- gonadal somatic factors in Atlantic salmon. - Since it is challenging to develop a successful treatment against a gene product already existing in the body, alternative targets are being explored. - Our aim was to identify and characterize novel germ cell and gonadal somatic factors in Atlantic salmon.. - 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. - Atlantic salmon is a highly valuable commercial species, to which a substantial amount of research has been dedi- cated during the recent years. - Since it is challenging to develop a success- ful sterility treatment against a gene product already present in the body, alternative novel targets are also being investigated. - Such proteins could potentially be expressed in the germ cells, such as Bmp15 or Gdf9 [10–13]. - Thus, blocking the function of proteins located in these nurturing cells or in the germ cells could potentially lead to sterility.. - As for now, some information is avail- able on genes exclusively expressed in germ cells in Atlantic salmon [7–9], and only one study has so far re- vealed a function of such proteins in salmon [3]. - Limited information is available for genes expressed exclusively in the somatic gonad of salmon. - The most known is the sex determining gene sdy, which is expressed in the male somatic gonad [15, 16]. - The GCF Atlantic salmon model represents a unique opportunity to identify genes that are exclusively expressed in gonadal somatic cells in this species. - Gonad specific genes preferentially expressed in gonadal somatic cells. - genes with more than 50 reads in the GCF group, and transcripts with less than 100 reads in the WT group, were excluded. - How- ever, in the case of the gene annotated to dsl1l (Gen- Bank a homology search had previously been performed using EST databases (GenBank CK897686.1 for salmon is identical to GenBank suggesting that this gene is gonadal somatic- derived factor (gsdf) [24]. - In the case of inha, a phylogenetic analysis (Additional file 4) was performed by comparing Inha protein sequences from tetrapods, teleosts and Spotted gar, a. - Expression profiles, shown as number of reads, of 11 genes with expression confined to gonadal somatic cells in adult immature Atlantic salmon testis tissue. - germ cell-free testis (n = 4). - Expression of gsdf and inha in Atlantic salmon tissues A qPCR tissue screen was performed to confirm that gsdf and inha are specifically expressed in gonads of At- lantic salmon. - In the case of inha, we also observed some staining within the ooplasm of immature oocytes (Fig. - In this study we have made available transcriptome data on genes expressed in the germ cell-free (GCF) Atlantic salmon testis tissue, which can also be compared to genes that are expressed in the same type of tissue with germ cells present. - Validation of tissue specificity of the identified transcripts In this study we characterized in more detail two genes with a high expression (read counts) in gonadal somatic cells, gsdf and inha. - A similar expression pattern has recently been shown in salmon testis tissue for Table 1 Gonad specific genes with expression in testicular somatic cells of Atlantic salmon. - 100 reads) in testicular somatic cells of Atlantic salmon. - Expression profiles, shown as number of reads, of 194 genes with expression confined to germ cells in adult immature Atlantic salmon testis tissue. - In the case of germ cell-specific genes identified in this study, several of these including piwil1, dazl, la-related protein 6-like (larp6l), bone morphogenetic protein 15-like (bmp15l) and folliculogenesis specific bHLH transcription factor (figla) have been validated by us using qPCR and ISH in previous publications [8, 9].. - When applying these criteria for in silico filtering, several well- known 1) gonad-specific genes were identified in the list of gonad-specific genes [Additional File 1], 2) well- known germ cell specific and gonadal somatic specific genes were identified in the lists of genes expressed in germ cells [Additional File 2] and in gonadal somatic cells (Table 1), which validates the chosen thresholds.. - They also represented the two genes with the highest expres- sion in testicular somatic cells, gsdf and inha, two genes that we have studied and discussed in more detail in the next sections. - TGF-β proteins are all secreted ligands known to be essential for many processes in gonad de- velopment including diverse functions in the testis asso- ciated with germ cells, Sertoli cells, growth and fertility [28]. - preferentially expressed in germ cells. - somatic gonad may be a result of sub-functionalization and a specialized function of this protein in the gonad of salmon. - function of this protein in the fish gonad. - This may indicate that the presence of germ cells has an inhibitory effect on nodal expression in Sertoli cells of immature testis tissue, and that Nodal is part of the germ-somatic cell communication in Atlantic salmon. - All values were calibrated to the average Δ Ct of the group with the highest expression (testis). - The identity of Atlantic salmon gsdf has previously been shown by phylogenetic analysis [24]. - Likewise, expression of gsdf in the same cell types has been observed in several fish species including rainbow trout (Oncorhynchus mykiss), medaka (Oryzias latipes), zebrafish, Nile tilapia (Oreochromis niloticus), Olive flounder (Paralichthys olivaceus), Spot- ted scat (Scatophagus argus) and Japanese flounder (Paralichthys olivaceus . - The fact that we detected a 3.5- fold lower number of gsdf reads in testis tissue devoid of germ cells compared to intact testis tissue may suggest a role for gsdf in the communication between germ cells and testicular somatic cells in Atlantic salmon.. - All values were calibrated to the average Δ Ct of the immature WT group. - GCF, germ cell-free. - This may be explained by a difference in the maturation stage, since the rainbow trout ovaries in the previous study were more advanced than the salmon ovar- ies applied in the current study. - In this study, Atlantic salmon inha was confirmed by chromosomal synteny to other fish species and by its. - In the current study it was difficult to distin- guish Leydig cells from the clearly stained and numerous Sertoli cells. - In the case of females, this is supported by studies where inha expression increased during folliculogenesis and peaked during final oocyte maturation in zebrafish [61] and coho salmon [62]. - Expression of gsdf and inha in Atlantic salmon ovary (gsdf: b, c, g, h. - Lysine degradation is a pathway essential for metabolic function in the cell and can in this way have a special function in germ cells. - Another speculation of an enrich- ment of these pathways in the gonad containing germ cells may be a potentially higher content of blood vessels in gonads with germ cells, since these pathways are asso- ciated with blood activity.. - The use of the experimental animals in this study was performed in strict accordance with the Norwegian. - Group 1: 7 Atlantic salmon males, 1 year old, reared and sampled as described previously [3]. - Group 2: 6 Atlantic salmon (3 females, 3 males), 1–2 years old, reared and sampled as described previously [9]. - Group 3: 83 Atlantic salmon (24 GCF/dnd-knockout, 10 immature and 9 early vitellogenic females. - The following tissues were included for qPCR analysis to reveal if inha and gsdf expression in the gonads change through puberty: GCF, immature and early vitellogenic ovary, and GCF, immature and mature testis (Fig. - Group 4: 3 Atlantic salmon (1 immature and 1 vitello- genic female. - The following tissues were included for ISH to reveal the cellular localization of gsdf and inha transcripts in the gonads: immature testis and ovary, and vitellogenic ovary (Fig. - Secondly, to identify which of the gonad specific genes that were expressed in the somatic part of the gonads, and which of them that were expressed within germ cells, we sequenced total RNA from 3 WT and 4 GCF testis (raw sequence reads accession no. - RNA-seq paired end sequences were mapped with Bowtie2 against the gene model transcripts of Atlantic salmon genome (ICSASG_v2) with standard Bowtie2 parameters [66]. - The read counts were normalized to the total reads in the sample with the smallest number of reads. - KEGG path- way analysis was performed by mapping the KEGG annotated genes to KEGG pathways as described in the KEGG Mapper tool [68]. - Consequently, germ cell spe- cific genes could be identified due to lack of expression in the GCF group, while gonadal somatic genes could be identified due to expression in both the GCF and WT group.. - Atlantic salmon (GenBank XP rainbow trout (GenBank XP and NP . - The following fish species were included in the analysis:. - The Atlantic salmon sequences and annotation were obtained from the official genome annotation (NCBI Salmo salar Annotation Release 100).. - All values were normalized to ef1a and calibrated to the average ΔCt of the testis tissue (group 2 samples) or the immature WT gonads (group 3 samples).. - The returned sequences were blasted in the NCBI database (https://www.ncbi.. - Probe size and qual- ity were checked with a Bioanalyzer (Agilent Technolo- gies), and the DIG incorporation in the probes was inspected by performing a spot-test. - 50 reads in extragonadal tissues and ≥ 100 reads in ovary and testis) genes in adult Atlantic salmon. - 50 reads in germ cell-free testis and ≥ 100 reads in WT testis) of adult Atlantic salmon males. - Full list of KEGG pathways annotated to gonad specific genes with expression preferentially within germ cells [Additional File 2] of adult Atlantic salmon males. - Phylogenetic analysis of Inha protein sequences from Atlantic salmon (GenBank XP rainbow trout (GenBank XP and NP . - The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test are shown next to the branches [71]. - The evolutionary distances were computed using the Poisson correction method [74] and are in the units of the number of amino acid substitutions per site. - A total of 432 positions were in the final dataset. - Completed “ The ARRIVE Guidelines Checklist ” for reporting information on experimental animals in this study. - Summary of the mapping of RNA-seq paired end sequences against the gene model tran- scripts of the Atlantic salmon genome (ICSASG_v2). - GCF: Germ cell-free. - Writing of the paper:. - The gene lists resulting from filtering of RNA sequencing data are available in the published paper and its additional files.. - Atlantic salmon populations invaded by farmed escapees: quantifying genetic introgression with a Bayesian approach and SNPs. - Three decades of farmed escapees in the wild: a spatio-temporal analysis of Atlantic salmon population genetic structure throughout Norway. - Dnd knockout ablates germ cells and demonstrates germ cell independent sex differentiation in Atlantic salmon.. - Gonad specific genes in Atlantic salmon (Salmo salar L. - bmp15l, figla, smc1bl, and larp6l are preferentially expressed in germ cells in Atlantic salmon (Salmo salar L. - Pharmacological characterization, localization and quantification of expression of gonadotropin receptors in Atlantic salmon (Salmo salar L.) ovaries. - Molecular cloning and characterization of FSH and LH receptors in Atlantic salmon (Salmo salar L. - Cloning, pharmacological characterization, and expression analysis of Atlantic salmon (Salmo salar L.) nuclear progesterone receptor.. - Sex-specific expression, synthesis and localization of aromatase regulators in one-year-old Atlantic salmon ovaries and testes.. - Regulation and expression of sexual differentiation factors in embryonic and extragonadal tissues of Atlantic salmon. - The Atlantic salmon genome provides insights into rediploidization. - Expression analysis of sex-determining pathway genes during development in male and female Atlantic salmon (Salmo salar). - Entry into puberty is reflected in changes in hormone production but not in testicular receptor expression in Atlantic salmon (Salmo salar). - Spatiotemporal expression of bone morphogenetic protein family ligands and receptors in the zebrafish ovary: a potential paracrine- signaling mechanism for oocyte-follicle cell communication. - Germ cell-Sertoli cell interactions: regulation by germ cells of the stage-specific expression of CP- 2/cathepsin L mRNA by Sertoli cells. - Gonadal soma-derived factor (gsdf), a TGF-beta superfamily gene, induces testis differentiation in the teleost fish Oreochromis niloticus.. - Autosomal gsdf acts as a male sex initiator in the fish medaka. - gsdf is a downstream gene of dmrt1 that functions in the male sex determination pathway of the Nile tilapia. - Characterization of gonadal soma-derived factor expression during sex change in the protogynous wrasse, Halichoeres trimaculatus. - Differential expression and cellular localization of activin and inhibin mRNA in the rainbow trout ovary and testis. - Characterization of inhibin alpha subunit (inha) in the zebrafish: evidence for a potential feedback loop between the pituitary and ovary. - Continuous light and elevated temperature can trigger maturation both during and immediately after smoltification in male Atlantic salmon (Salmo salar). - Evaluation of potential reference genes in real-time RT-PCR studies of Atlantic salmon. - Identification and localization of eight distinct hormone-producing cell types in the pituitary of male Atlantic halibut (Hippoglossus hippoglossus L.).
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