Aquatic pollution from anthropogenic discharges modulates gene expression in liver of rainbow trout (Oncorhynchus mykiss)

With the aim to characterise the biological impact of anthropogenic discharge in a river, gene expression in fish was evaluated as a biomarker for mixture effects of potential toxic compounds in the aquatic environment. Adult male rainbow trout (Oncorhynchus mykiss) were used as monitoring organisms. Within the German experimental set up, trout were exposed actively for 28 days in biologically purified sewage as well as in river water up- and downstream of the sewage treatment plant (STP) effluent. In a different approach, wild trout were captured in southern Chile near anthropogenic discharges. Fish were taken from river Maullin receiving sewage from a settlement and from river Pescado influenced by effluents from a fish farm. In vivo effects were assessed by RT-qPCR analyses of biomarker gene expression, vitellogenin (vg), metallothionein (mt) and cytochrome 1A (cyp1A) in liver samples with primers amplifying specific sequences previously confirmed by cloning and sequencing. The modulation of expression of marker genes involved in metal stress, reproduction and detoxifying systems in the liver of male rainbow trout revealed organismal response to anthropogenic contamination in two different study areas, such as Chile and Germany, thereby indicating a potential risk on the aquatic ecosystems

The Proteasomal Deubiquitinating Enzyme PSMD14 Regulates Macroautophagy by Controlling Golgi-to-ER Retrograde Transport

Ubiquitination regulates several biological processes, however the role of specific members of the ubiquitinome on intracellular membrane trafficking is not yet fully understood. Here, we search for ubiquitin-related genes implicated in protein membrane trafficking performing a High-Content siRNA Screening including 1187 genes of the human “ubiquitinome” using amyloid precursor protein (APP) as a reporter. We identified the deubiquitinating enzyme PSMD14, a subunit of the 19S regulatory particle of the proteasome, specific for K63-Ub chains in cells, as a novel regulator of Golgi-to-endoplasmic reticulum (ER) retrograde transport. Silencing or pharmacological inhibition of PSMD14 with Capzimin (CZM) caused a robust increase in APP levels at the Golgi apparatus and the swelling of this organelle. We showed that this phenotype is the result of rapid inhibition of Golgi-to-ER retrograde transport, a pathway implicated in the early steps of the autophagosomal formation. Indeed, we observed that inhibition of PSMD14 with CZM acts as a potent blocker of macroautophagy by a mechanism related to the retention of Atg9A and Rab1A at the Golgi apparatus. As pharmacological inhibition of the proteolytic core of the 20S proteasome did not recapitulate these effects, we concluded that PSMD14, and the K63-Ub chains, act as a crucial regulatory factor for macroautophagy by controlling Golgi-to-ER retrograde transport

Structurally conserved C-RFa revealed prolactin releasing activity in vitro and gene expression changes in pituitary of seasonally acclimatized carp.

Here we show the cloning and characterization of a novel homolog of prepro C-RFa cDNA from Cyprinus carpio. The deduced preprohormone precursor of 115 amino acids leads to a mature bioactive peptide of 20 amino acids with identical sequence to other teleost C-RFa. Modeling of the mature C-RFa peptide highlighted significant similarity to homologous human PrRP20, specifically the conserved amphipathic system defined by the C-terminal alpha-helix. Clearly, the synthetic C-RFa peptide stimulated prolactin release from primary cultured fish pituitary cells. For the first time, significant variation was shown in C-RFa mRNA and protein levels in the hypothalamus and pituitary between summer- and winter-acclimatized carp. Furthermore, C-RFa protein distribution in carp central nervous tissue was visualized by immunodetection in fibers and cells in hypothalamus, olfactory tract, cerebellum and pituitary stalk. In conclusion, we demonstrated the structure conservation of C-RFa in teleosts and mammals and immunopositive cells and fibers for C-RFa in brain areas. Finally, the increase of C-RFa expression suggests the participation of this hypothalamic factor in the mechanism of modulation in PRL expression in carp

In situ hybridization of somatolactin transcripts in the pituitary glands from acclimatized carp (Cyprinus carpio)

We isolated and cloned a carp somatolactin SL DNA fragment, of which 78% of the nucleotides were identical to the corresponding salmon SL sequence. The results obtained upon Northern blot hybridization of carp pituitary RNA allowed the identification of two transcripts as described for other fish. When the content of SL transcripts in pituitary sections from summer- and winter- acclimatized carp was quantified by in situ hybridization assays, we found no significant differences between the two seasons. In salmonids, plasma SL reaches higher levels in summer than in winter in synchrony with the water temperature cycle; in the eurythermal carp, however, the complex adaptive responses imposed by seasonal environmental changes do not seem to include the regulation of the somatolactin detected with the probe used at the transcriptional level in pituitary gland

Epigenetic traits related to vitellogenin gene expression reveal clues for organismal response to estrogen in Cyprinus carpio

peer reviewedEpigenetic mechanisms orchestrate gene expression during development and lifelong adaptation to changing environments. In adult male fish estrogen exposure leads to feminization where vitellogenin (vtg) expression serves as robust marker for exposure to estrogenic endocrine disruptor compounds. With the aim to reveal related epigenetic traits and novel blood markers, we analyzed response of male carp to 17-estrogen (E2). After three-day E2 treatment, DNA methylation bisulfite analysis of 8 CpGs in the vtg gene promoter in liver revealed diminished C-methylation in E2- relative to control carp, which correlated with increased vtg mRNA levels. We further studied male carp with high and low vtg expression in liver characterizing blood extracellular vesicles (EV), which seem to be released from different cells playing regulatory roles in an intricate communication network coordinating whole organism response. Small RNA libraries were prepared from EV enriched blood serum of male carp, presenting 50.1% miRNAs, 42.9% piRNAs, 7% snoRNA and snRNAs. From high versus low vtg carp, a set of 18 miRNAs differentially present in serum EVs was determined and target genes predicted. Comparison with in silico processed data sets from liver mRNAs of estrogen treated male zebrafish revealed 121 differentially expressed genes in liver in response to E2 in male fish. Finally, the integrative analyses of experimental and in silico data visualized a network of miRNAs and target genes involved in oxidoreductase pathways, response to chemical stimulus and small molecule and lipid biosynthetic processes. These epigenetic markers might help to better understand organismal responses to potential harmful exposure to endocrine disruptors in the aquatic environment. Acknowledgement: Conicyt 21130511 GV, 21150665 FS. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska Curie grant agreement No 722634.PROTECTE

Effect of seasonal acclimatization on the expression of the carp transcription factor Pit-1

We isolated a clone comprising four exons of the carp Pit-1 gene. Using synthetic oligonucleotide probes derived from the carp Pit-1 sequence Pit-1 expression was assessed by in situ hybridization in pituitary sections from summer- and winter-acclimatized carp. Semiquantitative analyses of the hybridization signals revealed a significant higher Pit-1 expression in the proximal pars distalis (PPD) and pars intermedia (PI) of the pituitary glands from summer-acclimatized carp, compared to the winter-acclimatized fish. In both adaptive states, relative to the PPD and PI, only a basal Pit-1 expression was detected in the rostral pars distalis. Thus, during seasonal acclimatization of an eurythermal fish, Pit-1 seems to be involved in the mechanisms that underlie the compensatory response