Identification of gonadal miRNAs in zebrafish exposed to high temperature during early stages of development

IX Jornada de Cromatina i Epigenètica organizada por la Secció de Cromatina i Epigenètica de la Societat Catalana de Biologia (SCB) y el Barcelona Chromatin Club (BCC) y cebrada el 22 de marzo de 2019.-- 1 page

MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression in a wide variety of physiological processes. In some fish species (e.g., rainbow trout and medaka), it has been shown that miRNAs play regulatory functions in the reproductive system and that some of them are specific to the fish gonads. However, how miRNAs change their gonadal dynamics in fish subjected to environmental factors (i.e., high-temperature) during early stages of life, has not been studied yet. In order to understand the role of the epigenetic regulation mediated by miRNAs in the gonads, we exposed zebrafish (Danio rerio) to elevated temperatures during early development (18-32 days post fertilization, dpf), a treatment that is known to result in male-skewed sex ratios. Once the fish reached adulthood (90 dpf), 8 ovaries and 8 testes were dissected and kept at -80ºC. MiRNAs were isolated and specific small RNA libraries were prepared and sequenced by Illumina technology (50 bp 1x50, v4, HiSeq). About 8 million reads were obtained from gonadal samples. Sequencing results were analyzed by miRDeep2 software that allowed, with high accuracy, to trim the sequencing data, to detect known miRNA from several databases and to identify novel miRNAs. Analysis of the expression levels of the miRNA identified a total of 25 and 1 unique miRNAs in ovaries and testes, respectively. After retrieving UTR regions, we analyzed these 26 identified miRNA by Miranda software and after filtering, we obtained almost 400 potential RNA targets. Currently, validation of the differentially expressed miRNAs by qPCR analysis is being performed. This study will provide a catalogue of both sex-specific and thermosensitive miRNAs in the zebrafish gonads that might be used as potential molecular biomarkers of the effect of temperature during sex differentiation