The flame retardant triphenyl phosphate alters the epigenome of embryonic cells in an aquatic in vitro model.

Triphenyl phosphate (TPhP) is an organophosphate flame retardant and plasticizer that is added to a wide variety of consumer and industrial products. It is also a ubiquitous environmental pollutant. Exposure to TPhP has been shown to alter gene expression in metabolic and estrogenic signaling pathways in in vitro and in vivo models of a variety of species, and as such, is considered to be an endocrine disrupting chemical. Exposure to endocrine disrupting chemicals is increasingly being associated with changes to the epigenome, especially during embryonic development. The aim of this study was to evaluate whether TPhP exposure in aquatic ecosystems has the ability to alter the epigenome in two immortal cell lines derived from trout (Oncorhynchus mykiss). This study assessed whether 24 h exposure to TPhP resulted in changes to histone modification and DNA methylation profiles in steelhead trout embryonic cells and rainbow trout gill epithelial cells. Results show that several epigenetic modifications on histone H3 and DNA methylation are altered in the embryonic cells following TPhP exposure, but not in the gill epithelial cells. Specifically, histone H3 acetylation, histone H3 mono-methylation and global DNA methylation were found to be reduced. The alterations of these epigenetic modification profiles in the embryonic cells suggest that exposure to TPhP during fetal development may alter gene expression in the developing embryo, likely in metabolic and estrogenic pathways. The impacts to the epigenome determined in this study may even carry multigenerational detrimental effects on human and ecosystem health, which requires further investigation.

Effect of increased embryonic temperature during developmental windows on survival, morphology and oxygen consumption of rainbow trout (Oncorhynchus mykiss).

Temperature is a crucial environmental factor that influences physiological functions in fishes, and increased temperature during development can shape an organism's phenotype. An active line of inquiry in comparative developmental physiology is whether short-term exposure to thermal changes have lasting phenotypic effects. This is the first study to apply a developmental 3-dimensional critical window experimental design for a vertebrate, using time, temperature, and phenotypic response (i.e., variables measured). Rainbow trout (Oncorhynchus mykiss) are an anadromous species for which resident populations occupy freshwater environments that are likely impacted by variable and rising temperatures, particularly during embryonic development. To assess thermal effects on fish development, we examined trout hatchling phenotypes following rearing in constant temperatures (5, 10, 15 and 17.5 °C) and following exposure to increased temperature above 5 °C during specific developmental windows. Time to 50% hatch, hatchling mass and body length showed general trends of decreasing with increasing constant temperature, and survival was highest in constant 10 °C incubation. Thermally shifting embryos into 17.5 °C during gastrulation and organogenesis reduced survival at hatch compared to 10 °C, and exposure to 15 and 17.5 °C only late in development produced lighter and shorter hatchlings. Oxygen consumption rate (V̇o2) at organogenesis differed between embryos incubated constantly in increased temperature or exposed only during organogenesis, but generally we found limited temperature effects on V̇o2 that may be due to high data variability. Collectively, these results suggest that survival of rainbow trout hatchlings is most sensitive to 17.5 °C exposure during gastrulation and organogenesis, while warm water exposure later in development has greater impacts on morphology. Thus, trait-specific critical windows of thermal sensitivity exist for rainbow trout embryos that alter the hatching phenotype.

Breathing with fins: do the pectoral fins of larval fishes play a respiratory role?

Convective water flow across respiratory epithelia in water-breathing organisms maintains transcutaneous oxygen (O2) partial pressure (Po2) gradients that drive O2 uptake. Following hatch, larval fishes lack a developed gill and the skin is the dominant site of gas transfer, yet few studies have addressed the contribution of convective water flow to cutaneous O2 uptake in larvae. We hypothesized that the pectoral fins, which can generate water flow across the skin in larvae, promote transcutaneous O2 transfer and thus aid in O2 uptake. In zebrafish (Danio rerio), the frequency of pectoral fin movements increased in response to hypoxia at 4 days postfertilization (dpf), but the response was blunted by 15 dpf, when the gills become the dominant site of O2 uptake, and was absent by 21 dpf. In rainbow trout (Oncorhynchus mykiss), Po2 measured at the skin surface of ventilating larvae was lower when the pectoral fins had been surgically removed, directly demonstrating that fins contribute to convective flow that dissipates cutaneous Po2 boundary layers. Lack of pectoral fins compromised whole animal O2 consumption in trout during hypoxia, but this effect was absent in zebrafish. Overall, our findings support a respiratory role of the pectoral fins in rainbow trout, but their involvement in zebrafish remains equivocal.

Endogenous regulation of 11-deoxycorticosterone (DOC) and corticosteroid receptors (CRs) during rainbow trout early development and the effects of corticosteroids on hatching.

Clear evidence for a physiological role of the mineralocorticoid-like hormone 11-deoxycorticosterone (DOC) and the mineralocorticoid receptor (MR) in fish is still lacking. Efforts to demonstrate an osmoregulatory role for this hormone has so far not been conclusive, while a few scattered studies have indicated a role for DOC in development and reproduction. In this study, we investigate the onset of de novo DOC synthesis in parallel with endogenous corticosteroid receptor mRNA production from fertilization to the swim-up stage in rainbow trout. Whole egg DOC content decreased from fertilization until hatching followed by an increase to pre-fertilization levels just after hatching. Onset of de novo transcription of corticosteroid receptor mRNA's was observed shortly after the midblastula transition; initially glucocorticoid receptor 2 (GR2) followed by MR and then GR1. Non-invasive introduction of DOC or cortisol at fertilization resulted in altered corticosteroid receptor regulation and accelerated hatching date, suggesting a regulatory role in trout ontogenesis of both hormones through MR signaling pathway. The results presented in this study suggest a possible physiological role of the DOC-MR signaling pathway during fish ontogenesis, at fertilization and just after hatching.

Formation of intramuscular connective tissue network in fish: first insight from the rainbow trout (Oncorhynchus mykiss).

The formation of the intramuscular connective tissue was investigated in rainbow trout Oncorhynchus mykiss by combining histological and in situ gene-expression analysis. Laminin, a primary component of basement membranes, surrounded superficial slow and deep fast muscle fibres in O. mykiss as soon as the hatching stage (c. 30 days post fertilization (dpf)). In contrast, type I collagen, the primary fibrillar collagen in muscle of vertebrates, appeared at the surface of individual slow and fast muscle fibres only at c. 90 and 110 dpf, respectively. The deposition of type I collagen in laminin-rich endomysium ensheathing individual muscle fibres correlated with the late appearance of collagen type 1 α 1 chain (col1α1) expressing fibroblasts inside slow and then fast-muscle masses. Double in situ hybridization indicated that coll1α1 expressing muscle resident fibroblasts also expressed collagen type 5 α 2 chain (col5α2) transcripts, showing that these cells are a major cellular source of fibrillar collagens within O. mykiss muscle. At c. 140 dpf, the formation of perimysium-like structure was manifested by the increase of type I collagen deposition around bundles of myofibres concomitantly with the alignment and elongation of some collagen-expressing fibroblasts. Overall, this study shows that the formation of O. mykiss intramuscular connective tissue network is completed only in aged fry when fibroblast-like cells expressing type I and V collagens arise inside of the growing myotome.

Interspecific germ cell transplantation: a new light in the conservation of valuable Balkan trout genetic resources?

Interspecific transplantation of germ cells from the brown trout Salmo trutta m. fario and the European grayling Thymallus thymallus into rainbow trout Oncorhynchus mykiss recipients was carried out in order to improve current practices in conservation of genetic resources of endangered salmonid species in the Balkan Peninsula. Current conservation methods mainly include in situ efforts such as the maintenance of purebred individuals in isolated streams and restocking with purebred fingerlings; however, additional ex situ strategies such as surrogate production are needed. Steps required for transplantation such as isolation of high number of viable germ cells and fluorescent labeling of germ cells which are to be transplanted have been optimized. Isolated and labeled brown trout and grayling germ cells were intraperitoneally transplanted into 3 to 5 days post hatch rainbow trout larvae. Survival of the injected larvae was comparable to the controls. Sixty days after transplantation, fluorescently labeled donor cells were detected within the recipient gonads indicating successful incorporation of germ cells (brown trout spermatogonia and oogonia-27%; grayling spermatogonia-28%; grayling oogonia-23%). PCR amplification of donor mtDNA CR fragments within the recipient gonads additionally corroborated the success of incorporation. Overall, the transplantation method demonstrated in this study presents the first step and a possible onset of the application of the germ cell transplantation technology in conservation and revitalization of genetic resources of endangered and endemic species or populations of salmonid fish and thus give rise to new or improved management strategies for such species.

Modifications in the proteome of rainbow trout (Oncorhynchus mykiss) embryo and fry as an effect of triploidy induction.

Two-dimensional gel electrophoresis (2-DE), matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) mass spectrometry, and database searching were used to analyze the effects of triploidization heat shock treatment on protein expression in rainbow trout eyed embryo and fry. After fertilization, the eggs were incubated at 10 °C for 10 min. Half of the eggs were then subjected to heat shock for 10 min submerged in a 28 °C water bath to induce triploidy. The remainder was incubated normally and used as diploid controls. Specimens of eyed embryos and fry were taken on 18 and 76 days post-fertilization, respectively. In the eyed embryo extracts, seven protein spots were significantly changed in abundance between the control and heat-shocked groups and one of these was decreased while the others were increased in the heat shock-treated group. Of the spots that were shown to change in abundance in the eyed embryos with heat shock treatment, two were identified as vitellogenin, while the others were creatine kinase and angiotensin I. In the 2-DE from the fry muscle extraction, 23 spots were significantly changed in abundance between the diploid and triploid groups. Nineteen of these showed a decreased abundance in diploids, while the remaining four spots had an increased abundance. Triploidization caused differential expression of muscle metabolic proteins including triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, and beta-enolase. Myosin heavy chain as a structural protein was also found to change in abundance in triploids. The altered expression of both structural and metabolic proteins in triploids was consistent with their increased cell size and lower growth performance.

Generation of juvenile rainbow trout derived from cryopreserved whole ovaries by intraperitoneal transplantation of ovarian germ cells.

Cryopreservation of fish sperm offers the practical applications in the selective breeding and biodiversity conservation. However, because of the lack of cryopreservation methods for fish eggs and embryos, maternally inherited cytoplasmic compartments cannot be successfully preserved. We previously developed an alternative method to derive functional eggs and sperm from cryopreserved whole testis by transplanting testicular cells into female and male recipients. However, if target fish had ovaries, the previous method employing male-derived germ cells would be ineffective. Here, we aimed to generate functional gametes from cryopreserved whole ovaries by transplanting ovarian germ cells into peritoneal cavity of sterile hatchlings. Cryopreservation conditions for rainbow trout ovaries (1.0 M DMSO, 0.1 M trehalose, and 10% egg yolk) were optimized by testing several different cryoprotective agents. Ovarian germ cells from thawed ovaries were intraperitoneally transplanted into allogeneic triploid hatchlings. Transplanted germ cells migrated toward and were incorporated into recipient gonads, where they underwent gametogenesis. Transplantation efficiency of ovarian germ cells remained stable after cryopreservation period up to 1185 days. Although all triploid recipients that did not undergo transplantation were functionally sterile, 5 of 25 female recipients and 7 of 25 male recipients reached sexual maturity at 2.5 years post-transplantation. Inseminating the resultant eggs and sperm generated viable offspring displaying the donor characteristics of orange body color, green fluorescence, and chromosome numbers. This method is thus a breakthrough tool for the conservation of endangered fish species that are crucial to cryopreserve the genetic resources of female fish.

Physiological and molecular ontogeny of branchial and extra-branchial urea excretion in posthatch rainbow trout (Oncorhynchus mykiss).

All teleost fish produce ammonia as a metabolic waste product. In embryos, ammonia excretion is limited by the chorion, and fish must detoxify ammonia by synthesizing urea via the ornithine urea cycle (OUC). Although urea is produced by embryos and larvae, urea excretion (J(urea)) is typically low until yolk sac absorption, increasing thereafter. The aim of this study was to determine the physiological and molecular characteristics of J(urea) by posthatch rainbow trout (Oncorhynchus mykiss). Following hatch, whole body urea concentration decreased over time, while J(urea) increased following yolk sac absorption. From 12 to 40 days posthatch (dph), extra-branchial routes of excretion accounted for the majority of J(urea), while the gills became the dominant site for J(urea) only after 55 dph. This represents the most delayed branchial ontogeny of any process studied to date. Urea transporter (UT) gene expression in the gills and skin increased over development, consistent with increases in branchial and extra-branchial J(urea). Following exposure to 25 mmol/l urea, the accumulation and subsequent elimination of exogenous urea was much greater at 55 dph than 12 dph, consistent with increased UT expression. Notably, UT gene expression in the gills of 55 dph larvae increased in response to high urea. In summary, there is a clear increase in urea transport capacity over posthatch development, despite a decrease in OUC activity.

Glucose metabolism ontogenesis in rainbow trout (Oncorhynchus mykiss) in the light of the recently sequenced genome: new tools for intermediary metabolism programming.

The rainbow trout (Oncorhynchus mykiss), a carnivorous fish species, displays a 'glucose-intolerant' phenotype when fed a high-carbohydrate diet. The importance of carbohydrate metabolism during embryogenesis and the timing of establishing this later phenotype are currently unclear. In addition, the mechanisms underlying the poor ability of carnivorous fish to use dietary carbohydrates as a major energy substrate are not well understood. It has recently been shown in trout that duplicated genes involved in glucose metabolism may participate in establishing the glucose-intolerant phenotype. The aim of this study was therefore to provide new understanding of glucose metabolism during ontogenesis and nutritional transition, taking into consideration the complexity of the trout genome. Trout were sampled at several stages of development from fertilization to hatching, and alevins were then fed a non-carbohydrate or a high-carbohydrate diet during first feeding. mRNA levels of all glucose metabolism-related genes increased in embryos during the setting up of the primitive liver. After the first meal, genes rapidly displayed expression patterns equivalent to those observed in the livers of juveniles. g6pcb2.a (a glucose 6-phosphatase-encoding gene) was up-regulated in alevins fed a high-carbohydrate diet, mimicking the expression pattern of gck genes. The g6pcb2.a gene may contribute to the non-inhibition of the last step of gluconeogenesis and thus to establishing the glucose-intolerant phenotype in trout fed a high-carbohydrate diet as early as first feeding. This information is crucial for nutritional programming investigations as it suggests that first feeding would be too late to programme glucose metabolism in the long term.

Stressor timing, not cortisol, is an important embryo viability determinant in female rainbow trout Oncorhynchus mykiss.

The objective of this study was to determine whether the timing of elevated cortisol levels in adult female rainbow trout Oncorhynchus mykiss, achieved by silastic implants within the body cavity, had an effect on embryo viability. Cortisol-implanted fish experienced a significant increase in circulating levels of plasma cortisol compared with sham-implanted fish, and plasma cortisol remained elevated relative to sham-implanted fish for 4 months in each of the three treatment groups (0-4, 4-8 and 8-12 months). There were no significant differences in embryo viability, egg diameters or plasma 17ß-oestradiol levels between the cortisol and sham-implanted treatments in any of the groups. There was a significant difference in the number of subfertile females (<80% embryo viability) when the three treatments were compared. The majority of the females (75%) implanted immediately postspawn (0-4 months) produced subfertile eggs, which was significantly higher than those treated 4-8 (33%) or 8-12 (17%) months postspawn. These results imply that silastic implantation can affect oocyte development, independent of plasma cortisol levels, leading to a reduction in embryo viability.

Ontogenetic changes in embryonic and brain gene expression in progeny produced from migratory and resident Oncorhynchus mykiss.

Little information has been gathered regarding the ontogenetic changes that contribute to differentiation between resident and migrant individuals, particularly before the onset of gross morphological and physiological changes in migratory individuals. The aim of this study was to evaluate gene expression during early development in Oncorhynchus mykiss populations with different life histories, in a tissue known to integrate environmental cues to regulate complex developmental processes and behaviours. We sampled offspring produced from migrant and resident parents, collecting whole embryos prior to the beginning of first feeding, and brain tissue at three additional time points over the first year of development. RNA sequencing for 32 individuals generated a reference transcriptome of 30 177 genes that passed count thresholds. Differential gene expression between migrant and resident offspring was observed for 1982 genes. The greatest number of differentially expressed genes occurred at 8 months of age, in the spring a full year before the obvious physiological transformation from stream-dwelling parr to sea water-adaptable smolts begins for migrant individuals. Sex and age exhibited considerable effects on differential gene expression between migrants and resident offspring. Differential gene expression was observed in genes previously associated with migration, but also in genes previously unassociated with early life history divergence. Pathway analysis revealed coordinated differential expression in genes related to phototransduction, which could modulate photoperiod responsiveness and variation in circadian rhythms. The role for early differentiation in light sensitivity and biological rhythms is particularly intriguing in understanding early brain processes involved in differentiation of migratory and resident life history types.

Inhibition of zygotic DNA repair: transcriptome analysis of the offspring in trout (Oncorhynchus mykiss).

Zygotic repair of the paternal genome is a key event after fertilization. Spermatozoa accumulate DNA strand breaks during spermatogenesis and can suffer additional damage by different factors, including cryopreservation. Fertilization with DNA-damaged spermatozoa (DDS) is considered to promote implantation failures and abortions, but also long-term effects on the progeny that could be related with a defective repair. Base excision repair (BER) pathway is considered the most active in zygotic DNA repair, but healthy oocytes contain enzymes for all repairing pathways. In this study, the effects of the inhibition of the BER pathway in the zygote were analyzed on the progeny obtained after fertilization with differentially DDS. Massive gene expression (GE; 61 657 unique probes) was analyzed after hatching using microarrays. Trout oocytes are easily fertilized with DDS and the high prolificacy allows live progeny to be obtained even with a high rate of abortions. Nevertheless, the zygotic inhibition of Poly (ADP-ribose) polymerase, upstream of BER pathway, resulted in 810 differentially expressed genes (DEGs) after hatching. DEGs are related with DNA repair, apoptosis, telomere maintenance, or growth and development, revealing a scenario of impaired DNA damage signalization and repair. Downregulation of the apoptotic cascade was noticed, suggesting a selection of embryos tolerant to residual DNA damage during embryo development. Our results reveal changes in the progeny from defective repairing zygotes including higher malformations rate, weight gain, longer telomeres, and lower caspase 3/7 activity, whose long-term consequences should be analyzed in depth.

Analysis of muscle fibre input dynamics using a myog:GFP transgenic trout model.

The dramatic increase in myotomal muscle mass in teleosts appears to be related to their sustained ability to produce new fibres in the growing myotomal muscle. To describe muscle fibre input dynamics in trout (Oncorhynchus mykiss), we generated a stable transgenic line carrying green fluorescent protein (GFP) cDNA driven by the myogenin promoter. In this myog:GFP transgenic line, muscle cell recruitment is revealed by the appearance of fluorescent, small, nascent muscle fibres. The myog:GFP transgenic line displayed fibre formation patterns in the developing trout and showed that the production of new fluorescent myofibres (muscle hyperplasia) is prevalent in the juvenile stage but progressively decreases to eventually cease at approximately 18 months post-fertilisation. However, fluorescent, nascent myofibres were formed de novo in injured muscle of aged trout, indicating that the inhibition of myofibre formation associated with trout ageing cannot be attributed to the lack of recruitable myogenic cells but rather to changes in the myogenic cell microenvironment. Additionally, the myog:GFP transgenic line demonstrated that myofibre production persists during starvation.

Influence of the forms and levels of dietary selenium on antioxidant status and oxidative stress-related parameters in rainbow trout (Oncorhynchus mykiss) fry.

Se is an essential micronutrient required for normal growth, development and antioxidant defence. The objective of the present study was to assess the impact of dietary Se sources and levels on the antioxidant status of rainbow trout (Oncorhynchus mykiss) fry. First-feeding fry (initial body weight: 91 mg) were fed either a plant- or fishmeal-based diet containing 0·5 or 1·2 mg Se/kg diet supplemented or not with 0·3 mg Se/kg diet supplied as Se-enriched yeast or sodium selenite for 12 weeks at 17°C. Growth and survival of rainbow trout fry were not significantly affected by dietary Se sources and levels. Whole-body Se was raised by both Se sources and to a greater extent by Se-yeast. The reduced:oxidised glutathione ratio was raised by Se-yeast, whereas other lipid peroxidation markers were not affected by dietary Se. Whole-body Se-dependent glutathione peroxidase (GPX) activity was enhanced in fish fed Se-yeast compared to fish fed sodium selenite or non-supplemented diets. Activity and gene expression of this enzyme as well as gene expression of selenoprotein P (SelP) were reduced in fish fed the non-supplemented plant-based diet. Catalase, glutamate-cysteine ligase and nuclear factor-erythroid 2-related factor 2 (Nrf2) gene expressions were reduced by Se-yeast. These results suggest the necessity to supplement plant-based diets with Se for rainbow trout fry, and highlight the superiority of organic form of Se to fulfil the dietary Se requirement and sustain the antioxidant status of fish. GPX and SelP expression proved to be good markers of Se status in fish.

The onset of stress response in rainbow trout Oncorhynchus mykiss embryos subjected to density and handling.

The present study made an attempt to measure the cortisol content, as an indicator of stress response, in rainbow trout embryos which were exposed to different densities and handling stress (air exposure) during incubation. The three densities of experimental embryos at early development stages were considered as 2.55 embryos/cm(2) (low density), 5.10 embryos/cm(2) (normal density) and 7.65 embryos/cm(2) (high density). The cortisol content of eggs (5.09 ± 0.12 ng/g) decreased to 3.68 ± 0.14 ng/g in newly fertilized eggs. Resting level of cortisol dropped at three densities by day 18 of post fertilization. Then, cortisol increased at hatching stage to 1.16 ± 0.11, 1.20 ± 0.12 and 1.21 ± 0.14 ng/g at low, normal and high densities, respectively. There were no statistically significant differences between cortisol concentrations in three densities. The acute handling stress test (5-min out-of-water), conducted on embryos (48 h post fertilization, organogenesis and eyed stage) in three densities, revealed no differences in whole-body cortisol levels between stressed and unstressed experimental groups. At hatching stage in low-density group, level of cortisol increased but the difference with the pre-stress levels was not statistically significant. Furthermore, significant differences in cortisol levels of stressed and unstressed embryos were detected on hatching in normal and high density groups [1.20 ± 0.12 at time 0-1.49 ± 0.11 ng/g at 1 hps (hours post stress) and from 1.21 ± 0.14 at time 0 to 1.53 ± 0.10 ng/g at 3 hps, respectively]. The results showed no difference in profile of cortisol in different densities, but acute stress conducted on embryos, incubated in different densities, revealed differences in cortisol stress response at hatching between normal and high density, which lead to cortisol increase at hatching time. It indicates that the lag time in the cortisol response to stressors immediately after hatching does not occur when the siblings were stressed during the embryo stage. Results, finally, indicated that hypothalamus-pituitary-interrenal axis was active and responded to an acute stressor under normal and high density, but it is unresponsive to a stressor around hatching under low density.

Estrogen and glucocorticoid receptor agonists and antagonists in oocytes modulate the pattern of expression of genes that encode nuclear receptor proteins in very early stage rainbow trout (Oncorhynchus mykiss) embryos.

Previous studies show that changes in estrogen (ER) and glucocorticoid receptor (GR) function in rainbow trout (Oncorhynchus mykiss) oocytes modulate the growth performance phenotype of embryo and juvenile progeny; the present study was undertaken to determine whether this altered growth performance is associated with changes in the expression of several growth-related genes in early-stage embryos. Unfertilized oocytes were incubated in the presence of various combinations of GR and ER agonists and antagonists; the oocytes were then fertilized and the expression of genes that encode for six nuclear receptor superfamily (NRS) proteins (GR1, GR2, ERα, ERß, TRα, and TRß) and the two IGF peptides (IGF1 and IGF2) were measured in the 7-, 13-, and 26-dpf embryos. By day 26 of embryogenesis, the expression of the six NRS-related genes of interest and that of igf2 were significantly enhanced in embryos reared from ER agonist- or ER antagonist-treated oocytes, regardless of whether the GR agonist, cortisol, was also included in the initial oocyte incubation medium. Conversely, the igf1 expression pattern among treatment groups was significantly enhanced in the cortisol-only treatment group and in the ER antagonist and GR antagonist groups that were co-incubated with cortisol. Additionally, in the ER agonist treatment groups igf1 expression was significantly inhibited when cortisol was included in the oocyte incubation medium. The findings show that a single in ovo exposure to the receptor agonists/antagonists markedly changed the programming of the expression of NRS-related and IGF-related genes of the early-stage trout embryos.

Kpna7 interacts with egg-specific nuclear factors in the rainbow trout (Oncorhynchus mykiss).

Nuclear proteins are required for the initiation of transcription in early embryos before embryonic genome activation. The regulated transport of nuclear proteins is mediated by factors known as importins (karyopherins). Kpna7, a newly discovered member of the importin α family, is critical for early development in mammals. In this study, we characterize rainbow trout Kpna7. The cDNA for rainbow trout Kpna7 encodes a 519 amino acid protein that contains a conserved importin ß binding (IBB) domain and seven armadillo/beta-catenin-like repeat (ARM) motifs. Reverse-transcriptase PCR and Western blot analyses revealed that Kpna7 is specifically expressed in eggs/ovary. Real-time PCR analysis demonstrated that expression of Kpna7 mRNA is high in unfertilized eggs, gradually decreases in early-stage embryos until 3 days post-fertilization, and declines sharply thereafter, reaching a level that is barely detectable in 4-day-old embryos. Using a yeast two-hybrid screening system, we identified two Kpna7-interacting proteins from a rainbow trout egg cDNA library: Stl3 (rhamnose-binding lectin 3) and an uncharacterized protein. Both genes appear to be expressed specifically in eggs/testis. Co-immunoprecipitation assays confirmed the interaction between Kpna7 and Stl3, and co-transfection experiments using EGFP-tagged Stl3 showed that Kpna7 facilitates the nuclear transport of Stl3 through an interaction with the predicted nuclear-localization signal cluster at the carboxy-terminus of Stl3. Our data suggest that Kpna7 may function in early embryonic development as a unique nuclear transporter for egg-specific proteins.

Novel sex-determining genes in fish and sex chromosome evolution.

Although the molecular mechanisms underlying many developmental events are conserved across vertebrate taxa, the lability at the top of the sex-determining (SD) cascade has been evident from the fact that four master SD genes have been identified: mammalian Sry; chicken DMRT1; medaka Dmy; and Xenopus laevis DM-W. This diversity is thought to be associated with the turnover of sex chromosomes, which is likely to be more frequent in fishes and other poikilotherms than in therian mammals and birds. Recently, four novel candidates for vertebrate SD genes were reported, all of them in fishes. These include amhy in the Patagonian pejerrey, Gsdf in Oryzias luzonensis, Amhr2 in fugu and sdY in rainbow trout. These studies provide a good opportunity to infer patterns from the seemingly chaotic picture of sex determination systems. Here, we review recent advances in our understanding of the master SD genes in fishes.

Expression profile of ribosomal protein L10a throughout gonadal development in rainbow trout (Oncorhynchus mykiss).

Ribosomal protein L10a (RpL10A) has been previously established as a stimulator during the early stages of ovarian development in both the banana prawn and the fruit fly. In order to develop a greater understanding of the role of this protein in vertebrates, the present study aimed to characterize the expression profile of rpl10a during gonadal development in fish. It was determined that the expression of rpl10a within genital ridges increased during embryonic development. Although rpl10a expression was observed in both gonadal somatic cells and primordial germ cells, higher levels of both transcript and protein expression were detected in somatic cells. rpl10a transcripts were observed in all of the adult tissues examined. Cellular level expression of rpl10a was subsequently characterized across various maturational stages using in situ hybridization and immunohistochemistry of both testes and ovaries. Analysis of tissue derived from the testis showed high levels of rpl10a expression within spermatogonia and the Sertoli cells attached to them. In ovarian tissue, rpl10a was strongly expressed in chromatin-nucleolus-stage and peri-nucleolus-stage oocytes. The relationship between rpl10a expression and regulation of gonadal development was confirmed using real-time PCR, which was performed in order to analyze rpl10a expression in testicular and ovarian tissues subsequent to incubation with salmon pituitary extract and various sex steroids for 24 h. Among them, 11-ketotestosterone at 100 ng/mL effectively up-regulated expression of rpl10a in testicular tissues, while 17ß-estradiol down-regulated rpl10a expression in ovarian tissues. These results suggested that rpl10a played a role in the regulation of gonadal development in fish.