Quantitative proteome profiling reveals molecular hallmarks of egg quality in Atlantic halibut: impairments of transcription and protein folding impede protein and energy homeostasis during early development.

BACKGROUND: Tandem mass tag spectrometry (TMT labeling-LC-MS/MS) was utilized to examine the global proteomes of Atlantic halibut eggs at the 1-cell-stage post fertilization. Comparisons were made between eggs judged to be of good quality (GQ) versus poor quality (BQ) as evidenced by their subsequent rates of survival for 12 days. Altered abundance of selected proteins in BQ eggs was confirmed by parallel reaction monitoring spectrometry (PRM-LC-MS/MS). Correspondence of protein levels to expression of related gene transcripts was examined via qPCR. Potential mitochondrial differences between GQ and BQ eggs were assessed by transmission electron microscopy (TEM) and measurements of mitochondrial DNA (mtDNA) levels. RESULTS: A total of 115 proteins were found to be differentially abundant between GQ and BQ eggs. Frequency distributions of these proteins indicated higher protein folding activity in GQ eggs compared to higher transcription and protein degradation activities in BQ eggs. BQ eggs were also significantly enriched with proteins related to mitochondrial structure and biogenesis. Quantitative differences in abundance of several proteins with parallel differences in their transcript levels were confirmed in egg samples obtained over three consecutive reproductive seasons. The observed disparities in global proteome profiles suggest impairment of protein and energy homeostasis related to unfolded protein response and mitochondrial stress in BQ eggs. TEM revealed BQ eggs to contain significantly higher numbers of mitochondria, but differences in corresponding genomic mtDNA (mt-nd5 and mt-atp6) levels were not significant. Mitochondria from BQ eggs were significantly smaller with a more irregular shape and a higher number of cristae than those from GQ eggs. CONCLUSION: The results of this study indicate that BQ Atlantic halibut eggs are impaired at both transcription and translation levels leading to endoplasmic reticulum and mitochondrial disorders. Observation of these irregularities over three consecutive reproductive seasons in BQ eggs from females of diverse background, age and reproductive experience indicates that they are a hallmark of poor egg quality. Additional research is needed to discover when in oogenesis and under what circumstances these defects may arise. The prevalence of this suite of markers in BQ eggs of diverse vertebrate species also begs investigation.

Potential of Genome Editing to Improve Aquaculture Breeding and Production.

Aquaculture is the fastest growing food production sector and is rapidly becoming the primary source of seafood for human diets. Selective breeding programs are enabling genetic improvement of production traits, such as disease resistance, but progress is limited by the heritability of the trait and generation interval of the species. New breeding technologies, such as genome editing using CRISPR/Cas9 have the potential to expedite sustainable genetic improvement in aquaculture. Genome editing can rapidly introduce favorable changes to the genome, such as fixing alleles at existing trait loci, creating de novo alleles, or introducing alleles from other strains or species. The high fecundity and external fertilization of most aquaculture species can facilitate genome editing for research and application at a scale that is not possible in farmed terrestrial animals.

Single nucleotide replacement in the Atlantic salmon genome using CRISPR/Cas9 and asymmetrical oligonucleotide donors.

BACKGROUND: New breeding technologies (NBT) using CRISPR/Cas9-induced homology directed repair (HDR) has the potential to expedite genetic improvement in aquaculture. The long generation time in Atlantic salmon makes breeding an unattractive solution to obtain homozygous mutants and improving the rates of perfect HDR in founder (F0) fish is thus required. Genome editing can represent small DNA changes down to single nucleotide replacements (SNR). This enables edits such as premature stop codons or single amino acid changes and may be used to obtain fish with traits favorable to aquaculture, e.g. disease resistance. A method for SNR has not yet been demonstrated in salmon. RESULTS: Using CRISPR/Cas9 and asymmetrical ODNs, we were able to perform precise SNR and introduce a premature stop codon in dnd in F0 salmon. Deep sequencing demonstrated up to 59.2% efficiency in single embryos. In addition, using the same asymmetrical ODN design, we inserted a FLAG element into slc45a2 and dnd, showing high individual perfect HDR efficiencies (up to 36.7 and 32.7%, respectively). CONCLUSIONS: In this work, we demonstrate that precise SNR and knock-in (KI) can be performed in F0 salmon embryos using asymmetrical oligonucleotide (ODN) donors. We suggest that HDR-induced SNR can be applied as a powerful NBT, allowing efficient introgression of favorable alleles and bypassing challenges associated with traditional selective breeding.

Transcriptomic analysis of dead end knockout testis reveals germ cell and gonadal somatic factors in Atlantic salmon.

BACKGROUND: Sustainability challenges are currently hampering an increase in salmon production. Using sterile salmon can solve problems with precocious puberty and genetic introgression from farmed escapees to wild populations. Recently sterile salmon was produced by knocking out the germ cell-specific dead end (dnd). Several approaches may be applied to inhibit Dnd function, including gene knockout, knockdown or immunization. Since it is challenging to develop a successful treatment against a gene product already existing in the body, alternative targets are being explored. Germ cells are surrounded by, and dependent on, gonadal somatic cells. Targeting genes essential for the survival of gonadal somatic cells may be good alternative targets for sterility treatments. Our aim was to identify and characterize novel germ cell and gonadal somatic factors in Atlantic salmon. RESULTS: We have for the first time analysed RNA-sequencing data from germ cell-free (GCF)/dnd knockout and wild type (WT) salmon testis and searched for genes preferentially expressed in either germ cells or gonadal somatic cells. To exclude genes with extra-gonadal expression, our dataset was merged with available multi-tissue transcriptome data. We identified 389 gonad specific genes, of which 194 were preferentially expressed within germ cells, and 11 were confined to gonadal somatic cells. Interestingly, 5 of the 11 gonadal somatic transcripts represented genes encoding secreted TGF-ß factors; gsdf, inha, nodal and two bmp6-like genes, all representative vaccine targets. Of these, gsdf and inha had the highest transcript levels. Expression of gsdf and inha was further confirmed to be gonad specific, and their spatial expression was restricted to granulosa and Sertoli cells of the ovary and testis, respectively. Finally, we show that inha expression increases with puberty in both ovary and testis tissue, while gsdf expression does not change or decreases during puberty in ovary and testis tissue, respectively. CONCLUSIONS: This study contributes with transcriptome data on salmon testis tissue with and without germ cells. We provide a list of novel and known germ cell- and gonad somatic specific transcripts, and show that the expression of two highly active gonadal somatic secreted TGF-ß factors, gsdf and inha, are located within granulosa and Sertoli cells.

Targeted mutagenesis of ∆5 and ∆6 fatty acyl desaturases induce dysregulation of lipid metabolism in Atlantic salmon (Salmo salar).

BACKGROUND: With declining wild fish populations, farmed salmon has gained popularity as a source for healthy long-chain highly unsaturated fatty acids (LC-HUFA). However, the introduction of plant oil in farmed salmon feeds has reduced the content of these beneficial LC-HUFA. The synthetic capability for LC-HUFAs depends upon the dietary precursor fatty acids and the genetic potential, thus there is a need for in-depth understanding of LC-HUFA synthetic genes and their interactions with other genes involved in lipid metabolism. Several key genes of LC-HUFA synthesis in salmon belong to the fatty acid desaturases 2 (fads2) family. The present study applied whole transcriptome analysis on two CRISPR-mutated salmon strains (crispants), 1) Δ6abc/5Mt with mutations in Δ5fads2, Δ6fads2-a, Δ6fads2-b and Δ6fads2-c genes, and 2) Δ6bcMt with mutations in Δ6fads2-b and Δ6fads2-c genes. Our purpose is to evaluate the genetic effect fads2 mutations have on other lipid metabolism pathways in fish, as well as to investigate mosaicism in a commercial species with a very long embryonal period. RESULTS: Both Δ6abc/5Mt and Δ6bcMt crispants demonstrated high percentage of indels within all intended target genes, though different indel types and percentage were observed between individuals. The Δ6abc/5Mt fish displayed several disruptive indels which resulted in over 100 differentially expressed genes (DEGs) enriched in lipid metabolism pathways in liver. This includes up-regulation of srebp1 genes which are known key transcription regulators of lipid metabolism as well as a number of down-stream genes involved in fatty acid de-novo synthesis, fatty acid ß-oxidation and lipogenesis. Both elovl5 and elovl2 genes were not changed, suggesting that the genes were not targeted by Srebp1. The mutation of Δ6bcMt surprisingly resulted in over 3000 DEGs which were enriched in factors encoding genes involved in mRNA regulation and stability. CONCLUSIONS: CRISPR-Cas9 can efficiently mutate multiple fads2 genes simultaneously in salmon. The results of the present study have provided new information on the transcriptional regulations of lipid metabolism genes after reduction of LC-HUFA synthesis pathways in salmon.

Development of supermale and all-male Atlantic salmon to research the vgll3 allele - puberty link.

BACKGROUND: Farmed Atlantic salmon are one of the most economically significant global aquaculture products. Early sexual maturation of farmed males represents a significant challenge to this industry and has been linked with the vgll3 genotype. However, tools to aid research of this topic, such as all-male and clonal fish, are still lacking. The present 6-year study examined if all-male production is possible in Atlantic salmon, a species with heteromorphic sex chromosomes (males being XY, females XX), and if all-male fish can be applied to further explore the vgll3 contribution on the likelihood of early maturation. RESULTS: Estrogen treatment of mixed sex yolk sac larvae gave rise to one sexually mature hermaphrodite with a male genotype (XY) that was used to produce both self-fertilized offspring and androgenetic double haploid (dh) offspring following egg activation with UV treated sperm and pressure shock to block the first mitotic division. There were YY supermales among both offspring types, which were crossed with dh females. Between 1 and 8% of the putative all-male offspring from the eight crosses with self-fertilized supermales were found to have ovaries, and 95% of these phenotypic females were also genetically female. None of the offspring from the one dh supermale cross had ovaries. When assessing the general contribution of the vgll3 locus on the likelihood of early post-smolt sexual maturation (jacking) in the all-male populations we found individuals that were homozygous for the early maturing genotype (97%) were more likely to enter puberty than individuals that were homozygous for the late maturing genotype (26%). However, the likelihood of jacking within individuals with an early/late heterozygous genotype was higher when the early allele came from the dam (94%) compared to the sire (45%). CONCLUSIONS: The present results show that supermale Atlantic salmon are viable and fertile and can be used as a research tool to study important aspects of sexual maturation, such as to further explore the sex dependent parental genetic contribution to age at puberty in Atlantic salmon. In addition, we report the production of viable double haploid supermale fish.

Application of genome editing in aquatic farm animals: Atlantic salmon.

Gene editing offers opportunities to solve fish farming sustainability issues that presently hampers expansion of the aquaculture industry. In for example Atlantic salmon farming, there are now two major bottlenecks limiting the expansion of the industry. One is the genetic impact of escaped farmed salmon on wild populations, which is considered the most long-term negative effect on the environment. Secondly and the utmost acute problem is the fish parasite salmon lice, which is currently causing high lethality in wild salmonids due to high concentrations of the parasite in the sea owing to sea cage salmon farming. There are also sustainability issues associated with increased use of vegetable-based ingredients as replacements for marine products in fish feed. This transition comes at the expense of the omega-3 content both in fish feed and the fish filet of the farmed fish. Reduced fish welfare represents another obstacle, and robust farmed fish is needed to avoid negative stress associated phenotypes such as cataract, bone and fin deformities, precocious maturity and higher disease susceptibility. Gene editing could solve some of these problems as genetic traits can be altered positively to reach phenotype of interest such as for example disease resistance and increased omega-3 production.

The influence of vgll3 genotypes on sea age at maturity is altered in farmed mowi strain Atlantic salmon.

BACKGROUND: In Atlantic salmon in the wild, age at maturity is strongly influenced by the vgll3 locus. Under farming conditions, light, temperature and feeding regimes are known significantly advance or delay age at maturity. However, the potential influence of the vgll3 locus on the maturation of salmon reared under farming conditions has been rarely investigated, especially in females. RESULTS: Here, we reared domesticated salmon (mowi strain) with different vgll3 genotypes under standard farming conditions until they matured at either one, two or more than two sea winters. Interestingly, and in contrast to previous findings in the wild, we were not able to identify a link between vgll3 and age at maturity in females when reared under farming conditions. For males however, we found that the probability of delaying maturation from one to two sea winters was significantly lower in fish homozygous for the early allele compared to homozygous fish for the late allele, while the probability for heterozygous fish was intermediate. These data also contrast to previous findings in the wild where the early allele has been reported as dominant. However, we found that the probability of males delaying maturation from two to three sea winters was regulated in the same manner as the wild. CONCLUSIONS: Collectively, our data suggest that increased growth rates in mowi salmon, caused by high feed intake and artificial light and temperature regimes together with other possible genetic/epigenetic components, may significantly influence the impact that the vgll3 locus has on age at maturity, especially in females. In turn, our results show that the vgll3 locus can only to a large extent be used in selective breeding to control age at maturation in mowi males. In summary, we here show that in contrast to the situation in wild salmon, under farming conditions vgll3 does not seem to influence age at maturity in mowi females whereas in mowi males, maturing as one or two sea winters it alters the early allele effect from dominant to intermediate.

The vgll3 Locus Controls Age at Maturity in Wild and Domesticated Atlantic Salmon (Salmo salar L.) Males.

Wild and domesticated Atlantic salmon males display large variation for sea age at sexual maturation, which varies between 1-5 years. Previous studies have uncovered a genetic predisposition for variation of age at maturity with moderate heritability, thus suggesting a polygenic or complex nature of this trait. The aim of this study was to identify associated genetic loci, genes and ultimately specific sequence variants conferring sea age at maturity in salmon. We performed a genome wide association study (GWAS) using a pool sequencing approach (20 individuals per river and phenotype) of male salmon returning to rivers as sexually mature either after one sea winter (2009) or three sea winters (2011) in six rivers in Norway. The study revealed one major selective sweep, which covered 76 significant SNPs in which 74 were found in a 370 kb region of chromosome 25. Genotyping other smolt year classes of wild and domesticated salmon confirmed this finding. Genotyping domesticated fish narrowed the haplotype region to four SNPs covering 2386 bp, containing the vgll3 gene, including two missense mutations explaining 33-36% phenotypic variation. A single locus was found to have a highly significant role in governing sea age at maturation in this species. The SNPs identified may be both used as markers to guide breeding for late maturity in salmon aquaculture and in monitoring programs of wild salmon. Interestingly, a SNP in proximity of the VGLL3 gene in humans (Homo sapiens), has previously been linked to age at puberty suggesting a conserved mechanism for timing of puberty in vertebrates.

Functional Annotation of All Salmonid Genomes (FAASG): an international initiative supporting future salmonid research, conservation and aquaculture.

We describe an emerging initiative - the 'Functional Annotation of All Salmonid Genomes' (FAASG), which will leverage the extensive trait diversity that has evolved since a whole genome duplication event in the salmonid ancestor, to develop an integrative understanding of the functional genomic basis of phenotypic variation. The outcomes of FAASG will have diverse applications, ranging from improved understanding of genome evolution, to improving the efficiency and sustainability of aquaculture production, supporting the future of fundamental and applied research in an iconic fish lineage of major societal importance.

bmp15l, figla, smc1bl, and larp6l are preferentially expressed in germ cells in Atlantic salmon (Salmo salar L.).

Atlantic salmon is a valuable commercial aquaculture species that would benefit economically and environmentally by controlling precocious puberty and preventing escapees from reproducing with wild populations. One solution to both these challenges is the production of sterile individuals by inhibiting the formation of germ cells, but achieving this requires more information on the specific factors that control germ cell formation. Here, we identified and characterized novel factors that are preferentially expressed in Atlantic salmon germ cells by screening for gonad-specific genes using available adult multi-tissue transcriptomes. We excluded genes with expression in tissues other than gonads based on quantity of reads, and then a subset of genes was selected for verification in a multi-tissue PCR screen. Four gonad-specific genes (bmp15l, figla, smc1bl, and larp6l) were chosen for further characterization, namely: germ cell specificity, investigated by comparing mRNA abundance in wild-type and germ cell-free gonads by quantitative real-time PCR, and cellular location, visualized by in situ hybridization. All four genes were expressed in both testis and ovary, and preferentially within the germ cells of both sexes. These genes may be essential players in salmon germ cell development, and could be important for future studies aiming to understand and control reproduction. Mol. Reprod. Dev. 84: 76-87, 2017. © 2016 Wiley Periodicals, Inc.

Rapid growth causes abnormal vaterite formation in farmed fish otoliths.

Sagittal otoliths are essential components of the sensory organs that enable all teleost fish to hear and maintain balance, and are primarily composed of calcium carbonate. A deformity, where aragonite (the normal crystal form) is replaced with vaterite, was first noted over 50 years ago but its underlying cause is unresolved. We evaluated the prevalence of vateritic otoliths from two captive rearing studies which suggested that fast growth, due to environmental rather than genetic control, led to vaterite development. We then tested this by varying light and temperature to create phenotypes with different growth rates, which resulted in fast growers (5 times larger) having 3 times more vaterite than slow growers. A decrease in either the ratio of otolith matrix proteins (otolin-1/OMM-64) or [Ca2+]/[CO32-] may explain why fast growth causes vaterite deposition. As vaterite decreases hearing sensitivity, reducing growth rates in hatcheries may improve the welfare of farmed fish and increase the success of conservation efforts.

Atlantic salmon populations reveal adaptive divergence of immune related genes - a duplicated genome under selection.

BACKGROUND: Populations of Atlantic salmon display highly significant genetic differences with unresolved molecular basis. These differences may result from separate postglacial colonization patterns, diversifying natural selection and adaptation, or a combination. Adaptation could be influenced or even facilitated by the recent whole genome duplication in the salmonid lineage which resulted in a partly tetraploid species with duplicated genes and regions. RESULTS: In order to elucidate the genes and genomic regions underlying the genetic differences, we conducted a genome wide association study using whole genome resequencing data from eight populations from Northern and Southern Norway. From a total of ~4.5 million sequencing-derived SNPs, more than 10 % showed significant differentiation between populations from these two regions and ten selective sweeps on chromosomes 5, 10, 11, 13-15, 21, 24 and 25 were identified. These comprised 59 genes, of which 15 had one or more differentiated missense mutation. Our analysis showed that most sweeps have paralogous regions in the partially tetraploid genome, each lacking the high number of significant SNPs found in the sweeps. The most significant sweep was found on Chr 25 and carried several missense mutations in the antiviral mx genes, suggesting that these populations have experienced differing viral pressures. Interestingly the second most significant sweep, found on Chr 5, contains two genes involved in the NF-KB pathway (nkap and nkrf), which is also a known pathogen target that controls a large number of processes in animals. CONCLUSION: Our results show that natural selection acting on immune related genes has contributed to genetic divergence between salmon populations in Norway. The differences between populations may have been facilitated by the plasticity of the salmon genome. The observed signatures of selection in duplicated genomic regions suggest that the recently duplicated genome has provided raw material for evolutionary adaptation.

A comparison between egg trancriptomes of cod and salmon reveals species-specific traits in eggs for each species.

Fish in use in aquaculture display large variation in gamete biology. To reach better understanding around this issue, this study aims at identifying if species specific "egg life history traits" can be hidden in the unfertilized egg. This was done by investigating egg transcriptome differences between Atlantic salmon and Atlantic cod. Salmon and cod eggs were selected due to their largely differencing phenotypes. An oligo microarray analysis was performed on ovulated eggs from cod (n = 8) and salmon (n = 7). The arrays were normalized to a similar spectrum for both arrays. Both arrays were re-annotated with SWISS-Prot and KEGG genes to retrieve an official gene symbol and an orthologous KEGG annotation, in salmon and cod arrays this represented 14,009 and 7,437 genes respectively. The probe linked to the highest gene expression for that particular KEGG annotation was used to compare expression between species. Differential expression was calculated for genes that had an annotation with score >300, resulting in a total of 2,457 KEGG annotations (genes) being differently expressed between the species (FD > 2). This analysis revealed that immune, signal transduction and excretory related pathways were overrepresented in salmon compared to cod. The most overrepresented pathways in cod were related to regulation of genetic information processing and metabolism. To conclude this analysis clearly point at some distinct transcriptome repertoires for cod and salmon and that these differences may explain some of the species-specific biological features for salmon and cod eggs.

Transcriptome sequencing of Atlantic salmon (Salmo salar L.) notochord prior to development of the vertebrae provides clues to regulation of positional fate, chordoblast lineage and mineralisation.

BACKGROUND: In teleosts such as Atlantic salmon (Salmo salar L.), segmentation and subsequent mineralisation of the notochord during embryonic stages are essential for normal vertebrae formation. However, the molecular mechanisms leading to segmentation and mineralisation of the notochord are poorly understood. The aim of this study was to identify genes/pathways acting in gradients over time and along the anterior-posterior axis during notochord segmentation and immediately prior to mineralisation of the vertebral bodies in Atlantic salmon. RESULTS: Notochord samples were collected from unsegmented, pre-segmented and segmented developmental stages. In each stage, the cellular core of the notochord was cut into three pieces along the longitudinal axis (anterior, mid, posterior). RNA was sequenced (22 million pair-end 100 bp/ library) and mapped to the salmon genome. 66569 transcripts were predicted and 55775 were annotated. In order to identify possible gradients leading to segmentation of the notochord, all 71 notochord-expressed hox genes were investigated, most of them displaying a typical anterior-posterior expression pattern along the notochord axis. The clustering of hox genes revealed a pattern that could be related to notochord segmentation. We further investigated how mineralisation is initiated in the notochord, and several factors related to chondrogenic lineage were identified (sox9, sox5, sox6, tgfb3, ihhb and col2a1), suggesting a cartilage-like character of the notochord. KEGG analysis of differentially expressed genes between stages revealed down-regulation of pathways associated with ECM, cell division, metabolism and development at onset of notochord segmentation. This implies that inhibitory signals produce segmentation of the notochord. One such potential inhibitory signal was identified, col11a2, which was detected in segments of non-mineralising notochord. CONCLUSIONS: An incomplete salmon genome was successfully used to analyse RNA-seq data from the cellular core of the Atlantic salmon notochord. In transcriptome we found; hox gene patterns possibly linked to segmentation; down-regulation of pathways in the notochord at onset of segmentation; segmented expression of col11a2 in non-mineralised segments of the notochord; and a chondroblast-like footprint in the notochord.

Global transcriptome analysis identifies regulated transcripts and pathways activated during oogenesis and early embryogenesis in Atlantic cod.

The molecular mechanisms underlying oogenesis and maternally controlled embryogenesis in fish are not fully understood, especially in marine species. Our aim was to study the egg and embryo transcriptome during oogenesis and early embryogenesis in Atlantic cod. Follicles from oogenesis stages (pre-, early-, and late-vitellogenic), ovulated eggs, and two embryonic stages (blastula, gastrula) were collected from broodstock fish and fertilized eggs. Gene expression profiles were measured in a 44 K oligo microarray consisting of 23,000 cod genes. Hundreds of differentially expressed genes (DEGs) were identified in the follicle stages investigated, implicating a continuous accumulation and degradation of polyadenylated transcripts throughout oogenesis. Very few DEGs were identified from ovulated egg to blastula, showing a more stable maternal RNA pool in early embryonic stages. The highest induction of expression was observed between blastula and gastrula, signifying the onset of zygotic transcription. During early vitellogenesis, several of the most upregulated genes are linked to nervous system signaling, suggesting increasing requirements for ovarian synaptic signaling to stimulate the rapid growth of oocytes. Highly upregulated genes during late vitellogenesis are linked to protein processing, fat metabolism, osmoregulation, and arrested meiosis. One of the genes with the highest upregulation in the ovulated egg is involved in oxidative phosphorylation, reflecting increased energy requirements during fertilization and the first rapid cell divisions of early embryogenesis. In conclusion, this study provides a large-scale presentation of the Atlantic cod's maternally controlled transcriptome in ovarian follicles through oogenesis, ovulated eggs, and early embryos.

Loss of Fshr Prevents Testicular Maturation in Atlantic Salmon (Salmo salar L.).

Early puberty poses a significant challenge for male Atlantic salmon in aquaculture due to its negative impact on growth and welfare. The regulation of puberty in vertebrates involves 2 key reproductive hormones: follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and their gonadal receptors. In male mice lacking FSH receptor, testes size is reduced, but fertility is maintained, while medaka and zebrafish with a disrupted fshr gene exhibit near normal testis size and fertility. In these fishes both Fsh and Lh are present during puberty and Lh may rescue fertility, while in salmonid fish only Fsh is present in the circulation during puberty. Using CRISPR-Cas9, we produced crispants with a high prevalence of fshr mutations at the target site, which remained fertile, although more than half showed a testis development deviating from wild-type (wt) males. Crossing out these F0 crispants to each other produced a viable F1 generation showing frameshift (fshr-/-) or in-frame mutations (fshrif/if). Nearly all wt males matured while all fshr-/- males remained immature with small testes containing A spermatogonia as the furthest developed germ cell type and prepubertal plasma androgen levels. Also, the pituitary transcript levels of gnrhr2bba and lhb, but not for fshb, were reduced in the fshr-/- males compared with maturing males. More than half of the fshrif/if mutant males showed no or a delayed maturation. In conclusion, Atlantic salmon show the unique characteristic that loss of Fshr function alone results in male infertility, offering new opportunities to control precocious puberty or fertility in salmon.

Mineralization of the vertebral bodies in Atlantic salmon (Salmo salar L.) is initiated segmentally in the form of hydroxyapatite crystal accretions in the notochord sheath.

We performed a sequential morphological and molecular biological study of the development of the vertebral bodies in Atlantic salmon (Salmo salar L.). Mineralization starts in separate bony elements which fuse to form complete segmental rings within the notochord sheath. The nucleation and growth of hydroxyapatite crystals in both the lamellar type II collagen matrix of the notochord sheath and the lamellar type I collagen matrix derived from the sclerotome, were highly similar. In both matrices the hydroxyapatite crystals nucleate and accrete on the surface of the collagen fibrils rather than inside the fibrils, a process that may be controlled by a template imposed by the collagen fibrils. Apatite crystal growth starts with the formation of small plate-like structures, about 5 nm thick, that gradually grow and aggregate to form extensive multi-branched crystal arborizations, resembling dendritic growth. The hydroxyapatite crystals are always oriented parallel to the long axis of the collagen fibrils, and the lamellar collagen matrices provide oriented support for crystal growth. We demonstrate here for the first time by means of synchroton radiation based on X-ray diffraction that the chordacentra contain hydroxyapatite. We employed quantitative real-time PCR to study the expression of key signalling molecule transcripts expressed in the cellular core of the notochord. The results indicate that the notochord not only produces and maintains the notochord sheath but also expresses factors known to regulate skeletogenesis: sonic hedgehog (shh), indian hedgehog homolog b (ihhb), parathyroid hormone 1 receptor (pth1r) and transforming growth factor beta 1 (tgfb1). In conclusion, our study provides evidence for the process of vertebral body development in teleost fishes, which is initially orchestrated by the notochord.

Cortisol treatment of prespawning female cod affects cytogenesis related factors in eggs and embryos.

A stable supply of viable eggs and embryos is crucial for successful farming of Atlantic cod. Stress during broodstock rearing can have negative effects on offspring, but little is known about the molecular mechanisms that cause abnormal development. Maternally transferred mRNAs have been shown to be essential for normal development, and stress may therefore influence their expression and the subsequent embryonic development. We investigated if mimicked stress in cod females affects mRNA concentrations in eggs/embryos, and if this can be linked to viability of embryos. Three weeks before peak spawning, 20 fish were intraperitoneally implanted with either cortisol-containing or cortisol-free (sham) osmotic pumps. At peak spawning all individuals were stripped and eggs were fertilized and incubated until hatching. Samples were collected from unfertilized eggs and embryos for analysis of gene expression (microarray), viability, steroids and vitellogenin. Plasma concentration of cortisol (ng/ml) in treated females was significantly higher at spawning (127.1±20.9) than that of sham control (11.3±6.7). This difference was also reflected in eggs and embryos. Percent fertilization, asymmetric cell division and hatching were not affected. However, numerous genes were differentially expressed in eggs and embryos in response to elevated cortisol, especially in maternal (oocyte and blastula) stages. Among these differentially expressed genes, some were found to be linked to cytogenesis (stxbp6, fbxw2, capn12, thbs4, sytl2, coro1c, sel1l3), induction of mesodermal fate (fgfrl1) and import of the glucocorticoid receptor to the cell nucleus (ipo7). Gene ontology overrepresentation analysis on the whole set of differentially expressed genes at maternal stages (539 genes) revealed enriched activity in membrane associated regions, which largely corresponds to cytogenesis related processes. These results suggest that despite no visible phenotypic effects in early embryos, broodstock stress affects the egg/embryonic transcriptome, especially in relation to cytogenesis. Furthermore, effects related to egg/embryo phenotypes are difficult to measure at early stages of development, and instead might become apparent at later life stages.

Maternal 3'UTRs: from egg to onset of zygotic transcription in Atlantic cod.

BACKGROUND: Zygotic transcription in fish embryos initiates around the time of gastrulation, and all prior development is initiated and controlled by maternally derived messenger RNAs. Atlantic cod egg and embryo viability is variable, and it is hypothesized that the early development depends upon the feature of these maternal RNAs. Both the length and the presence of specific motifs in the 3'UTR of maternal RNAs are believed to regulate expression and stability of the maternal transcripts. Therefore, the aim of this study was to characterize the overall composition and 3'UTR structure of the most common maternal RNAs found in cod eggs and pre-zygotic embryos. RESULTS: 22229 Sanger-sequences were obtained from 3'-end sequenced cDNA libraries prepared from oocyte, 1-2 cell, blastula and gastrula stages. Quantitative PCR revealed that EST copy number below 9 did not reflect the gene expression profile. Consequently genes represented by less than 9 ESTs were excluded from downstream analyses, in addition to sequences with low-quality gene hits. This provided 12764 EST sequences, encoding 257 unique genes, for further analysis. Mitochondrial transcripts accounted for 45.9-50.6% of the transcripts isolated from the maternal stages, but only 12.2% of those present at the onset of zygotic transcription. 3'UTR length was predicted in nuclear sequences with poly-A tail, which identified 191 3'UTRs. Their characteristics indicated a more complex regulation of transcripts that are abundant prior to the onset of zygotic transcription. Maternal and stable transcripts had longer 3'UTR (mean 187.1 and 208.8 bp) and more 3'UTR isoforms (45.7 and 34.6%) compared to zygotic transcripts, where 15.4% had 3'UTR isoforms and the mean 3'UTR length was 76 bp. Also, diversity and the amount of putative polyadenylation motifs were higher in both maternal and stable transcripts. CONCLUSIONS: We report on the most pronounced processes in the maternally transferred cod transcriptome. Maternal stages are characterized by a rich abundance of mitochondrial transcripts. Maternal and stable transcripts display longer 3'UTRs with more variation of both polyadenylation motifs and 3'UTR isoforms. These data suggest that cod eggs possess a complex array of maternal RNAs which likely act to tightly regulate early developmental processes in the newly fertilized egg.