soybeans and their derived products. These flavonoids can be present in agricultural, domestic and industrial wastewater effluents as a result of anthropogenic activities and may be discharged in the environment. Due to the large growth of the aquaculture sector in recent decades, new and cost-effective fish feeds are being sought, but there is also a particular need to determine the effects of exposed flavonoids on fish in the aquatic environment, as this is the main route of exposure of organisms to endocrine disruptors. This study evaluated the possible effects of these isoflavones on juveniles of Solea senegalensis and Solea solea. After 48-96 h of exposure, the acetylcholinesterase activity in the sole head tissues was measured, and the cholinesterase activity in juveniles of common sole (S. solea) was determined. Experiments were carried out to determine the optimal pH, investigate the specificity of three substrates (acetylthiocholine, butyrylthiocholine, propionylthiocholine) on cholinesterase activity and determine the kinetic parameters (Vmax and Km ). The results obtained showed that neither genistein nor daidzein exposure to S. senegalensis and S. solea inhibited the activity of acetylcholinesterase at the tested concentrations (genistein: 1.25, 2.5, 5, 10 and 20 mg/L; daidzein: 0.625, 1.25, 2.5, 5 and 10 mg/L).

Toxicity of malathion during Senegalese sole, Solea senegalensis larval development and metamorphosis: Histopathological disorders and effects on type B esterases and CYP1A enzymatic systems.

The toxicity of malathion to Solea senegalensis was studied in a static renewal bioassay during its first month of larval life (between 4 and 30 dph). Through the use of different biomarkers and biochemical, cellular and molecular approaches (inhibition of cholinesterases [ChEs], changes in cytochrome P450-1A [CYP1A] and the study of histopathological alterations), the effects of three concentrations of malathion (1.56, 3.12, and 6.25 µg/L) have been analyzed. In subacute exposure, malathion inhibited cholinesterase activities (AChE, BChE, CbE) in a dose- and time-dependent manner, ranging the inhibition percentage from 20% to 90%. However, the expression levels of CYP1A and AChE transcripts or proteins were not modified. Additionally, exposure to malathion provoked histopathological alterations in several organ systems of Senegalese sole in a time- and dose dependent way, namely disruption of parenchymal architecture in the liver, epithelial desquamation, pyknotic nuclei and steatosis in the intestine, disorganization of supporting cartilage, and sings of hyperplasia and hypertrophy in the gills and degeneration of the epithelial cells from the renal tubules. Malathion exposure also provoked strong disorganization of cardiac fibers from the heart. The findings provide evidence that exposure to sublethal concentrations of malathion that provoked serious injury to the fish S. senegalensis, were below the expected environmental concentrations reported in many other ecosystems and different fish species,revealing a higher sensitivity for Solea senegalensis to malathion exposure, thus reinforcing its use as sentinel species for environmental pollution in coastal and estuarine environments.

Effects of the soya isoflavone genistein in early life stages of the Senegalese sole, Solea senegalensis: Thyroid, estrogenic and metabolic biomarkers.

This study examines the effects induced by environmentally relevant concentrations of the isoflavone genistein (3mg/L and 10mg/L) during early life stages of the Senegalese sole. Throughout the hypothalamus-pituitary-thyroid (HPT) axis, several neurohormonal regulatory thyroid signalling patterns (thyroglobulin/Tg, thyroid peroxidase/TPO, transthyretin/TTR, thyroid receptors/TRß, and iodothrynonine deiodinases, Dio2 and Dio3) were analysed. Furthermore, the expression patterns of estrogen receptor ERß and haemoprotein Cyp1a were also evaluated. In the control larvae, progressive increases of constitutive hormonal signalling pathways have been evidenced from the pre-metamorphosis phase onwards, reaching the highest expression basal levels at the metamorphosis (Tg, TPO, Dio2) and/or during post-metamorphosis (TTR, TRß, ERß). When the early larvae were exposed to both genistein concentrations (3mg/L and 10mg/L), a statistically significant down-regulation of TPO, TTR and Tg mRNA levels was clearly detected at the metamorphic stages. In addition, the Dio2 and Dio3 transcript expression levels were also down and up-regulated when exposed to both genistein concentrations. In the larvae exposed to genistein, no statistically significant responses were recorded for the TRß expression patterns. Nevertheless, the ERß and Cyp1a transcript levels were up-regulated at the middle metamorphic stage (S2, at 16 dph) in the larvae exposed to high genistein concentrations and, only the ERß was down-regulated (S1, at 12dph) at the lower doses. Finally, all these pointed out imbalances were only temporarily disrupted by exposure to genistein, since most of the modulated transcriptional signals (i.e. up or down-regulation) were quickly restored to the baseline levels. Additionally, the control and genistein-exposed Senegalese sole specimens showed characteristic ontogenetic patterns and completely suitable for an optimal development, metamorphosis, and growth.

Molecular characterization and transcriptional regulation by GH and GnRH of insulin-like growth factors I and II in white seabream (Diplodus sargus).

Insulin-like growth factors (IGF) I and II are key regulators of development, growth and reproduction in fish. In the present study we have cloned and characterized the cDNA and genomic sequences of IGF-I and IGF-II in the white seabream (Diplodus sargus). The igf1 and igf2 genes were encoded putatively by five and four exons, respectively. Moreover, the 5'-flanking upstream region of the igf1 gene contained highly conserved regulatory elements including HNF-1α, HNF-3ß, CCAAT/enhancer binding protein (C/EBP) and the TATA box. The full-length cDNAs were 1225 and 1666 nucleotides long for igf1 and igf2, respectively. Sequence analysis identified the A-E domains as well as three spliced forms involving the E domain in exons 3-5. ORF identities were higher than 83% with respect to other fish orthologs. Expression analysis demonstrated that igf1 and its spliced forms were mostly expressed in liver, whereas the igf2 was expressed ubiquitously not detecting significant differences among the ten tissues analyzed. Hormonal treatments using the porcine GH demonstrated a sharply increase of both igf1 and igf2 mRNA levels in liver and gills at 30 min and 1h after injection. In the gonads, igf1 mRNA levels increased steadily with testis and ovary maturation. In contrast, igf2 transcript amounts were higher in immature stages (S1-S2). Hormonal treatments using GH and GnRH demonstrated that igf1 and igf2 expression were upregulated in the gonads. Overall, these data demonstrate that IGF-I and IGF-II are locally expressed in several tissues and regulated by key hormones of the somatotropic and gonadotropic axes.

Molecular regulation of both dietary vitamin A and fatty acid absorption and metabolism associated with larval morphogenesis of Senegalese sole (Solea senegalensis).

The present study aimed to deepen the understanding of molecular mechanisms governing the absorption and metabolism of some nutrients, growth and development in larvae of Senegalese sole (Solea senegalensis) fed with Artemia enriched with Easy Selco (ES, INVE) or Aquagrow Gold (AGG, ABN), which mainly differed in their vitamin A (VA) content and fatty acid composition. The expression profile of genes involved in VA metabolism (crbp2, rbp, crabp1), lipid transport (i-fabp, l-fabp), nuclear receptors for VA and fatty acids (rarα1, rxrα, pparß), growth (igf1, igf2 and their receptor igf1r) and development (bgp) was analyzed at 22, 30 and 38 days post hatching. The main results suggested that the amount of VA absorbed by larvae is controlled at the intestinal level by crbp2 in both groups, preventing excessive accumulation of this vitamin in the target tissues. The stable expression of i-fabp in the ES group with age could cause an excessive fat accumulation in the intestine inducing, in turn, the steatosis found in the liver and vascular system of these specimens. In liver, the regulation of rbp and fabp expression reflected the status of the physiological functions demanding VA and lipids. The findings revealed that dietary composition induced different strategies for VA and lipid absorption and metabolism affecting, in turn, larval development, growth and health.