Joan Cerdà

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.

Fish proteome analysis: model organisms and non-sequenced species.

In the last decade, proteomic technologies have been increasingly used in fish biology research. Proteomics has been applied primarily to investigate the physiology, development biology and the impact of contaminants in fish model organisms, such as zebrafish (Danio rerio), as well as in some commercial species produced in aquaculture, mainly salmonids and cyprinids. However, the lack of previous genetic information on most fish species has been a major drawback for a more general application of the different proteomic technologies currently available. Also, many teleosts of interest in biological research and with potential application in aquaculture hold unique physiological characteristics that cannot be directly addressed from the study of small laboratory fish models. This review describes proteomic approaches that have been used to investigate diverse biological questions in model and non‐model fish species. We will also evaluate the current possibilities to integrate fish proteomics with other “omic” approaches, as well as with additional complementary techniques, in order to address the future challenges in fish biology research.

The zebrafish genome encodes the largest vertebrate repertoire of functional aquaporins with dual paralogy and substrate specificities similar to mammals

BackgroundAquaporins are integral membrane proteins that facilitate the transport of water and small solutes across cell membranes. These proteins are vital for maintaining water homeostasis in living organisms. In mammals, thirteen aquaporins (AQP0-12) have been characterized, but in lower vertebrates, such as fish, the diversity, structure and substrate specificity of these membrane channel proteins are largely unknown.ResultsThe screening and isolation of transcripts from the zebrafish (Danio rerio) genome revealed eighteen sequences structurally related to the four subfamilies of tetrapod aquaporins, i.e., aquaporins (AQP0, -1 and -4), water and glycerol transporters or aquaglyceroporins (Glps; AQP3 and AQP7-10), a water and urea transporter (AQP8), and two unorthodox aquaporins (AQP11 and -12). Phylogenetic analyses of nucleotide and deduced amino acid sequences demonstrated dual paralogy between teleost and human aquaporins. Three of the duplicated zebrafish isoforms have unlinked loci, two have linked loci, while DrAqp8 was found in triplicate across two chromosomes. Genomic sequencing, structural analysis, and maximum likelihood reconstruction, further revealed the presence of a putative pseudogene that displays hybrid exons similar to tetrapod AQP5 and -1. Ectopic expression of the cloned transcripts in Xenopus laevis oocytes demonstrated that zebrafish aquaporins and Glps transport water or water, glycerol and urea, respectively, whereas DrAqp11b and -12 were not functional in oocytes. Contrary to humans and some rodents, intrachromosomal duplicates of zebrafish AQP8 were water and urea permeable, while the genomic duplicate only transported water. All aquaporin transcripts were expressed in adult tissues and found to have divergent expression patterns. In some tissues, however, redundant expression of transcripts encoding two duplicated paralogs seems to occur.ConclusionThe zebrafish genome encodes the largest repertoire of functional vertebrate aquaporins with dual paralogy to human isoforms. Our data reveal an early and specific diversification of these integral membrane proteins at the root of the crown-clade of Teleostei. Despite the increase in gene copy number, zebrafish aquaporins mostly retain the substrate specificity characteristic of the tetrapod counterparts. Based upon the integration of phylogenetic, genomic and functional data we propose a new classification for the piscine aquaporin superfamily.

Piscine aquaporins: an overview of recent advances.

Aquaporins are a superfamily of integral membrane proteins that facilitate the rapid and yet highly selective flux of water and other small solutes across biological membranes. Since their discovery, they have been documented throughout the living biota, with the majority of research focusing on mammals and plants. Here, we review available data for piscine aquaporins, including Agnatha (jawless fish), Chondrichthyes (chimaeras, sharks, and rays), Dipnoi (lungfishes), and Teleostei (ray-finned bony fishes). Recent evidence suggests that the aquaporin superfamily has specifically expanded in the chordate lineage consequent to serial rounds of whole genome duplication, with teleost genomes harboring the largest number of paralogs. The selective retention and dichotomous clustering of most duplicated paralogs in Teleostei, with differential tissue expression profiles, implies that novel or specialized physiological functions may have evolved in this clade. The recently proposed new nomenclature of the piscine aquaporin superfamily is discussed in relation to the phylogenetic signal and genomic synteny, with the teleost aquaporin-8 paralogs used as a case study to illustrate disparities between the underlying codons, molecular phylogeny, and physical locus. Structural data indicate that piscine aquaporins display similar channel restriction residues found in the tetrapod counterparts, and hence their functional properties seem to be conserved. However, emerging evidence suggests that regulation of aquaporin function in teleosts may have diverged in some cases. Cell localization and experimental studies imply that the physiological roles of piscine aquaporins extend at least to osmoregulation, reproduction, and early development, although in most cases their specific functions remain to be elucidated.

Influence of nutritional composition of diet on sea bass, Dicentrarchus labrax L., reproductive performance and egg and larval quality

Abstract Two isocaloric formulated diets varying in protein and carbohydrate content (D1 = 51% (P)rotein, 13% (L)ipid, 10% (C)arbohydrate; D2 = 34% P, 14% L, 32% C) were fed to 2-year-old sea bass broodstock starting 6 months before spawning to determine their effects on reproductive performance and egg and larval survival. Fish were distributed in 8-m3 tanks, supplied with aerated running sea water (salinity 37.8‰, pH 8.3) and maintained under ambient photoperiod and temperature conditions. Before the experiment, fish were acclimated to the artificial diets by feeding D1 for 30 days. After this period, fish were fed D1 and D2 twice a day (1.5% of body weight per day). Fish fed D2, especially females, showed significantly diminished growth (weight, length and CF) and specific growth rates (P ≤ 0.05) over the first months. However, the dietary treatments did not have any effect on the histomorphology of ovarian development or gonad proximate composition. At spawning, D2 fish exhibited different reproductive performances compared to D1 fish. They did not have any peak in the production of eggs and showed a slightly extended spawning period. The mean number of eggs produced per female and the relative fecundity of fish given D2 diet were also appreciably lower than found for the D1 group. Although the egg proximate composition was the same regardless of dietary treatment, the eggs of the D2 fed fish had significantly lower buoyancy and hatchability (P ≤ 0.05) than those from D1 fish. In addition, more than 50% of hatched larvae from the D2 group showed body deformities. Taken together, these results indicate that the reproduction of sea bass and the survival of the progeny are greatly affected by the levels of energy-yielding nutrients in broodstock diets, and that a dietary protein level and a carbohydrate content of 32–33% have adverse consequences on sea bass reproduction. The sea bass dietary requirements to produce viable eggs seem also to be very specific since the hatching rates observed for both treatment groups were lower than those reported in other feeding trials in which natural diets were used.

Dormancy and resistance in harsh environments

Many organisms have evolved the ability to enter into and revive from a dormant state. They can survive for long periods in this state (often even months to years), yet can become responsive again within minutes or hours. This is often, but not necessarily, associated with desiccation. Preserving one’s body and reviving it in future generations is a dream of mankind. To date, however, we have failed to learn how cells, tissues or entire organisms can be made dormant or be effectively revived at ambient temperatures. In this book studies on organisms, ranging from aquatic cyanobacteria that produce akinetes to hibernating mammals, are presented, and reveal common but also divergent physiological and molecular pathways for surviving in a dormant form or for tolerating harsh environments. Attempting to learn the functions associated with dormancy and how they are regulated is one of the great future challenges. Its relevance to the preservation of cells and tissues is one of the key concerns of this book

Molecular Characterization and Expression Pattern of Zona Pellucida Proteins in Gilthead Seabream (Sparus aurata)

Abstract The developing oocyte is surrounded by an acellular envelope that is composed of 2–4 isoforms of zona pellucida (ZP) proteins. The ZP proteins comprise the ZP1, ZP2, ZP3, and ZPX isoforms. While ZP1 (ZPB) and ZP3 (ZPC) are present in all species, ZP2 (ZPA) is not found in teleost fish and ZPX is not found in mammals. In the present study, we identify and characterize the ZP1, ZP3 and ZPX isoforms of gilthead seabream. Furthermore, by analyzing the conserved domains, which include the external hydrophobic patch and the internal hydrophobic patch, we show that ZP2 and ZPX are closely related isoforms. ZP proteins are synthesized in either the liver or ovary of most teleosts. Only in rainbow trout has it been shown that zp3 has dual transcription sites. In gilthead seabream, all four mRNA isoforms are transcribed in both the liver and ovary, with zp1a, zp1b, and zp3 being highly expressed in the liver, and zpx being primarily expressed in the ovary. However, determination of the ZP proteins in plasma showed high levels of ZP1b, ZP3, and ZPX, with low or non-detectable levels of ZP1a. In similarity to other teleost ZPs, the hepatic transcription of all four ZP isoforms is under estrogenic control. Previously, we have shown that cortisol can potentiate estrogen-induced ZP synthesis in salmonids, and now we show that this is not the case in the gilthead seabream. The present study shows for the first time the endocrine regulation of a teleost ZPX isoform, and demonstrates the dual-organ transcriptional activities of all the ZP proteins in one species.

Short- and long-term dietary effects on female sea bass (Dicentrarchus labrax): seasonal changes in plasma profiles of lipids and sex steroids in relation to reproduction

Abstract Sea bass were fed a natural diet (ND) (trash fish) or two commercial diets ( CD 1 = 56% (P)rotein, 11% (L)ipids; CD 2 = 47% P, 7% L) during two consecutive reproductive cycles in order to assess the effects on the reproductive performance. CD1 and CD2 had lower energetic and PUFA (polyunsaturated fatty acids) content, especially n -3 fatty acids, with respect to ND. During the first spawning, the levels of sex steriods (17β-estradiol [E2] and testosterone [T], the percentage of good quality (i.e. buoyant) eggs and the hatching rates were similar between the ND and CD1 groups. Females fed CD2 did not show significant variations in E2 or T, and had few spawnings, low egg quality and no egg survival. At second spawning, the fish fed the commercial diets had reduced fecundity and egg viability, together with lower levels of E2 and T, altered patterns of plasma lipids and early presence of atresia in the ovary, with respect to the females fed ND. The present study provides evidence for the importance of dietary lipid for the reproductive processes in the sea bass. The data suggest that long-term dietary deficiences in n -3 fatty acids may affect the patterns of plasma lipids and may induce early gonadal atresia. For example, follicular atresia could reduce the production of gonadal steroids, the fecundity and the subsequent egg survival.

New insights into molecular pathways associated with flatfish ovarian development and atresia revealed by transcriptional analysis

BackgroundThe Senegalese sole (Solea senegalensis) is a marine flatfish of increasing commercial interest. However, the reproduction of this species in captivity is not yet controlled mainly because of the poor knowledge on its reproductive physiology, as it occurs for other non-salmonid marine teleosts that exhibit group-synchronous ovarian follicle development. In order to investigate intra-ovarian molecular mechanisms in Senegalese sole, the aim of the present study was to identify differentially expressed genes in the ovary during oocyte growth (vitellogenesis), maturation and ovarian follicle atresia using a recently developed oligonucleotide microarray.ResultsMicroarray analysis led to the identification of 118 differentially expressed transcripts, of which 20 and 8 were monitored by real-time PCR and in situ hybridization, respectively. During vitellogenesis, many up-regulated ovarian transcripts had putative mitochondrial function/location suggesting high energy production (NADH dehydrogenase subunits, cytochromes) and increased antioxidant protection (selenoprotein W2a), whereas other regulated transcripts were related to cytoskeleton and zona radiata organization (zona glycoprotein 3, alpha and beta actin, keratin 8), intracellular signalling pathways (heat shock protein 90, Ras homolog member G), cell-to-cell and cell-to-matrix interactions (beta 1 integrin, thrombospondin 4b), and the maternal RNA pool (transducer of ERBB2 1a, neurexin 1a). Transcripts up-regulated in the ovary during oocyte maturation included ion transporters (Na+-K+-ATPase subunits), probably required for oocyte hydration, as well as a proteinase inhibitor (alpha-2-macroglobulin) and a vesicle calcium sensor protein (extended synaptotagmin-2-A). During follicular atresia, few transcripts were found to be up-regulated, but remarkably most of them were localized in follicular cells of atretic follicles, and they had inferred roles in lipid transport (apolipoprotein C-I), chemotaxis (leukocyte cell-derived chemotaxin 2,), angiogenesis (thrombospondin), and prevention of apoptosis (S100a10 calcium binding protein).ConclusionThis study has identified a number of differentially expressed genes in the ovary that were not previously found to be regulated during ovarian development in marine fish. Specifically, we found evidence, for the first time in teleosts, of the activation of chemoattractant, angiogenic and antiapoptotic pathways in hypertrophied follicular cells at the onset of ovarian atresia.

Follicle-Stimulating Hormone and Luteinizing Hormone Mediate the Androgenic Pathway in Leydig Cells of an Evolutionary Advanced Teleost

ABSTRACT The endocrine pathways controlling vertebrate spermatogenesis are well established in mammals where the pituitary gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) exclusively activate the FSH receptor (FSHR) in Sertoli cells and the LH/choriogonadotropin receptor (LHCGR) in Leydig cells, respectively. In some teleosts, however, it has been shown that Lh can cross-activate the Fshra ortholog, and that Leydig cells coexpress the Lhcgrba and Fshra paralogs, thus mediating the androgenic function of Fsh in the testis. Here, we investigated whether these proposed mechanisms are conserved in an evolutionary advanced pleuronectiform teleost, the Senegalese sole (Solea senegalensis). Transactivation assays using sole Fshra- and Lhcgrba-expressing cells and homologous single-chain recombinant gonadotropins (rFsh and rLh) showed that rFsh exclusively activated Fshra, whereas rLh stimulated both Lhcgrba and Fshra. The latter cross-activation of Fshra by rLh occurred with an EC50 4-fold higher than for rFsh. Both recombinant gonadotropins elicited a significant androgen release response in vitro and in vivo, which was blocked by protein kinase A (PKA) and 3beta-hydroxysteroid dehydrogenase inhibitors, suggesting that activation of steroidogenesis through the cAMP/PKA pathway is the major route for both Lh- and Fsh-stimulated androgen secretion. Combined in situ hybridization and immunocytochemistry using cell-specific molecular markers and antibodies specifically raised against sole Fshra and Lhcgrba demonstrated that both receptors are expressed in Leydig cells, whereas Sertoli cells only express Fshra. These data suggest that Fsh-mediated androgen production through the activation of cognate receptors in Leydig cells is a conserved pathway in Senegalese sole.