Gene Expression and Phenotypic Assessment of Egg Quality across Developmental Stages of Atlantic Cod throughout the Spawning Season.

Egg quality in fishes is commonly determined by fertilisation success and cleavage patterns as a phenotypic outcome of underlying regulatory mechanisms. Although these phenotypic estimators of egg quality are useful in farming conditions, these "good quality" egg batches do not always translate to good larval growth and survival. The identification of genes involved in embryonic development may help find links between genetic factors of maternal origin and egg quality. Herein, the relative expression of seven stage-specific developmental genes of Atlantic cod was analysed using quantitative PCR to understand the function during embryogenesis and its relationship with egg quality. Genes ccnb2 and pvalb1 showed significant differential expression between developmental stages and significant upregulation from blastula and somite stages, respectively. The comparison of spawning batches showed that the relative gene expression of genes ccnb2, acta, tnnt3 and pvalb1 was significantly higher from the middle of the spawning season where phenotypic quality estimators establish the best egg quality. Moreover, a positive significant correlation was observed between quality estimators based on egg morphology and the genetic expression of genes acta and acta1 during somitogenesis. This study suggests that the combination of quality estimators, genetics and batch timing could help optimise reproductive protocols for commercial stocks of Atlantic cod.

Ultrasonic imaging as a means of monitoring gonadal development in lumpfish (Cyclopterus lumpus).

The commercial farming of juvenile lumpfish requires monitoring of gonadal development to achieve synchronized production. Conventional methods such as gonadosomatic index (GSI), sex hormone analyses, gonadal histology, endoscopy, and gene expression analyses are costly, invasive, and often involve sacrificing the fish. We assessed the efficiency of ultrasound as a non-invasive method for monitoring gonadal development in lumpfish. Based on ultrasound observations, we categorized the fish into six stages; F0 to F5 for females and M0 to M5 for males, that represented maturity levels from immature to spent. Importantly, the ultrasound gonadal stages aligned with histological gonadal stages. Additionally, ultrasound stages aligned with profiles of GSI, testosterone (T), 11-ketotestosterone, and 17ß-estradiol throughout gonadal development including the spawning period. Moreover, these parameters exhibited significant positive correlations with each other reflecting their parallel trends during gonadal development. To minimize the frequency of ultrasound usage and fish handling, we established F3 and M3/M4 as arbitrary thresholds for identifying ripe females and males, respectively. By using these thresholds, the need for regular ultrasound monitoring could be reduced during most of the rearing period. Ultrasound proves to be useful and reliable for monitoring gonadal development in lumpfish, enabling synchronized production of juvenile fish.

Impact of germ cell ablation on the activation of the brain-pituitary-gonadal axis in precocious Atlantic salmon (Salmo salar L.) males.

The germ cells are essential for sexual reproduction by giving rise to the gametes, but the importance of germ cells for gonadal somatic functions varies among vertebrates. The RNA-binding dead end (Dnd) protein is necessary for the specification and migration of primordial germ cells to the future reproductive organs. Here, we ablated the gametes in Atlantic salmon males and females by microinjecting dnd antisense gapmer oligonucleotides at the zygotic stage. Precocious maturation was induced in above 50% of both germ cell-depleted and intact fertile males, but not in females, by exposure to an off-season photoperiod regime. Sterile and fertile males showed similar body growth, but maturing fish tended to be heavier than their immature counterparts. Pituitary fshß messenger RNA levels strongly increased in maturing sterile and fertile males concomitant with the upregulated expression of Sertoli and Leydig cell markers. Plasma concentrations of 11-ketotestosterone and testosterone in maturing sterile males were significantly higher than the basal levels in immature fish, but lower than those in maturing fertile males. The study demonstrates that germ cells are not a prerequisite for the activation of the brain-pituitary-gonad axis and sex steroidogenesis in Atlantic salmon males, but may be important for the maintenance of gonadal somatic functions.

Sex steroid dynamics and mRNA transcript profiles of growth- and development-related genes during embryogenesis following induced follicular maturation in European eel.

Hormones and mRNA transcripts of maternal origin deposited in the egg may affect early embryonic development in oviparous species. These hormones include steroids, such as estradiol-17ß (E2), testosterone (T), 11-ketotestosterone (11-kt), 17α,20ß-dihydroxy-4-pregnen-3-one (DHP), and cortisol, which also play an important role in fish reproduction. In European eel, Anguilla anguilla, which does not reproduce naturally in captivity, vitellogenesis in female broodstock is commonly induced by administration of salmon or carp pituitary extract (PE) as an exogenous source of gonadotropins, while follicular maturation is stimulated by a priming dose of PE followed by provision of DHP as a maturation inducing hormone. In this regard, the main purpose of the present study was to evaluate effects of induced follicular maturation on reproductive success in European eel, focusing on maternal transfer and dynamics of steroids and mRNA transcripts of growth- and development-related genes throughout embryogenesis. The results showed that maternal blood plasma concentrations of E2, T and DHP were reflected in the unfertilized eggs. Moreover, a negative relationship between concentrations of E2 and DHP in eggs and embryos and quality parameters measured as fertilization success, cleavage abnormalities, embryonic survival, and hatch success was found. Concomitant mRNA transcript abundance analysis including genes involved in stress response (hsp70, hsp90), somatotropic axis (gh, igf1, igf2a, igf2b), lipid (cpt1a, cpt1b, pigf5) and thyroid metabolism (dio1, dio2, dio3, thrαb, thrßa, thrßb) varied among unfertilized egg batches. For the majority of genes, mRNA abundance increased during the maternal-to-zygotic transition in connection to activation of the transcription of the embryos own genome. mRNA abundance of dio1, cpt1a and cpt1b throughout embryogenesis was related to embryonic developmental competence. Notably, mRNA abundance of dio3 was positively associated with E2 concentrations, while the mRNA abundance of thrαb was negatively related to T concentrations in the unfertilized eggs, which may suggest an interaction between the thyroid and steroid hormone systems. Altogether, maternal plasma concentrations of E2 and DHP were reflected in the eggs, with high concentrations of these steroids in the eggs being negatively associated with embryonic developmental competence. Additionally, high transcript levels of two of the investigated genes (dio1, cpt1b) were positively associated with embryonic developmental competence. This study reveals maternal transfer of steroids and mRNA transcripts to the eggs, which may be significant contributors to the variability in embryonic survival observed in European eel captive reproduction.

Low Oxygen Stress During Early Development Influences Regulation of Hypoxia-Response Genes in Farmed Atlantic Salmon (Salmo salar).

Survival and growth of developing salmonids are negatively affected by low oxygen levels within gravel nests in natural streams, and hypoxic stress is often experienced by farmed Atlantic salmon (Salmo salar) within hatcheries. Exposure to hypoxia during early development may have long-lasting effects by altering epigenetic marks and gene expression in oxygen regulatory pathways. Here, we examine the transcriptomic response to low dissolved oxygen (DO) in post-hatch salmon reared continuously in 30%, 60% or 100% DO from fertilization until start of feeding. RNA sequencing revealed multiple differentially expressed genes, including oxygen transporting hemoglobin embryonic α subunit (hbae) and EGLN3 family hypoxia-inducible factor 3 (egln3) which regulates the stability of hypoxia inducible factor 1α (HIF-1α). Both hbae and egln3 displayed expression levels inversely correlated to oxygen concentration, and DNA methylation patterns within the egln3 promoter were negatively associated with the transcript levels. These results suggest that epigenetic processes are influenced by low oxygen levels during early development in Atlantic salmon to upregulate hypoxia-response genes.

Cold seawater induces early sexual developmental stages in the BPG axis of European eel males.

BACKGROUND: The impossibility of closing the life cycle of the European eel (Anguilla anguilla) in captivity troubles the future of this critically endangered species. In addition, the European eel is a highly valued and demanded resource, thus the successful closing of its life cycle would have a substantial economic and ecological impact. With the aim of obtaining the highest gamete quality, the study of the effects of environmental factors, such as temperature, on reproductive performance may prove valuable. This is especially true for the exposure to cold water, which has been reported to improve sexual development in multiple other Actinopterygii species. RESULTS: European eel males treated with cold seawater (10 °C, T10) for 2 weeks showed an increase in the proliferation and differentiation of spermatogonial cells until the differentiated spermatogonial type A cell stage, and elevated testosterone and 11-ketotestosterone plasma levels. Transcriptomes from the tissues of the brain-pituitary-gonad (BPG) axis of T10 samples revealed a differential gene expression profile compared to the other experimental groups, with clustering in a principal component analysis and in heat maps of all differentially expressed genes. Furthermore, a functional analysis of differentially expressed genes revealed enriched gene ontology terms involved in the regulation of circadian rhythm, histone modification, meiotic nuclear division, and others. CONCLUSIONS: Cold seawater treatment had a clear effect on the activity of the BPG-axis of European eel males. In particular, our cold seawater treatment induces the synchronization and increased proliferation and differentiation of specific spermatogonial cells. In the transcriptomic results, genes related to thermoception were observed. This thermoception may have caused the observed effects through epigenetic mechanisms, since all analysed tissues further revealed differentially expressed genes involved in histone modification. The presented results support our hypothesis that a low temperature seawater treatment induces an early sexual developmental stage in European eels. This hypothesis is logical given that the average temperature experienced by eels in the early stages of their oceanic reproductive migration is highly similar to that of this cold seawater treatment. Further studies are needed to test whether a cold seawater treatment can improve the response of European eels to artificial hormonal treatment, as the results suggest.

Early Developmental Stress Affects Subsequent Gene Expression Response to an Acute Stress in Atlantic Salmon: An Approach for Creating Robust Fish for Aquaculture?

Stress during early life has potential to program and alter the response to stressful events and metabolism in later life. Repeated short exposure of Atlantic salmon to cold water and air during embryonic (E), post-hatch (PH) or both phases of development (EPH) has been shown to alter the methylome and transcriptome and to affect growth performance during later life compared to untreated controls (CO). The aim of this study was to investigate how the transcriptome of these fish responds to subsequent acute stress at the start feeding stage, and to describe methylation differences that might steer these changes. EPH treated fish showed the strongest down-regulation of corticotropin releasing factor 1, up-regulation of glucocorticoid receptor and 3-oxo-5-alpha-steroid 4-dehydrogenase 2 gene expression and a suppressed cortisol response 3 hr after the acute stress, differences that could influence hormesis and be affecting how EPH fish cope and recover from the stress event. Growth hormone 2 and insulin-like growth factor 1 were more strongly down-regulated following acute stress in EPH treated fish relative to E, PH and CO fish. This indicates switching away from growth toward coping with stress following stressful events in EPH fish. Genes implicated in immune function such as major histocompatibility class 1A, T-cell receptor and toll-like receptor also responded to acute stress differently in EPH treated fish, indicating that repeated stresses during early life may affect robustness. Differential DNA methylation was detected in regions mapping <500 bases from genes differentially responding to acute stress suggesting the involvement of epigenetic mechanisms. Stress treatments applied during early development therefore have potential as a husbandry tool for boosting the productivity of aquaculture by affecting how fish respond to stresses at critical stages of production.

Differential expression of gonadotropin and estrogen receptors and oocyte cytology during follicular maturation associated with egg viability in European eel (Anguilla anguilla).

In captivity, oogenesis and ovarian follicle maturation in European eel can be induced experimentally using hormonal therapy. The follicle's ability to respond effectively to the induction of maturation and ovulation, resulting in viable eggs, depends on the oocyte stage at the time of induction. We hypothesized that variation in the expression of key hormone receptors in the ovary and size of oocyte lipid droplets are associated with changes in oocyte stage. Thus, we induced ovarian follicle maturation using a priming dose of fish pituitary extract followed by the administration of a 17α, 20ß-dihydroxy-4-pregnen-3-one (DHP) injection. Females were then strip-spawned, the eggs were fertilized in vitro, incubated and larval survival was recorded at 3 days post hatch (dph). The expression of gonadotropin receptors (fshr, lhcgr1 and lhcgr2) and estrogen receptors (esr1, esr2a, esr2b, gpera and gperb) was quantified and the size of oocyte lipid droplets measured. Larval survival at 3 dph was used to differentiate high- and low-quality egg batches. Results showed significantly higher abundance of lhcgr1 and esr2a at priming for high-quality egg batches whereas fshr and gperb transcripts were significantly higher at DHP injection for low-quality egg batches. Therefore, high levels of lhcgr1 and esr2a may be important for attaining follicular maturational competence, while high fshr and gperb mRNA levels may indicate inadequate maturational competence. Furthermore, lipid droplet size at DHP and in ovulated eggs was significantly smaller in high-quality egg batches than in low-quality, which indicates that droplet size may be a useful marker of follicular maturational stage.

Resolving the complexity of vitellogenins and their receptors in the tetraploid Atlantic salmon (Salmo salar): Ancient origin of the phosvitin-less VtgC in chondrichthyean fishes.

Egg yolk proteins are mainly derived from vitellogenin (Vtg), and serve as essential nutrients during early development in oviparous organisms. Vertebrate Vtgs are predominantly synthesized in the liver of maturing females, and are internalized by the oocyte after binding to specific surface receptors (VtgR). Here, we clarify the evolutionary history of vertebrate Vtgs, including the teleost VtgC, which lacks phosvitin, and investigate the repertoire of Vtgs and VtgRs in the tetraploid Atlantic salmon (Salmo salar). Conserved synteny of the vtg genes in elephant fish (Callorhinchus milii) strongly indicates that the vtg gene cluster was present in the ancestor of tetrapods and ray-finned fish. The shortened phosvitin in the VtgC ortholog of this chondrichthyean fish may have resulted from early truncation events that eventually allowed the total disappearance of phosvitin in teleost VtgC. In contrast, the tandem-duplicated VtgCs identified in the spotted gar (Lepisosteus oculatus) both contain the phosvitin domain. The Atlantic salmon genome harbors four vtg genes encoding the complete VtgAsa1, phosvitin-less VtgC, and truncated VtgAsb proteins; vtgAsa2 is a pseudogene. The three vtg genes were mainly expressed in the liver of maturing females, and the vtgAsa1 transcript predominated prior to spawning. The splice variant lacking the O-linked sugar domain dominated ovarian expression of vtgr1 and vtgr2. Strongly increased vtgAsa1 expression during vitellogenesis contrasted with the peaks of vtgr1 and vtgr2 in the previtellogenic oocytes, which gradually decreased over the same period. Recycling of the oocyte VtgRs is probably not sufficient to maintain receptor number during vitellogenesis.

Impacts of Early Life Stress on the Methylome and Transcriptome of Atlantic Salmon.

Exposure to environmental stressors during early-life stages can change the rate and timing of various developmental processes. Epigenetic marks affecting transcriptional regulation can be altered by such environmental stimuli. To assess how stress might affect the methylome and transcriptome in salmon, fish were treated using cold-shock and air-exposure from the eye-stage until start-feeding. The fish were either stressed prior to hatching (E), post-hatching (PH), pre- and post-hatching (EPH) or not stressed (CO). Assessing transcriptional abundances just prior to start feeding, E and PH individuals were found to have modified the expression of thousands of genes, many with important functions in developmental processes. The EPH individuals however, showed expression similar to those of CO, suggesting an adaptive response to extended periods of stress. The methylome of stressed individuals differed from that of the CO, suggesting the importance of environment in shaping methylation signatures. Through integration of methylation with transcription, we identified bases with potential regulatory functions, some 10s of kb away from the targeted genes. We then followed fish growth for an additional year. Individuals in EPH showed superior growth compared to other treatment groups, highlighting how stress can potentially have long-lasting effects on an organism's ability to adapt to environmental perturbations.

The expression of nuclear and membrane estrogen receptors in the European eel throughout spermatogenesis.

Estradiol (E2) can bind to nuclear estrogen receptors (ESR) or membrane estrogen receptors (GPER). While mammals possess two nuclear ESRs and one membrane GPER, the European eel, like most other teleosts, has three nuclear ESRs and two membrane GPERs, as the result of a teleost specific genome duplication. In the current study, the expression of the three nuclear ESRs (ESR1, ESR2a and ESR2b) and the two membrane GPERs (GPERa and GPERb) in the brain-pituitary-gonad (BPG) axis of the European eel was measured, throughout spermatogenesis. The eels were first transferred from freshwater (FW) to seawater (SW), inducing parallel increases in E2 plasma levels and the expression of ESRs. This indicates that salinity has a stimulatory effect on the E2 signalling pathway along the BPG axis. Stimulation of sexual maturation by weekly injections of human chorionic gonadotropin (hCG) induced a progressive decrease in E2 plasma levels, and different patterns of expression of ESRs and GPERs in the BPG axis. The expression of nuclear ESRs increased in some parts of the brain, suggesting a possible upregulation due to a local production of E2. In the testis, the highest expression levels of the nuclear ESRs were observed at the beginning of spermatogenesis, possibly mediating the role of E2 as spermatogonia renewal factor, followed by a sharply decrease in the expression of ESRs. Conversely, there was a marked increase observed in the expression of both membrane GPERs throughout spermatogenesis, suggesting they play a major role in the final stages of spermatogenesis.

Multiplicity of Buc copies in Atlantic salmon contrasts with loss of the germ cell determinant in primates, rodents and axolotl.

BACKGROUND: The primordial germ cells (PGCs) giving rise to gametes are determined by two different mechanisms in vertebrates. While the germ cell fate in mammals and salamanders is induced by zygotic signals, maternally delivered germ cell determinants specify the PGCs in birds, frogs and teleost fish. Assembly of the germ plasm in the oocyte is organized by the single Buc in zebrafish, named Velo1 in Xenopus, and by Oskar in Drosophila. Secondary loss of oskar in several insect lineages coincides with changes in germline determination strategies, while the presence of buc in mammals suggests functions not associated with germline formation. RESULTS: To clarify the evolutionary history of buc we searched for the gene in genomes available from various chordates. No buc sequence was found in lamprey and chordate invertebrates, while the gene was identified in a conserved syntenic region in elephant shark, spotted gar, teleosts, Comoran coelacanth and most tetrapods. Rodents have probably lost the buc gene, while a premature translation stop was found in primates and in Mexican axolotl lacking germ plasm. In contrast, several buc and buc-like (bucL) paralogs were identified in the teleosts examined, including zebrafish, and the tetraploid genome of Atlantic salmon harbors seven buc and bucL genes. Maternal salmon buc1a, buc2a and buc2b mRNAs were abundant in unfertilized eggs together with dnd and vasa mRNAs. Immunostained salmon Buc1a was restricted to cleavage furrows in 4-cell stage embryos similar to a fluorescent zebrafish Buc construct injected in salmon embryos. Salmon Buc1a and Buc2a localized together with DnD, Vasa and Dazl within the Balbiani body of early oocytes. CONCLUSIONS: Buc probably originated more than 400 million years ago and might have played an ancestral role in assembling germ plasm. Functional redundancy or subfunctionalization of salmon Buc paralogs in germline formation is suggested by the maternally inherited mRNAs of three salmon buc genes, the localized Buc1a in the cleavage furrows and the distribution of Buc1a and Buc2a in the Balbiani body during oogenesis. The extra-ovarian expression of salmon buc genes and the presence of a second zebrafish bucL gene suggest additional functions not related to germ cell specification.

Interactive effects of dietary composition and hormonal treatment on reproductive development of cultured female European eel, Anguilla anguilla.

Farmed female eels were fed two experimental diets with similar proximate composition but different n-3 polyunsaturated fatty acid (PUFA) levels. Both diets had similar levels of arachidonic acid (ARA), while levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in one diet were approximately 4.5 and 2.6 times higher compared to the other diet, respectively. After the feeding period, each diet group was divided into two and each half received one of two hormonal treatments using salmon pituitary extract (SPE) for 13 weeks: i) a constant hormone dose of 18.75mg SPE/kg initial body weight (BW) and ii) a variable hormone dosage that increased from 12.5mg SPE/kg initial BW to 25mg SPE/kg initial BW. Results showed a significant interaction between diets and hormonal treatments on gonadosomatic index (GSI), indicating that the effect of broodstock diets on ovarian development depends on both nutritional status and hormonal regime. Females fed with higher levels of n-3 series PUFAs and stimulated with the constant hormonal treatment reached higher GSIs than those receiving the variable hormonal treatment. However, when females were fed lower levels of n-3 series PUFAs there was no difference in the effect of hormonal treatments on GSI. We also found that, independent of hormonal treatment, the diet with higher levels of n-3 series PUFAs led to the most advanced stages of oocyte development, such as germinal vesicle migration. Concentration of sex steroids (E2, T, and 11-KT) in the plasma did not differ between diets and hormonal treatments, but was significantly correlated with ovarian developmental stage. In conclusion, increasing dietary levels of n-3 PUFAs seemed to promote oocyte growth, leading to a more rapid progression of ovarian development in European eel subjected to hormonal treatment.

Temperature modulates testis steroidogenesis in European eel.

This study evaluates the effects of temperature on hCG-induced spermatogenesis in European eel (Anguilla anguilla), subjected to three thermal regimes: T10: 10°C (first 4weeks), 15°C (next 3weeks) and 20°C (last 6weeks); T15: 15°C (first 4weeks) and 20°C (last 9weeks); and T20: constant 20°C for the duration of the experiment. At 10°C, maturation stopped in the A spermatogonial stage (SPG1), and no further maturation was observed until the temperature was ≥15°C. With the aim of explaining these results, the influence of temperature on steroidogenic enzyme gene expression and steroid synthesis was tested. The initial synthesis of androgens (T and 11-KT) increased at SPG1, and was not influenced by temperature. Likewise, the gene expression of the steroidogenic enzymes linked to androgen synthesis (aacyp11a1, aacyp17-I and aa11ßHSD) also increased at SPG1. In contrast, no correlation was seen between the increase in E2 and the aacyp19a1 gene expression peak in the testes, with E2 increasing as a consequence of the seawater acclimation carried out before hormonal treatment, and peaking the aacyp19a1 gene expression at B spermatogonial stage (SPG2). Aacyp21 gene expression was also higher at SPG2, and this stage was only reached when the rearing temperature was ≥15°C. In conclusion, androgen synthesis is not dependent on temperature, but further maturation requires higher temperatures in order to induce a change in the steroidogenic pathway towards estrogen and progestin synthesis. This study demonstrates that temperature plays a crucial role in European eel maturation, even perhaps controlling gonad development during the reproductive migration.

Effect of the probiotic Lactobacillus rhamnosus on the expression of genes involved in European eel spermatogenesis.

Positive effects of probiotics on fish reproduction have been reported in several species. In the present study, 40 male European eels were weekly treated with recombinant hCG for 9 weeks and with three different concentrations (10(3), 10(5), and 10(6) CFU/mL) of probiotic Lactobacillus rhamnosus IMC 501 (Sinbyotec, Italy). The probiotics were daily added to the water from the sixth week of the hCG treatment. Males from the treated and control groups were sacrificed after 1, 2, and 3 weeks of probiotic treatment (seventh-ninth weeks of hCG treatment); at this point, sperm and testis samples were also collected. Sperm volume was estimated, and motility was analyzed by computer-assisted sperm analysis software. Alternations in transcription of specific genes involved in reproductive process such as activin, androgen receptors α and ß (arα and arß), progesterone receptor 1 (pr1), bone morphogenetic protein 15 (bmp15), and FSH receptor (fshr) were analyzed in the testis. After 2 weeks of probiotic treatment, sperm production and sperm motility parameters (percentage of motile cells and percentage of straight-swimming spermatozoa) were increased in the European eel treated with 10(5) CFU/mL compared to controls or to the other probiotic doses. These changes were associated with increases in messenger RNA expression of activin, arα, arß, pr1, and fshr. Conversely, after 3 weeks, activin and pr1 expression decreased. No significant changes were observed on bmp15 expression throughout the duration of the treatment with 10(5) CFU/mL dose. The lowest and highest probiotic dose (10(3) and 10(6) CFU/mL, respectively) inhibited the transcription of all genes along all the experiment, except for arα and arß after 1 week of probiotic treatment when compared to controls. The changes observed by transcriptomic analysis and the sperm parameters suggest that a treatment with L rhamnosus at 10(5) CFU/mL for 2 weeks could improve spermatogenesis process in Anguilla anguilla.

Identification of cortisol metabolites in the bile of Atlantic cod Gadus morhua L.

Interpretation of plasma cortisol levels in wild-caught fish is confounded by the stress of capture. Measurement of cortisol metabolites in fish bile could provide a method for assessing the stress level of wild fish because the time-lag for metabolism, conjugation and excretion into bile avoids the effects of sampling stress. To determine which biliary metabolite(s) to target, four Atlantic cod, Gadus morhua L., were injected with radioactive cortisol. After 22 h, the bile was collected and found to contain 30% of the injected activity. Cortisol metabolites were extracted from diluted bile samples using solid phase extraction and the radioactive metabolites separated by several different chromatographic procedures. The metabolites were predominantly present as sulfates (95%) with the remainder being glucuronidated. Chromatography split the sulfates into at least seven peaks, and acid solvolysis (which removes sulfate groups from steroids) generated four major radioactive steroids. These were identified, using microchemical reactions and re-crystallization to constant specific activity, as: 11ß,17,21-trihydroxypregn-4-ene-3,20-dione (cortisol), 3α,11ß,17,21-tetrahydroxy-5ß-pregnan-20-one (tetrahydrocortisol; THF), 3α,17,21-trihydroxy-5ß-pregnane-11,20-dione (tetrahydrocortisone; THE) and 3α,17,20ß,21-tetrahydroxy-5ß-pregnan-11-one (ß-cortolone). The last of these was the most abundant, and thus a likely target for a biliary stress assay. Studies were also carried out to determine the best method for extraction and solvolysis of sulfates. Solid phase extraction (i.e. using octadecylsilane) was found to be too unreliable for routine use. Even though the extraction efficiency could be improved by acidifying the bile, this caused premature solvolysis of sulfated steroids. Acid solvolysis of unextracted bile worked best (c. 90% converted to free steroids) on volumes that were 1 µL or lower. Aryl sulfatase digestion of unextracted bile did not work well (only 20% of radioactivity was converted to free steroids).

Knockdown of the germ cell factor Dead end induces multiple transcriptional changes in Atlantic cod (Gadus morhua) hatchlings.

The RNA binding protein Dead end (DnD) is essential for maintaining viable germ cells in vertebrates and silencing of the gene has been demonstrated to cause sterility in several mammalian and fish species. Here we investigated transcriptome changes in hatched larvae of Atlantic cod induced by DnD knockdown using morpholino oligonucleotides (MO) injected in two-cell embryos. Whereas no fluorescently labeled germ cells were shown in embryos coinjected with dnd MO and nanos3 3'UTR coupled to green fluorescent protein, DnD knockdown had no visible effect on the number and location of Vasa protein positive cells in larvae. However, quantitative real-time RT-PCR (qPCR) revealed decreased vasa, nanos3 and tudor domain containing protein 7 mRNA expression and genome-wide oligonucleotide microarray analyses indicated profound suppression of genes involved in development and regulation of the reproductive system. DnD morphants showed lowered expression of genes encoding proteins involved in lipid, retinoid, cholesterol and steroid metabolism, including those with roles in sex hormone metabolism. Biotransformation of lipophilic compounds appeared suppressed too, as evidenced by down-regulation of several key genes from the phases 1 and 2 detoxification pathways. Effects of DnD silencing were highly pleiotropic and consisted of endocrine and metabolic changes, massive induction of histones and suppression of diverse developmental processes, including erythropoiesis and formation of extracellular matrix. While transient inhibition of dnd mRNA translation did not block development of primordial germ cells until hatch, results suggested that ablation of DnD might have major indirect consequences, including suppression of reproductive functions.

Conserved mechanisms for germ cell-specific localization of nanos3 transcripts in teleost species with aquaculture significance.

The importance of the aquaculture production is increasing with the declining global fish stocks, but early sexual maturation in several farmed species reduces muscle growth and quality, and escapees could have a negative impact on wild populations. A possible solution to these problems is the production of sterile fish by ablation of the embryonic primordial germ cells (PGCs), a technique developed in zebrafish. Cell-specific regulation of mRNA stability is crucial for proper specification of the germ cell lineage and commonly involves microRNA (miRNA)-mediated degradation of targeted mRNAs in somatic cells. This study reports on the functional roles of conserved motifs in the 3' untranslated region (UTR) of the miRNA target gene nanos3 identified in Atlantic cod, Atlantic salmon, and zebrafish. The 3'UTR of cod nanos3 was sufficient for targeting the expression of green fluorescent protein (GFP) to the presumptive PGCs in injected embryos of the three phylogenetically distant species. 3'UTR elements of importance for PGC-specific expression were further examined by fusing truncated 3'UTR variants of cod nanos3 to GFP followed by injections in zebrafish embryos. The expression patterns of the GFP constructs in PGCs and somatic cells suggested that the proximal U-rich region is responsible for the PGC-specific stabilization of the endogenous nanos3 mRNA. Morpholino-mediated downregulation of the RNA-binding protein Dead end (DnD), a PGC-specific inhibitor of miRNA action, abolished the fluorescence of the PGCs in cod and zebrafish embryos, suggesting a conserved DnD-dependent mechanism for germ cell survival and migration.

Divergent and sex-dimorphic expression of the paralogs of the Sox9-Amh-Cyp19a1 regulatory cascade in developing and adult atlantic cod (Gadus morhua L.).

The factors of the Sox9-Amh-Cyp19a1 cascade play a crucial role in the complex process of sex differentiation in mammals. The involvement of Sox9 and Cyp19a1 paralogs and the single Amh ortholog in sex differentiation and development of the gonads and the brain in Atlantic cod was examined by analyzing bimodal and sex-dimorphic gene expression patterns, respectively, during early stages and in maturing males and females. Expression of sox9a and sox9b were initiated at blastulation, and both paralogs were expressed in chondrogenic tissue in the hatched larvae. The male-specific expression of sox9a in the adult gonads supports a conserved role in testis function, while sox9b was expressed in the maturing testes and ovaries at similar levels. Amh was expressed at low, but variable, levels from late gastrulation prior to the onset of cyp19a1a and cyp19a1b expression. Male-biased amh expression was found in the maturing gonads, but the increased ovarian levels during maturation suggest a role also in females. The larval expression of cyp19a1a and cyp19a1b increased at the expected time of sex differentiation, but showed large individual variation. The ovarian expression of cyp19a1a and amh increased concomitant with increased plasma estradiol levels during vitellogenesis. The testis-specific cyp19a1b expression supports the importance of estrogen in the spermatogenesis, while abundant expression in the male and female brain is probably related to the continuous neurogenesis in fish. These divergent and sex-dimorphic expression patterns of the cod sox9 and cyp19a1 paralogs demonstrate the complexity of the genetic network regulating sexual development in fish.

Molecular and physiological study of the artificial maturation process in European eel males: from brain to testis.

European eel males can be artificially matured (1.5IU hCG/g fish), but the regulatory mechanisms of their reproductive development are practically unknown. Spermatogenic stages (S1-S6), biometric characters [eye index (EI), gonadosomatic index (GSI), hepatosomatic index (HSI)] and sperm quality parameters (motility, viability and head spermatozoa morphometry) were analysed. Moreover, the present study evaluated the expression of GnRHs (mammal and chicken II Gonadotropin Release Hormone I) and gonadotrophins (FSHbeta and LHbeta) during hormonal treatment, as well as 11-ketotestosterone (11-KT) and 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P) plasma levels. One week was enough to observe the S2 of gonad development, but it was necessary to reach the 7th week of treatment to obtain animals that presented the most advanced stage of development (S6). Differential regulation of the two GnRH expressions was found, supporting the main role of mGnRH in the control of gonadotrophin release. One hCG injection was enough to dramatically decrease the FSHbeta expression, being close to zero during the rest of the treatment. LHbeta expression and 17,20beta-P registered a significant increase in the same stage of development, S3/4, confirming the role of this gonadotrophin in the last steps of maturation and 17,20beta-P in the spermatozoa maturation. The 11-KT increased with GSI, and the highest 11-KT values coincided with the advanced steps of spermatogenesis prior to spermiation. Being consistent with the known role of the steroid in these processes. Furthermore, this study supports a role for 11-KT in stimulating eye growth, presenting high values when EI increased. Sperm production was obtained from the 4th week of treatment, but it was in the 8th week when a significant increase was observed in sperm quality [viability, high motility (>75%)].