Julien Bobe

Oogenesis in teleosts: How fish eggs are formed

One of the major objectives of the aquaculture industry is the production of a large number of viable eggs with high survival. Major achievements have been made in recent years in improving protocols for higher efficiency of egg production and viability of progeny. Main gaps remain, however, in understanding the dynamic processes associated with oogenesis, the formation of an egg, from the time that germ cells turn into oogonia, until the release of ova during spawning in teleosts. Recent studies on primordial germ-cells, yolk protein precursors and their processing within the developing oocyte, the deposition of vitamins in eggs, structure and function of egg envelopes and oocyte maturation processes, further reveal the complexity of oogenesis. Moreover, numerous circulating endocrine and locally-acting paracrine and autocrine factors regulate the various stages of oocyte development and maturation. Though it is clear that the major regulators during vitellogenesis and oocyte maturation are the pituitary gonadotropins (LH and FSH) and sex steroids, the picture emerging from recent studies is of complex hormonal cross-talk at all stages between the developing oocyte and its surrounding follicle layers to ensure coordination of the various processes that are involved in the production of a fertilizable egg. In this review we aim at highlighting recent advances on teleost fish oocyte differentiation, maturation and ovulation, including those involved in the degeneration and reabsorption of ovarian follicles (atresia). The role of blood-borne and local ovarian factors in the regulation of the key steps of development reveal new aspects associated with egg formation.

The rainbow trout genome provides novel insights into evolution after whole-genome duplication in vertebrates

Vertebrate evolution has been shaped by several rounds of whole-genome duplications (WGDs) that are often suggested to be associated with adaptive radiations and evolutionary innovations. Due to an additional round of WGD, the rainbow trout genome offers a unique opportunity to investigate the early evolutionary fate of a duplicated vertebrate genome. Here we show that after 100 million years of evolution the two ancestral subgenomes have remained extremely collinear, despite the loss of half of the duplicated protein-coding genes, mostly through pseudogenization. In striking contrast is the fate of miRNA genes that have almost all been retained as duplicated copies. The slow and stepwise rediploidization process characterized here challenges the current hypothesis that WGD is followed by massive and rapid genomic reorganizations and gene deletions.

Egg and sperm quality in fish.

Fish egg quality can be defined as the ability of the egg to be fertilized and subsequently develop into a normal embryo. Similarly, sperm quality can be defined as its ability to successfully fertilize an egg and subsequently allow the development of a normal embryo. In the wild or under aquaculture conditions, the quality of fish gametes can be highly variable and is under the influence of a significant number of external factors or broodstock management practices. For these reasons, the topic of gamete quality has received increasing attention. Despite the significant efforts made towards a better understanding of the factors involved in the control of gamete quality, the picture is far from being complete and the control of gamete quality remains an issue in the aquaculture industry. Some of the factors responsible for the observed variability of gamete quality remain largely unknown or poorly understood. In addition very little is known about the cellular and molecular mechanisms involved in the control of egg and sperm quality. In the present review, the molecular and cellular characteristics of fish gametes are presented with a special interest for the mechanisms that could participate in the regulation of gamete quality. Then, after defining egg and sperm quality, and how can it can be accurately estimated or predicted, we provide an overview of the main factors that can impact gamete quality in teleosts.

The spotted gar genome illuminates vertebrate evolution and facilitates human-teleost comparisons

To connect human biology to fish biomedical models, we sequenced the genome of spotted gar (Lepisosteus oculatus), whose lineage diverged from teleosts before teleost genome duplication (TGD). The slowly evolving gar genome has conserved in content and size many entire chromosomes from bony vertebrate ancestors. Gar bridges teleosts to tetrapods by illuminating the evolution of immunity, mineralization and development (mediated, for example, by Hox, ParaHox and microRNA genes). Numerous conserved noncoding elements (CNEs; often cis regulatory) undetectable in direct human-teleost comparisons become apparent using gar: functional studies uncovered conserved roles for such cryptic CNEs, facilitating annotation of sequences identified in human genome-wide association studies. Transcriptomic analyses showed that the sums of expression domains and expression levels for duplicated teleost genes often approximate the patterns and levels of expression for gar genes, consistent with subfunctionalization. The gar genome provides a resource for understanding evolution after genome duplication, the origin of vertebrate genomes and the function of human regulatory sequences.

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

BackgroundThe 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.MethodsNylon 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.ResultsFrom 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.ConclusionWe 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.

Molecular cloning and expression of a TNF receptor and two TNF ligands in the fish ovary.

Using degenerative primers, partial cDNAs of a TNF (tumor necrosis factor) receptor and two TNF ligands were obtained by PCR of zebrafish and trout cDNAs, or cDNA libraries. These fragments were then used to screen cDNA libraries of appropriate tissues to obtain clones containing full coding sequences. A zebrafish cDNA was obtained that presumably codes for a 438 amino acid ovarian TNF receptor (OTR) that was identified as a death-domain-containing member of the TNF receptor family. On Northern blots, the OTR cDNA hybridized with a 3.4-kb transcript that is abundant in the zebrafish ovary but lightly detected in all other tissues tested. A zebrafish cDNA presumably coding for a 214 amino acid protein with sequence similarity to mammalian TRAIL (TNF-related apoptosis inducing ligand), was also isolated. In addition, a fragment of the brook trout TRAIL homologue was obtained. Finally, a full-length brook trout cDNA, that presumably codes for a 255 amino acid protein with sequence similarity to mammalian TNF-alpha and lymphotoxin-alpha, was isolated. This study is the first report of a death-domain-containing TNF receptor and the first published report of a TNF ligand in fish.

Targeted Gene Expression Profiling in the Rainbow Trout (Oncorhynchus mykiss) Ovary During Maturational Competence Acquisition and Oocyte Maturation

Abstract A real-time polymerase chain reaction-based gene expression survey was performed using 37 target genes and 22 female rainbow trout sampled during follicular maturational competence (FMC) acquisition or during oocyte maturation. In females sampled before meiosis resumption, FMC was estimated using an in vitro assay. Several growth factors, bone morphogenetic proteins, steroidogenic enzymes, cathepsins, genes known to play a role in the fish preovulatory ovary, as well as previously unstudied genes, were analyzed in this survey. Gene expression profiling was performed using a supervised clustering analysis in order to identify groups of genes exhibiting similar expression profiles in the ovary during FMC acquisition and follicular maturation. From the clustering analysis, three clusters exhibiting a specific expression during FMC acquisition or at the time of oocyte maturation were identified. Cluster 1 was characterized by a progressive increase in gene expression during FMC acquisition, whereas cluster 2 exhibited an increased expression at the time of oocyte maturation. In contrast, cluster 3 was characterized by a decreased mRNA expression at the time of oocyte maturation. Among the 37 target genes used in this survey, 18 were significantly regulated during maturational competence acquisition or at the time of oocyte maturation. Among these 18 genes, 16 belonged to one of the three clusters identified. Although the results allowed a global description of gene expression profiles, they also suggest an important role for several factors, including some previously unstudied bone morphogenetic proteins, in the paracrine control of FMC acquisition and meiosis resumption.

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

BackgroundThe 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.ResultsThe 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.ConclusionFirst, 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.

Nme protein family evolutionary history, a vertebrate perspective.

BackgroundThe Nme family, previously known as Nm23 or NDPK, is involved in various molecular processes including tumor metastasis and some members of the family, but not all, exhibit a Nucleoside Diphosphate Kinase (NDPK) activity. Ten genes are known in humans, in which some members have been extensively studied. In non-mammalian species, the Nme protein family has received, in contrast, far less attention. The picture of the vertebrate Nme family remains thus incomplete and orthology relationships with mammalian counterparts were only partially characterized. The present study therefore aimed at characterizing the Nme gene repertoire in vertebrates with special interest for teleosts, and providing a comprehensive overview of the Nme gene family evolutionary history in vertebrates.ResultsIn the present study, we present the evolutionary history of the Nme family in vertebrates and characterize the gene family repertoire for the first time in several non-mammalian species. Our observations show that vertebrate Nme genes can be separated in two evolutionary distinct groups. Nme1, Nme2, Nme3, and Nme4 belong to Group I while vertebrate Nme5, Nme6, Nme7, Nme8, and Nme9 belong to Group II. The position of Nme10 is in contrast more debatable due to its very specific evolutionary history. The present study clearly indicates that Nme5, Nme6, Nme7, and Nme8 originate from duplication events that occurred before the chordate radiation. In contrast, Nme genes of the Group I have a very different evolutionary history as our results suggest that they all arise from a common gene present in the chordate ancestor. In addition, expression patterns of all zebrafish nme transcripts were studied in a broad range of tissues by quantitative PCR and discussed in the light of the function of their mammalian counterparts.ConclusionThis work offers an evolutionary framework that will pave the way for future studies on vertebrate Nme proteins and provides a unified vertebrate Nme nomenclature that is consistent with the nomenclature in use in mammals. Based on protein structure and expression data, we also provide new insight into molecular functions of Nme proteins among vertebrates and raise intriguing questions on the roles of Nme proteins in gonads.

Post-ovulatory ageing and egg quality: A proteomic analysis of rainbow trout coelomic fluid

BackgroundIn fish, oocyte post-ovulatory ageing is associated with egg quality decrease. During this period, eggs are held in the body cavity where they bath in a semi-viscous liquid known as coelomic fluid (CF). CF components are suspected to play a role in maintaining oocyte fertility and developmental competence (egg quality). However, CF proteic composition remains poorly studied. Thus rainbow trout CF proteome was studied during the egg quality decrease associated with oocyte post-ovulatory ageing.MethodsHigh resolution two-dimensional gel electrophoresis was used to analyze the proteome of rainbow trout (Oncorhynchus mykiss) CF in relationship with the egg quality decrease associated with oocyte post-ovulatory ageing. A first experiment was performed using CF pools originating from 17 females sampled at ovulation as well as 7, 14 and 21 days later. These observations were verified using a second set of CF pools originating from 22 females sampled 5 and 16 days following ovulation.ResultsApproximately 200 protein spots of 10–105 kDa molecular mass and 3–10 pI were detected in CF samples. Several protein spots, while undetected at the time of ovulation, exhibited a progressive and strong accumulation in CF during post-ovulatory ageing. After silver-staining and Matrix-Assisted Laser Desorption Time Of Flight (MALDI-TOF) mass spectrometer analysis, some of these protein spots were identified as lipovitellin II fragments.ConclusionsThese observations suggest that egg protein fragments accumulate in the CF during the post-ovulatory period and could therefore be used to detect egg quality defects associated with oocyte post-ovulatory ageing.