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Understanding microRNA (miRNA) functions has been hampered by major difficulties in identifying their biological target(s). Currently, the main limitation is the lack of a suitable strategy to identify biologically relevant targets among a high number of putative targets. Here we provide a proof of concept of successful de novo (i.e. without prior knowledge of its identity) miRNA phenotypic target (i.e. target whose de-repression contributes to the phenotypic outcomes) identification from RNA-seq data. Using the medaka mir-202 knock-out (KO) model in which inactivation leads to a major organism-level reproductive phenotype, including reduced egg production, we introduced novel criteria including limited fold-change in KO and low interindividual variability in gene expression to reduce the list of 2853 putative targets to a short list of 5. We selected tead3b, a member of the evolutionarily-conserved Hippo pathway, known to regulate ovarian functions, due to its remarkably strong and evolutionarily conserved binding affinity for miR-202-5p. Deleting the miR-202-5p binding site in the 3' UTR of tead3b, but not of other Hippo pathway members sav1 and vgll4b, triggered a reduced egg production phenotype. This is one of the few successful examples of de novo functional assignment of a miRNA phenotypic target in vivo in vertebrates., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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., (© 2022. The Author(s).)

Background: Circulating miRNAs (c-miRNAs) are found in most, if not all, biological fluids and are becoming well-established non-invasive biomarkers of many human pathologies. However, their features in non-pathological contexts and whether their expression profiles reflect normal life history events have received little attention, especially in non-mammalian species. The aim of the present study was to investigate the potential of c-miRNAs to serve as biomarkers of reproductive and metabolic states in fish., Results: The blood plasma was sampled throughout the reproductive cycle of female rainbow trout subjected to two different feeding regimes that triggered contrasting metabolic states. In addition, ovarian fluid was sampled at ovulation, and all samples were subjected to small RNA-seq analysis, leading to the establishment of a comprehensive miRNA repertoire (i.e., miRNAome) and enabling subsequent comparative analyses to a panel of RNA-seq libraries from a wide variety of tissues and organs. We showed that biological fluid miRNAomes are complex and encompass a high proportion of the overall rainbow trout miRNAome. While sharing a high proportion of common miRNAs, the blood plasma and ovarian fluid miRNAomes exhibited strong fluid-specific signatures. We further revealed that the blood plasma miRNAome significantly changed depending on metabolic and reproductive states. We subsequently identified three evolutionarily conserved muscle-specific miRNAs or myomiRs (miR-1-1/2-3p, miR-133a-1/2-3p, and miR-206-3p) that accumulated in the blood plasma in response to high feeding rates, making these myomiRs strong candidate biomarkers of active myogenesis. We also identified miR-202-5p as a candidate biomarker for reproductive success that could be used to predict ovulation and/or egg quality., Conclusions: Together, these promising results reveal the high potential of c-miRNAs, including evolutionarily conserved myomiRs, as physiologically relevant biomarker candidates and pave the way for the use of c-miRNAs for non-invasive phenotyping in various fish species., (© 2021. The Author(s).)

Female gamete production relies on coordinated molecular and cellular processes that occur in the ovary throughout oogenesis. In fish, as in other vertebrates, these processes have been extensively studied both in terms of endocrine/paracrine regulation and protein expression and activity. The role of small non-coding RNAs in the regulation of animal reproduction remains however largely unknown and poorly investigated, despite a growing interest for the importance of miRNAs in a wide variety of biological processes. Here, we analyzed the role of miR-202, a miRNA predominantly expressed in male and female gonads in several vertebrate species. We studied its expression in the medaka ovary and generated a mutant line (using CRISPR/Cas9 genome editing) to determine its importance for reproductive success with special interest for egg production. Our results show that miR-202-5p is the most abundant mature form of the miRNA and that it is expressed in granulosa cells and in the unfertilized egg. The knock out (KO) of mir-202 gene resulted in a strong phenotype both in terms of number and quality of eggs produced. Mutant females exhibited either no egg production or produced a dramatically reduced number of eggs that could not be fertilized, ultimately leading to no reproductive success. We quantified the size distribution of the oocytes in the ovary of KO females and performed a large-scale transcriptomic analysis approach to identified dysregulated molecular pathways. Together, cellular and molecular analyses indicate that the lack of miR-202 impairs the early steps of oogenesis/folliculogenesis and decreases the number of large (i.e. vitellogenic) follicles, ultimately leading to dramatically reduced female fecundity. This study sheds new light on the regulatory mechanisms that control the early steps of follicular development, including possible targets of miR-202-5p, and provides the first in vivo functional evidence that a gonad-predominant microRNA may have a major role in female reproduction., Competing Interests: The authors have declared that no competing interests exist.

MicroRNAs (miRNAs) are small, highly conserved non-coding RNAs that play important roles in the regulation of many physiological processes. However, the role of miRNAs in vertebrate oocyte formation (i.e., oogenesis) remains poorly investigated. To gain new insights into the roles of miRNAs in oogenesis, we searched for ovarian-predominant miRNAs. Using a microarray displaying 3,800 distinct miRNAs originating from different vertebrate species, we identified 66 miRNAs that are expressed predominantly in the ovary. Of the miRNAs exhibiting the highest overabundance in the ovary, 20 were selected for further analysis. Using a combination of QPCR and in silico analyses, we identified 8 novel miRNAs that are predominantly expressed in the ovary, including 2 miRNAs (miR-4785 and miR-6352) that exhibit strict ovarian expression. Of these 8 miRNAs, 7 were previously uncharacterized in fish. The strict ovarian expression of miR-4785 and miR-6352 suggests an important role in oogenesis and/or early development, possibly involving a maternal effect. Together, these results indicate that, similar to protein-coding genes, a significant number of ovarian-predominant miRNA genes are found in fish.

Oogenesis is a complex process requiring the coordinated sequential expression of specific genes and ultimately leading to the release of the female gamete from the ovary. In the present study we aimed to investigate the contribution of miRNAs to the regulation of this key biological process in teleosts using a model in which growing oocytes develop simultaneously. Taking advantage of the strong sequence conservation of miRNAs among phylogenetically distant species, we designed a generic microarray displaying most known chordate miRNAs. It allowed us to provide an overview of the ovarian miRNome during oogenesis for the first time in any vertebrate species. We identified 13 differentially expressed miRNAs, and a differential expression of at least one miRNA was observed at each step of oogenesis. A surprisingly high differential expression of several miRNAs was observed at several stages of oogenesis and subsequently confirmed using quantitative PCR. By refining in silico prediction of target genes with gene expression data obtained within the same sample set, we provide strong evidence that miRNAs target major players of oogenesis, including genes involved in rate-limiting steps of steroidogenesis and those involved in gonadotropic control of oocyte development, as well as genes involved in ovulation, oocyte hydration, and acquisition of the ability of the oocyte to support further development once fertilized (i.e., oocyte developmental competence). Together, these observations stress the importance of miRNAs in the regulation and success of female gamete formation during oogenesis.