Biology subjects

Montfort, J.

Publications and source records attributed to Montfort, J..

12 recordsLinked to original sources

bioRxiv2026

Identification of miR-187 as a modulator of early oogenesis and female fecundity in medaka

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/{beta}-catenin signaling, and TGF-{beta} 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. AUTHOR SUMMARYSuccessful fish reproduction depends on the continuous production of oocytes within the ovary. This process depends on the coordinated development of germ cells and the supporting somatic cells. Although microRNAs (miRNAs) are known to post-transcriptionally regulate gene expression, their physiological roles in the fish ovary and female reproduction remain poorly understood. In this study, we used the medaka to investigate the function of miR-187, a miRNA strongly enriched in the ovary. By generating a CRISPR/Cas9 knockout line, we showed that females lacking miR-187 produce fewer eggs and display defects in early follicle development. Using whole-ovary 3D imaging, we found that mutant females accumulate early follicles that fail to progress through normal growth stages. Transcriptomic analyses revealed broad gene-expression changes affecting pathways involved in follicle activation, somatic cell maturation, and ovarian signaling. Our results demonstrate that miR-187 is a previously unrecognized regulator of early folliculogenesis and female fecundity in medaka. This work expands the landscape of miRNA-mediated regulation in the ovary and highlights the importance of post-transcriptional mechanisms in controlling reproductive success in teleosts. Grant supportThe DYNAMO project (French National Research Agency, ANR-18-CE20-0004). The OVOPAUSE project (French National Research Agency, ANR-22-CE45-00017-02). The IMMO project (INRAE Metaprogramme DIGIT-BIO).

developmental biology↗

bioRxiv2024

FEVER: An interactive web-based resource for evolutionary transcriptomics across fishes

Teleost fish represent one of the largest and most diverse clades of vertebrates, which makes them great models in various research areas such as ecology and evolution. Recent sequencing endeavors provided high-quality genomes for species covering the main fish evolutionary lineages, opening up large-scale comparative genomics studies. However, transcriptomic data across fish species and organs are heterogenous and have not been integrated with newly sequenced genomes making gene expression quantification and comparative analyses particularly challenging. Thus, resources integrating genomic and transcriptomic data across fish species and organs are still lacking. Here, we present FEVER, a web-based resource allowing evolutionary transcriptomics across species and tissues. First, based on query genes FEVER reconstructs gene trees providing orthologous and paralogous relationships as well as their evolutionary dynamics across 13 species covering the major fish lineages, and 4 model species as evolutionary outgroups. Second, it provides unbiased gene expression across 11 tissues using up-to-date fish genomes. Finally, genomic and transcriptomic data are combined together allowing the exploration of gene expression evolution following speciation and duplication events. FEVER is freely accessible at https://fever.sk8.inrae.fr/.

evolutionary biology↗

bioRxiv2024

Identification of sex-biased miRNA markers informative of heat-past events

Elevated temperatures during early developmental stages play a pivotal role in the fate of the final sexual phenotype of fish populations, particularly towards male-skewed sex ratios. This is the case with European sea bass (Dicentrarchus labrax), one of the most important European species in the aquaculture industry. To unveil informative markers of the past thermal events, we investigated changes in the miRNome within the gonads of this species. Consequently, we exposed European sea bass to elevated temperatures during early development and conducted miRNA-sequencing analysis in the ovaries and testes one year post-heat treatment. The examination of miRNA expression levels identified three and twelve miRNAs in ovaries and testes, respectively, reflecting past thermal events. To assess the evolutionary conservation of these identified miRNAs in gonads, we cross-referenced our data with miRNome public information from ovaries and testes in nine additional fish species. This analysis uncovered 43 potential sex-biased markers present in at least five studied species along the evolutionary timeline. For instance, miR-155, miR-429, and miR-140 were female-skewed while miR-184, miR-499, and miR-135 were male-skewed. In addition, among these markers, eight conserved sex-skewed miRNAs proved informative regarding past thermal events in both the ovaries (e.g., miR-192-5p and miR-143-3p) and testes (miR-129-5p, miR-2187a-3p, miR-724-5p, miR-143-3p, miR-194a-5p and miR-223-3p). Notably, miR-223-3p and miR-194a-5p were conserved female-skewed, but showed upregulation in males exposed to high temperature. These miRNAs could serve as markers of heat-induced masculinization of genetic female-prone fish. The current research broadens the inventory of sex-specific miRNAs along evolution in fish and, elucidates thermosensitive miRNAs in the gonads. These findings hold promise as potential tools for predicting historical environmental events associated with masculinization due to high-temperature treatments in cultured species but also perhaps for natural populations exposed to a climate change scenario.

molecular biology↗

bioRxiv2023

Looking for a needle in a haystack: de novo phenotypic target identification reveals Hippo pathway-mediated miR-202 regulation of egg production

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.

genomics↗

bioRxiv2023

Evolutionarily conserved ovarian fluid proteins are responsible for extending egg viability in salmonid fish

In contrast to most fish species, salmonids exhibit the unique ability to hold their eggs for several days after ovulation without significant loss of viability. During this period, eggs are held in the body cavity in a biological fluid, the coelomic fluid (CF) that is responsible for preserving egg viability. To identify CF proteins responsible for preserving egg viability, a proteomic comparison was performed using 3 salmonid species and 3 non-salmonid species to identify salmonid-specific highly abundant proteins. In parallel, rainbow trout CF fractions were purified and used in a biological test to estimate their egg viability preservation potential. The most biologically active CF fractions were then subjected to mass spectrometry analysis. We identified 50 CF proteins that are overabundant in a salmonid-specific manner and present in analytical fractions exhibiting the highest egg viability preservation potential. Here we show that salmonid CF is a complex biological fluid and that several proteins are responsible for preserving egg viability. Among these key players are proteins related to immunity, calcium binding, lipid metabolism, proteolysis, extracellular matrix and sialic acid metabolic pathway.

evolutionary biology↗

bioRxiv2022

Genome structures resolve the early diversification of teleost fishes

Accurate species phylogenies are a prerequisite for evolutionary research. Teleosts are by far the largest and the most diversified group of extant vertebrates, but relationships among the three oldest lineages of extant teleosts remain unresolved. Based on seven high-quality new genome assemblies in Elopomorpha (tarpons, eels), we revisited the topology of the deepest branches of the teleost phylogeny using independent gene sequence and chromosomal rearrangement phylogenomic approaches. These analyses converged to a single scenario that unambiguously places the Elopomorpha and Osteoglossomorpha (bony-tongues) in a monophyletic group sister to all other teleosts, i.e., the Clupeocephala lineage. This finding resolves over 50 years of controversy on the evolutionary relationships of these lineages and highlights the power of combining different levels of genome-wide information to solve complex phylogenies. One-Sentence SummaryWhole-genome analyses place Elopomorpha (tarpons, eels) and Osteoglossomorpha (bony-tongues) as sister groups at the deepest branching of crown teleosts.

evolutionary biology↗

bioRxiv2022

An ancient truncated duplication of the anti-Mullerian hormone receptor type 2 gene is a potential conserved master sex determinant in the Pangasiidae catfish family

The evolution of sex determination (SD) mechanisms in teleost fishes is amazingly dynamic, as reflected by the variety of different master sex-determining genes identified, even sometimes among closely related species. Pangasiids are a group of economically important catfishes in many South-Asian countries, but little is known about their sex determination system. Here, we generated novel genomic resources for 12 Pangasiid species and provided a first characterization of their SD system. Based on an Oxford Nanopore long-read chromosome-scale high quality genome assembly of the striped catfish Pangasianodon hypophthalmus, we identified a duplication of the anti-Mullerian hormone receptor type II gene (amhr2), which was further characterized as being sex-linked in males and expressed only in testicular samples. These first results point to a male-specific duplication on the Y chromosome (amhr2by) of the autosomal amhr2a. Sequence annotation revealed that the P. hypophthalmus Amhr2by is truncated in its N-terminal domain, lacking the cysteine-rich extracellular part of the receptor that is crucial for ligand binding, suggesting a potential route for its neofunctionalization. Short-read genome sequencing and reference-guided assembly of 11 additional Pangasiid species, along with sex-linkage studies, revealed that this truncated amhr2by duplication is also conserved as a male-specific gene in many Pangasiids. Reconstructions of the amhr2 phylogeny suggested that amhr2by arose from an ancient duplication / insertion event at the root of the Siluroidei radiation that is dated around 100 million years ago. Altogether these results bring multiple lines of evidence supporting that amhr2by is an ancient and conserved master sex-determining gene in Pangasiid catfishes, a finding that highlights the recurrent usage of the transforming growth factor {beta} pathway in teleost sex determination and brings another empirical case towards the understanding of the dynamics or stability of sex determination systems.

genomics↗

bioRxiv2021

Deciphering sex-specific miRNAs as heat-recorders in zebrafish

MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression in a wide variety of physiological processes, including those related to the reproductive system. Although in the last decade a plethora of miRNAs has been reported, the miRNA alterations occurred by environmental cues and their biological functions have not yet been elucidated. With the aim to identify epigenetic regulations mediated by miRNAs in the gonads in a climate change scenario, zebrafish (Danio rerio) were subjected to high temperatures during sex differentiation (18-32 days post fertilization, dpf), a treatment that results in male-skewed sex ratios. Once the fish reached adulthood (90 dpf), ovaries and testes were sequenced by high-throughput technologies. About 101 million high-quality reads were obtained from gonadal samples. Analyses of the expression levels of the miRNAs identified a total of 23 and 1 differentially expressed (DE) miRNAs in ovaries and testes, respectively, two months after the heat treatment. Most of the identified miRNAs were involved in human sex-related cancer. After retrieving 3 UTR regions, ~400 predicted targets of the 24 DE miRNAs were obtained, some with reproduction-related functions. Their synteny in the zebrafish genome was, for more than half of them, in the chromosomes 7, 2, 4, 3 and 11 in the ovaries, chromosome 4 being the place where the predicted sex-associated-region (sar) is localized in wild zebrafish. Further, spatial localization in the gonads of two selected 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.

genomics↗

bioRxiv2021

Circulating miRNA diversity, origin and response to changing metabolic and reproductive states, new insights from the rainbow trout

Circulating miRNAs (c-miRNAs) are found in most, if not all, biological fluids and are becoming well established biomarkers of many human pathologies. The aim of the present study was to investigate the potential of c-miRNAs as biomarkers of reproductive and metabolic states in fish, a question that has received little attention. Plasma was collected throughout the reproductive cycle from rainbow trout females subjected to two different feeding levels to trigger contrasting metabolic states; ovarian fluid was sample at ovulation. Fluid samples were subjected to small RNA-seq analysis followed by quantitative PCR validation for a subset of promising c-miRNA biomarkers. A comprehensive miRNA repertoire, which was lacking in trout, was first established to allow subsequent analysis. We first showed that biological fluids miRNAomes are complex and encompass a high proportion of the overall species miRNAome. While sharing a high proportion of common miRNAs, plasma and ovarian fluid miRNAomes exhibited strong fluid-specific signatures. We further showed that the plasma miRNAome exhibited major significant changes depending on metabolic and reproductive state. We subsequently identified three (miR-1-1/2-3p, miR-133-a-1/2-3p and miR-206-3p) evolutionarily conserved muscle-specific miRNA that accumulate in the 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. These highly promising results reveal the high potential of c-miRNAs as physiologically relevant biomarkers and pave the way for the use of c-miRNAs for non-invasive phenotyping in various fish species.

genomics↗

bioRxiv2021

Neurodevelopment vs. the immune system: complementary contributions of maternally-inherited gene transcripts and proteins to successful egg development in fish

BackgroundIn Metazoans, embryonic development relies on maternally-inherited mRNAs and proteins that are critical for early developmental success and known to play major roles at later stages, beyond zygotic genome activation. However, very poor concordance between transcript and protein levels in oocytes and embryos of vertebrates suggest that maternally-inherited proteins and maternally-inherited mRNAs are playing different roles in unfertilized eggs, not considered to date comprehensively. The aim of this study was to investigate the respective contribution of maternally-inherited mRNAs and maternally-inherited proteins to egg molecular cargo and to its developmental competence using pikeperch, an ecologically and commercially relevant freshwater fish species, as a model. ResultsOur data shed new light on the importance of maternally-inherited mRNAs in nervous system development suggesting that neurogenesis is a major mRNA-dependent non-genetic inheritance factor. In contrast, our results highlight a specific role of maternally-inherited proteins in immune response in ovulated eggs suggesting that maternal proteins would rather contribute to developmental success through protection of the embryo against pathogens. Further analysis revealed susceptibility of the transcriptome to modifications during the post-vitellogenic processes (i.e., final oocyte maturation and ovulation), whereas proteomic cargo remains unaffected. This may negatively affect developmental competence of the egg and possibly influence further nervous system development of the embryo. ConclusionsOur study provides novel insights into the understanding of type-specific roles of maternally-inherited molecules in fish. Here we show, for the first time, that transcripts and proteins have distinct, yet complementary, functions in the egg of teleost fish. Maternally-inherited mRNAs would shape embryo neurodevelopment and possibly the future behavior of the fish, while maternally-inherited proteins would rather be responsible for protecting the embryo against pathogens. Additionally, we observed that processes directly preceding ovulation may considerably affect the reproductive success by modifying expression level of genes crucial for proper embryonic development, being novel fish egg quality markers (e.g., smarca4 or h3f3a). These results are of major importance for understanding the influence of external factors on reproductive fitness in both captive and wild-type fish species.

developmental biology↗

bioRxiv2018

MicroRNA-202 (miR-202) controls female fecundity by regulating medaka oogenesis

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 engineering) to determine its importance for reproductive success with special interest for egg production. Our results show that miR-202-5p is the biologically active form of the miRNA and that it is expressed in granulosa cells and in the unfertilized egg. The knock out (KO) of miR-202 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 drastically 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 genome-wide transcriptomic analysis approach to identified dysregulated molecular pathways. Together, cellular and molecular analyses indicate that 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 and provides the first in vivo functional evidence that an ovarian-predominant microRNA may have a major role in female reproduction.\n\nAuthor summaryThe role of small non-coding RNAs in the regulation of animal reproduction remains 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 gonads in vertebrate. We studied its expression in the medaka ovary and knocked out the miR-202 genes to study its importance for reproductive success. We showed that the lack of miR-202 results in the sterility of both females and males. In particular, it lead to a drastic reduction of both the number and the quality of eggs produced by females. Mutant females exhibited either no egg production or produced a drastically reduced number of eggs that could not be fertilized, ultimately leading to no reproductive success. Quantitative histological and molecular analyses indicated that miR-202 KO impairs oocyte development and is also associated with the dysregulation of many genes that are critical for reproduction. This study sheds new light on the regulatory mechanisms that control oogenesis and provides the first in vivo functional evidence that an ovarian-predominant microRNA may have a major role in female reproduction.

genetics↗

bioRxiv2017

Evolution of gene expression after whole-genome duplication: new insights from the spotted gar genome

Whole genome duplications (WGD) are important evolutionary events. Our understanding of underlying mechanisms, including the evolution of duplicated genes after WGD, however remains incomplete. Teleost fish experienced a common WGD (teleost-specific genome duplication, or TGD) followed by a dramatic adaptive radiation leading to more than half of all vertebrate species. The analysis of gene expression patterns following TGD at the genome level has been limited by the lack of suitable genomic resources. The recent concomitant release of the genome sequence of spotted gar (a representative of holosteans, the closest lineage of teleosts that lacks the TGD) and the tissue-specific gene expression repertoires of over 20 holostean and teleostean fish species, including spotted gar, zebrafish and medaka (the PhyloFish project), offered a unique opportunity to study the evolution of gene expression following TGD in teleosts. We show that most TGD duplicates gained their current status (loss of one duplicate gene or retention of both duplicates) relatively rapidly after TGD (i.e. prior to the divergence of medaka and zebrafish lineages). The loss of one duplicate is the most common fate after TGD with a probability of approximately 80%. In addition, the fate of duplicate genes after TGD, including subfunctionalization, neofunctionalization, or retention of two similar copies occurred not only before, but also after the radiation of species tested, in consistency with a role of the TGD in speciation and/or evolution of gene function. Finally, we report novel cases of TGD ohnolog subfunctionalization and neofunctionalization that further illustrate the importance of these processes.

evolutionary biology↗