Biology subjects

Bobe, J.

Publications and source records attributed to Bobe, J..

At least 19 recordsLinked to original sources

bioRxiv2026

miR-202 drives medaka fertility by targeting antagonistic Yap-dependent transcriptional regulators tead3b and vgll4b in a sex-dependent manner

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 3UTR of tead3b, but not vgll4b, results in a significant decrease in female fertility. In contrast, disrupting miR-202-5p target site in the 3UTR 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 3UTR 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.

genetics↗

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↗

bioRxiv2025

Circulating microRNAs reveal egg-brain crosstalk and a brain-specific microRNA linked to the onset of the next reproductive cycle in iteroparous salmonids

Mechanisms regulating the transition between two consecutive reproductive cycles are complex and remain poorly understood, mostly because they involve a dialog between the ovary and the central nervous system that is difficult to disentangle. In rainbow trout (Oncorhynchus mykiss), an iteroparous species spawning every year, removal of the eggs from the body cavity was used as a switch to trigger the onset of the next reproductive cycle. Changes in circulating miRNAs (c-miRNAs) levels in blood plasma and ovarian fluid were then monitored over time. Upon removal of the eggs from the body cavity we observed the dramatic down regulation of the blood plasma levels of a single c-miRNA (miR-139-5p) that is predominantly expressed in the brain. In contrast, very distinct c-miRNAs profiles were observed in blood plasma when eggs are retained in the body cavity. Among plasma c-miRNAs showing dynamic changes with egg retention, miR-135c is strongly expressed in the brain and pituitary, while miR-457a is predominant in the postovulatory ovary. In addition, egg retention in the body cavity triggers a dramatic drop in ovarian fluid levels of miR-202-5p, a miRNA known to regulate egg production in fish. Our observations reveal that the transition between two successive reproductive cycles involves a crosstalk between the eggs and the central system and that a single miRNA, miR-139, predominantly expressed in the brain, is associated with the onset of the next reproductive cycle. We identified possible miR-139-5p functional targets in rainbow trout and other iteroparous species that have been lost in semelparous salmonids. Our results offer new research perspectives to better understand the mechanisms triggering the next reproductive cycle in iteroparous fish species, including post-transcriptional regulations by miR-139 in the brain.

physiology↗

bioRxiv2025

Salmonids reveal principles of regulatory evolution following autotetraploidization

Early vertebrate autotetraploidization events may have enabled major innovations by expanding the genetic material for functional diversification, yet their ancient timing obscures how genome doubling reshaped gene regulatory evolution. Salmonids provide a unique window to these mechanisms, because they experienced a comparatively recent autotetraploidization and are earlier in the rediploidization process - which creates new genes and regulatory elements during evolution. Here, using large-scale multiomics spanning embryonic and adult tissues in two salmonids, we investigate gene regulatory evolution following genome doubling and rediploidization, which we show is governed by developmental and tissue-specific context, with a period of maximal constraint at advanced stages of embryogenesis. This work advances understanding of vertebrate genome evolution, while providing an open resource supporting salmonid aquaculture and conservation.

evolutionary biology↗

bioRxiv2024

Inter-individual gene expression variability implies stable regulation of brain-biased genes across organs

Phenotypic variation among individuals provides the raw material for evolution, and gene expression is a key mediator between genetic and phenotypic variation. As for phenotypes, the range of gene expression is limited and biased by evolutionary and developmental constraints. Observed expression variability due to biomolecular stochasticity and cell-to-cell heterogeneity has been well-studied in isogenic populations of unicellular organisms. However, for multicellular organisms with a diversity of cells and tissues sharing the same genetic background, the interplay between expression variability, gene and organ function, and gene regulation remains an open question. Here, we use highly multiplexed 3-end bulk RNA sequencing to generate transcriptome profiles spanning at least nine organs in outbred individuals of three ray-finned fish species: zebrafish, Northern pike, and spotted gar. Per organ, we quantify individual-to-individual gene expression variability independent of mean expression level. Lowly variable genes are enriched in cellular housekeeping functions whereas highly variable genes are enriched in stimulus-response functions. Furthermore, highly variable genes evolve under weaker purifying selection at the protein-coding sequence, indicating that intra-species expression variability predicts inter-species protein sequence divergence. Genes that are broadly expressed across organs are both highly expressed and lowly variable, whereas organ-biased genes are typically highly variable within their top organ. Among organ-biased genes, patterns of expression variance are dependent on the top organ. Specifically, brain- and gonads-biased genes have lowly variable expression across different organs, suggesting stabilizing selection. These patterns suggest that gene regulatory mechanisms evolve under organ-specific selective pressures.

evolutionary biology↗

bioRxiv2024

Identifying sexually dimorphic circulating microRNAs in gonochoristic and hermaphroditic marine fish species.

In many fish species, males and females are hard to distinguish at the juvenile stage and size differences appear during maturation, often favoring females. Molecular tools for sexing live fish would benefit aquaculture, fisheries, and conservation research. Here we aimed to explores to what extent circulating microRNAs (miRNAs) can decipher phenotypic males from phenotypic females in gonochoristic fish species (the European seabass Dicentrarchus labrax, the Turbot Scophthalmus maximus, the Red drum Sciaenops ocellatus, and the Blue Runner Caranx crysos) as well as sexual stages in a protandrous hermaphrodite species (the Gilthead seabream Sparus aurata). We collected and extracted total RNA from 258 plasma samples, of which 96 samples with satisfactory RNA quality were sequenced using small RNA-seq. Circulating miRNAs detected in the plasma allowed to easily discriminate species, but some miRNAs were significantly correlated to one sex, independently of the species. In immature fishes, 3 miRNAs: miR-21a-3p, miR-18a-3p, and miR-29-1a-5p were overexpressed in females compared to males, while in mature fish, miR-21a-3p exhibited an opposite pattern. In the Gilthead seabream, we detected that both the miR-21a-3p and the miR-125b-2/3-5p were likely involved in the sexual transition from male to female. A complementary analysis on the 3'UTR sequences of all fish species allowed to predict potential mRNA targets of those two miRNAs, some of them being particularly relevant regarding sexual development (i.e. wnt4, esrrb, esrrga and hsd17b1). The identification of miRNAs like miR-21a-3p and miR-125b-2/3-5p as potential sex markers could offer a new, poorly-invasive method to monitor sex and developmental stages in fishes.

molecular 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↗

bioRxiv2023

Genomic disruption of type II vitellogenin leads to vitellin membrane deficiencies andsignificant mortalities at early stages of embryonic development in zebrafish (Danio rerio)

Type II vitellogenin (Vtg2), the second most abundant type of vitellogenin in zebrafish eggs, is a major source of nutrients for early embryonic development. The main objective of this study was to determine the specific functions and essentiality of vtg2 in zebrafish early development using CRISPR/Cas9 genome editing tool. A 2811 bp deletion on gDNA was detected in vtg2-mutant zebrafish via PCR genotyping and sequencing. Introduced mutation caused vitelline membrane deficiencies and significant mortalities of mutant offspring. Further effects on female fecundity, egg fertilization rate and vtg2 gene expression and Vtgs abundance in liver were observed in F2, while effects on embryo hatching, survival rates, proteomic profiles and abundances were observed in F3 generations of the vtg2-mutant line. No change in vtg2 transcript has been detected, however, Vtg2 abundance in F2 female liver was 5x, and in 1 hpf F3 vtg2-mutant embryos was 3.8x less than Wt (p < 0.05). All Vtgs, except Vtg7, declined in abundance in 1 hpf F3 vtg2-mutant embryos (p < 0.05). Fecundity was unaffected while fertilization rate was more than halved in F2 vtg2-mutant females (p < 0.05). Hatching rate was significantly higher in F3 vtg2-mutant embryos in comparison to Wt embryos. Survival rate declined drastically to 29 % and 18 % at 24 hpf and 20 dpf, respectively, in F3 vtg2-mutant embryos. Pericardial, yolk sac/abdominal edema and spinal lordosis were evident at later stages in the surviving F3 vtg2-mutant larvae. Overrepresentation and high expression of histones, zona pellucida proteins, lectins, and protein degradation related proteins in F3 vtg2-mutant embryos provide evidence to impaired mechanisms involved in vitellin membrane formation. Findings of this study imply a potential function of Vtg2 in acquisition of vitellin membrane integrity, among other reproductive functions, and therefore, its essentiality in early zebrafish embryo development. AUTHOR SUMMARYVitellogenins (Vtgs) are major yolk nutrient precursors supporting early vertebrate development. Most species have multiple forms of Vtg, but little is known about their individual roles in reproduction and it is uncertain which forms are essential for successful development or at what stage(s) of development they are required. This study employed a CRISPR/Cas9 gene knock out (KO) to assess essentiality and functionality of Vtg2 in zebrafish, in continuation to a previously published work on type I and type III vtgs KO. The findings of this study, in combination with the previous findings, present a new model of Vtg functionality. Accordingly, Vtg2 contribute to regulation of fecundity and fertilization in female reproduction while make essential contributions to embryonic morphogenesis, hatching and embryonic and larval kinetics and survival. In addition, Vtg2 is critically important to proper formation of the vitellin membrane, and thus, to embryogenesis and later development. Our novel findings provide, for the first time, empirical evidence that the Vtg2 are essential, having critical requisite functions during oogenesis and embryonic and larval development. The overall results substantiate the concept that each type of Vtg is specialized to play unique roles in reproduction and development.

molecular biology↗

bioRxiv2023

Circulating MicroRNAs indicative of sex and stress in the European seabass (Dicentrarchus labrax): toward the identification of new biomarkers.

MicroRNAs (miRNAs) constitute a new category of biomarkers. Studies on miRNAs in non-mammalian species have drastically increased in the last few years. Here, we explored the use of miRNAs as potential, poorly-invasive markers, to identify sex and characterize acute stress in fish. The European seabass (Dicentrarchus labrax) was chosen as model because of its rapid response to stress and its specific sex determination system, devoid of sexual chromosomes. We performed a small RNA-sequencing analysis in the blood plasma of males and females European seabass (mature and immature) as well as in the blood plasma of juveniles submitted to an acute stress and sampled throughout the recovery period (at 0h, 0.5h, 1.5h and 6h). In immature individuals, both miR-1388-3p and miR-7132a-5p were up-regulated in females, while miR-499a-5p was more abundant in males. However, no miRNAs were found to be differentially expressed between sexes in the blood plasma of mature individuals. For the acute stress analysis, five miRNAs (miR-155-5p, miR-200a-3p, miR-205-1-5p, miR-143-3p and miR-223-3p) followed cortisol production over time. All miRNAs identified were tested and validated by RT-qPCR on sequenced samples. A complementary analysis on the 3UTR sequences of the European seabass allowed to predict potential mRNA targets, some of them being particularly relevant regarding stress regulation, e.g. the glucocorticoid and the mineralocorticoid receptor. The present study provides new avenues and recommendations on the use of miRNAs as biomarkers of sex or stress of the European seabass, with potential application on other fish species.

physiology↗

bioRxiv2023

Yeast extract improves growth in rainbow trout (Oncorhynchus mykiss) fed a fishmeal-free diet and modulates the hepatic and distal intestine transcriptomic profile

Replacing fishmeal with alternative protein sources and improving new ingredients diets with feed additives are major objectives in aquaculture. The aim of this study was to evaluate benefits for rainbow trout (Oncorhynchus mykiss) of supplementing a fishmeal-free diet, composed of processed animal proteins, with yeast extract. Juvenile rainbow trout (initial weight 37 {+/-} 2 g) were fed either with a control diet (19% fishmeal) or with a diet based on terrestrial animal by-products (17%) supplemented or not with 3% of yeast extract. Effects of the diets were evaluated in a 4-week digestibility trial and a 12-week growth experiment. Fish health was investigated by measuring plasma immune markers and performing histological study of the gut. Underlying molecular responses were investigated using unbiased transcriptomic analysis of the liver and distal intestine. Results indicated that supplementing with 3% yeast extract did not influence nutrient digestibility substantially. Nevertheless, fish fed the supplemented fishmeal-free diet grew more than those fed the non-supplemented processed animal protein diet. Plasma and structural parameters indicated no exacerbated immune response or signs of intestinal inflammation in fish fed the fishmeal-free diets. However, plasma total immunoglobulin M levels and intestinal villi were significantly higher in fish fed the diet supplemented with yeast extract. The transcriptomic analysis revealed that the diets influenced immune, inflammatory, pathogen fighting and coagulation gene-related expressions. These results suggest that the dietary inclusion of yeast can enhance a fishmeal-free diet by improving rainbow trout performances and potentially their robustness.

physiology↗

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

A non-functional copy of the salmonid sex determining gene (sdY) is responsible for the "apparent" XY females in Chinook salmon, Oncorhynchus tshawytscha.

Many salmonids have a male heterogametic (XX/XY) sex determination system, and they are supposed to have a conserved master sex determining gene (sdY), that interacts at the protein level with Foxl2 leading to the blockage of the synergistic induction of Foxl2 and Nr5a1 of the cyp19a1a promoter. However, this hypothesis of a conserved master sex determining role of sdY in salmonids is still challenged by a few exceptions, one of them being the presence of some naturally occurring "apparent" XY Chinook salmon females. Here we show that XY Chinook salmon females have a sdY gene (sdY-N183), which has one missense mutation leading to a substitution of a conserved isoleucine to an asparagine (SdY I183N). In contrast, Chinook salmon males have both a non-mutated sdY-I183 gene and the missense mutation sdY-N183 gene. The 3D model of SdY-N183 predicts that the I183N hydrophobic to hydrophilic amino acid change leads to a local modification of the {beta}-sandwich structure of SdY. Using in vitro cell transfection assays we found that SdY-N183, like SdY-I183, is preferentially localized in the cytoplasm. However, compared to SdY-I183, SdY-N183 is more prone to degradation, its nuclear translocation by Foxl2 is reduced and SdY-N183 is unable to significantly repress the synergistic Foxl2/Nr5a1 induction of the cyp19a1a promoter. Altogether our results suggest that the sdY-N183 gene of XY Chinook females is a non-functional gene and that SdY-N183 is no longer able to promote testicular differentiation by impairing the synthesis of estrogens in the early differentiating gonads of wild Chinook salmon XY females.

genetics↗

bioRxiv2021

Effect of a low water salinity on oocyte maturation, oocyte hydration, ovulation and egg quality in rainbow trout.

Water salinity is an important environmental factor known to have detrimental effects on salmonid reproduction, mostly when migrating female broodfish are held in sea water. In contrast, data obtained in freshwater are scarce and the impact of low water salinity, and more specifically of low water concentrations in sodium, chloride and potassium, during reproduction in freshwater is currently unknown. For this reason, and because ion and water fluxes are critical for the final steps of the female gamete formation, including oocyte hydration and ovulation, the aim of the present study was to investigate the impact of low salinity water on final oocyte maturation, ovulation and, ultimately, on egg quality, using rainbow trout as a physiological model and relevant aquaculture species. Fish from the same commercial strain were raised either in a site characterized by low concentrations of Na+, K+, and Cl- ions in the water or in a closely located control site exhibiting higher concentration in these elements. Egg quality and duration of final oocyte maturation were investigated using innovative phenotyping tools such as automatic assessment of egg viability using the VisEgg system and non-invasive echograph-based monitoring of final oocyte maturation duration, respectively. Oocyte hydration during final oocyte maturation and after ovulation was also investigated. Finally, molecular phenotyping was performed using real-time PCR-based monitoring of several key players of final oocyte maturation and ovulation associated with ion and water transport, inflammation, proteolytic activity, and coagulation. Oocyte hydration and gene expression data were analyzed in the light of the duration of final oocyte maturation. Here we show that low water salinity (i.e., low water concentration in chloride, sodium and potassium) negatively influences final oocyte maturation, ovulation and, ultimately, egg quality. Low water salinity triggered delayed ovulation and lower oocyte viability. When investigating the impact of low water salinity on final oocyte maturation duration, individuals presenting the most severe phenotypes exhibited impaired oocyte hydration and abnormally reduced gene expression levels of several key players of the ovulatory process. While the under expression of water (i.e., aquaporins) and ion (i.e., solute carriers) transporters is consistent with impaired oocyte hydration, our observations also indicate that the overall ovulatory gene expression program is disrupted. Our results raise the question of the mechanisms underlying the negative influence of low salinity water on the dynamics of the preovulatory phase, on the control of the oocyte homeostasis, including hydration, and on the overall success of the maturation-ovulation process. HighlightsO_LILow water salinity impairs final oocyte maturation and egg quality in rainbow trout C_LIO_LILow water salinity induces delayed ovulation and impaired oocyte hydration C_LIO_LILow water salinity induces a dysregulation of several key ovulatory genes C_LIO_LIMonitoring of final oocyte maturation can be performed using ultrasound staging C_LI

physiology↗

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↗