Microarray-based analysis of fish egg quality after natural or controlled ovulation

BACKGROUND: The preservation of fish egg quality after ovulation-control protocols is a major issue for the development of specific biotechnological processes (e.g. nuclear transfer). Depending on the species, it is often necessary to control the timing of ovulation or induce the ovulatory process. The hormonal or photoperiodic control of ovulation can induce specific egg quality defects that have been thoroughly studied. In contrast, the impact on the egg transcriptome as a result of these manipulations has received far less attention. Furthermore, the relationship between the mRNA abundance of maternally-inherited mRNAs and the developmental potential of the egg has never benefited from genome-wide studies. Thus, the present study aimed at studying the rainbow trout (Oncorhynchus mykiss) egg transcriptome after natural or controlled ovulation using 9152-cDNA microarrays. RESULTS: The analysis of egg transcriptome after natural or controlled ovulation led to the identification of 26 genes. The expression patterns of 17 of those genes were monitored by real-time PCR. We observed that the control of ovulation by both hormonal induction and photoperiod manipulation induced significant changes in the egg mRNA abundance of specific genes. A dramatic increase of Apolipoprotein C1 (APOC1) and tyrosine protein kinase HCK was observed in the eggs when a hormonal induction of ovulation was performed. In addition, both microarray and real-time PCR analyses showed that prohibitin 2 (PHB2) egg mRNA abundance was negatively correlated with developmental success. CONCLUSION: First, we showed, for the first time in fish, that the control of ovulation using either a hormonal induction or a manipulated photoperiod can induce differences in the egg mRNA abundance of specific genes. While the impact of these modifications on subsequent embryonic development is unknown, our observations clearly show that the egg transcriptome is affected by an artificial induction of ovulation. Second, we showed that the egg mRNA abundance of prohibitin 2 was reflective of the developmental potential of the egg. Finally, the identity and ontology of identified genes provided significant hints that could result in a better understanding of the mechanisms associated with each type of ovulation control (i.e. hormonal, photoperiodic), and in the identification of conserved mechanisms triggering the loss of egg developmental potential

Two unrelated putative membrane-bound progestin receptors, progesterone membrane receptor component 1 (PGMRC1) and membrane progestin receptor (mPR) beta, are expressed in the rainbow trout oocyte and exhibit similar ovarian expression patterns

BACKGROUND: In lower vertebrates, steroid-induced oocyte maturation is considered to involve membrane-bound progestin receptors. Two totally distinct classes of putative membrane-bound progestin receptors have been reported in vertebrates. A first class of receptors, now termed progesterone membrane receptor component (PGMRC; subtypes 1 and 2) has been studied since 1996 but never studied in a fish species nor in the oocyte of any animal species. A second class of receptors, termed membrane progestin receptors (mPR; subtypes alpha, beta and gamma), was recently described in vertebrates and implicated in the progestin-initiated induction of oocyte maturation in fish. METHODS: In the present study, we report the characterization of the full coding sequence of rainbow trout PGMRC1 and mPR beta cDNAs, their tissue distribution, their ovarian expression profiles during oogenesis, their hormonal regulation in the full grown ovary and the in situ localization of PGMRC1 mRNA in the ovary. RESULTS: Our results clearly show, for the first time in any animal species, that rainbow trout PGMRC1 mRNA is present in the oocyte and has a strong expression in ovarian tissue. In addition, we show that both mPR beta and PGMRC1, two members of distinct membrane-bound progestin receptor classes, exhibit highly similar ovarian expression profiles during the reproductive cycle with maximum levels during vitellogenesis and a down-expression during late vitellogenesis. In addition, the mRNA abundance of both genes is not increased after in vitro hormonal stimulation of full grown follicles by maturation inducing hormones. CONCLUSION: Together, our findings suggest that PGMRC1 is a new possible participant in the progestin-induced oocyte maturation in fish. However, its participation in the process of oocyte maturation, which remains to be confirmed, would occur at post-transcriptional levels

Androgen-induced masculinization in rainbow trout results in a marked dysregulation of early gonadal gene expression profiles

<p>Abstract</p> <p>Background</p> <p>Fish gonadal sex differentiation is affected by sex steroids treatments providing an efficient strategy to control the sexual phenotype of fish for aquaculture purposes. However, the biological effects of such treatments are poorly understood. The aim of this study was to identify the main effects of an androgen masculinizing treatment (11β-hydroxyandrostenedione, 11βOHΔ4, 10 mg/kg of food for 3 months) on gonadal gene expression profiles of an all-female genetic population of trout. To characterize the most important molecular features of this process, we used a large scale gene expression profiling approach using rainbow trout DNA microarrays combined with a detailed gene ontology (GO) analysis.</p> <p>Results</p> <p>2,474 genes were characterized as up-regulated or down-regulated in trout female gonads masculinized by androgen in comparison with control male or female gonads from untreated all-male and all-female genetic populations. These genes were classified in 13 k-means clusters of temporally correlated expression profiles. Gene ontology (GO) data mining revealed that androgen treatment triggers a marked down-regulation of genes potentially involved in early oogenesis processes (GO 'mitotic cell cycle', 'nucleolus'), an up-regulation of the translation machinery (GO 'ribosome') along with a down-regulation of proteolysis (GO 'proteolysis', 'peptidase' and 'metallopeptidase activity'). Genes considered as muscle fibres markers (GO 'muscle contraction') and genes annotated as structural constituents of the extracellular matrix (GO 'extracellular matrix') or related to meiosis (GO 'chromosome' and 'meiosis') were found significantly enriched in the two clusters of genes specifically up-regulated in androgen-treated female gonads. GO annotations 'Sex differentiation' and 'steroid biosynthesis' were enriched in a cluster of genes with high expression levels only in control males. Interestingly none of these genes were stimulated by the masculinizing androgen treatment.</p> <p>Conclusion</p> <p>This study provides evidence that androgen masculinization results in a marked dysregulation of early gene expression profiles when compared to natural testicular or ovarian differentiation. Based on these results we suggest that, in our experimental conditions, androgen masculinization proceeds mainly through an early inhibition of female development.</p

Photoperiodic treatments and reproduction in farm animals

Farm animal productions are frequently tied to the seasons, which explain the seasonal variations in availability of fresh animal products (meat, milk, cheese and eggs). Among farmed species, fishes and birds are generally considered as more directly sensitive to external factors (mainly temperature in fish and photoperiod in birds). Artificial photoperiodic treatments are widely used in numerous species including mammals; they consist in providing extra-light during natural short days (chickens, turkeys, guinea fowl, sheep and goats), or in the administration of melatonin during long days (goats, sheep). These measures help adjust the breeding season to the requirements of the breeder and/or cancel completely seasonal variations of sperm production in semen production centres (mammals) as well as in poultry and fish farms. Additionally, pure light treatments (without melatonin), especially when there is outside access, are non invasive and preserve the animals’ integrity and welfare.Les productions issues d'animaux d'élevage sont fréquemment rythmées sur les saisons, avec pour conséquence une disponibilité saisonnière en produits frais sur les marchés (viandes, lait et fromages, oeufs). Parmi les espèces d'intérêt en production animale, les poissons et les oiseaux sont généralement considérés comme plus directement sensibles aux facteurs externes, essentiellement les variations de la température pour les poissons et de la durée de la phase claire du jour, ou photopériode, pour les oiseaux. Chez de nombreuses espèces incluant des mammifères, nous utilisons largement des traitements photopériodiques artificiels qui consistent en un éclairement supplémentaire pendant les jours courts naturels (chez le poulet, la dinde, la pintade, le mouton et la chèvre) ou en l'administration de mélatonine pendant les jours longs (chez le mouton et la chèvre); ils permettent d'ajuster la période de reproduction aux souhaits de l'éleveur et/ou d'abolir complètement les variations saisonnières de la production spermatique dans les centres producteurs de semence destinée à l'insémination artificielle (mammifères), et dans les élevages (volailles, poissons). Par ailleurs, des traitements photopériodiques « purs » (sans mélatonine), en particulier lorsqu'ils sont appliqués en bâtiments ouverts, sont non invasifs et donc respectent l'intégrité et le bien-être des animaux

Identification of new participants in the rainbow trout (Oncorhynchus mykiss) oocyte maturation and ovulation processes using cDNA microarrays

BACKGROUND: The hormonal control of oocyte maturation and ovulation as well as the molecular mechanisms of nuclear maturation have been thoroughly studied in fish. In contrast, the other molecular events occurring in the ovary during post-vitellogenesis have received far less attention. METHODS: Nylon microarrays displaying 9152 rainbow trout cDNAs were hybridized using RNA samples originating from ovarian tissue collected during late vitellogenesis, post-vitellogenesis and oocyte maturation. Differentially expressed genes were identified using a statistical analysis. A supervised clustering analysis was performed using only differentially expressed genes in order to identify gene clusters exhibiting similar expression profiles. In addition, specific genes were selected and their preovulatory ovarian expression was analyzed using real-time PCR. RESULTS: From the statistical analysis, 310 differentially expressed genes were identified. Among those genes, 90 were up-regulated at the time of oocyte maturation while 220 exhibited an opposite pattern. After clustering analysis, 90 clones belonging to 3 gene clusters exhibiting the most remarkable expression patterns were kept for further analysis. Using real-time PCR analysis, we observed a strong up-regulation of ion and water transport genes such as aquaporin 4 (aqp4) and pendrin (slc26). In addition, a dramatic up-regulation of vasotocin (avt) gene was observed. Furthermore, angiotensin-converting-enzyme 2 (ace2), coagulation factor V (cf5), adam 22, and the chemokine cxcl14 genes exhibited a sharp up-regulation at the time of oocyte maturation. Finally, ovarian aromatase (cyp19a1) exhibited a dramatic down-regulation over the post-vitellogenic period while a down-regulation of Cytidine monophosphate-N-acetylneuraminic acid hydroxylase (cmah) was observed at the time of oocyte maturation. CONCLUSION: We showed the over or under expression of more that 300 genes, most of them being previously unstudied or unknown in the fish preovulatory ovary. Our data confirmed the down-regulation of estrogen synthesis genes during the preovulatory period. In addition, the strong up-regulation of aqp4 and slc26 genes prior to ovulation suggests their participation in the oocyte hydration process occurring at that time. Furthermore, among the most up-regulated clones, several genes such as cxcl14, ace2, adam22, cf5 have pro-inflammatory, vasodilatory, proteolytics and coagulatory functions. The identity and expression patterns of those genes support the theory comparing ovulation to an inflammatory-like reaction

Évolution de la place de l’animal et des points de vue sur son élevage dans la société française : quels enjeux pour la recherche agronomique ?

Plusieurs travaux sociologiques ont permis d’identifier des sujets de contestation des élevages en France. Cette contestation s’accompagne d’une baisse de la consommation moyenne par habitant des produits carnés, à l’exception de la volaille, avec une évolution des comportements alimentaires. Sans nier les effets du prix des produits carnés et du revenu des ménages sur les décisions d’achat des consommateurs, nous nous intéressons ici à d’autres éléments pouvant influencer les points de vue des citoyens sur les élevages. Avec quelques repères historiques, l’évolution de la place de l’animal dans la société est examinée. Sont ensuite passés en revue : poids économique de l’élevage, conditions d’élevage et de mise à mort de l’animal, débats philosophiques, choix religieux, choix alimentaires, choix culturels, coexistence des animaux et des citadins, intérêt des médias pour l’animal et son élevage, militantisme. Pour finir, nous évoquons brièvement les pistes explorées par la recherche agronomique, que ce soit au niveau de l’évolution des systèmes d’élevage et de leurs impacts négatifs et positifs, ou au niveau de la biologie de l’animal. Des données génériques sont acquises à ces différents niveaux et des approches interdisciplinaires sont développées pour mieux appréhender des phénomènes complexes et proposer des innovations. Enfin, plusieurs méthodologies sont utilisées pour différents types d’évaluation à différentes échelles permettant d’affiner les diagnostics, d’anticiper les conséquences d’une innovation technologique, de repérer objectivement les progrès ou les retards par rapport à des objectifs d’amélioration des systèmes, et aussi d’aborder la question des impacts de la recherche en agriculture sur la société.Sociological studies have identified several points of dispute regarding animal farming in France. Furthermore, the consumption of meat products is decreasing except for poultry meat. In this paper, the possible reasons for this trend are analyzed by taking into consideration the views of citizens on animal farming without denying the influence of product prices and household incomes on customers’ buying decisions. The evolution of animal status is examined by considering the historical background. Then, various factors which could be involved in developing points of views are considered: economical weight of the animal farming sector, farming and slaughter methods, philosophical debates, religious options, food choices, cultural attitudes, animals in the city, information sources and activism. Finally, some new strategies of agricultural research are highlighted. Interdisciplinary generic knowledge is produced both in animal biology and the livestock farming systems in order to decipher complex mechanisms to propose innovative solutions. In addition, various evaluation methods are developed to improve diagnostics, anticipate the effects of technological innovations, follow the evolution of farming systems according to improvement objectives, and analyze the impact of agricultural research on society

Broodstock management and hormonal manipulations of fish reproduction

93 p., il., y bibliografíaControl of reproductive function in captivity is essential for the sustainability of commercial aquaculture production, and in many fishes it can be achieved by manipulating photoperiod, water temperature or spawning substrate. The fish reproductive cycle is separated in the growt(gametogenesis) and maturation phase (oocyte maturation and spermiation), both controlled by the reproductive hormones of the brain, pituitary and gonad. Although the growth phase of reproductive development is concluded in captivity in most fishes-the major exemption being the freshwater eel (Anguilla spp.), oocyte maturation (OM) and ovulation in females, and spermiation in males may require exogenous hormonal therapies. In some fishes, these hormonal manipulations are used only as a management tool to enhance the efficiency of egg production and facilitate hatchery operations, but in others exogenous hormones are the only way to produce fertilized eggs reliably. Hormonal manipulations of reproductive function in cultured fishes have focused on the use of either exogenous luteinizing hormone(LH) preparations that act directly at the level of the gonad, or synthetic agonists of gonadotropin-releasing hormone (GnRHa) that act at the level of the pituitary to induce release of the endogenous LH stores, which, in turn act at the level of the gonad to induce steroidogenesis and the process of OM and spermiation. After hormonal induction of maturation, broodstock should spawn spontaneously in their rearing enclosures, however, the natural breeding behavior followed by spontaneous spawning may be lost in aquaculture conditions. Therefore, for many species it is also necessary to employ artificial gamete collection and fertilization. Finally, a common question in regards to hormonal therapies is their effect on gamete quality, compared to naturally maturing or spawning broodfish. The main factors that may have significant consequences on gamete quality-mainly on eggs-and should be considered when choosing a spawning induction procedure include (a) the developmental stage of the gonads at the time the hormonal therapy is applied, (b) the type of hormonal therapy, (c) the possible stress induced by the manipulation necessary for the hormone administration and (d) in the case of artificial insemination, the latency period between hormonal stimulation and stripping for in vitro fertilization.Peer reviewe

Modes de perturbation de la stéroïdogenèse testiculaire et de la spermatogenèse chez le poisson zèbre (Danio rerio) par des fongicides azolés

Les azoles sont des fongicides présents dans les milieux aquatiques et connus pour inhiber des activités enzymatiques de cytochromes P450 (CYP). L'objectif de ce travail de thèse est de caractériser le mécanisme d'action d'un fongicide pharmaceutique, le clotrimazole sur la stéroïdogenèse testiculaire chez le poisson zèbre au travers l'étude d'un réseau de gènes fonctionnels le long de l'axe cerveau-hypophyse-gonade, et d'évaluer la capacité du clotrimazole à perturber la spermatogenèse. Nous montrons que le clotrimazole est capable d'affecter la stéroïdogenèse de manière différente in vitro et in vivo (i) des expositions d'explants testiculaires in vitro conduisent à l'inhibition de la synthèse de 11-kétotestostérone (11-KT), montrant une action directe de la molécule sur le testicule et (ii) l'exposition in vivo provoque une augmentation de l'expression de gènes impliqués dans le processus de la stéroïdogenèse. Nous avons ainsi mis en évidence un système de compensation biologique au niveau de l'organisme, avec un rôle prépondérant de la voie Fsh/FshR dans la médiation des effets du clotrimazole. Enfin, des effets sur la spermatogenèse ont été observés in vivo suite à une exposition chronique au clotrimazole, avec notamment une augmentation de la masse gonadique et du nombre de cellules de Leydig. Les effets tissulaires observés sont cohérents avec des effets mesurés au niveau moléculaire. L'ensemble de ces données montre l'intérêt de la démarche expérimentale utilisée pour caractériser le mécanisme d'action du clotrimazole. Ce travail ouvre de nombreuses perspectives, en premier lieu sur l'étude de l'impact fonctionnel du clotrimazole sur la reproduction.Azole fungicides are detected in the aquatic environment and can inhibit enzymatic activities of cytochrome P450 (CYP). This thesis aims to characterize the mechanism of action of the pharmaceutical clotrimazole on testicular steroidogenesis in zebrafish using a network of functional genes along the brain-pituitary-gonad axis and to evaluate the effect of clotrimazole on spermatogenesis. We show that clotrimazole is able to affect steroidogenesis differently in vitro and in vivo (i) the exposure of testicular explants in vitro leads to inhibition of the synthesis of 11-ketotestosterone, showing a direct action on the testis, and (ii) in vivo exposure increases the transcript levels of genes involved in steroidogenesis. An integrative approach of gene expression measurements along the pituitary-gonad axis highlights a mechanism of biological compensation, with a critical role of the Fsh/FshR pathway in mediating the effects of clotrimazole on testicular steroidogenesis. Finally, effects on spermatogenesis were observed in vivo following chronic exposure to clotrimazole, with an increase in gonado-somatic index as well as in number of Leydig cells. These observations are consistent with measurements at molecular level. Taken together, these data show the interest of an integrative approach. This work raises further concerns, primarily on the study of the functional impact of clotrimazole on reproduction, including male and female studies.RENNES1-BU Sciences Philo (352382102) / SudocSudocFranceF

The follicular sensitivity in vitro to maturation-inducing hormones in rainbow trout, Salmo gairdneri: Role of oestradiol-17β

Trout ovaries were processed in vitro to determine relationships between the following parameters — oocyte sensitivity to the maturational steroid 17α-hydroxy-20β-dihydroprogesterone (17α,20β-OH-P); follicular sensitivity to the maturational gonadotropin s-GtH; inhibitory potency of exogenous oestradiol-17β (E2) during s-GtH-induced maturation; and level of E2 in the plasma. The sensitivity to hormones was estimated by the median efficient dose (MED) for oocyte maturation in vitro.The peripheral migration of the germinal vesicle coincided with an increase in oocyte sensitivity to 17α,20β-OH-P. A significant correlation was observed between plasma E2 level and the follicular sensitivity to s-GtH, but not between plasma E2 and the oocyte sensitivity to 17α,20β-OH-P. It was concluded that the peripheral migration of the GV is a morphological event which coincides with an increase in oocyte sensitivity, and that E2 is a physiological regulator of follicular sensitivity to GtH