Ovarian transcriptome analysis of diploid and triploid rainbow trout revealed new pathways related to gonadal development and fertility.

Triploidisation represents several advantages (e.g. sterility) and therefore is routinely applied in aquaculture of several commercially important fish species, including rainbow trout. The comparative transcriptomic analysis of ovaries of triploid (3N) and diploid (2N) female rainbow trout revealed a total of 9 075 differentially expressed genes (DEGs; 4 105 genes upregulated in 2N and 4 970 genes upregulated in 3N ovaries, respectively). Identified clusters for DEGs upregulated in 3N and 2N ovaries were different, including carbohydrate and lipid metabolic process and transport, protein modification, signalling (related to folliculogenesis) and response to stimulus for DEGs upregulated in 2N, and developmental process, signalling (related to apoptosis, cellular senescence and adherence junctions) and regulation of RNA metabolic process for DEGs upregulated in 3N. The enrichment of processes involved in carbohydrate and lipid metabolism in 2N ovaries indicated high metabolism of ovarian tissue and the energy reservoir generation indispensable during the earliest stages of development. Our results highlight the importance of oocyte hydration along with oestrogen, insulin, leptin, fibroblast growth factor, and Notch signalling and pathways related to the regulation of cyclic adenosine monophosphate (cAMP) levels in proper oocyte meiotic maturation prior to ovulation in 2N ovaries. Conversely, triploidisation may lead to an increase in ovarian cellular senescence and apoptosis, which in turn can result in abnormal gonadal morphology and fibrosis. The downregulation of genes responsible for the precise regulation of meiosis and proper chromosome segregation during meiosis probably affects meiotic maturation via irregular meiotic division of chromosomes. The induction of triploidy of the rainbow trout genome resulted in enhanced expression of male-specific genes, genes responsible for re-establishing the transcriptional balance after genome reorganisation and genes involved in regulatory mechanisms, including gene silencing and DNA methylation. To the best of our knowledge, this is the first genome-wide investigation providing in-depth comprehensive and comparative gene expression patterns in the ovary from 2N and 3N rainbow trout females helping in elucidating the molecular mechanisms leading to impaired gonadal development and sterility of female triploids.

Chromosomal characteristics and distribution of rDNA sequences in the brook trout Salvelinus fontinalis (Mitchill, 1814).

Brook trout Salvelinus fontinalis (Mitchill, 1814) chromosomes have been analyzed using conventional and molecular cytogenetic techniques enabling characteristics and chromosomal location of heterochromatin, nucleolus organizer regions (NORs), ribosomal RNA-encoding genes and telomeric DNA sequences. The C-banding and chromosome digestion with the restriction endonucleases demonstrated distribution and heterogeneity of the heterochromatin in the brook trout genome. DNA sequences of the ribosomal RNA genes, namely the nucleolus-forming 28S (major) and non-nucleolus-forming 5S (minor) rDNAs, were physically mapped using fluorescence in situ hybridization (FISH) and primed in situ labelling. The minor rDNA locus was located on the subtelo-acrocentric chromosome pair No. 9, whereas the major rDNA loci were dispersed on 14 chromosome pairs, showing a considerable inter-individual variation in the number and location. The major and minor rDNA loci were located at different chromosomes. Multichromosomal location (3-6 sites) of the NORs was demonstrated by silver nitrate (AgNO3) impregnation. All Ag-positive i.e. active NORs corresponded to the GC-rich blocks of heterochromatin. FISH with telomeric probe showed the presence of the interstitial telomeric site (ITS) adjacent to the NOR/28S rDNA site on the chromosome 11. This ITS was presumably remnant of the chromosome rearrangement(s) leading to the genomic redistribution of the rDNA sequences. Comparative analysis of the cytogenetic data among several related salmonid species confirmed huge variation in the number and the chromosomal location of rRNA gene clusters in the Salvelinus genome.

Induction of gynogenetic and androgenetic haploid and doubled haploid development in the brown trout (Salmo trutta Linnaeus 1758).

Gynogenetic and androgenetic brown trout (Salmo trutta Linnaeus 1758) haploids (Hs) and doubled haploids (DHs) were produced in the present research. Haploid development was induced by radiation-induced genetic inactivation of spermatozoa (gynogenesis) or eggs (androgenesis) before insemination. To provide DHs, gynogenetic and androgenetic haploid zygotes were subjected to the high pressure shock to suppress the first mitotic cleavage. Among haploids, gynogenetic embryos were showing lower mortality when compared to the androgenetic embryos; however, most of them die before the first feeding stage. Gynogenetic doubled haploids provided in the course of the brown trout eggs activation performed by homologous and heterologous sperm (rainbow trout) were developing equally showing hatching rates of 14.76 ± 2.4% and 16.14 ± 2.90% and the survival rates at the first feeding stage of 10.48 ± 3.48% and 12.78 ± 2.18%, respectively. Significantly, lower survival rate was observed among androgenetic progenies from the diploid groups with only few specimens that survived to the first feeding stage. Cytogenetic survey showed that among embryos from the diploid variants of the research, only gynogenetic individuals possessed doubled sets of chromosomes. Thus, it is reasonable to assume that radiation employed for the genetic inactivation of the brown trout eggs misaligned mechanism responsible for the cell divisions and might have delayed or even arrested the first mitotic cleavage in the androgenetic brown trout zygotes. Moreover, protocol for the radiation-induced inactivation of the paternal and maternal genome should be adjusted as some of the cytogenetically surveyed gynogenetic and androgenetic embryos exhibited fragments of the irradiated chromosomes.

Effect of postthaw storage time and sperm-to-egg ratio on fertility of cryopreserved brook trout sperm.

The aim of this study was to test the influence of postthaw storage time on sperm motility parameters of brook trout (n = 9). Furthermore, we examined the effect of sperm-to-egg ratios of 300,000:1 and 600,000:1 on fertility of postthaw, cryopreserved, brook trout sperm. The application of a cryopreservation procedure produced very high postthaw sperm motility (56.8 ± 4.0%). The cryopreserved sperm of brook trout could be stored up to 60 minutes without loss of the percentage of sperm motility (52.0 ± 9.0%). The fertilization capacity of brook trout postthaw sperm was comparable with the fertilization rate of fresh semen at a sperm-to-egg ratio as low as 300,000:1 (42.4 ± 14.0% and 36.5 ± 11.0% for eyed and hatched stages, respectively). The possibility of postthaw semen storage for the prolonged time and the obtainment of high fertilization rate at low sperm-to-egg ratio can lead to the significant improvement of brook trout semen cryopreservation procedure.

Inhibition of ß-N-acetylglucosaminidase by acetamide affects sperm motility and fertilization success of rainbow trout (Oncorhynchus mykiss) and Siberian sturgeon (Acipenser baerii).

ß-N-Acetylglucosaminidase (ß-NAGase) is an enzyme found in the sperm acrosome of numerous animal species including fish. Fish spermatozoa differ in their morphology including acrosome or acrosomeless aquasperm in chondrostean (e.g., sturgeon) and teleostean (e.g., rainbow trout). It has been shown that ß-NAGase exists with high activity in both eggs and sperm of these species. The present study shows the potency of ß-NAGase in fertilization. In rainbow trout, increase in sperm motility parameters (VAP and MOT) were observed in the presence of acetamide, an inhibitor for ß-NAGase. In contrast, sperm motility parameters (VCL, VSL, VAP, MOT, and PRG) were reduced on the Siberian sturgeon in the presence of acetamide. The inhibition of the activity of ß-NAGase in rainbow trout spermatozoa was led to a reduction in the number of fertilized eggs from 79% to 40%, whereas in sturgeon no change was observed in fertilization. Moreover, inhibition of ß-NAGase in both spermatozoa and eggs of trout and sturgeon resulted in significant decrease in fertilization rate from 79% to 1% in rainbow trout and from 84% to 12% in Siberian sturgeon. Our research proves that ß-NAGase can play a significant role in the fertilization process in teleosteans.

Use of eggs derived from the interspecific charr hybrids to induce androgenetic development of the brook charr (Salvelinus fontinalis Mitchill 1814).

Although, brook charr (Salvelinus fontinalis Mitchill 1814) and Arctic charr (Salvelinus alpinus Linnaeus 1758) are able to cross and give fertile offspring, their androgenetic nucleocytoplasmic hybrids are not viable. To overcome incompatibility between the egg cytoplasm of one charr species and the sperm nucleus of another charr species, application of F1 interspecific hybrids as egg donors for the purpose of androgenesis has been proposed. Here, androgenetic development of the brook charr was successfully induced in the brook charr eggs and the eggs derived from the reciprocal brook charr × Arctic charr F1 hybrids. A working androgenesis protocol included inactivation of the maternal nuclear DNA achieved by irradiation of the eggs with 420 Gy of X-rays, insemination of such treated eggs with the haploid sperm cells and exposition of the haploid androgenetic zygotes to the high hydrostatic pressure shock (51.711 MPa for 4 min) applied 420 min after insemination. Androgenetic larvae that hatched from the brook charr and the hybrid eggs were shown to be homozygous brook charr individuals. Androgenetic individuals exhibited 84 chromosomes and 100 chromosome arms (FN), values characteristic for the brook charr diploid cells. Strategy hybridize first than induce androgenesis should be tested in order to provide androgenetic offspring in other salmonids that are able to cross and produce fertile offspring.

Influence of zearalenone on selected biochemical parameters in juvenile rainbow trout (Oncorhynchus mykiss).

Zearalenone (ZEA) is a mycoestrogen frequently found in food and animal feed materials all over the world. Despite its hydrophobic character, ZEA is also found in surface and ground waters which suggests an environmental risk for aquatic animals. Knowledge concerning mycotoxin-related mechanisms of toxicity is still incomplete, e.g. little is known about the influence of ZEA exposure on fish. The aim of this study was to investigate the effect of ZEA on selected biochemical parameters in juvenile rainbow trout after 24, 72, and 168 h of intraperitoneal exposure (10 mg/kg of body weight). The analysis showed a slight tendency towards prolonged blood clotting time and significant iron deficiency in the liver and ovary of exposed animals. However, no differences in aminotransferase (AlaAT, AspAT) activity or glucose levels in fish plasma was observed. The results of this study suggest that although trout exposed to ZEA did not exhibit any distinct symptoms of liver damage, the mycotoxin tested was able interfere with blood coagulation and iron-storage processes.

Distribution of telomeric DNA sequences on the X-radiation-induced chromosome fragments observed in the genome of androgenetic brook trout (Salvelinus fontinalis, Mitchill 1814).

Cytogenetic screening of the androgenetic brook trout (Salvelinus fontinalis, Mitchill 1814) offspring hatched from eggs exposed to 420 Gy of X-radiation before insemination exhibited residues of the irradiated maternal nuclear genome in the form of small chromosome fragments. Remnants of the irradiated chromosomes had different sizes, and their number varied intraindividually from 1 to 15. To efficiently pass through the series of the cell divisions, such chromosome fragments must have had functional kinetochores. Distribution patterns of the telomeric hybridization signals on the chromosome fragments enabled us to distinguish their 3 groups: (i) telomere-less ring chromosomes with fused broken chromosome arms, (ii) rings formed in the course of fusion of the radiation-broken chromosome arm with the opposite telomeric region and exhibiting interstitial telomeric signals at the fusion point, and (iii) chromosome fragments with fused unprotected sister chromatids of 1 broken arm and intact telomeres from the other arm. Disturbances during segregation of such fragments, mainly breakages during anaphase, may partially explain intraindividual variation in the number and size of the chromosome fragments observed in the androgenetic brook trout.

Changes in sperm parameters of sex-reversed female rainbow trout during spawning season in relation to sperm parameters of normal males.

The production of all-female populations has important economic benefits in commercial rainbow trout aquaculture. The procedure commonly implemented to produce all-female stocks centers on the sex reversal of rainbow trout females via the administration of androgens in the early developmental stages, followed by the egg fertilization of normal females with semen from sex-reversed females (srf). However, there is no information regarding the quality of semen from srf rainbow trout throughout the spawning season. This information is critical because the quality of srf semen is highly variable. The aim of the study was to determine the changes in the semen parameters of srf rainbow trout throughout the duration of the spawning season. Sperm concentration, sperm motility parameters, and the biochemical parameters of seminal plasma (protein concentration, antitrypsin activity, osmolality, and lactate dehydrogenase activity) from srf were monitored during the spawning season and compared with normal male rainbow trout. The observed values of sperm, protein concentration, antitrypsin activity, osmolality, and lactate dehydrogenase activity of seminal plasma were all higher in comparison with normal males. Semen from srf was therefore characterized by a lower sperm motility during each period of the spawning season, in comparison with normal males, approximately 1.8, 1.5, and 1.7 times, respectively for the beginning, middle, and end of the spawning season. The percentage of sperm motility from srf and normal males were affected by the spawning season in the same way, as the highest values in the middle of the spawning season demonstrate (60% and 91% for srf and normal males, respectively). Spermatozoa of srf are characterized by a lower speed and a more curvilinear trajectory of movement as compared with that of normal males. The patterns of changes during the spawning season in sperm concentration, sperm motility parameters, as well as osmolality, and lactate dehydrogenase activity of the seminal plasma of srf were different in comparison with normal males. Our results could be important for fish breeders in regard to the spawning control of srf rainbow trout, as well as for the development of short- and long-term sperm storage procedures.

Chromosome rearrangements and survival of androgenetic rainbow trout (Oncorhynchus mykiss).

The purpose of this work was to quantify the impact of spontaneous and X-radiation-induced chromosome rearrangements on survival rate of androgenetic rainbow trout (Oncorhynchus mykiss). Various doses of X irradiation (50, 150, 250, 350 Gy) were used for inactivation of nuclear DNA in oocytes. After the irradiation, eggs were inseminated with normal sperm from 4 males derived from a strain characterized by Robertsonian rearrangements and length polymorphism of the Y chromosome. The haploid zygotes were exposed to a high hydrostatic pressure (7000 psi) to duplicate the paternal DNA. Neither Robertsonian chromosome polymorphism nor the Y chromosome morphology impaired the viability of the androgenetic embryos and alevins. Moreover, survival of eyed embryos of the androgenetic rainbow trout increased significantly with increasing doses of oocyte X irradiation. After 6 months of rearing, only specimens from the 250 and 350 Gy variants survived. The number of fingerlings with remnants of the maternal genome in the forms of chromosome fragments was higher in the 250 Gy group. Intraindividual variation of chromosome fragment number was observed, and some individuals exhibited haploid/diploid mosaicism and body malformations. Individuals irradiated with less than 250 Gy died, presumably because of the conflict between intact paternally derived chromosomes and the residues of maternal genome in the form of chromosome fragments.

Internal frequency conversion extreme ultraviolet interferometer using mutual coherence properties of two high-order-harmonic sources.

We report on an innovative two-dimensional imaging extreme ultraviolet (XUV) interferometer operating at 32 nm based on the mutual coherence of two laser high order harmonics (HOH) sources, separately generated in gas. We give the first evidence that the two mutually coherent HOH sources can be produced in two independent spatially separated gas jets, allowing for probing centimeter-sized objects. A magnification factor of 10 leads to a micron resolution associated with a subpicosecond temporal resolution. Single shot interferograms with a fringe visibility better than 30% are routinely produced. As a test of the XUV interferometer, we measure a maximum electronic density of 3x10(20) cm(-3) 1.1 ns after the creation of a plasma on aluminum target.

Karyotype variation in the albino rainbow trout (Oncorhynchus mykiss (Walbaum)).

A Robertsonian polymorphism resulting in diploid chromosome number ranging from 59 to 61 and constant chromosome arm number (fundamental number = 104) was observed in the albino rainbow trout (Oncorhynchus mykiss (Walbaum)) from the yellow color strain. In one individual, 90 mitotic chromosomes and 156 chromosome arms were counted, indicating the fish as a triploid. Morphology of the chromosomes, DAPI staining, and the cytogenetic location of 5S rDNA sequences showed sex-related chromosomal heteromorphism in the specimens. Additionally, length polymorphism of the X chromosome was detected in the studied individuals and two morphs of the X chromosome were described, XL and XS, according to the size of its short arm (p). The XS was observed in the female as well as male albino rainbow trout; however, among females, no XSXS genotype was found. After primed in situ labeling with 5S rDNA primers, the p-arms of both types of the X chromosome showed similar hybridization signals. On the other hand, fluorescence in situ hybridization with telomeric PNA (peptide nucleic acid) probe exhibited weak hybridization spots on the p-arm of the XS chromosome compared with the distinct hybridization spots observed on the XL p-arm. This could reflect a different telomere length on the p-arm of the XS and XL chromosomes. Partial translocation and deletion of the X chromosome p-arm are considered to be responsible for the p-arm length difference between the two morphological variants of X chromosome.

Formation of chromosome aberrations in androgenetic rainbow trout Oncorhynchus mykiss.

Residues of maternal nuclear DNA in the form of chromosome fragments were observed in the healthy and morphologically normal androgenetic rainbow trout Oncorhynchus mykiss. A hypothetical model for formation of chromosome re-arrangements caused by the incomplete maternal nuclear DNA inactivation in the androgenetic rainbow trout was proposed in the present paper.

Intense gamma-ray source in the giant-dipole-resonance range driven by 10-TW laser pulses.

A gamma-ray source with an intense component around the giant dipole resonance for photonuclear absorption has been obtained via bremsstrahlung of electron bunches driven by a 10-TW tabletop laser. 3D particle-in-cell simulation proves the achievement of a nonlinear regime leading to efficient acceleration of several sequential electron bunches per each laser pulse. The rate of the gamma-ray yield in the giant dipole resonance region (8

Coherent wake emission of high-order harmonics from overdense plasmas.

We present a new mechanism for high-order harmonic generation by reflection of a laser beam from an overdense plasma, efficient even at moderate laser intensities (down to Igamma2 approximately 4x10(15) W cm-2 microm2). In this mechanism, a transient phase matching between the electromagnetic field and plasma oscillations within a density gradient leads to the emission of harmonics up to the plasma frequency. These plasma oscillations are periodically excited in the wake of attosecond electron bunches which sweep across the density gradient. This process leads to a train of unevenly spaced chirped attosecond pulses and, hence, to broadened and chirped harmonics. This last effect is confirmed experimentally.

Probing hot and dense laser-induced plasmas with ultrafast XUV pulses.

In this Letter, we demonstrate the instantaneous creation of a hot solid-density plasma generated by focusing an intense femtosecond, high temporal contrast laser on an ultrathin foil (100 nm) in the 10(18) W/cm2 intensity range. The use of high-order harmonics generated in a gas jet, providing a probe beam of sufficiently short wavelengths to penetrate such a medium, enables the study of the dynamics of this plasma on the 100 fs time scale. The comparison of the transmission of two successive harmonics permits us to determine the electronic density and the temperature with accuracies better than 15%, never achieved up to this date in the regime of laser pulses at relativistic intensity.

Effects of UV irradiation and hydrogen peroxide on DNA fragmentation, motility and fertilizing ability of rainbow trout (Oncorhynchus mykiss) spermatozoa.

Preservation of DNA integrity is essential for protection of sperm quality. This study examined, with the use of comet assay, DNA fragmentation of rainbow trout (Oncorhynchus mykiss) spermatozoa subjected to UV irradiation (2,075 microW/cm(2), 0-15 min) or oxidative stress induced by hydrogen peroxide (0-20mM). Sperm motility and fertilizing ability were also measured. A dramatic increase in DNA fragmentation was recorded after 5 min UV irradiation but no significant changes in sperm motility were observed at this time. Longer irradiation resulted in a decrease in motility parameters and further increase of DNA fragmentation. UV irradiation caused a clear decrease in the percentage of eyed embryos and most of the embryos did not hatch. When highly diluted sperm suspensions (50,000-fold) were exposed to 0.1mM H(2)O(2) evident increase in DNA fragmentation was observed. On the other hand, when more concentrated sperm suspensions (diluted only 40-fold) were employed (in order to conduct motility and fertilization measurements at the same time) 1-20mM H(2)O(2) caused only moderate increase in DNA fragmentation and dose-dependent decline in sperm motility and fertilizing ability. This suggests that toxic effects of H(2)O(2) were primarily related to inhibition of sperm motility. Our results demonstrate that comet assay can be used for monitoring the effectiveness of fish sperm DNA inactivation by UV irradiation. Therefore, the comet assay together with sperm motility analysis can be applied in optimization works of gynogenetic procedures in fish. Lack of effectiveness of H(2)O(2) in inducing major DNA fragmentation suggests presence of mechanisms of antioxidative defense in rainbow trout spermatozoa.

Blood cells in rainbow trout Oncorhynchus mykiss milt: relation to milt collection method and sampling period.

The presence of blood cells in milt of rainbow trout (Oncorhynchus mykiss) collected every week between the middle at the end of the spawning season, either by stripping or by catheterization was investigated. Basic sperm biological and biochemical characteristics were also evaluated. Because milt often becomes contaminated with blood during collection, we also studied the influence of experimental blood contamination on sperm motility and biochemical parameters of seminal plasma. We demonstrated the presence of blood cells (erythrocytes, lymphoid, and phagocytes) in rainbow trout milt collected by both methods. Both sampling period and collection method influenced sperm characteristics, however the relationship between these characteristics and blood cells are not clear at present. A high number of blood cells in milt was found in some samples, possibly due to inflammation, because at the same time we observed bacteria and elevated levels of protein and antiproteinase activity in contaminated samples. Experimental contamination of milt with blood did not influence sperm motility, protein concentration and LDH activity of the 5-day-stored semen. Our study demonstrated that blood cells were present in rainbow trout milt. Blood cells may also appear in milt as a result of bleeding and their elevated levels are present during inflammation.

The stability of telomereless chromosome fragments in adult androgenetic rainbow trout.

The study provides new data on the stability of gamma radiation-induced chromosome fragments of a putative maternal nuclear genome in an androgenetic vertebrate, rainbow trout (Oncorhynchus mykiss Walbaum). The fragments were found in five of 16 examined individuals and they were mostly centromeric parts of metacentric or subtelocentric chromosomes. Chromosome fragments were identical in all cells of a given androgenetic individual, indicating that segregation of chromosome fragments is active from the early cell divisions. Most of the fragments were telomereless, i.e. they had no telomeric sequences on their ends. This shows that telomeres are not necessary for stability of chromosomal structures in a vertebrate genome. In one individual, the interstitial telomeric sites were found in chromosomes, which could be the effect of joining chromosome fragments.