Violette Thermes

MicroRNAs (miRNAs) are known regulators of ovarian function in vertebrates, yet their physiological roles in fish reproduction remain poorly understood. Here, we identified miR - 187 as one of the most ovarian-enriched miRNAs in medaka ( Oryzias latipes ) and we uncovered its function in vivo using CRISPR/Cas9-mediated gene inactivation. We showed that miR-187-3p is expressed in oocytes and granulosa cells in the ovary, and in discrete brain areas. Its loss-of-function led to a significant reduction in female fecundity. High-resolution 3D imaging of whole ovaries revealed that mir-187 mutants accumulate early stage I follicles and fewer advanced follicles, suggesting a defect in follicle growth. Transcriptomic profiling of mutant ovaries revealed extensive gene-expression changes consistent with the altered follicular-stage composition, including downregulation of key regulators of steroidogenesis, Wnt/β-catenin signaling, and TGF-β pathways, and upregulation of genes associated with early germ cells and immature ovarian states. Using an expression-based target-prediction pipeline, we selected several putative miR-187-3p targets, including nr6a1a ( gcnf ) and dpagt1 , two genes previously implicated in oocyte differentiation and female fertility in mammals. Together, our results demonstrate that miR-187 acts as a previously unrecognized regulator of early folliculogenesis and female reproductive capacity in medaka, expanding the repertoire of miRNAs with essential in vivo roles in teleost oogenesis and female fecundity.

How miRNAs can sometimes drive major organism-level phenotypes by targeting a single gene and by triggering limited changes in mRNA levels remains poorly understood, especially in vertebrates. In medaka, the knockout of miR-202-5p, a gonad-specific miRNA in vertebrates, leads to impaired male and female fertility, including drastically reduced egg production and low developmental success. Here we show that miR-202-5p drives gamete formation by targeting antagonist Yap-dependent transcriptional regulators tead3b and vgll4b in a sex-dependent manner. Disruption of the miR-202-5p binding site in the 3’UTR of tead3b , but not vgll4b , results in a significant decrease in female fertility. In contrast, disrupting miR-202-5p target site in the 3’UTR of vgll4b , but not tead3b , results in impaired male fertility. In females, 3D ovary imaging and RNA-seq analysis of isolated ovarian follicles revealed that disrupting miR-202-5p binding to the 3’ UTR of tead3b results in a polycystic ovarian syndrome (PCOS)-like phenotype and the expression of many PCOS-associated marker, including androgen signaling and estrogen metabolism genes. In males, disrupting miR-202-5p binding to the 3’UTR of vgll4b triggers severe phenotypes, including reduced sperm motility and abnormal testicular development. No effects on sex ratio were observed, indicating that miR-202-5p drives gamete formation by regulating mechanisms acting down-stream of the sex-determining cascade. The analysis of miR-202-5p target sites in 3’ UTRs suggests long-term conservation of antagonistic TEAD and VGLL targeting across vertebrate species, including mammals. Together, our results show that miR-202-5p drives fertility by leveraging antagonistic Yap-dependent transcriptional regulators in a sex dependent manner.

MicroRNAs (miRNAs) are known regulators of ovarian function in vertebrates, yet their physiological roles in fish reproduction remain poorly understood. Here, we identified miR-187 as one of the most ovarian-enriched miRNAs in medaka (Oryzias latipes) and we uncovered its function in vivo using CRISPR/Cas9-mediated gene inactivation. miR-187-3p is expressed in oocytes and granulosa cells in the ovary, and in discrete brain areas. Its loss-of-function leads to a significant reduction in female fecundity. High-resolution 3D imaging of whole ovaries revealed that mir-187 mutants accumulate early stage I follicles and show reduced progression to later stages, indicating a defect in early follicle recruitment and growth. Transcriptomic profiling of mutant ovaries revealed extensive gene-expression remodeling, including downregulation of key regulators of steroidogenesis, Wnt/β-catenin signaling, and TGF-β pathways, and upregulation of genes associated with early follicle activation and immature somatic cell states. Using an expression-based target-prediction pipeline, we identified several putative miR-187-3p targets, including nr6a1a (gcnf) and dpagt1, two genes previously implicated in oocyte differentiation and female fertility in mammals. Together, our results demonstrate that miR-187 acts as a previously unrecognized regulator of early folliculogenesis and female reproductive capacity in medaka, expanding the repertoire of miRNAs with essential in vivo roles in teleost oogenesis and female fecundity.

Different adipose tissues (AT) have been described, including subcutaneous and visceral tissues (SCAT and VAT). They display different morphological structures, physiological and metabolic functions. Imaging adipocytes in the whole AT was not feasible because of the large adipocyte sizes and the lipid-full content of the droplets that increased the refractive index. Tissue clearing is then required mainly through a delipidation step, which induces also a tissue shrinkage. Our aim was to image in 3D freshly extracted adipocytes and compare them to those within their tissues. Trout ATs were stained with 5DTAF (extracellular matrix) and Nile Red (lipids). After clearing with Histodenz, 3D images were obtained using a confocal microscope, and adipocytes were segmented and measured. In situ, major differences in adipocyte size and shape were observed between the VAT and SCAT. Ex situ, only the size mattered because all cells were round outside their tissues. This method can be applied to other species, such as mice. In situ, adipocyte sphericity was even higher in the SCAT from a Swiss and a C57Bl6. This approach demonstrates that 3D adipocyte imaging with lipid labeling enables accurate morphological characterization, provides insights into depot-specific structural features, and supports optimization of cell isolation protocols.

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.

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 2,853 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.

Computational analysis of bio-images by deep learning (DL) algorithms has made exceptional progress in recent years and has become much more accessible to non-specialists with the development of ready-to-use tools. The study of oogenesis mechanisms and female reproductive success has also recently benefited from the development of efficient protocols for three-dimensional (3D) imaging of ovaries. Such datasets have a great potential for generating new quantitative data but are, however, complex to analyze due to the lack of efficient workflows for 3D image analysis. Here, we have integrated two existing open-source DL tools, Noise2Void and Cellpose, into an analysis pipeline dedicated to 3D follicular content analysis, which is available on Fiji. Our pipeline was developed on larvae and adult medaka ovaries but was also successfully applied to different types of ovaries (trout, zebrafish and mouse). Image enhancement, Cellpose segmentation and post-processing of labels enabled automatic and accurate quantification of these 3D images, which exhibited irregular fluorescent staining, low autofluorescence signal or heterogeneous follicles sizes. In the future, this pipeline will be useful for extensive cellular phenotyping in fish or mammals for developmental or toxicology studies.

Computational analysis of bio-images by deep learning (DL) algorithms has made exceptional progress in recent years and has become much more accessible to non-specialists with the development of ready-to-use tools. The study of oogenesis mechanisms and female reproductive success in fish has also recently benefited from the development of efficient three-dimensional (3D) imaging protocols on entire ovaries. Such large datasets have a great potential for the generation of new quantitative data on oogenesis but are, however, complex to analyze due to imperfect fluorescent signals and the lack of efficient image analysis workflows. Here, we applied two open-source DL tools, Noise2Void and Cellpose, to analyze the oocyte content of medaka ovaries at larvae and adult stages. These tools were integrated into end-to-end analysis pipelines that include image pre-processing, cell segmentation, and image post-processing to filter and combine labels. Our pipelines thus provide effective solutions to accurately segment complex 3D images of entire ovaries with either irregular fluorescent staining or low autofluorescence signal. In the future, these pipelines will be applicable to extensive cellular phenotyping in fish for developmental or toxicology studies. Summary statement An accessible image analysis method for biologists, which includes easy-to-use deep learning algorithms, designed for accurate quantitative measurement of ovarian content from complex 3D fluorescent images.

In the Mediterranean Sea, a demographic substructure of the Atlantic bluefin tuna Thunnus thynnus has emerged over the last decade, with old and young individuals exhibiting different horizontal movements and spatial–temporal patterns of gonad maturation. In the present study, histology and molecular reproductive markers were integrated with the gonad-specific mir-202 gene expression and ovarian localization to provide a comprehensive picture of the reproductive performances in young and old females and investigate the role played by the mir-202 during gonadal maturation. During the reproductive period, old females (>100 kg; 194.6 ± 33.9 cm straight fork length; 11.3 ± 2.7 years old) were found to have greater reproductive performances than younger females (star, lhr) and both histological and molecular (casp3) levels of follicular atresia. Overall, old females exhibited greater reproductive performances than younger females, likely reflecting different reproductive dynamics linked to the physical condition, habitat usage and migratory behaviour. These results highlight the importance of preserving large and old females in the context of fishery management. Finally, the mir-202 appears to be a good candidate to regulate the reproductive output of this species in an autocrine/paracrine manner through either stage- or age-dependent processes.

Deciphering mechanisms of oocyte development in female fishes still remains challenging and a comprehensive overview of this process at the level of the organ is still needed. The recent development of optical tissue clearing methods has tremendously boosted the 3D imaging of large size biological samples that are naturally opaque. However, no attempt of clearing on fish ovary that accumulates extremely high concentration of lipids within oocytes has been reported to date. To face with this ovarian-specific challenge, we combined two existing clearing methods, the non-toxic solvent-based ECi method for efficient clearing and the CUBIC method to enhance lipid removal and reduce non-specific staining. The methyl green fluorescent dye was used to stain nuclei and delineate the follicular structures that include oocytes. Using this procedure (named C-ECi), ovaries of both medaka and trout could be imaged in 3D and follicles analyzed. To our knowledge this is the first procedure elaborated for clearing and imaging fish ovary in 3D. The C-ECi method thus provides an interesting tool for getting precise quantitative data on follicular content in fish ovary and promises to be useful for further developmental and morphological studies.