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. We have from these data identified genes that are preferentially expressed in both gonadal somatic cells (Table 1) and germ cells of salmon [Additional File 2], which adds novel factors to the list of genes that have been reported previously [7-9]. This dataset therefore provides valuable information for further studies on potential sterility targets in Atlantic salmon, but also serves as an interesting resource for the field of reproductive biology in a wider context, such as cell-cell communication within the testis.
In silico filtering thresholds
To be able to say that a gene is expressed or not in a given tissue sample, a threshold for number of reads needs to be determined. In this study we applied the same thresholds as a previous similar study ; firstly, genes with 50 or less reads are not considered expressed, while fewer reads are considered as background/noise. This choice is supported by the fact that for example vasa, known to be expressed exclusively in germ cells, still had 70 and 57 reads in eye and gill tissues. Secondly, we chose 100 reads as a minimum for genes considered to be expressed; this creates a clear distinction from genes considered not expressed (50 or less reads). 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. Nevertheless, cutoffs most likely also cause some relevant genes to be excluded from the analysis.
Genes with expression preferentially in gonadal somatic cells
To our knowledge, this is the first study to screen for genes with expression exclusively in gonadal somatic cells in Atlantic salmon, using a knockout model. Only 11 genes (gsdf, inha, ctsl, nodal, two bmp6l, ctssl, slc25a12l, two unknown genes and one unknown ncRNA) had 100 or more reads in GCF and WT testis (Table 1) while at the same time being exclusive to gonadal tissue. The most striking finding was that around half of these genes, (gsdf, inha, nodal and the two bmp6l) are associated with the TGF-β pathway. They also represented the two genes with the highest expression 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 development including diverse functions in the testis associated with germ cells, Sertoli cells, growth and fertility . However in our case, the finding of TGF-β transcripts restricted to gonadal somatic cells is truly promising for future targeting since TGF-β proteins are secreted proteins that may likely have functions confined to the gonad, which is a prerequisite for 1) functional studies of the gonad without using conditional mutants, and 2) finding potential target proteins for a sterility vaccine in salmon. Interestingly, bmp6 plays a role in human Sertoli cell proliferation and apoptosis . In fish, Bmp6 has been linked to fin regeneration, iron metabolism, tooth patterning and viability and growth [29-32]. Furthermore, bmp6 has been linked to ovarian function in tounge sole and zebrafish [33; 34]. The diverse roles of this protein may be explained by its widespread expression in many tissues, however our finding of a unique bmp6-like transcript confined largely to the somatic gonad may be a result of sub-functionalization and a specialized function of this protein in the gonad of salmon. Nodal, another TGF-β protein, is involved in various processes within mammalian testes. More specifically, Nodal signaling plays a role in regulation of pluripotency factor expression, proliferation and survival of germ cells, as well as establishment of the somatic niche through seminiferous cord formation, steroidogenesis and Sertoli cell function (reviewed by ). In human testis, Nodal has been shown to regulate Sertoli cell proliferation . In zebrafish Nodal is involved in dorso-ventral patterning of the embryo , while to our knowledge no specific studies have investigated the function of this protein in the fish gonad. A high nodal expression restricted to gonads in salmon as observed in this study suggests a role of this gene in reproduction in fish. Interestingly, we observed a 3-fold higher nodal expression in salmon testis lacking germ cells, compared to WT testis. 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. Two Cathepsins, Ctsl and Ctssl, had gonad specific transcripts and were also expressed within testicular somatic cells in salmon. These are Cysteine Cathepsins, proteases involved in a number of physiological processes like protein breakdown and immune responses [38; 39]. Interestingly, the stage-specific expression of ctsl in rat Sertoli cells is influenced by the presence or absence of germ cells, suggesting a role of ctsl in germ-somatic cell interaction in testis tissue . A higher number of ctsl as well as ctssl reads in GCF compared to WT testis tissue as observed in this study, may suggest possible involvement for these Cathepsins in germ-somatic cell signaling in salmon.
The identity of Atlantic salmon gsdf has previously been shown by phylogenetic analysis . Gsdf is a growth factor and as mentioned above, a member of the Transforming growth factor beta (TGF-β) superfamily . Gsdf has mainly been detected in teleosts, but recently also in the coelacanth Latimeria menadoensis  and the West African lungfish Protopterus annectens . Expression of gsdf is gonad specific and predominantly localized to gonadal somatic cells surrounding primordial germ cells in early life stages, and in testicular Sertoli and ovarian granulosa cells in adults of several fish species such as rainbow trout (Oncorhynchus mykiss), medaka (Oryzias latipes), zebrafish, Nile tilapia (Oreochromis niloticus), Olive flounder (Paralichthys olivaceus), Spotted scat (Scatophagus argus) and Japanese flounder (Paralichthys olivaceus) [24; 43-49). Transcripts of gsdf have also been detected within oocytes in Olive flounder , and within spermatogonia and spermatids in Chinese tongue sole (Cynoglossus semilaevis) . The gsdf gene was first described as an essential player in proliferation of primordial germ cells and spermatogonia in rainbow trout . In Oryzias luzonensis, gsdf is a sex determining gene , and gsdf knockdown in Chinese tounge sole testicular somatic cell lines influenced the expression of several genes that are involved in sex differentiation . In medaka it was shown that in homozygous gsdf XY mutants, ovaries developed at an early developmental stage. However, some of these mutants developed testis tissue at adult stages. Thus, in medaka gsdf is essential for directing the bipotential gonad at early developmental stages . The role of gsdf as an endogenous inducer of testis differentiation in medaka has also been shown . In Nile tilapia homozygous mutation of gsdf in XY individuals induced complete sex reversal to female, which could be rescued by aromatase inhibitor treatment . Our observation of a high gsdf expression in testis tissue, specifically localized to Sertoli cells, suggests that Gsdf has an essential role in testis development in Atlantic salmon as well. Furthermore, Gsdf expression is high in immature compared to mature testis stages. this finding is similar to what has been observed in other species including Japanese flounder , medaka , wrasse (Halichoeres trimaculatus)  and rice field eel (Monopterus albus) . Although numerous studies are implying the importance of Gsdf in spermatogenesis in teleosts, including salmonids, the exact function(s) is not yet clear. 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.
Limited information exists on the role of gsdf in females. Targeted disruption of gsdf in medaka causes sterility (abnormal folliculogenesis with restrained oocyte growth and maturation) in 33 out of 38 females. Here it was suggested that Gsdf is involved in proliferation and differentiation of early primordial follicle formation . Another study showed that in zebrafish gsdf transcripts are present in somatic cells of bipotential gonads, and later during adult stages in both ovarian granulosa and testicular Sertoli cells. Furthermore, gsdf mutant males are fertile, while mutant females gradually become sterile. The authors suggested that Gsdf regulates ovarian follicle maturation and expression of genes involved in steroid biosynthesis, obesity, diabetes, and female fertility . Lack of Gsdf in medaka has been linked to a dysregulation of Igf2bp3-mediated oocyte development . In coho salmon (Oncorhynchus kisutch) ovarian tissue, gsdf transcript levels are significantly increased during early secondary growth (cortical alveoli are abundant, but oil drops have not yet appeared). The authors speculated that Gsdf may be involved in granulosa cell proliferation . The current study shows a clear gsdf expression in adult Atlantic salmon ovarian tissue, specifically localized to granulosa cells in both previtellogenic and vitellogenic follicles. The gsdf mRNA localization to ovarian somatic cells is further confirmed by the presence of gsdf transcripts in GCF ovaries, although at a lower level. Based on previous suggestions that Gsdf is involved in teleost folliculogenesis, and our observation of gsdf transcripts specifically located in granulosa cells, we speculate that Gsdf plays a similar role Atlantic salmon folliculogenesis.
The identity of inha was confirmed by chromosomal synteny to other fish species and by its close phylogenetic relationship within teleost species. Inha is the specific α-subunit which together with a β-subunit (βA or βB) comprise Inhibin A or B protein complexes . Inhibins are, as mentioned above, secreted TGF-β proteins. In mammals, inhibins are produced by Sertoli cells in the testis and granulosa cells in the ovary in response to Follicle stimulating hormone (FSH) (which regulates spermatogenesis and oogenesis), and subsequently target pituitary gonadotropic cells to reduce the synthesis and secretion of FSH . A loss-of function study in mice has demonstrated that Inha acts as a negative regulator of gonadal stromal cell proliferation . In rats, inha has been shown to be expressed in several extragonadal tissues, suggesting diverse functions in the body . In agreement with our findings in salmon, inha expression was restricted to the gonads in zebrafish, suggesting that this gene is more confined to reproduction in fish .
Further characterization of inha in salmon revealed that the transcript level increased during puberty in both females and males, suggesting a role in sexual maturation. In the case of females, this is supported by studies where inha expression increased during folliculogenesis and peaked during final oocyte maturation in zebrafish  and coho salmon . Studies on rainbow trout  and zebrafish  females have shown that inha expression is restricted to the somatic follicle cells surrounding the oocytes, which agrees to what was observed in the current study. Similar spatiotemporal transcript expression patterns suggest a similar functional role for Inha in the different species. One suggestion to how Inha functions in fish oogenesis was presented by a study on zebrafish performed by Li and Ge , where oocyte-derived Bone Morphogenetic Proteins (BMPs), including zfBMP2b and zfBMP4, suppressed mRNA levels of all activin β subunits, but stimulated the expression of inha and follistatin (fst). The full-grown oocyte (where inha expression peaks ) may release BMPs, which in turn stimulates inhibin production, as a signal to the brain-pituitary-gonadal axis of its readiness to mature. Inhibin may in turn suppress the activity of pituitary Activin to inhibit Fsh and increase Lh production .
In salmon males, inha was expressed in Sertoli cells of the testis. Likewise, in rainbow trout it has been shown that inha transcripts are located to Sertoli cells, but expression was also detected in Leydig cells . In the current study it was difficult to distinguish Leydig cells from the clearly stained and numerous Sertoli cells; thus, a conclusion could not be made on whether the Leydig cells contained inha transcripts. Limited information exists on the function of Inha in teleost testis, however some insight has been generated from studies in rainbow trout. It was shown that testicular inha expression is upregulated by Fsh and Lh , and that the response in inha transcript levels to Fsh is mediated through the production of sex steroids . This interaction of inha transcript levels with Fsh and sex steroids in trout, together with the localization of inha mRNA in Sertoli cells and increase of testicular inha expression during spermatogenesis (this study), suggest an essential role of Inha in spermatogenesis in salmonids.
Genes with expression preferentially in germ cells
In our dataset we also had the opportunity to identify transcripts confined to germ cells, based on their expression in WT and absence in GCF testis. We cannot rule out however, that some of these genes may be expressed in gonadal somatic cells, but are downregulated as a response to the absence of germ cells. Nevertheless, numerous candidates were identified [Additional File 2], including several transcripts encoding well-known germ cell specific proteins such as Piwi, Dazl and Tdrd6. We did not assay this group of genes in detail, however KEGG pathways annotated to the genes revealed that several of them are involved in lysine degradation and complement and coagulation cascades. 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 enrichment 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 associated with blood activity.