In the last decade, a plethora of microRNAs (miRNAs) has been reported in a wide variety of physiological processes, including reproduction, in many aquatic organisms. However, miRNAome alterations occurred by environmental cues due to water temperature increment have not yet been elucidated. With the aim to identify epigenetic regulations mediated by miRNAs in the gonads in a climate change scenario, the animal model zebrafish (Danio rerio) were subjected to high temperatures during sex differentiation, a treatment that results in male-skewed sex ratios in the adulthood. Once the fish reached adulthood, gonads were sequenced by high-throughput technologies and a total of 23 and 1 differentially expressed miRNAs in ovaries and testes, respectively, were identified two months after the heat treatment. Most of these heat-recorder miRNAs were involved in human sex-related cancer and about 400 predicted-target genes were obtained, some with reproduction-related functions. Their synteny in the zebrafish genome was, for more than half of the predicted target genes, in the chromosomes 7, 2, 4, 3 and 11 in the ovaries, chromosome 4 being the place where the sex-associated-region (sar) is localized in wild zebrafish. Further, spatial localization in the gonads of two selected heat-recorder miRNAs (miR-122-5p and miR-146-5p) showed exclusive expression in the ovarian germ cells. The present study expands the catalog of sex-specific miRNAs and deciphers, for the first time, thermosensitive miRNAs in the zebrafish gonads that might be used as potential epimarkers to predict environmental past events.
MicroRNAs (miRNAs) are small, non-coding RNAs that are involved in post-transcriptional gene regulation in many cellular functions and are highly conserved throughout evolution. In teleost fish species, miRNAs are believed to play a role in the reproductive system, but more research is needed to better understand the functions of miRNAs in fish gonads. Furthermore, miR-210 has previously been described to be involved in many processes, such as hypoxia response, angiogenesis, cell proliferation and male infertility. The aim of this study was, first, to develop an in vitro model in fish to study the functions of miRNAs and second, to identify target genes of dre-miR-210-5p by establishing a primary ovarian cell culture in zebrafish (Danio rerio). The cell culture was performed by isolating ovaries from adult female fish (n=4) which were incubated with dre-miR-210-5p mimic or scramble miRNA mimic. Cell survival was studied by flow cytometry analysis and fluorescent microscopy. Following, the effect of dre-miR-210-5p on ovarian cells was uncovered by RNA-sequencing, identifying ~6,000 targeted-genes differentially expressed (DEGs), of which ~2,600 downregulated and ~3,400 upregulated. GO term and KEGG pathway analyses showed downregulated genes involved in cell cycle processes and reproduction-related pathways while, in contrast, immune-related pathways were upregulated after miR-210 mimic treatment in the ovarian cells. To provide the molecular mechanisms triggered by miRNA-210, seed regions of targeted genes were identified and in silico analysis classified DEGs as potential biomarkers in reproduction or immune cell types. These results support the crosstalk between the reproductive and immune system in which dre-miR-210-5p plays a key role in the transcriptomic alteration in the fish ovaries.
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