Jérôme Bugeon

Characteristics and metabolism of different adipose tissues in fish

Lipids are the predominant source of energy for fish and are stored in fat depots in different parts of the body regions. This review focuses on visceral, subcutaneous and intramuscular adipose tissues that interfere with carcass and fillet yields and with flesh quality. The morphological, cellular and biochemical characteristics of these tissues are discussed as well as the different mechanisms involved in the regulation of their lipid metabolism. Particular emphasis is given to the modulation of these characteristics and mechanisms by different extrinsic (food composition, water parameters) and intrinsic (selective breeding, life cycle status) factors. This review focuses on recent studies that take into account the present challenges of fin-fish aquaculture, which are principally (1) the replacement of fish oil and meal by vegetable oil and meal due to the need for sustainability and the limited availability of fish to prepare food pellets, and (2) selective breeding programs to improve fish growth and flesh quality. These studies apply various modern technologies to different fish species, including the development of cell culture systems and transcriptomic and proteomic techniques. This review highlights that fish adipose tissues differ in their localization and their morphological characteristics and that they show a large plasticity in their responses to variations of both extrinsic and intrinsic factors. These different responses reinforce the idea of their differential participation in fish lipid homeostasis.

How Muscle Structure and Composition Influence Meat and Flesh Quality.

Skeletal muscle consists of several tissues, such as muscle fibers and connective and adipose tissues. This review aims to describe the features of these various muscle components and their relationships with the technological, nutritional, and sensory properties of meat/flesh from different livestock and fish species. Thus, the contractile and metabolic types, size and number of muscle fibers, the content, composition and distribution of the connective tissue, and the content and lipid composition of intramuscular fat play a role in the determination of meat/flesh appearance, color, tenderness, juiciness, flavor, and technological value. Interestingly, the biochemical and structural characteristics of muscle fibers, intramuscular connective tissue, and intramuscular fat appear to play independent role, which suggests that the properties of these various muscle components can be independently modulated by genetics or environmental factors to achieve production efficiency and improve meat/flesh quality.

Rapid quantification of muscle fat content and subcutaneous adipose tissue in fish using MRI

The potentiality of MRI to quantify fat content in flesh and subcutaneous fat in fish cutlets was investigated. Low measurement time was aimed at in a view to handling large number of samples needed in selective breeding programs for example. Results on fresh and frozen-thawed cutlets were compared to assess this way of conservation. As MRI generates unwanted spatial variations of the signal, a correction method was developed enabling the measurement on several cutlets simultaneously in less than 3 min per sample. For subcutaneous fat, the results were compared with vision measurements. High correlations between both techniques were found (R(2)=0.77 and 0.87 for the ventral and dorsal part). Fat in flesh was validated vs NMR measurements. No statistical difference was found between fresh and frozen-thawed cutlets. RMSE was respectively 0.8% and 0.89%. These results confirmed the potentiality of MRI for fat measurement in fish particularly for a large number of samples.

Myostatin induces atrophy of trout myotubes through inhibiting the TORC1 signaling and promoting Ubiquitin–Proteasome and Autophagy-Lysosome degradative pathways

Myostatin (MSTN) is well known as a potent inhibitor of muscle growth in mammals and has been shown to both inhibit the growth promoting TORC1 signaling pathway and promote Ubiquitin-Proteasomal and Autophagy-Lysosomal degradative routes. In contrast, in non-mammalian species, despite high structural conservation of MSTN sequence, functional conservation is only assumed. Here, we show that treatment of cultured trout myotubes with human recombinant MSTN (huMSTN) resulted in a significant decrease of their diameter by up to 20%, validating the use of heterologous huMSTN in our in vitro model to monitor the processes by which this growth factor promotes muscle wasting in fish. Accordingly, huMSTN stimulation prevented the full activation by IGF1 of the TORC1 signaling pathway, as revealed by the analysis of the phosphorylation status of 4E-BP1. Moreover, the levels of the proteasome-dependent protein Atrogin1 exhibited an increase in huMSTN treated cells. Likewise, we observed a stimulatory effect of huMSTN treatment on the levels of LC3-II, the more reliable marker of the Autophagy-Lysosomal degradative system. Overall, these results show for the first time in a piscine species the effect of MSTN on several atrophic and hypertrophic pathways and support a functional conservation of this growth factor between lower and higher vertebrates.

ATOL: The Multi-species Livestock Trait Ontology

This paper presents the multi-species Animal Trait Ontology for Livestock (ATOL) and the methodology used for its design. ATOL has been designed as a reference source for indexing phenotype databases and scientific papers. It covers five major topics related to animal productions: growth and meat quality, animal nutrition, milk production, reproduction and welfare. It is composed of species-independent concepts subsuming species-specific ones so that cross-species and species-specific reasoning can be performed consistently. In order to ensure a large consensus, three complementary approaches have successively been applied to its design: reuse of existing ontologies, integration of production-specific livestock traits by a large team of domain experts and curators and terminology analysis of scientific papers. It resulted in a detailed taxonomy of 1,654 traits that is available at http://www.atol-ontology.com

Differentially expressed proteins in rainbow trout adipocytes isolated from visceral and subcutaneous tissues

In rainbow trout, subcutaneous (in dorsal and ventral positions) and visceral fat deposits are known to influence the yield of edible flesh, whilst their respective roles in metabolism, storage and release of fatty acids have not, so far, been directly studied. The present work aimed to identify, by using 2D electrophoresis, proteins differentially expressed in isolated mature adipocytes originating from these various localizations in prepubescent females. A total of nine proteins were estimated to be differentially expressed according to the localisation of the adipocytes. Seven protein spots were considered to be present in the three fat deposits at differing abundances, and among them only six were estimated as being specific to fat tissues. Among these, five were more abundant in subcutaneous adipocytes of both sites compared to perivisceral adipocytes. Four were identified: three as H-FABP, ATP synthase, serum deprivation-response protein, indicating higher metabolic activity in subcutaneous adipocytes, while the latter, annexin, indicative of a higher proportion of less mature adipocytes, as also suggested by their smaller mean diameter. The more abundant protein in visceral isolated adipocytes is actin, known to be involved in cytoskeleton structure and to increase during adipogenesis. This allows us to suggest their more mature stage of development, in relation with their higher mean diameter.

The genetics of overwintering performance in two-year old common carp and its relation to performance until market size

Using farmed common carp, we investigated the genetic background of the second year overwintering performance and its relation to the performance during the third growing season and at market size. The experimental stock was established by partial factorial design with a series of 4 factorial matings of 5 dams and 10 sires each. The families were reared communally and pedigree was re-constructed with 93.6% success using 12 microsatellites on 2008 offspring. Three successive recordings (second autumn, third spring, and third autumn—market size) covering two periods (second overwintering, third growing season) were included. Body weight, Fulton’s condition factor and percent muscle fat content were recorded at all times and headless carcass yield and fillet yield were recorded at market size. Specific growth rate, absolute and relative fat change and overall survival were calculated for each period. Heritability estimates were significantly different from zero and almost all traits were moderately to highly heritable (h2 = 0.36–1.00), except survival in both periods and fat change (both patterns) during overwintering (h2 = 0.12–0.15). Genetic and phenotypic correlations imply that selection against weight loss and fat loss during overwintering is expected to lead to a better winter survival, together with a positive effect on growth in the third growing season. Interestingly, higher muscle fat content was genetically correlated to lower survival in the following period (rg = -0.59; -0.53, respectively for winter and the third summer). On the other hand, higher muscle fat was also genetically linked to better slaughter yields. Moreover, selection for higher condition factor would lead to better performance during winter, growing season and at market size.

Naa15 knockdown enhances c2c12 myoblast fusion and induces defects in zebrafish myotome morphogenesis

The comprehension of muscle tissue formation and regeneration is essential to develop therapeutic approaches against muscle diseases or loss in muscle mass and strength during ageing or cancer. One of the critical steps in muscle formation is the fusion of muscle cells to form or regenerate muscle fibres. To identify new genes controlling myoblast fusion, we undertook an siRNA screen in c2c12 myoblasts and found that N-alpha-acetyltransferase 15 (Naa15) knockdown enhanced c2c12 myoblast fusion suggesting that Naa15 negatively regulated myogenic cell fusion. We identified two Naa15 orthologous genes in zebrafish genome: naa15a and naa15b. These two orthologs are both expressed in myogenic domain of the somite. Knockdown of zebrafish naa15a and naa15b genes induced a “U” shaped segmentation of the myotome and alteration of myotome boundaries resulting in the formation of abnormally long myofibres spanning adjacent somites. Taken together these results show that Naa15 regulates myotome formation and myogenesis in fish.

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. Author summary The 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.

Histological, transcriptomic and in vitro analysis reveal an intrinsic activated state of myogenic precursors in hyperplasic muscle of trout

Background The dramatic increase in myotomal muscle mass in post-hatching fish is related to their ability to lastingly produce new muscle fibres, a process termed hyperplasia. The molecular and cellular mechanisms underlying fish muscle hyperplasia largely remain unknown. In this study, we aimed to characterize intrinsic properties of myogenic cells originating from fish hyperplasic muscle. For this purpose, we compared in situ proliferation, in vitro cell behavior and transcriptomic profile of myogenic precursors originating from hyperplasic muscle of juvenile trout (JT) and from non-hyperplasic muscle of fasted juvenile trout (FJT) and adult trout (AT). Results For the first time, we showed that myogenic precursors proliferate in hyperplasic muscle from JT as shown by in vivo BrdU labeling. This proliferative rate was very low in AT and FJT muscle. Transcriptiomic analysis revealed that myogenic cells from FJT and AT displayed close expression profiles with only 64 differentially expressed genes (BH corrected p-val < 0.001). In contrast, 2623 differentially expressed genes were found between myogenic cells from JT and from both FJT and AT. Functional categories related to translation, mitochondrial activity, cell cycle, and myogenic differentiation were inferred from genes up regulated in JT compared to AT and FJT myogenic cells. Conversely, Notch signaling pathway, that signs cell quiescence, was inferred from genes down regulated in JT compared to FJT and AT. In line with our transcriptomic data, in vitro JT myogenic precursors displayed higher proliferation and differentiation capacities than FJT and AT myogenic precursors. Conclusions The transcriptomic analysis and examination of cell behavior converge to support the view that myogenic cells extracted from hyperplastic muscle of juvenile trout are intrinsically more potent to form myofibres than myogenic cells extracted from non-hyperplasic muscle. The generation of gene expression profiles in myogenic cell extracted from muscle of juvenile trout may yield insights into the molecular and cellular mechanisms controlling hyperplasia and provides a useful list of potential molecular markers of hyperplasia.